Ecologically-sustainable fertility management for the...
Transcript of Ecologically-sustainable fertility management for the...
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Ecologically-sustainable fertility management for the maintenance of species-rich hay meadows a 12 year fertilizer and lime experiment Francis W Kirkham1 Robert M Dunn2 Jerry RB Tallowin2 Anne Bhogal3 Brian J Chambers3 and Richard D Bardgett4 1Ecological Research amp Consultancy Far View Nymet Rowland Crediton Devon EX17 6AL UK 2Rothamsted Research North Wyke Okehampton Devon EX20 2SB 3ADAS Gleadthorpe Meden Vale Mansfield Nottingham NG20 9PF UK 4Lancaster Environment Centre Lancaster University Lancaster LA1 4YQ UK
Summary
1 The aim of this study was to determine vegetation and soil microbial responses to
fertilizers and lime applied in a 12 year study at species-rich upland (Cumbria UK) and
lowland (Monmouthshire UK) mesotrophic hay meadows
2 Treatments were 6 12 or 24 tonnes farmyard manure (FYM) ha-1
applied annually or
triennially inorganic fertilizers giving equivalent amounts of N P and K to 12 or 24 t
FYM ha-1
applied annually or triennially and lime applied either alone or with 12 t FYM
ha-1
applied annually or triennially
3 Annual FYM at 24 tonnes ha-1
reduced species-richness and the richness of positive
indicator species at both meadows and increased the proportional cover of negative
indicator species while 12 t FYM ha-1
applied annually was detrimental at the lowland
species-rich meadow but not at the upland meadow Inorganic fertilizers were no more
damaging to plant species-richness than equivalent FYM treatments High vegetation
quality was maintained by FYM at le12 t ha-1
year-1
in the upland meadow where such
levels had previously been used with some improvement in botanical diversity at lower
levels but modeling suggested that only levels of about le 4 tonnes FYM ha-1
year-1
were
sustainable at the lowland meadow with no recent history of fertilizer use Fertilizers of
either type had no detectable impact on the soil microbial communities
4 Between-meadow differences in response to the treatments are most likely attributable
to differences in site-specific factors including past management rather than to innate
differences in plant community type although there was little evidence of vegetation
adapting to initially-damaging fertility inputs
5 Synthesis and applications Knowledge of soil physical and chemical status and past
fertility management is important in deciding what level of fertilizer use might be
tolerable Relatively modest inputs can reduce the ecological value of sensitive vegetation
in meadows with no recent history of such inputs whereas moderate inputs of fertilizer
and lime are likely to be ecologically sustainable where there is a long history of such
inputs Inorganic fertilizers are no more damaging than FYM when applied at equivalent
amounts of N P and K
Correspondence author Emailfranciskirkhamgmailcom
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Key-words farmyard manure inorganic fertilizer vegetation composition hay
meadows species-rich grassland
Introduction
Widespread losses of species-rich grasslands occurred throughout Britain and mainland
Europe during the second half of the 20th
Century due to agricultural intensification
(Fuller 1987 Ellenberg 1988 Bakker 1989) with associated severe declines in many
once common plant and animal species (Ratcliffe 1977 1984 Fuller 1987 Rich amp
Woodruff 1996 Goulson et al 2005) These losses many of which have been
associated with increased use of artificial fertilizers (Ellenberg 1988 Bakker 1989
Berendse et al 1992 Myklestad amp Saeligtersdal 2004) have made it vitally important both
to develop methods for restoring diversity to species-impoverished grasslands (Ormerod
2003) and to define appropriate vegetation management to maintain those high nature
value grasslands that remain or that have been created
Cynosurus cristatus ndash Centaurea nigra grassland the MG5 community of the British
National Vegetation Classification (NVC ndash Rodwell 1992) is the most widespread
species-rich lowland meadow community in the UK (Blackstock et al 1999) whilst the
MG3 Anthoxanthum odoratum ndash Geranium sylvaticum grassland is the characteristic
hay meadow of the upland fringes of northern England (Rodwell 1992) MG5 grassland
has close affinity to Atlantic and Sub-Atlantic Cynosurion grassland in Europe whilst
MG3 grassland has clear affinities with meadows growing at higher altitudes throughout
northern and central mainland Europe (Rodwell et al 2007)
Communities such as these are the result of traditional management maintained over a
long period of time A combination of hay cutting and grazing is the most common form
of vegetation management in both MG3 and MG5 habitats although some MG5
communities are managed by extensive grazing only (Smith amp Jones 1991 Rodwell
1992 Smith amp Rushton 1994 Smith et al 1996 Crofts amp Jefferson 1999 Jefferson
2005) Use of farmyard manure (FYM) and occasional liming are also traditional
practices for hay meadows (Smith 1988 Simpson amp Jefferson 1996 Tallowin 1998
Crofts amp Jefferson 1999 Jefferson 2005) but what constitutes sustainable practices to
maintain the nature conservation value of some species-rich communities is ill defined
There is a large body of evidence showing the detrimental effects of inorganic fertilizers
on species-rich meadow vegetation (eg Berendse et al 1992 Mountford Lakhani amp
Kirkham 1993 Kirkham Mountford amp Wilkins 1996 Silvertown et al 2006) and on
components of the soil microbial community including decomposer and arbuscular
mycorrhizal fungi which are known to play key roles in nutrient cycling and plant
nutrition in species-rich grasslands (Bardgett amp McAlister 1999 Donnison Griffith amp
Bardgett 2000a Donisson et al 2000b) However no previous study has examined the
impacts of FYM treatments on vegetation and soil microbial communities when matched
with inorganic fertilizers supplying equivalent amounts of inorganic nitrogen (N)
phosphorus (P) and potassium (K) Changes in farming practice in the UK have reduced
the availability of FYM so there is a need to ascertain if or when inorganic fertilizers
can be suitable alternatives
These questions need to be resolved as a basis for refining management guidelines
applied within statutory sites and agri-environment schemes for the conservation of
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biodiversity within existing species-rich semi-natural plant communities and within
meadows that have undergone restoration of biodiversity
An experiment involving several FYM or matched inorganic equivalent fertilizer
treatments and treatments incorporating initial liming with or without annual or
intermittent (triennial) FYM applications was established in 1999 on in species-rich
meadows at a lowland site in Monmouthshire UK and an upland site in Cumbria UK
Treatment effects on botanical composition were monitored annually to address the
following specific hypotheses for species-rich meadows
H1 ndash increasing rates of fertilizer reduce species richness and nature value
H2 ndash inorganic fertilizer and FYM treatments supplying equivalent amounts of N P and
K differ in their effects on the botanical composition
H3 ndash fertilizers (whether inorganic or FYM) applied triennially differ in their impact on
botanical composition compared with correspondingly lower amounts applied annually
H4 ndash applying lime to raise soil pH to 60 with or without annual or triennial FYM is
consistent with the maintenance or enhancement of vegetation quality
H5 ndash applying FYM or inorganic fertilizers over a period of 12 years will alter the
composition of soil microbial communities
Preliminary agronomic and botanical results have been reported elsewhere (Tallowin et
al 2002 Kirkham et al 2002 Kirkham et al 2008) This paper describes botanical and
soil microbial responses over the period 1999-2010 Nomenclature of vascular plants
follows Stace (2010) except for NVC community names which follow Rodwell (1992)
Materials and methods
EXPERIMENT SITES
Experimental plots were established in 1999 in two agriculturally unimproved meadows one at
an upland site at in Cumbria North West England (Raisbeck) and the other at a lowland site in
Monmouthshire South Wales (Pentwyn) The upland meadow is located at 54ordm 27rsquo N and 2ordm 34rsquo
W (National Grid Reference NY635069) near the village of Orton whilst the lowland meadow is
close to Monmouth at 51ordm 46rsquo N and 2ordm 41rsquo W (NGR SO524093)
Annual and seasonal rainfall
Average rainfall differed between the two areas 1999-2009 means (recorded at Meteorological
Office weather stations at Newton Rigg in Cumbria (NGR NY493308) and Ross-on-Wye (NGR
SO598238) in Monmouthshire) were 982 mm and 797 mm for annual rainfall for the upland and
lowland sites respectively and 525 mm and 462 mm respectively for the growing season (April-
October) These values were somewhat higher than the longer term (1980-2009) averages - by
3 and 6 for annual and seasonal rainfall respectively at the upland site and by 8 and 13
respectively at the lowland site
Plant communities
The vegetation of the upland meadow in 1999 (Kirkham et al 2002) corresponded to the MG3b
(Anthoxanthum odoratum-Geranium sylvaticum grassland Briza media sub-community) of the
NVC whilst that at the lowland meadow was close to both MG5a (Cynosurus cristatus-
Centaurea nigra grassland Lathyrus pratensis sub-community) and MG5c (Danthonia
decumbens sub-community) although lacking some of the preferential species differentiating
MG5c from the MG5a (Rodwell 1992)
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Past management
Both meadows had previously been managed over a long period by cutting for hay with aftermath
grazing Cutting occurred after 1 July at the upland meadow and after the second week in July at
the lowland meadow The upland meadow had received about 12 t FYM ha-1
each year usually in
late April but occasionally in mid summer following the hay harvest and had received periodic
applications of lime (amounts not known) the last one in about 1993 Details of past fertilizer
application at the lowland meadow are uncertain although this site had not received any form of
fertilizer or lime in the preceding 20 years or more there is some evidence to suggest that basic
slag and lime may have been applied at some time before that
Soil properties
Soils at the upland meadow were clay-loam in texture whilst those at the lowland meadow were
a mixture of sandy silt loam to silty clay-loam The upland meadow soils were notably higher in
total N carbon and organic matter (Table 1)
Table 1 Soil chemical properties (top 75 cm) at experimental sites in Cumbria (the upland
meadow) and Wales (the lowland meadow) in 1999
Upland meadow Lowland meadow
Mean Range Mean Range
pH 518 512-527 501 494-511
Total N 065 059-071 034 029-039
C 676 615-776 315 266-351
Organic matter 1718 1577-2069 903 801-973
Olsen extractable P (mg l-1
) 550 444-679 512 410-600
Exchangeable K (mg l-1
) 1971 1629-2479 1747 1323-2205
Exchangeable Mg (mg l-1
) 1548 1368-1957 2014 1606-2389
Exchangeable Ca (mg l-1
) 1480 1334-1723 1047 852-1214
Measured by loss on ignition (LOI)
EXPERIMENTAL DESIGN
All experimental treatments (see Table 2) were applied by hand to individual 7m x 5m plots laid
out in a randomized block design with three replicate blocks at each site Treatments were applied
between March and late April Due to access restrictions resulting from the national outbreak of
Foot and Mouth Disease (FMD) no treatment was applied in 2001 This meant that treatments
requiring annual applications only received a total of 1112 (92) of the intended amounts over
the 12 year period 1999-2010 Triennial treatments were applied in 1999 2002 2005 and 2008
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Table 2 Treatments applied 1999-2010 at the upland and lowland meadows Values are the mean
amounts applied (kg ha-1
year-1
elemental N P and K) either as FYM (estimated) or in inorganic
form (actual) averaged over 12 yearsa 1999-2010 Treatments 13-15 were limed only in both
1999 and 2005 at the upland site but in 1999 only at the lowland site Treatments 2-12 received
lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Upland meadow Lowland meadow
Treatment N P K N P K
1 Untreated control 00 00 00 00 00 00
2 Limed (in 2005) control 00 00 00 00 00 00
3 FYM at 6 t ha-1
annual 44 50 345 29 38 292
4+ FYM at 12 t ha-1
annual 88 100 689 57 76 585
5 + FYM at 24 t ha
-1 annual 176 199 1378
114 151 1169
6 FYM at 6 t ha-1
triennially 15 22 113 09 13 110
7 + FYM at 12 t ha
-1 triennially 30 45 226
19 26 221
8 + FYM at 24 t ha
-1 triennially 61 90 452
38 52 441
9 + Inorg equivalent to Tr 4 85 84 689 61 63 585
10 + Inorg equivalent to Tr 5 170 168 1378
121 125 1169
11 + Inorg equivalent to Tr 7 30 36 226
24 21 221
12 + Inorg equivalent to Tr 8 61 72 452
48 42 441
13 Lime in years 1 (and 7) 00 00 00 00 00 00
14 Lime as Tr 13 + FYM as Tr 4 88 100 689 57 76 585
15 Lime as tr 13 + FYM as Tr 7 30 45 226 19 26 221 aNote that no fertilizer was applied in 2001 so that for annual treatments the amounts shown
are 1112ths of the mean amount applied in each of the remaining years
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime
in 1999 x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
The FYM used in the experiment was sourced locally from the same farms each year and was
sampled at the time of application for subsequent chemical analysis The ADAS Manure Nitrogen
Evaluation Routine (MANNER Chambers et al 1999) was used between 1999 and 2006 to
predict the amount of plant available N supplied by the FYM after which a refined model
(MANNER-NPK Nicholson et al 2010) was used Refinements to the model included more
detailed N mineralisation functions and predictions of N availability in the year following
application Availabilities of 60 and 90 of total P and K respectively (Anon 2010) were
assumed between 1999 and 2006 and this assumption for K was maintained throughout the 1999-
2010 period From 2007 onwards the assumed availability of P from FYM was increased to 80
of total P in line with information on longer term release of plant-available P from FYM (Smith
et al 1998) For both N and P the mean amounts assumed to have been applied in FYM
treatments averaged over 1999-2010 and shown in Table 2 are based upon revised estimates for
the whole period The inorganic fertilizer N treatments applied each year ie the inorganic
lsquoequivalentsrsquo for specific FYM treatments (see Table 2) were based upon estimates made using
the original MANNER model from 1999-2006 and MANNER-NPK from 2007-2010 Similarly
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the inorganic fertilizer P applied each year corresponded to 60 of the total P in FYM between
1999 and 2006 and 80 from 2007-2010
Inorganic N P and K were applied as ammonium nitrate triple super-phosphate and muriate of
potash respectively to the relevant treatments The FYM treatments were designed to encompass
the range of inputs traditionally applied to species-rich hay meadows upon which current agri-
environment guidelines are based (Simpson and Jefferson 1996 Crofts and Jefferson 1999)
Lime was applied to treatments 13 14 and 15 only at each site in March-April 1999 and to most
treatments in 2005 (Table 2) The latter included the three previously limed treatments at the
upland meadow but not at the lowland one since soil pH levels had not fallen significantly by
2005 on these plots at the latter site Based on assessments of soil texture and soil pH tests
conducted in February-March the amounts of lime were calculated to be sufficient to raise the
soil pH of the experimental plots to 60 (Anon 2010)
At each site one plot per replicate block received no fertilizer or lime (untreated control)
between 1999 and 2005 whilst another plot per replicate was designated as a continuation of past
fertilizer inputs At the lowland meadow the latter was identical to the untreated control
treatment but at the upland meadow it was identical to Treatment 4 (12 t ha-1
FYM annually) In
2005 a decision was made to treat occasional liming as lsquobackgroundrsquo management for all
fertilizer treatments at both sites to be triggered for each treatment in future years once mean pH
for that treatment had declined to 55 whilst retaining both an untreated control (ie nil fertilizer
nil lime ndash Treatment 1) and a nil fertilizer (limed in 2005) control treatment (Treatment 2) at each
site The rationale behind this decision is addressed in the Discussion section At the lowland
meadow one of the two hitherto untreated control plots per replicate (Treatment 2) was therefore
limed in 2005 along with other treatments Only one nil input plot per replicate was available at
the upland meadow but since the site had a history of liming the existing nil input plots were
limed in 2005 (Treatment 2) and new untreated control plots (Treatment 1) were established
adjacent to each replicate block with the end at which each was located chosen randomly for
each block
The experimental plots were cut for hay after 1 July each year at the upland site and after 15
July at the lowland site the actual cutting date being dependent on weather conditions The hay
aftermath growth was grazed each year with experimental plots being grazed with the remainder
of the field At the upland meadow mature sheep were used normally commencing in September-
October continuing until March or late April The lowland meadow was grazed by beef store
cattle from mid Octoberearly November to late February
Botanical assessments were carried out during May each year except in 2001 at the upland
meadow due to FMD restrictions The percentage cover of each species present was estimated
visually in three 1m2 quadrats positioned at random within each plot in 1999 and then fixed for
the project duration
Soils were sampled in March 1999 2002 2004 2007 and 2010 by taking five 35 mm diameter x
75mm deep soil cores at random from within each treatment plot but outside the fixed positions
of the three 1m2 botanical survey quadrats The five samples per plot were combined dried at
30oC and then ground prior to analysis for organic carbon (C) total nitrogen (N) Olsen
extractable P exchangeable potassium (K) magnesium (Mg) calcium (Ca) sodium (Na) and pH
(in H2O) using standard laboratory methods (Allen 1974 MAFF 1986)
Soils were further sampled in June 1999 2002 2004 2007 and 2010 for soil microbial
assessment by taking five 35 mm diameter x 100 mm deep cores which were subsequently bulked
for each plot sieved (5 mm) and stored at 4oC prior to analysis Microbial community structure
was assessed using phospholipid fatty acid analysis (PLFA) as described by Bardgett et al
(1996) The fatty acids i150 a150 150 i160 170 i170 cy170 cis181ω7 and cy190 were
chosen to represent bacterial PLFAs (Federle 1986 Frostegaringrd Tunlid amp Baringaringth 1993) and
182ω6 was used as an indicator of fungal biomass (Federle 1986) The ratio of 182ω6bacterial
7
PLFAs was taken to represent the ratio of fungal-to-bacterial biomass in soil (Bardgett Hobbs amp
Frostegaringrd 1996 Frostegaringrd amp Baringaringth 1996)
DERIVED BOTANICAL VARIABLES
Estimates of cover for individual species were converted to a percentage of the total live
vegetation cover present in each quadrat in order to minimize the effects of year-to-year variation
and variation between treatments in vegetation density and total cover Several composite
variables were calculated to characterize the vegetation and these were then averaged across the
three quadrats in each plot to give plot mean values for each variable Plot means were then used
as the basic units for subsequent analyses of treatment effects
Key variables
Five key variables were used to investigate the effects of treatment and time on botanical
composition total species-richness (the total number of vascular plant species per m2) the
number per m2 of MG3 and MG5 positive indicator species (the upland and lowland meadows
respectively) the aggregate cover of these species as a percentage of total vegetation cover the
number per m2 of negative indicator species and the aggregate cover of negative indicator
species as a percentage of total vegetation cover Positive mesotrophic indicator species were
identified from lists produced by Robertson and Jefferson (2000) for use in monitoring the
condition of grassland Sites of Special Scientific Interest (SSSIs) in MG3 (Anthoxanthum
odoratum-Geranium sylvaticum grassland) and MG5 (Cynosurus cristatus-Centaurea nigra
grassland) communities of the NVC (Rodwell 1992) A single list of negative indicator species
was compiled from the generic negative indicators for MG3 and MG5 communities (Robertson amp
Jefferson 2000) augmented by species shown in previous work (eg Mountford et al 1993
Kirkham et al 1996) to be favoured by nutrient addition and for which an increase in abundance
would indicate a negative effect on plant community quality Positive and negative indicator
species are listed in Supporting Information Table S3
Additional variables
Among other derived variables was the weighted Ellenberg N score (Smith et al 2003 Kirkham
et al 2008) referred to hereafter as the fertility score The Ellenberg N index represents the
degree of association with soil fertility (not specifically soil N) of a particular species (Ellenberg
1988 Hill et al 1999) Fertility scores were calculated for each quadrat as the average N index
of the component species weighted according to the proportional contribution of each species to
total vegetation cover Plot mean fertility scores were highly correlated at both sites with the
aggregate cover of negative indicator species (NI) (Pearson r=0845 and r=0927 at the upland
and lowland sites respectively in 2010 both Plt0001) The latter variable was preferred to
fertility scores for statistical analyses in this paper because it proved more responsive to fertilizer
treatments although fertility scores are referred to in the Discussion section in relation to results
from elsewhere
DATA ANALYSIS
ANOVAs for treatment effects
For each of the key variables botanical data for all years 1999-2010 except 2001 at the upland
meadow and for all treatments except Treatment 1 at the same meadow were analyzed by
repeated measures analysis of variance (ANOVA - Genstat V Committee 1997) with Year as the
repeated measures factor Separate ANOVAs were carried out for each variable within each site
using three separate ANOVA models in each case In each model variation between replicate
8
blocks was accounted for by treating replicate as a blocking factor The first model included all
the treatments except Treatment 1 at the upland meadow whilst the other two models used
balanced factorial subsets (series) of treatments (see Table 2) the Form x Rate x Frequency
(FRF) series and the Lime x FYM frequency (LFF) series
The FRF series tested the effects of form of fertilizer (ie FYM or inorganic) the rate at which
fertilizers were applied (12 or 24 t FYM ha-1
and inorganic treatments corresponding to these)
the frequency at which treatments were applied (ie annual or triennially) and all two- and three-
way interactions between these factors
The LFF series tested the effects of liming regime ie lime in 1999 (and 2005 at the upland site)
versus lime in 2005 only the frequency of FYM application (ie nil annual or triennially) and the
Lime x FYM frequency interaction All FYM treatments within this series were applied at 12 t ha-
1 At both sites the LFF series consisted of Treatments 2 4 7 13 14 and 15 (see Table 2) The
liming comparison therefore differed slightly between the two sites since liming was repeated in
2005 on plots previously limed in 1999 at the upland meadow but not repeated at the lowland
one
In both the LFF and the FRF models for each variable the first stratum of the ANOVA
consisted of the effects of all factors and interactions averaged over time with the same factors
and interactions repeated in the second stratum with time as an additional factor
In all repeated measures ANOVAs to account for the repeated measures aspect all degrees of
freedom in the Year x Treatment stratum were adjusted to account for departure from
homogeneity of the population covariance matrix using ε-coefficients calculated by the
Greenhouse-Geisser method (Genstat V Committee 1997)
In most years including 2010 all species were recorded individually but at both sites
bryophytes were recorded only as a single group in some years This had only a small influence
on the data since there were seldom more than two bryophyte species present in a quadrat and
more often less Nevertheless for consistency between years bryophytes were treated as a single
group in all years when calculating total species-richness for data for repeated measures
ANOVAs
In addition to the above analyses 2010 data for the key variables were tested alone for treatment
effects by ANOVA and by analysis of covariance (ANCOVA) the latter using 1999 data as the
covariate (Genstat 5 Committee 1997) All three models as described above were tested The
ANCOVAs for the upland meadow excluded Treatment 1 (untreated control plots established in
2005) Where an ANCOVA showed significant treatment covariance with 1999 data the adjusted
results were used In other cases the unadjusted analyses were used Individual bryophyte species
were included in the calculations of species-richness at both sites in these analyses
In all analyses in order to satisfy the conditions for ANOVA (normality of residuals and
homogeneity of residual variation) all percentage data were transformed before analysis by
arcsine(radicp) where p is the percentage value expressed as a proportion All results quoted for
percentage data refer to analyses of arcsine-square root transformed data
Mixed modeling
Mixed model analysis (Residual Estimated Maximum Likelihood ndash REML Genstat V
Committee 1997) was used to identify any effect of form rate or frequency of application on
vegetation composition at each site in 2010 not simply attributable to the total (or mean per year)
amount of fertilizer applied over the study period A variable representing this amount was
included as a continuous explanatory variable in each analysis along with other fixed factors
form rate and frequency of application (Block was included as a random factor) Since the total
(or mean) amounts of N P and K applied over the whole period were completely correlated
across treatments at each site it was not feasible to include all three elements as separate terms in
the model Nor was it feasible to simply use a single notional variable for fertilizer amount (eg t
9
FYM ha-1
year-1
or inorganic equivalent) since there was lack of equivalence of N and P supply
(but not K) between individual FYM treatments and their corresponding inorganic treatments For
each dependent variable tested two separate models were therefore developed using either N or
P ha-1
year-1
as the continuous variable In each case the presence of any non-linearity in response
was tested by including a quadratic term (ie N2 or P
2) or where more appropriate by logn-
transformation of the explanatory variable
For each analysis an initial model was constructed that included all five main effects and all
interactions The importance of each term was evaluated initially by a χ2 test on the Wald statistic
(Genstat V Committee 1997) The number and order of terms in the model were varied but with
N or P amount always included until a model was developed in which each term was significant
The model was then refined by testing the contribution of each term to the model by the more
conservative deviance test ie a χ2 test on the difference in deviance between the model
containing the term and a sub-model from which it was dropped (Welham amp Thompson 1997)
Where there was an interaction between form of fertilizer and the quadratic term but no overall
effect of form - implying that the difference between the two forms of fertilizer lay primarily in a
difference in the curvilinearity of response - a refined model was tested with the P2 (or N
2) x
Form term replaced by Form nested within the quadratic term (eg P2Form) This has the effect
of absorbing the overall effect of form of fertilizer within the interaction term and returns the two
terms P2 and P
2 x Form (Galwey 2006)
These modeling approaches were used with total species-richness and PI species-richness as
(separate) response variables and included data for all treatments limed in 2005 but not in 1999
(ie Trs 2-12)
Results
TEMPORAL CHANGES 1999-2010
Changes over time in total species-richness (number per m2) richness and proportional
aggregate cover of positive indicator (PI) species and proportional aggregate cover of
negative indicator (NI) species at the upland and lowland meadows are represented in
Figs 1 and 2 respectively for ten of the fifteen treatments applied Repeated measures
ANOVA statistics for each of the four variables are given in Supporting Information
Tables S4 S6 S8 and S10 respectively for the upland meadow and Tables S12 S14 S16
and S18 respectively for the lowland meadow Corresponding mean values for each
treatment in each year are given in Tables S5 S7 S9 S11 S13 S15 S17 and S19
respectively Changes in individual species abundance contributing to the temporal trends
in aggregated PI and NI species described below are outlined under Supporting
Information
The upland meadow
The overall effects of both treatment and time were highly significant in the repeated
measures ANOVA for total species-richness at the upland meadow (both P=0001) but
there was no treatment x time interaction (P=0647)
After an initial decline between 1999 and 2000 species-richness remained fairly
constant for most treatments until 2003 after which there was a general increase with the
notable exception of the highest rate annual FYM treatment (Tr 5) (Fig 1a) The latter
treatment declined further between 2005 and 2007 after which it appeared to recover
slightly and averaged over all years this treatment was significantly less species-rich than
all others (Plt001 compared to Tr 14 Plt0001 the remainder) None of the remaining
10
treatments differed significantly from the limed control treatment (Tr2) averaged over all
years However the more species-rich treatments ie nil fertilizer plus lime applied in
both 1999 and 2005 (Tr 13) low rate annual and medium rate triennial FYM (Tr 3 and 7
respectively) and the inorganic equivalent to the latter (Tr 11) were all more species-rich
averaged over all years (Plt005) than annual FYM at the medium rate under either
liming regime (ie Trs 4 and 14 the former representing a continuation of past
management)
The effect of the high rate annual FYM treatment (Tr 5) was largely responsible for
several main effects within the repeated measures ANOVA of treatments within the Form
x Rate x Frequency (FRF) factorial series namely overall effects (averaged over all
years) of rate (mediumgthigh P=0002) and frequency (triennialgtannual P=0001) of
application and a not quite significant effect of form of fertilizer (FYMltinorganic
P=0057) Both the form x rate and the form x frequency interactions were significant
(both Plt005) high rate FYM was significantly less species-rich than all other
combinations averaged over annual and three yearly treatments and all years (Plt0001
compared to medium rate FYM Plt001 compared to both medium and high rate
inorganic fertilizer) and annual FYM was less species-rich than three yearly FYM and
than inorganic fertilizer either annually or triennial (all Plt0001) averaged over the two
application rates and all years The rate x frequency interaction was not significant
(P=0105) but there was a marginally non-significant three-way interaction between
form rate and frequency (P=0070) with a low mean for high rate annual FYM (Tr 5
211 species m-2
) contrasting markedly with all other treatments within the series
particularly with low rate triennial FYM (Tr 6 267 species m-2
)
As with the full treatment ANOVA the overall time factor was highly significant
(Plt0001) but only the effect of application frequency varied significantly with time
(interaction Plt005) Species-richness declined slightly with annual treatments overall
and increased slightly with triennial treatments such that differences between these were
significant in all years from 2005 onwards (Plt001 for 2006-2009 Plt005 in 2005 and
2010)
Both the richness and aggregate cover of PI species (PI) increased generally with time
(Fig 1b and 1c) although in both cases there was a progressive separation between
species The temporal trend was particularly marked for PI although the time factor
was highly significant (Plt0001) in both ANOVAs The overall treatment effect was
marginally non-significant for PI richness (P=0069) with no indication of a time x
treatment interaction (P=0703) whereas for PI there was both a significant treatment
effect (P=0002) and a marginally non-significant treatment x time interaction (P=0075)
Mean PI was significantly lower averaged over all years for annual FYM treatments at
both 12 and 24 t ha-1
(ie Trs 4 14 and 5) compared to several other treatments notably
nil fertilizer treatments limed either in 2005 only (Tr 2 limed control) or in both 1999
and 2005 (Tr 13) (all differences Plt0001 except Tr 2 vs Tr 4 Plt001) and triennial
inorganic treatments (Trs 11 and 12 Plt001 except for Tr 4 vs Tr 11 Plt005) Within
the FRF series ANOVA the increase in PI over time was greater with triennial
treatments compared to annual averaged over fertilizer forms and rates of application
(time x frequency interaction P=0016) with a marked overall difference due to
application frequency averaged over forms rates and all years (P=0005)
11
PI species-richness showed a similar overall effect of application frequency within the
FRF series (triennialgtannual P=0013) and also a significant overall effect of rate of
application (medium rategthigh rate P=0022) The overall effect of form of fertilizer was
marginally non-significant (FYMltinorganic P=0059) and there was no significant
interaction among any of the three factors averaged over all years nor any interaction
with time
16
18
20
22
24
26
28
30
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Specie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime in Year 1+ ann FYM (Tr 14) Nil fert + Lime in Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr1)
a Species richness (total)
3
4
5
6
7
8
9
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
70
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
0
5
10
15
20
25
30
35
40
45
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
Fig 1 Changes 1999-2010 in vegetation composition an upland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control
The mean number of negative indicator (NI) species per m2 averaged over all treatments
varied significantly over time at the upland meadow (P=0002 data not shown) though
with no consistent trend - it was lowest overall in 2002 (499 per m2) and highest in both
1999 and 2008 (548 per m2) The treatment x time interaction was not significant
(P=0469) but the overall treatment effect was highly significant (Plt0001) mean
number of NI species per m2 was higher than the limed control (Tr 1 453 per m
2) with
all fertilizer treatments except Treatments 11 (triennial inorganic fertilizer at the lower
rate) and 13 (nil fertilizer limed in both 1999 and 2005) (Plt0001 for all except Trs 5
and 6 for which Plt001) The highest mean NI m-2
value was for high rate inorganic
12
fertilizer (Tr 10 565 per m2) which differed significantly (Plt005) from its FYM
counterpart Treatment 5 There was however no consistent difference between FYM
treatments and their inorganic counterparts and no overall effect of form of fertilizer
within the FRF series (P=0745) There was a significant form x rate interaction within
this ANOVA (P=0032) but only the difference between the high rate and lower rate
inorganic means was significant (high rategtlower rate Plt001)
The aggregate contribution to vegetation cover of negative indicator species (NI)
showed a very marked divergence between treatments over time (Fig 1d) with NI
either changing little or declining under most treatments again with the notable exception
of high rate annual FYM (Tr 5) under which NI increased over time Time treatment
and the treatment x time interaction were all highly significant (Plt0001) The
predominant temporal trend was downward (from 25 of live cover in 1999 to 14 in
2010 averaged over all treatments) but NI for Treatment 5 had significantly increased
compared to 1999 in 2002 2003 (Plt005) 2005 (Plt0001) 2008 (Plt0001) and 2009
(Plt001) but not in 2010 (Fig 1d) The most marked declines in NI occurred on the
limed control (Tr 2) nil fertilizer plus lime in both 1999 and 2005 (Tr 13) and the lower
rate triennial inorganic fertilizer treatments (Tr 11) differences compared to both 1999
and 2002 were highly significant (Plt0001) in most subsequent years for these
treatments A smaller decline also occurred after 2002 on the treatment equivalent to past
inputs (ie12 t ha-1
year-1
annual FYM Tr 4) In most years this treatment differed little
from the equivalent FYM treatment limed in both 1999 and 2005 (Tr 14)
These trends were reflected in highly significant overall effects on NI within the FRF
series of form (FYMgtinorganic) rate (mediumlthigh) and frequency (triennialltannual)
(all effects Plt0001) Time x rate and time x frequency interactions were both significant
(P=0019 and Plt0001 respectively) with a marginally non-significant rate x frequency
interaction averaged over all years (P=0054) NI declined over time under both
application rates averaged over forms and frequencies of application but the decline was
slower with high rate treatments compared to lower rate Difference between rates were
significant in all years from 2002 onwards (Plt005 in 2002 2006 and 2007 Plt0001 in
other years) Overall differences due to frequency of application were even more marked
Under annual application NI declined initially from 2003-2006 compared to 1999 but
subsequently recovered and did not differ from 1999 levels from 2007 onwards whilst
under triennial application differences were highly significant (Plt0001) compared to
1999 from 2003 onwards with significant differences between years during this same
period (Plt005 in 2003 and 2006 Plt0001 in remaining years)
Repeated-measures ANOVAs within the LFF treatment series at the upland meadow
showed no overall effect of liming regime on temporal trends for any of the above
variables nor any interaction of lime with either time or FYM frequency - note that FYM
was applied at the intermediate rate (12 t ha-1
) in all FYM treatments within this series
The overall effect of FYM frequency was significant for both PI (P=0003) and the
number of NI species m-2
(P=0001) PI was highest with nil FYM and lowest with
annual FYM with triennial intermediate and differing significantly (Plt005) from the
other two means NI species m-2
did not differ between triennial and annual application
within the LFF series but both were significantly higher than nil FYM (Plt001 for
triennial Plt0001 for annual application) There was also a significant time x FYM
frequency interaction for NI within this series (P=0001) reflecting a more rapid
13
decline with nil FYM than with triennial FYM and no significant overall trend with
annual FYM when averaged over both lime treatments
12
14
16
18
20
22
24
26
28
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Spe
cie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime Year 1 + ann FYM (Tr 14) Nil fert + Lime Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr 1)
a Species richness (total)
2
3
4
5
6
7
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
0
10
20
30
40
50
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
Fig 2 Changes 1999-2010 in vegetation composition at a lowland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control treatment and those that had received lime in year 1
Treatments 1 and 2 were identical (nil input) until 2005
The lowland meadow
Both time and treatment were significant within the ANOVA for total species-richness at
the lowland meadow (Plt0001 and P=0009 respectively and in contrast to the upland
site the treatment x time interaction was significant (P=0012) Averaged over all years
high rate annual FYM treatment (Tr 5) was significantly less species-rich than all others
except Treatment 14 (annual FYM at 12 t ha-1
limed in 1999) having declined
progressively between 1999 and 2009 (Fig 2a) with most differences significant at
Plt001 Treatment 14 did not differ significantly overall compared to the corresponding
FYM treatment not limed until 2005 (Tr 4) although these two treatments showed
14
markedly different temporal trends Treatment 14 was less species-rich than others during
the first five years of the experiment but did not change significantly throughout the
whole 12 year period whilst Treatment 4 changed little between 1999 and 2006 but
declined thereafter (Plt001 and Plt0001 for 2009 and 2010 respectively compared to
2006) possibly in response to liming in 2005 However there was no significant overall
effect on total species-richness of liming strategy within the LFF series and no interaction
between liming and either time or FYM application
There were marked differences in temporal trend in species-richness between FYM
treatments and their inorganic counterparts particularly with the high rate annual
treatments (Tr 5 and Tr 10) Whereas species-richness declined more or less continually
throughout under Treatment 5 at least until 2009 (Plt001 for 2004 compared to 1999 and
for both 2009 and 2010 compared to 2004) Treatment 10 declined between 2000 and
2001 (Plt005) and then remained relatively unchanged until 2008 after which it declined
again (2010 Plt005 compared to 2008)
Other treatments within the full treatment series including untreated control (Tr 1) and
limed control (Tr 2) showed no particular temporal trend in the full repeated measures
ANOVA although both the latter treatments showed a dip in species-richness in 2004
(Plt005 compared to both 2003 and 2005)
There was a strong overall effect of form of fertilizer within the FRF series ANOVA
(FYMltinorganic P=0003) a significant effect of frequency of application
(triennialgtannual P=0027) and a not quite significant effect of application rate (high
rateltmedium rate P=0064) but no significant interaction between any of these factors
The time x application frequency interaction was highly significant (Plt0001) species-
richness remained unchanged under triennial application but under annual application
was significantly lower compared to 1999 in each year from 2004 onwards with the
exception of 2006 (Plt005 in 2004 Plt001 in 2005 and 2008 Plt0001 the remainder)
Again with the exception of 2006 differences between triennial and annual application
were significant in each year from 2005 onwards (Plt005 in 2007 Plt001 in 2005 and
2008 Plt0001 in 2009 and 2010)
Both treatment time and their interaction were significant in the full treatment ANOVA
for PI species-richness at the lowland meadow (ie time and treatment both Plt0001
time x treatment P=0006) There was a general decline in PI-richness after 2005 (Fig
2b) with PI species m-2
averaged over all treatments lower from 2007 onwards (Plt0001)
than all years up to 2005 except 2001 Although the initiation of this trend corresponded
with the liming of most treatments in 2005 a similar pattern was shown by treatments
that did not receive this lime application ie the untreated control treatment (Tr 1) the
nil fertilizer treatment limed in 1999 (Tr 13) and the corresponding treatment also
receiving triennial FYM (Tr 15) In all these cases PI-richness showed no significant
difference between years until 2005 but was significantly (Plt001) lower by 2010 than in
2005 Treatment 14 (limed in 1999 plus annual FYM) did not show this pattern but PI
species-richness had been lowest under this treatment in most years between 1999 and
2005 (Fig 2b)
As with total species-richness PI richness declined more or less progressively under
Treatment 5 until 2008 (Plt005 compared to 1999 by 2001 Plt0001 from 2006
onwards) whereas Treatment 4 did not change significantly until 2006 after which it
declined (Plt005 in 2007 compared to 2006 Plt001 thereafter) Both these treatments
15
tended be less rich in PI species than the corresponding inorganic treatment (Trs 9 and
10 respectively) in most years consistently so for Tr 4 compared to Tr 9 but these
differences were not significant in any year nor when averaged over all years There was
however a significant overall effect of form of fertilizer in the FRF series ANOVA
(FYMltinorganic P=0002) and a highly significant effect of frequency of application
(triennialgtannual Plt0001) The time x frequency of application interaction was also
highly significant (Plt0001) Under triennial application PI species per m2 increased
slightly until 2005 (Plt005 compared to 1999) and declined thereafter so that by 2009
levels were significantly lower than in 1999 (Plt001 for 2009 Plt0001 for 2010) By
contrast under annual application PI species m-2
declined progressively until 2009
following a temporary though highly significant (Plt0001) dip in 2000 so that the
difference compared to 1999 was significant in every year from 2002 onwards and highly
significant (Plt0001) from 2005 With the exception of 2003 differences between
triennial and annual application were significant from 2002 onwards and highly
significant (Plt0001) in 2005 and from 2007 to 2010
There was no significant effect other than of time within the LFF treatment series
ANOVA although there was a marginally non-significant time x FYM frequency
interaction (P=0054) This reflected a more rapid decline in PI species-richness under
annual FYM application than with either nil or triennial FYM with the latter two showing
no discernible difference
By contrast with PI species m-2
PI cover percentage (PI) showed no overall treatment
effect within the full treatment ANOVA nor a time x treatment interaction although the
overall effect of time was highly significant (Plt0001) The temporal pattern in PI (Fig
2c) also differed from that shown by PI-richness with PI averaged over all treatments
declining between 1999 and 2005 in three highly significant increments (1999-2000
2000-2002 and 2002-2005 all Plt0001) and increasing again thereafter (2005-2009 and
2009-2010 both Plt0001) There was no significant effect within either the LFF or FRF
treatment series ANOVAs although their was a slight time x frequency of application
effect within the latter (P=0081) PI was slightly higher under annual application than
triennial in each year 1999-2004 after which the reverse was true although differences
were always small
There was no overall effect of treatment on the number of NI species per m2 within the
full treatment range at the lowland meadow (data not shown) but both the overall effect
of time and the time x treatment interaction were significant (Plt0001 and P=0009
respectively) Overall NI species m-2
increased over time from 24 species m-2
in 1999 to
a peak of 42 m-2
in 2008 averaged over all treatments NI species m-2
increased on the
untreated control treatment (Tr 1) from 21 species m-2
in 1999 to 34 species m-2
in 2007
(increase Plt001) then declined to the 1999 value by 2010 The limed (in 2005) control
(Tr 2) showed a somewhat similar pattern though from a slightly higher starting level
(31 species m-2
) and with the increase to 38 species m-2
in 2008 not reaching
significance
These temporal trends in NI species m-2
were much accentuated by annual fertilizer
application particularly by FYM treatments with peak numbers of NI species reaching
52 and 47 species m-2
for Treatments 4 and 5 respectively in 2008 (both Plt0001
compared to 1999) and despite slight declines in both cases remaining significantly
higher in 2010 than in 1999 (both Plt0001) Inorganic counterparts to these treatments
16
showed similar though slightly less marked trends and there was no significant
difference between all four treatments in the peak number reached in 2008 There was no
overall effect of either form of fertilizer or rate of application within the FRF series
ANOVA and only a marginally non-significant effect of frequency of application
(triennialltannual P=0061) Liming in 1999 (Trs 13 14 and 15) also appeared to
accentuate the generally increasing trend although peak numbers in 2008 did not differ
between these treatments and either untreated or limed control in the full treatment range
ANOVA The overall effects of liming regime (1999 or 2005) were not significant within
the LFF treatment series but the faster rate of increase with liming in 1999 was reflected
in a significant time x lime interaction in this analysis (P=0017) The mean number of NI
species m-2
increased very significantly (Plt0001) between 1999 and 2000 following
liming in 1999 with the increment increasing until 2008 whilst on plots un-limed until
2005 NI species m-2
did not increase significantly until 2003 (Plt005) although the
increase compared to 1999 was highly significant (Plt0001) in 2006 2008 and 2010
Nevertheless the differences in rate of increase was at least partly due to a lower 1999
mean for the 1999-limed treatments (170 species m-2
compared to 244 species m-2
difference NS) and although in all years from 2001 onwards mean NI species m-2
was
higher for 1999-limed treatments compared to those not limed until 2005 the difference
was not significant in any year
The contribution of NI species to vegetation cover (NI) showed a very marked
separation between treatments over time at the lowland meadow (Fig 2d) starting from
much lower levels than at the upland meadow (compare with Fig 1d) There were
noticeable differences in temporal trend between annual fertilizer treatments on the one
hand and triennial and nil fertilizer treatments on the other (Fig 2d) The effects of
treatment time and the treatment x time interaction were all highly significant (Plt0001)
in the full treatment range ANOVA The most marked increase in NI occurred with
annual FYM at the high rate (Tr 5) with NI increasing rapidly between 1999 and 2001
(Plt001) and a further highly significant increment between 2004 and 2009 (Plt0001)
(Fig 2d) A similar though less marked trend occurred with annual FYM at the medium
rate (Tr 4) with significant increases between 1999 and 2005 and between 2005 and
2010 (both Plt001) NI also increased markedly with the inorganic treatment
equivalent to Treatment 5 (Tr 10) although the overall rate of increase shown by this
treatment was closer to that shown by Treatment 4 than Treatment 5 There were
significant differences between Treatments 5 and 10 in most years from 2004 onwards
(Plt001 and Plt0001 in 2009 and 2010 respectively)
These effects were reflected in a significant overall effect of form of fertilizer
(FYMgtinorganic P=0008) and highly significant (Plt0001) overall effects of both rate
(high rategtlower rate) and frequency of application (annualgttriennial) within the FRF
series ANOVA and a significant form x rate interaction (P=0043) Mean NI was
highest for high rate annual FYM (Plt0001 compared to all three remaining means) and
lowest for lower rate inorganic (Plt005 compared to high rate inorganic NS compared
to lower rate FYM) Only frequency of application interacted significantly with time
(Plt0001) NI increased moderately under triennial application until 2008 (Plt005 in
2005 compared to 1999 Plt0001 each year 2006-2008) followed by a decline (Plt001
2010 compared to 2008) but with mean NI in 2010 still higher than 1999 (Plt005)
The increasing trend was very much more marked with annual fertilizer application for
17
which there were highly significant (Plt0001) increments between 1999 and 2002 and
between 2002 and 2008 but with no significant increase thereafter Differences between
annual and triennial treatment means were highly significant (Plt0001) from 2003
onwards
There was no significant change in NI over time either under the untreated control
(Tr 1) or the nil fertilizer (Tr 2) treatments in the full treatment ANOVA NI increased
until 2005 on plots limed in 1999 without fertilizer Tr 13 (Plt001 compared to 1999)
and declined slightly but not significantly thereafter Trends were similar with the
treatment limed in 1999 and receiving triennial FYM (Tr 15 ndash not illustrated in Fig 2)
although NI was higher with this treatment than with Tr 13 (Plt005) in both 2005 and
2008 By contrast applying the same rate of FYM annually after liming in 1999 (Tr 14)
caused a more rapid increase with levels by 2005 higher than either Tr 13 (Plt001) or
Tr 4 (Plt005) ndash note that the latter was equivalent to Tr 14 but without lime until 2005
NI declined after 2006 under Tr 14 but continued to increase with Tr 4 with the
difference between these two treatments reversed by 2010 (Plt005)
All main effects and interactions were significant in the LFF treatment series ANOVA
for NI except for lime x FYM frequency and the lime x FYM x time interactions Plots
limed in 1999 supported higher NI levels than those limed in 2005 averaged over the
three FYM frequencies and all years (P=0042) NI increased more rapidly after liming
in 1999 than where liming was delayed until 2005 (P=0033 for time x lime interaction)
with the overall difference due to liming in 1999 highly significant (Plt0001) by 2005
NI declined thereafter on the 1999-limed treatments whilst continuing to increase
following liming in 2005 of previously un-limed treatments both peaking in 2008 at a
very similar level Averaged over lime treatments NI increased much more rapidly
under annual FYM application than under either triennial or nil FYM (P=0002 for the
time x FYM frequency interaction) a highly significant increase had occurred by 2002
with annual FYM (Plt0001 compared to 1999) but increases of similar magnitude were
not reached until 2005 with triennial application and not until 2007 with nil FYM NI
subsequently declined under the latter two FYM regimes but not with annual FYM so
that by 2010 NI was significantly higher with annual FYM than with either triennial or
nil FYM (Plt0001) but with only a small non-significant difference between the latter
two treatments
TREATMENT EFFECTS AFTER 12 YEARS OF APPLICATION
The effects of all treatments on the five key variables at each site in 2010 are shown in
Table S20 with treatments differing significantly (Plt005) from the limed control
treatment (Tr 2) highlighted for each variable Treatment effects in 2010 are not
described in detail here but the modeled responses of total species-richness and of
richness of PI species in 2010 to the mean amounts of N and P applied per year averaged
over 1999-2010 are described below
Relationships with the amounts of fertilizer applied
The effect of neither rate nor frequency of application was significant in any of the
REML models for either total species-richness or PI species-richness at either site when
the amount of N or P applied ha-1
year-1
was included The effect of form of fertilizer was
still significant in most models however either as a main effect or in interaction with the
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
References
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Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
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Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
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Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
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Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
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Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
phosphorous vs nitrogen enrichment in plant species loss across North-west Europen
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Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
and losses following application of manures to arable land MANNER Soil Use and
Management 15 137-143
Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
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Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
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Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
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Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
2
Key-words farmyard manure inorganic fertilizer vegetation composition hay
meadows species-rich grassland
Introduction
Widespread losses of species-rich grasslands occurred throughout Britain and mainland
Europe during the second half of the 20th
Century due to agricultural intensification
(Fuller 1987 Ellenberg 1988 Bakker 1989) with associated severe declines in many
once common plant and animal species (Ratcliffe 1977 1984 Fuller 1987 Rich amp
Woodruff 1996 Goulson et al 2005) These losses many of which have been
associated with increased use of artificial fertilizers (Ellenberg 1988 Bakker 1989
Berendse et al 1992 Myklestad amp Saeligtersdal 2004) have made it vitally important both
to develop methods for restoring diversity to species-impoverished grasslands (Ormerod
2003) and to define appropriate vegetation management to maintain those high nature
value grasslands that remain or that have been created
Cynosurus cristatus ndash Centaurea nigra grassland the MG5 community of the British
National Vegetation Classification (NVC ndash Rodwell 1992) is the most widespread
species-rich lowland meadow community in the UK (Blackstock et al 1999) whilst the
MG3 Anthoxanthum odoratum ndash Geranium sylvaticum grassland is the characteristic
hay meadow of the upland fringes of northern England (Rodwell 1992) MG5 grassland
has close affinity to Atlantic and Sub-Atlantic Cynosurion grassland in Europe whilst
MG3 grassland has clear affinities with meadows growing at higher altitudes throughout
northern and central mainland Europe (Rodwell et al 2007)
Communities such as these are the result of traditional management maintained over a
long period of time A combination of hay cutting and grazing is the most common form
of vegetation management in both MG3 and MG5 habitats although some MG5
communities are managed by extensive grazing only (Smith amp Jones 1991 Rodwell
1992 Smith amp Rushton 1994 Smith et al 1996 Crofts amp Jefferson 1999 Jefferson
2005) Use of farmyard manure (FYM) and occasional liming are also traditional
practices for hay meadows (Smith 1988 Simpson amp Jefferson 1996 Tallowin 1998
Crofts amp Jefferson 1999 Jefferson 2005) but what constitutes sustainable practices to
maintain the nature conservation value of some species-rich communities is ill defined
There is a large body of evidence showing the detrimental effects of inorganic fertilizers
on species-rich meadow vegetation (eg Berendse et al 1992 Mountford Lakhani amp
Kirkham 1993 Kirkham Mountford amp Wilkins 1996 Silvertown et al 2006) and on
components of the soil microbial community including decomposer and arbuscular
mycorrhizal fungi which are known to play key roles in nutrient cycling and plant
nutrition in species-rich grasslands (Bardgett amp McAlister 1999 Donnison Griffith amp
Bardgett 2000a Donisson et al 2000b) However no previous study has examined the
impacts of FYM treatments on vegetation and soil microbial communities when matched
with inorganic fertilizers supplying equivalent amounts of inorganic nitrogen (N)
phosphorus (P) and potassium (K) Changes in farming practice in the UK have reduced
the availability of FYM so there is a need to ascertain if or when inorganic fertilizers
can be suitable alternatives
These questions need to be resolved as a basis for refining management guidelines
applied within statutory sites and agri-environment schemes for the conservation of
3
biodiversity within existing species-rich semi-natural plant communities and within
meadows that have undergone restoration of biodiversity
An experiment involving several FYM or matched inorganic equivalent fertilizer
treatments and treatments incorporating initial liming with or without annual or
intermittent (triennial) FYM applications was established in 1999 on in species-rich
meadows at a lowland site in Monmouthshire UK and an upland site in Cumbria UK
Treatment effects on botanical composition were monitored annually to address the
following specific hypotheses for species-rich meadows
H1 ndash increasing rates of fertilizer reduce species richness and nature value
H2 ndash inorganic fertilizer and FYM treatments supplying equivalent amounts of N P and
K differ in their effects on the botanical composition
H3 ndash fertilizers (whether inorganic or FYM) applied triennially differ in their impact on
botanical composition compared with correspondingly lower amounts applied annually
H4 ndash applying lime to raise soil pH to 60 with or without annual or triennial FYM is
consistent with the maintenance or enhancement of vegetation quality
H5 ndash applying FYM or inorganic fertilizers over a period of 12 years will alter the
composition of soil microbial communities
Preliminary agronomic and botanical results have been reported elsewhere (Tallowin et
al 2002 Kirkham et al 2002 Kirkham et al 2008) This paper describes botanical and
soil microbial responses over the period 1999-2010 Nomenclature of vascular plants
follows Stace (2010) except for NVC community names which follow Rodwell (1992)
Materials and methods
EXPERIMENT SITES
Experimental plots were established in 1999 in two agriculturally unimproved meadows one at
an upland site at in Cumbria North West England (Raisbeck) and the other at a lowland site in
Monmouthshire South Wales (Pentwyn) The upland meadow is located at 54ordm 27rsquo N and 2ordm 34rsquo
W (National Grid Reference NY635069) near the village of Orton whilst the lowland meadow is
close to Monmouth at 51ordm 46rsquo N and 2ordm 41rsquo W (NGR SO524093)
Annual and seasonal rainfall
Average rainfall differed between the two areas 1999-2009 means (recorded at Meteorological
Office weather stations at Newton Rigg in Cumbria (NGR NY493308) and Ross-on-Wye (NGR
SO598238) in Monmouthshire) were 982 mm and 797 mm for annual rainfall for the upland and
lowland sites respectively and 525 mm and 462 mm respectively for the growing season (April-
October) These values were somewhat higher than the longer term (1980-2009) averages - by
3 and 6 for annual and seasonal rainfall respectively at the upland site and by 8 and 13
respectively at the lowland site
Plant communities
The vegetation of the upland meadow in 1999 (Kirkham et al 2002) corresponded to the MG3b
(Anthoxanthum odoratum-Geranium sylvaticum grassland Briza media sub-community) of the
NVC whilst that at the lowland meadow was close to both MG5a (Cynosurus cristatus-
Centaurea nigra grassland Lathyrus pratensis sub-community) and MG5c (Danthonia
decumbens sub-community) although lacking some of the preferential species differentiating
MG5c from the MG5a (Rodwell 1992)
4
Past management
Both meadows had previously been managed over a long period by cutting for hay with aftermath
grazing Cutting occurred after 1 July at the upland meadow and after the second week in July at
the lowland meadow The upland meadow had received about 12 t FYM ha-1
each year usually in
late April but occasionally in mid summer following the hay harvest and had received periodic
applications of lime (amounts not known) the last one in about 1993 Details of past fertilizer
application at the lowland meadow are uncertain although this site had not received any form of
fertilizer or lime in the preceding 20 years or more there is some evidence to suggest that basic
slag and lime may have been applied at some time before that
Soil properties
Soils at the upland meadow were clay-loam in texture whilst those at the lowland meadow were
a mixture of sandy silt loam to silty clay-loam The upland meadow soils were notably higher in
total N carbon and organic matter (Table 1)
Table 1 Soil chemical properties (top 75 cm) at experimental sites in Cumbria (the upland
meadow) and Wales (the lowland meadow) in 1999
Upland meadow Lowland meadow
Mean Range Mean Range
pH 518 512-527 501 494-511
Total N 065 059-071 034 029-039
C 676 615-776 315 266-351
Organic matter 1718 1577-2069 903 801-973
Olsen extractable P (mg l-1
) 550 444-679 512 410-600
Exchangeable K (mg l-1
) 1971 1629-2479 1747 1323-2205
Exchangeable Mg (mg l-1
) 1548 1368-1957 2014 1606-2389
Exchangeable Ca (mg l-1
) 1480 1334-1723 1047 852-1214
Measured by loss on ignition (LOI)
EXPERIMENTAL DESIGN
All experimental treatments (see Table 2) were applied by hand to individual 7m x 5m plots laid
out in a randomized block design with three replicate blocks at each site Treatments were applied
between March and late April Due to access restrictions resulting from the national outbreak of
Foot and Mouth Disease (FMD) no treatment was applied in 2001 This meant that treatments
requiring annual applications only received a total of 1112 (92) of the intended amounts over
the 12 year period 1999-2010 Triennial treatments were applied in 1999 2002 2005 and 2008
5
Table 2 Treatments applied 1999-2010 at the upland and lowland meadows Values are the mean
amounts applied (kg ha-1
year-1
elemental N P and K) either as FYM (estimated) or in inorganic
form (actual) averaged over 12 yearsa 1999-2010 Treatments 13-15 were limed only in both
1999 and 2005 at the upland site but in 1999 only at the lowland site Treatments 2-12 received
lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Upland meadow Lowland meadow
Treatment N P K N P K
1 Untreated control 00 00 00 00 00 00
2 Limed (in 2005) control 00 00 00 00 00 00
3 FYM at 6 t ha-1
annual 44 50 345 29 38 292
4+ FYM at 12 t ha-1
annual 88 100 689 57 76 585
5 + FYM at 24 t ha
-1 annual 176 199 1378
114 151 1169
6 FYM at 6 t ha-1
triennially 15 22 113 09 13 110
7 + FYM at 12 t ha
-1 triennially 30 45 226
19 26 221
8 + FYM at 24 t ha
-1 triennially 61 90 452
38 52 441
9 + Inorg equivalent to Tr 4 85 84 689 61 63 585
10 + Inorg equivalent to Tr 5 170 168 1378
121 125 1169
11 + Inorg equivalent to Tr 7 30 36 226
24 21 221
12 + Inorg equivalent to Tr 8 61 72 452
48 42 441
13 Lime in years 1 (and 7) 00 00 00 00 00 00
14 Lime as Tr 13 + FYM as Tr 4 88 100 689 57 76 585
15 Lime as tr 13 + FYM as Tr 7 30 45 226 19 26 221 aNote that no fertilizer was applied in 2001 so that for annual treatments the amounts shown
are 1112ths of the mean amount applied in each of the remaining years
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime
in 1999 x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
The FYM used in the experiment was sourced locally from the same farms each year and was
sampled at the time of application for subsequent chemical analysis The ADAS Manure Nitrogen
Evaluation Routine (MANNER Chambers et al 1999) was used between 1999 and 2006 to
predict the amount of plant available N supplied by the FYM after which a refined model
(MANNER-NPK Nicholson et al 2010) was used Refinements to the model included more
detailed N mineralisation functions and predictions of N availability in the year following
application Availabilities of 60 and 90 of total P and K respectively (Anon 2010) were
assumed between 1999 and 2006 and this assumption for K was maintained throughout the 1999-
2010 period From 2007 onwards the assumed availability of P from FYM was increased to 80
of total P in line with information on longer term release of plant-available P from FYM (Smith
et al 1998) For both N and P the mean amounts assumed to have been applied in FYM
treatments averaged over 1999-2010 and shown in Table 2 are based upon revised estimates for
the whole period The inorganic fertilizer N treatments applied each year ie the inorganic
lsquoequivalentsrsquo for specific FYM treatments (see Table 2) were based upon estimates made using
the original MANNER model from 1999-2006 and MANNER-NPK from 2007-2010 Similarly
6
the inorganic fertilizer P applied each year corresponded to 60 of the total P in FYM between
1999 and 2006 and 80 from 2007-2010
Inorganic N P and K were applied as ammonium nitrate triple super-phosphate and muriate of
potash respectively to the relevant treatments The FYM treatments were designed to encompass
the range of inputs traditionally applied to species-rich hay meadows upon which current agri-
environment guidelines are based (Simpson and Jefferson 1996 Crofts and Jefferson 1999)
Lime was applied to treatments 13 14 and 15 only at each site in March-April 1999 and to most
treatments in 2005 (Table 2) The latter included the three previously limed treatments at the
upland meadow but not at the lowland one since soil pH levels had not fallen significantly by
2005 on these plots at the latter site Based on assessments of soil texture and soil pH tests
conducted in February-March the amounts of lime were calculated to be sufficient to raise the
soil pH of the experimental plots to 60 (Anon 2010)
At each site one plot per replicate block received no fertilizer or lime (untreated control)
between 1999 and 2005 whilst another plot per replicate was designated as a continuation of past
fertilizer inputs At the lowland meadow the latter was identical to the untreated control
treatment but at the upland meadow it was identical to Treatment 4 (12 t ha-1
FYM annually) In
2005 a decision was made to treat occasional liming as lsquobackgroundrsquo management for all
fertilizer treatments at both sites to be triggered for each treatment in future years once mean pH
for that treatment had declined to 55 whilst retaining both an untreated control (ie nil fertilizer
nil lime ndash Treatment 1) and a nil fertilizer (limed in 2005) control treatment (Treatment 2) at each
site The rationale behind this decision is addressed in the Discussion section At the lowland
meadow one of the two hitherto untreated control plots per replicate (Treatment 2) was therefore
limed in 2005 along with other treatments Only one nil input plot per replicate was available at
the upland meadow but since the site had a history of liming the existing nil input plots were
limed in 2005 (Treatment 2) and new untreated control plots (Treatment 1) were established
adjacent to each replicate block with the end at which each was located chosen randomly for
each block
The experimental plots were cut for hay after 1 July each year at the upland site and after 15
July at the lowland site the actual cutting date being dependent on weather conditions The hay
aftermath growth was grazed each year with experimental plots being grazed with the remainder
of the field At the upland meadow mature sheep were used normally commencing in September-
October continuing until March or late April The lowland meadow was grazed by beef store
cattle from mid Octoberearly November to late February
Botanical assessments were carried out during May each year except in 2001 at the upland
meadow due to FMD restrictions The percentage cover of each species present was estimated
visually in three 1m2 quadrats positioned at random within each plot in 1999 and then fixed for
the project duration
Soils were sampled in March 1999 2002 2004 2007 and 2010 by taking five 35 mm diameter x
75mm deep soil cores at random from within each treatment plot but outside the fixed positions
of the three 1m2 botanical survey quadrats The five samples per plot were combined dried at
30oC and then ground prior to analysis for organic carbon (C) total nitrogen (N) Olsen
extractable P exchangeable potassium (K) magnesium (Mg) calcium (Ca) sodium (Na) and pH
(in H2O) using standard laboratory methods (Allen 1974 MAFF 1986)
Soils were further sampled in June 1999 2002 2004 2007 and 2010 for soil microbial
assessment by taking five 35 mm diameter x 100 mm deep cores which were subsequently bulked
for each plot sieved (5 mm) and stored at 4oC prior to analysis Microbial community structure
was assessed using phospholipid fatty acid analysis (PLFA) as described by Bardgett et al
(1996) The fatty acids i150 a150 150 i160 170 i170 cy170 cis181ω7 and cy190 were
chosen to represent bacterial PLFAs (Federle 1986 Frostegaringrd Tunlid amp Baringaringth 1993) and
182ω6 was used as an indicator of fungal biomass (Federle 1986) The ratio of 182ω6bacterial
7
PLFAs was taken to represent the ratio of fungal-to-bacterial biomass in soil (Bardgett Hobbs amp
Frostegaringrd 1996 Frostegaringrd amp Baringaringth 1996)
DERIVED BOTANICAL VARIABLES
Estimates of cover for individual species were converted to a percentage of the total live
vegetation cover present in each quadrat in order to minimize the effects of year-to-year variation
and variation between treatments in vegetation density and total cover Several composite
variables were calculated to characterize the vegetation and these were then averaged across the
three quadrats in each plot to give plot mean values for each variable Plot means were then used
as the basic units for subsequent analyses of treatment effects
Key variables
Five key variables were used to investigate the effects of treatment and time on botanical
composition total species-richness (the total number of vascular plant species per m2) the
number per m2 of MG3 and MG5 positive indicator species (the upland and lowland meadows
respectively) the aggregate cover of these species as a percentage of total vegetation cover the
number per m2 of negative indicator species and the aggregate cover of negative indicator
species as a percentage of total vegetation cover Positive mesotrophic indicator species were
identified from lists produced by Robertson and Jefferson (2000) for use in monitoring the
condition of grassland Sites of Special Scientific Interest (SSSIs) in MG3 (Anthoxanthum
odoratum-Geranium sylvaticum grassland) and MG5 (Cynosurus cristatus-Centaurea nigra
grassland) communities of the NVC (Rodwell 1992) A single list of negative indicator species
was compiled from the generic negative indicators for MG3 and MG5 communities (Robertson amp
Jefferson 2000) augmented by species shown in previous work (eg Mountford et al 1993
Kirkham et al 1996) to be favoured by nutrient addition and for which an increase in abundance
would indicate a negative effect on plant community quality Positive and negative indicator
species are listed in Supporting Information Table S3
Additional variables
Among other derived variables was the weighted Ellenberg N score (Smith et al 2003 Kirkham
et al 2008) referred to hereafter as the fertility score The Ellenberg N index represents the
degree of association with soil fertility (not specifically soil N) of a particular species (Ellenberg
1988 Hill et al 1999) Fertility scores were calculated for each quadrat as the average N index
of the component species weighted according to the proportional contribution of each species to
total vegetation cover Plot mean fertility scores were highly correlated at both sites with the
aggregate cover of negative indicator species (NI) (Pearson r=0845 and r=0927 at the upland
and lowland sites respectively in 2010 both Plt0001) The latter variable was preferred to
fertility scores for statistical analyses in this paper because it proved more responsive to fertilizer
treatments although fertility scores are referred to in the Discussion section in relation to results
from elsewhere
DATA ANALYSIS
ANOVAs for treatment effects
For each of the key variables botanical data for all years 1999-2010 except 2001 at the upland
meadow and for all treatments except Treatment 1 at the same meadow were analyzed by
repeated measures analysis of variance (ANOVA - Genstat V Committee 1997) with Year as the
repeated measures factor Separate ANOVAs were carried out for each variable within each site
using three separate ANOVA models in each case In each model variation between replicate
8
blocks was accounted for by treating replicate as a blocking factor The first model included all
the treatments except Treatment 1 at the upland meadow whilst the other two models used
balanced factorial subsets (series) of treatments (see Table 2) the Form x Rate x Frequency
(FRF) series and the Lime x FYM frequency (LFF) series
The FRF series tested the effects of form of fertilizer (ie FYM or inorganic) the rate at which
fertilizers were applied (12 or 24 t FYM ha-1
and inorganic treatments corresponding to these)
the frequency at which treatments were applied (ie annual or triennially) and all two- and three-
way interactions between these factors
The LFF series tested the effects of liming regime ie lime in 1999 (and 2005 at the upland site)
versus lime in 2005 only the frequency of FYM application (ie nil annual or triennially) and the
Lime x FYM frequency interaction All FYM treatments within this series were applied at 12 t ha-
1 At both sites the LFF series consisted of Treatments 2 4 7 13 14 and 15 (see Table 2) The
liming comparison therefore differed slightly between the two sites since liming was repeated in
2005 on plots previously limed in 1999 at the upland meadow but not repeated at the lowland
one
In both the LFF and the FRF models for each variable the first stratum of the ANOVA
consisted of the effects of all factors and interactions averaged over time with the same factors
and interactions repeated in the second stratum with time as an additional factor
In all repeated measures ANOVAs to account for the repeated measures aspect all degrees of
freedom in the Year x Treatment stratum were adjusted to account for departure from
homogeneity of the population covariance matrix using ε-coefficients calculated by the
Greenhouse-Geisser method (Genstat V Committee 1997)
In most years including 2010 all species were recorded individually but at both sites
bryophytes were recorded only as a single group in some years This had only a small influence
on the data since there were seldom more than two bryophyte species present in a quadrat and
more often less Nevertheless for consistency between years bryophytes were treated as a single
group in all years when calculating total species-richness for data for repeated measures
ANOVAs
In addition to the above analyses 2010 data for the key variables were tested alone for treatment
effects by ANOVA and by analysis of covariance (ANCOVA) the latter using 1999 data as the
covariate (Genstat 5 Committee 1997) All three models as described above were tested The
ANCOVAs for the upland meadow excluded Treatment 1 (untreated control plots established in
2005) Where an ANCOVA showed significant treatment covariance with 1999 data the adjusted
results were used In other cases the unadjusted analyses were used Individual bryophyte species
were included in the calculations of species-richness at both sites in these analyses
In all analyses in order to satisfy the conditions for ANOVA (normality of residuals and
homogeneity of residual variation) all percentage data were transformed before analysis by
arcsine(radicp) where p is the percentage value expressed as a proportion All results quoted for
percentage data refer to analyses of arcsine-square root transformed data
Mixed modeling
Mixed model analysis (Residual Estimated Maximum Likelihood ndash REML Genstat V
Committee 1997) was used to identify any effect of form rate or frequency of application on
vegetation composition at each site in 2010 not simply attributable to the total (or mean per year)
amount of fertilizer applied over the study period A variable representing this amount was
included as a continuous explanatory variable in each analysis along with other fixed factors
form rate and frequency of application (Block was included as a random factor) Since the total
(or mean) amounts of N P and K applied over the whole period were completely correlated
across treatments at each site it was not feasible to include all three elements as separate terms in
the model Nor was it feasible to simply use a single notional variable for fertilizer amount (eg t
9
FYM ha-1
year-1
or inorganic equivalent) since there was lack of equivalence of N and P supply
(but not K) between individual FYM treatments and their corresponding inorganic treatments For
each dependent variable tested two separate models were therefore developed using either N or
P ha-1
year-1
as the continuous variable In each case the presence of any non-linearity in response
was tested by including a quadratic term (ie N2 or P
2) or where more appropriate by logn-
transformation of the explanatory variable
For each analysis an initial model was constructed that included all five main effects and all
interactions The importance of each term was evaluated initially by a χ2 test on the Wald statistic
(Genstat V Committee 1997) The number and order of terms in the model were varied but with
N or P amount always included until a model was developed in which each term was significant
The model was then refined by testing the contribution of each term to the model by the more
conservative deviance test ie a χ2 test on the difference in deviance between the model
containing the term and a sub-model from which it was dropped (Welham amp Thompson 1997)
Where there was an interaction between form of fertilizer and the quadratic term but no overall
effect of form - implying that the difference between the two forms of fertilizer lay primarily in a
difference in the curvilinearity of response - a refined model was tested with the P2 (or N
2) x
Form term replaced by Form nested within the quadratic term (eg P2Form) This has the effect
of absorbing the overall effect of form of fertilizer within the interaction term and returns the two
terms P2 and P
2 x Form (Galwey 2006)
These modeling approaches were used with total species-richness and PI species-richness as
(separate) response variables and included data for all treatments limed in 2005 but not in 1999
(ie Trs 2-12)
Results
TEMPORAL CHANGES 1999-2010
Changes over time in total species-richness (number per m2) richness and proportional
aggregate cover of positive indicator (PI) species and proportional aggregate cover of
negative indicator (NI) species at the upland and lowland meadows are represented in
Figs 1 and 2 respectively for ten of the fifteen treatments applied Repeated measures
ANOVA statistics for each of the four variables are given in Supporting Information
Tables S4 S6 S8 and S10 respectively for the upland meadow and Tables S12 S14 S16
and S18 respectively for the lowland meadow Corresponding mean values for each
treatment in each year are given in Tables S5 S7 S9 S11 S13 S15 S17 and S19
respectively Changes in individual species abundance contributing to the temporal trends
in aggregated PI and NI species described below are outlined under Supporting
Information
The upland meadow
The overall effects of both treatment and time were highly significant in the repeated
measures ANOVA for total species-richness at the upland meadow (both P=0001) but
there was no treatment x time interaction (P=0647)
After an initial decline between 1999 and 2000 species-richness remained fairly
constant for most treatments until 2003 after which there was a general increase with the
notable exception of the highest rate annual FYM treatment (Tr 5) (Fig 1a) The latter
treatment declined further between 2005 and 2007 after which it appeared to recover
slightly and averaged over all years this treatment was significantly less species-rich than
all others (Plt001 compared to Tr 14 Plt0001 the remainder) None of the remaining
10
treatments differed significantly from the limed control treatment (Tr2) averaged over all
years However the more species-rich treatments ie nil fertilizer plus lime applied in
both 1999 and 2005 (Tr 13) low rate annual and medium rate triennial FYM (Tr 3 and 7
respectively) and the inorganic equivalent to the latter (Tr 11) were all more species-rich
averaged over all years (Plt005) than annual FYM at the medium rate under either
liming regime (ie Trs 4 and 14 the former representing a continuation of past
management)
The effect of the high rate annual FYM treatment (Tr 5) was largely responsible for
several main effects within the repeated measures ANOVA of treatments within the Form
x Rate x Frequency (FRF) factorial series namely overall effects (averaged over all
years) of rate (mediumgthigh P=0002) and frequency (triennialgtannual P=0001) of
application and a not quite significant effect of form of fertilizer (FYMltinorganic
P=0057) Both the form x rate and the form x frequency interactions were significant
(both Plt005) high rate FYM was significantly less species-rich than all other
combinations averaged over annual and three yearly treatments and all years (Plt0001
compared to medium rate FYM Plt001 compared to both medium and high rate
inorganic fertilizer) and annual FYM was less species-rich than three yearly FYM and
than inorganic fertilizer either annually or triennial (all Plt0001) averaged over the two
application rates and all years The rate x frequency interaction was not significant
(P=0105) but there was a marginally non-significant three-way interaction between
form rate and frequency (P=0070) with a low mean for high rate annual FYM (Tr 5
211 species m-2
) contrasting markedly with all other treatments within the series
particularly with low rate triennial FYM (Tr 6 267 species m-2
)
As with the full treatment ANOVA the overall time factor was highly significant
(Plt0001) but only the effect of application frequency varied significantly with time
(interaction Plt005) Species-richness declined slightly with annual treatments overall
and increased slightly with triennial treatments such that differences between these were
significant in all years from 2005 onwards (Plt001 for 2006-2009 Plt005 in 2005 and
2010)
Both the richness and aggregate cover of PI species (PI) increased generally with time
(Fig 1b and 1c) although in both cases there was a progressive separation between
species The temporal trend was particularly marked for PI although the time factor
was highly significant (Plt0001) in both ANOVAs The overall treatment effect was
marginally non-significant for PI richness (P=0069) with no indication of a time x
treatment interaction (P=0703) whereas for PI there was both a significant treatment
effect (P=0002) and a marginally non-significant treatment x time interaction (P=0075)
Mean PI was significantly lower averaged over all years for annual FYM treatments at
both 12 and 24 t ha-1
(ie Trs 4 14 and 5) compared to several other treatments notably
nil fertilizer treatments limed either in 2005 only (Tr 2 limed control) or in both 1999
and 2005 (Tr 13) (all differences Plt0001 except Tr 2 vs Tr 4 Plt001) and triennial
inorganic treatments (Trs 11 and 12 Plt001 except for Tr 4 vs Tr 11 Plt005) Within
the FRF series ANOVA the increase in PI over time was greater with triennial
treatments compared to annual averaged over fertilizer forms and rates of application
(time x frequency interaction P=0016) with a marked overall difference due to
application frequency averaged over forms rates and all years (P=0005)
11
PI species-richness showed a similar overall effect of application frequency within the
FRF series (triennialgtannual P=0013) and also a significant overall effect of rate of
application (medium rategthigh rate P=0022) The overall effect of form of fertilizer was
marginally non-significant (FYMltinorganic P=0059) and there was no significant
interaction among any of the three factors averaged over all years nor any interaction
with time
16
18
20
22
24
26
28
30
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Specie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime in Year 1+ ann FYM (Tr 14) Nil fert + Lime in Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr1)
a Species richness (total)
3
4
5
6
7
8
9
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
70
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
0
5
10
15
20
25
30
35
40
45
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
Fig 1 Changes 1999-2010 in vegetation composition an upland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control
The mean number of negative indicator (NI) species per m2 averaged over all treatments
varied significantly over time at the upland meadow (P=0002 data not shown) though
with no consistent trend - it was lowest overall in 2002 (499 per m2) and highest in both
1999 and 2008 (548 per m2) The treatment x time interaction was not significant
(P=0469) but the overall treatment effect was highly significant (Plt0001) mean
number of NI species per m2 was higher than the limed control (Tr 1 453 per m
2) with
all fertilizer treatments except Treatments 11 (triennial inorganic fertilizer at the lower
rate) and 13 (nil fertilizer limed in both 1999 and 2005) (Plt0001 for all except Trs 5
and 6 for which Plt001) The highest mean NI m-2
value was for high rate inorganic
12
fertilizer (Tr 10 565 per m2) which differed significantly (Plt005) from its FYM
counterpart Treatment 5 There was however no consistent difference between FYM
treatments and their inorganic counterparts and no overall effect of form of fertilizer
within the FRF series (P=0745) There was a significant form x rate interaction within
this ANOVA (P=0032) but only the difference between the high rate and lower rate
inorganic means was significant (high rategtlower rate Plt001)
The aggregate contribution to vegetation cover of negative indicator species (NI)
showed a very marked divergence between treatments over time (Fig 1d) with NI
either changing little or declining under most treatments again with the notable exception
of high rate annual FYM (Tr 5) under which NI increased over time Time treatment
and the treatment x time interaction were all highly significant (Plt0001) The
predominant temporal trend was downward (from 25 of live cover in 1999 to 14 in
2010 averaged over all treatments) but NI for Treatment 5 had significantly increased
compared to 1999 in 2002 2003 (Plt005) 2005 (Plt0001) 2008 (Plt0001) and 2009
(Plt001) but not in 2010 (Fig 1d) The most marked declines in NI occurred on the
limed control (Tr 2) nil fertilizer plus lime in both 1999 and 2005 (Tr 13) and the lower
rate triennial inorganic fertilizer treatments (Tr 11) differences compared to both 1999
and 2002 were highly significant (Plt0001) in most subsequent years for these
treatments A smaller decline also occurred after 2002 on the treatment equivalent to past
inputs (ie12 t ha-1
year-1
annual FYM Tr 4) In most years this treatment differed little
from the equivalent FYM treatment limed in both 1999 and 2005 (Tr 14)
These trends were reflected in highly significant overall effects on NI within the FRF
series of form (FYMgtinorganic) rate (mediumlthigh) and frequency (triennialltannual)
(all effects Plt0001) Time x rate and time x frequency interactions were both significant
(P=0019 and Plt0001 respectively) with a marginally non-significant rate x frequency
interaction averaged over all years (P=0054) NI declined over time under both
application rates averaged over forms and frequencies of application but the decline was
slower with high rate treatments compared to lower rate Difference between rates were
significant in all years from 2002 onwards (Plt005 in 2002 2006 and 2007 Plt0001 in
other years) Overall differences due to frequency of application were even more marked
Under annual application NI declined initially from 2003-2006 compared to 1999 but
subsequently recovered and did not differ from 1999 levels from 2007 onwards whilst
under triennial application differences were highly significant (Plt0001) compared to
1999 from 2003 onwards with significant differences between years during this same
period (Plt005 in 2003 and 2006 Plt0001 in remaining years)
Repeated-measures ANOVAs within the LFF treatment series at the upland meadow
showed no overall effect of liming regime on temporal trends for any of the above
variables nor any interaction of lime with either time or FYM frequency - note that FYM
was applied at the intermediate rate (12 t ha-1
) in all FYM treatments within this series
The overall effect of FYM frequency was significant for both PI (P=0003) and the
number of NI species m-2
(P=0001) PI was highest with nil FYM and lowest with
annual FYM with triennial intermediate and differing significantly (Plt005) from the
other two means NI species m-2
did not differ between triennial and annual application
within the LFF series but both were significantly higher than nil FYM (Plt001 for
triennial Plt0001 for annual application) There was also a significant time x FYM
frequency interaction for NI within this series (P=0001) reflecting a more rapid
13
decline with nil FYM than with triennial FYM and no significant overall trend with
annual FYM when averaged over both lime treatments
12
14
16
18
20
22
24
26
28
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Spe
cie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime Year 1 + ann FYM (Tr 14) Nil fert + Lime Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr 1)
a Species richness (total)
2
3
4
5
6
7
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
0
10
20
30
40
50
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
Fig 2 Changes 1999-2010 in vegetation composition at a lowland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control treatment and those that had received lime in year 1
Treatments 1 and 2 were identical (nil input) until 2005
The lowland meadow
Both time and treatment were significant within the ANOVA for total species-richness at
the lowland meadow (Plt0001 and P=0009 respectively and in contrast to the upland
site the treatment x time interaction was significant (P=0012) Averaged over all years
high rate annual FYM treatment (Tr 5) was significantly less species-rich than all others
except Treatment 14 (annual FYM at 12 t ha-1
limed in 1999) having declined
progressively between 1999 and 2009 (Fig 2a) with most differences significant at
Plt001 Treatment 14 did not differ significantly overall compared to the corresponding
FYM treatment not limed until 2005 (Tr 4) although these two treatments showed
14
markedly different temporal trends Treatment 14 was less species-rich than others during
the first five years of the experiment but did not change significantly throughout the
whole 12 year period whilst Treatment 4 changed little between 1999 and 2006 but
declined thereafter (Plt001 and Plt0001 for 2009 and 2010 respectively compared to
2006) possibly in response to liming in 2005 However there was no significant overall
effect on total species-richness of liming strategy within the LFF series and no interaction
between liming and either time or FYM application
There were marked differences in temporal trend in species-richness between FYM
treatments and their inorganic counterparts particularly with the high rate annual
treatments (Tr 5 and Tr 10) Whereas species-richness declined more or less continually
throughout under Treatment 5 at least until 2009 (Plt001 for 2004 compared to 1999 and
for both 2009 and 2010 compared to 2004) Treatment 10 declined between 2000 and
2001 (Plt005) and then remained relatively unchanged until 2008 after which it declined
again (2010 Plt005 compared to 2008)
Other treatments within the full treatment series including untreated control (Tr 1) and
limed control (Tr 2) showed no particular temporal trend in the full repeated measures
ANOVA although both the latter treatments showed a dip in species-richness in 2004
(Plt005 compared to both 2003 and 2005)
There was a strong overall effect of form of fertilizer within the FRF series ANOVA
(FYMltinorganic P=0003) a significant effect of frequency of application
(triennialgtannual P=0027) and a not quite significant effect of application rate (high
rateltmedium rate P=0064) but no significant interaction between any of these factors
The time x application frequency interaction was highly significant (Plt0001) species-
richness remained unchanged under triennial application but under annual application
was significantly lower compared to 1999 in each year from 2004 onwards with the
exception of 2006 (Plt005 in 2004 Plt001 in 2005 and 2008 Plt0001 the remainder)
Again with the exception of 2006 differences between triennial and annual application
were significant in each year from 2005 onwards (Plt005 in 2007 Plt001 in 2005 and
2008 Plt0001 in 2009 and 2010)
Both treatment time and their interaction were significant in the full treatment ANOVA
for PI species-richness at the lowland meadow (ie time and treatment both Plt0001
time x treatment P=0006) There was a general decline in PI-richness after 2005 (Fig
2b) with PI species m-2
averaged over all treatments lower from 2007 onwards (Plt0001)
than all years up to 2005 except 2001 Although the initiation of this trend corresponded
with the liming of most treatments in 2005 a similar pattern was shown by treatments
that did not receive this lime application ie the untreated control treatment (Tr 1) the
nil fertilizer treatment limed in 1999 (Tr 13) and the corresponding treatment also
receiving triennial FYM (Tr 15) In all these cases PI-richness showed no significant
difference between years until 2005 but was significantly (Plt001) lower by 2010 than in
2005 Treatment 14 (limed in 1999 plus annual FYM) did not show this pattern but PI
species-richness had been lowest under this treatment in most years between 1999 and
2005 (Fig 2b)
As with total species-richness PI richness declined more or less progressively under
Treatment 5 until 2008 (Plt005 compared to 1999 by 2001 Plt0001 from 2006
onwards) whereas Treatment 4 did not change significantly until 2006 after which it
declined (Plt005 in 2007 compared to 2006 Plt001 thereafter) Both these treatments
15
tended be less rich in PI species than the corresponding inorganic treatment (Trs 9 and
10 respectively) in most years consistently so for Tr 4 compared to Tr 9 but these
differences were not significant in any year nor when averaged over all years There was
however a significant overall effect of form of fertilizer in the FRF series ANOVA
(FYMltinorganic P=0002) and a highly significant effect of frequency of application
(triennialgtannual Plt0001) The time x frequency of application interaction was also
highly significant (Plt0001) Under triennial application PI species per m2 increased
slightly until 2005 (Plt005 compared to 1999) and declined thereafter so that by 2009
levels were significantly lower than in 1999 (Plt001 for 2009 Plt0001 for 2010) By
contrast under annual application PI species m-2
declined progressively until 2009
following a temporary though highly significant (Plt0001) dip in 2000 so that the
difference compared to 1999 was significant in every year from 2002 onwards and highly
significant (Plt0001) from 2005 With the exception of 2003 differences between
triennial and annual application were significant from 2002 onwards and highly
significant (Plt0001) in 2005 and from 2007 to 2010
There was no significant effect other than of time within the LFF treatment series
ANOVA although there was a marginally non-significant time x FYM frequency
interaction (P=0054) This reflected a more rapid decline in PI species-richness under
annual FYM application than with either nil or triennial FYM with the latter two showing
no discernible difference
By contrast with PI species m-2
PI cover percentage (PI) showed no overall treatment
effect within the full treatment ANOVA nor a time x treatment interaction although the
overall effect of time was highly significant (Plt0001) The temporal pattern in PI (Fig
2c) also differed from that shown by PI-richness with PI averaged over all treatments
declining between 1999 and 2005 in three highly significant increments (1999-2000
2000-2002 and 2002-2005 all Plt0001) and increasing again thereafter (2005-2009 and
2009-2010 both Plt0001) There was no significant effect within either the LFF or FRF
treatment series ANOVAs although their was a slight time x frequency of application
effect within the latter (P=0081) PI was slightly higher under annual application than
triennial in each year 1999-2004 after which the reverse was true although differences
were always small
There was no overall effect of treatment on the number of NI species per m2 within the
full treatment range at the lowland meadow (data not shown) but both the overall effect
of time and the time x treatment interaction were significant (Plt0001 and P=0009
respectively) Overall NI species m-2
increased over time from 24 species m-2
in 1999 to
a peak of 42 m-2
in 2008 averaged over all treatments NI species m-2
increased on the
untreated control treatment (Tr 1) from 21 species m-2
in 1999 to 34 species m-2
in 2007
(increase Plt001) then declined to the 1999 value by 2010 The limed (in 2005) control
(Tr 2) showed a somewhat similar pattern though from a slightly higher starting level
(31 species m-2
) and with the increase to 38 species m-2
in 2008 not reaching
significance
These temporal trends in NI species m-2
were much accentuated by annual fertilizer
application particularly by FYM treatments with peak numbers of NI species reaching
52 and 47 species m-2
for Treatments 4 and 5 respectively in 2008 (both Plt0001
compared to 1999) and despite slight declines in both cases remaining significantly
higher in 2010 than in 1999 (both Plt0001) Inorganic counterparts to these treatments
16
showed similar though slightly less marked trends and there was no significant
difference between all four treatments in the peak number reached in 2008 There was no
overall effect of either form of fertilizer or rate of application within the FRF series
ANOVA and only a marginally non-significant effect of frequency of application
(triennialltannual P=0061) Liming in 1999 (Trs 13 14 and 15) also appeared to
accentuate the generally increasing trend although peak numbers in 2008 did not differ
between these treatments and either untreated or limed control in the full treatment range
ANOVA The overall effects of liming regime (1999 or 2005) were not significant within
the LFF treatment series but the faster rate of increase with liming in 1999 was reflected
in a significant time x lime interaction in this analysis (P=0017) The mean number of NI
species m-2
increased very significantly (Plt0001) between 1999 and 2000 following
liming in 1999 with the increment increasing until 2008 whilst on plots un-limed until
2005 NI species m-2
did not increase significantly until 2003 (Plt005) although the
increase compared to 1999 was highly significant (Plt0001) in 2006 2008 and 2010
Nevertheless the differences in rate of increase was at least partly due to a lower 1999
mean for the 1999-limed treatments (170 species m-2
compared to 244 species m-2
difference NS) and although in all years from 2001 onwards mean NI species m-2
was
higher for 1999-limed treatments compared to those not limed until 2005 the difference
was not significant in any year
The contribution of NI species to vegetation cover (NI) showed a very marked
separation between treatments over time at the lowland meadow (Fig 2d) starting from
much lower levels than at the upland meadow (compare with Fig 1d) There were
noticeable differences in temporal trend between annual fertilizer treatments on the one
hand and triennial and nil fertilizer treatments on the other (Fig 2d) The effects of
treatment time and the treatment x time interaction were all highly significant (Plt0001)
in the full treatment range ANOVA The most marked increase in NI occurred with
annual FYM at the high rate (Tr 5) with NI increasing rapidly between 1999 and 2001
(Plt001) and a further highly significant increment between 2004 and 2009 (Plt0001)
(Fig 2d) A similar though less marked trend occurred with annual FYM at the medium
rate (Tr 4) with significant increases between 1999 and 2005 and between 2005 and
2010 (both Plt001) NI also increased markedly with the inorganic treatment
equivalent to Treatment 5 (Tr 10) although the overall rate of increase shown by this
treatment was closer to that shown by Treatment 4 than Treatment 5 There were
significant differences between Treatments 5 and 10 in most years from 2004 onwards
(Plt001 and Plt0001 in 2009 and 2010 respectively)
These effects were reflected in a significant overall effect of form of fertilizer
(FYMgtinorganic P=0008) and highly significant (Plt0001) overall effects of both rate
(high rategtlower rate) and frequency of application (annualgttriennial) within the FRF
series ANOVA and a significant form x rate interaction (P=0043) Mean NI was
highest for high rate annual FYM (Plt0001 compared to all three remaining means) and
lowest for lower rate inorganic (Plt005 compared to high rate inorganic NS compared
to lower rate FYM) Only frequency of application interacted significantly with time
(Plt0001) NI increased moderately under triennial application until 2008 (Plt005 in
2005 compared to 1999 Plt0001 each year 2006-2008) followed by a decline (Plt001
2010 compared to 2008) but with mean NI in 2010 still higher than 1999 (Plt005)
The increasing trend was very much more marked with annual fertilizer application for
17
which there were highly significant (Plt0001) increments between 1999 and 2002 and
between 2002 and 2008 but with no significant increase thereafter Differences between
annual and triennial treatment means were highly significant (Plt0001) from 2003
onwards
There was no significant change in NI over time either under the untreated control
(Tr 1) or the nil fertilizer (Tr 2) treatments in the full treatment ANOVA NI increased
until 2005 on plots limed in 1999 without fertilizer Tr 13 (Plt001 compared to 1999)
and declined slightly but not significantly thereafter Trends were similar with the
treatment limed in 1999 and receiving triennial FYM (Tr 15 ndash not illustrated in Fig 2)
although NI was higher with this treatment than with Tr 13 (Plt005) in both 2005 and
2008 By contrast applying the same rate of FYM annually after liming in 1999 (Tr 14)
caused a more rapid increase with levels by 2005 higher than either Tr 13 (Plt001) or
Tr 4 (Plt005) ndash note that the latter was equivalent to Tr 14 but without lime until 2005
NI declined after 2006 under Tr 14 but continued to increase with Tr 4 with the
difference between these two treatments reversed by 2010 (Plt005)
All main effects and interactions were significant in the LFF treatment series ANOVA
for NI except for lime x FYM frequency and the lime x FYM x time interactions Plots
limed in 1999 supported higher NI levels than those limed in 2005 averaged over the
three FYM frequencies and all years (P=0042) NI increased more rapidly after liming
in 1999 than where liming was delayed until 2005 (P=0033 for time x lime interaction)
with the overall difference due to liming in 1999 highly significant (Plt0001) by 2005
NI declined thereafter on the 1999-limed treatments whilst continuing to increase
following liming in 2005 of previously un-limed treatments both peaking in 2008 at a
very similar level Averaged over lime treatments NI increased much more rapidly
under annual FYM application than under either triennial or nil FYM (P=0002 for the
time x FYM frequency interaction) a highly significant increase had occurred by 2002
with annual FYM (Plt0001 compared to 1999) but increases of similar magnitude were
not reached until 2005 with triennial application and not until 2007 with nil FYM NI
subsequently declined under the latter two FYM regimes but not with annual FYM so
that by 2010 NI was significantly higher with annual FYM than with either triennial or
nil FYM (Plt0001) but with only a small non-significant difference between the latter
two treatments
TREATMENT EFFECTS AFTER 12 YEARS OF APPLICATION
The effects of all treatments on the five key variables at each site in 2010 are shown in
Table S20 with treatments differing significantly (Plt005) from the limed control
treatment (Tr 2) highlighted for each variable Treatment effects in 2010 are not
described in detail here but the modeled responses of total species-richness and of
richness of PI species in 2010 to the mean amounts of N and P applied per year averaged
over 1999-2010 are described below
Relationships with the amounts of fertilizer applied
The effect of neither rate nor frequency of application was significant in any of the
REML models for either total species-richness or PI species-richness at either site when
the amount of N or P applied ha-1
year-1
was included The effect of form of fertilizer was
still significant in most models however either as a main effect or in interaction with the
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
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Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
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Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
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Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
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Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
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Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
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Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
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Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
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Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
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Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
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Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
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Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
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Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
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Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
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Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
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plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
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on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
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Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
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Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
3
biodiversity within existing species-rich semi-natural plant communities and within
meadows that have undergone restoration of biodiversity
An experiment involving several FYM or matched inorganic equivalent fertilizer
treatments and treatments incorporating initial liming with or without annual or
intermittent (triennial) FYM applications was established in 1999 on in species-rich
meadows at a lowland site in Monmouthshire UK and an upland site in Cumbria UK
Treatment effects on botanical composition were monitored annually to address the
following specific hypotheses for species-rich meadows
H1 ndash increasing rates of fertilizer reduce species richness and nature value
H2 ndash inorganic fertilizer and FYM treatments supplying equivalent amounts of N P and
K differ in their effects on the botanical composition
H3 ndash fertilizers (whether inorganic or FYM) applied triennially differ in their impact on
botanical composition compared with correspondingly lower amounts applied annually
H4 ndash applying lime to raise soil pH to 60 with or without annual or triennial FYM is
consistent with the maintenance or enhancement of vegetation quality
H5 ndash applying FYM or inorganic fertilizers over a period of 12 years will alter the
composition of soil microbial communities
Preliminary agronomic and botanical results have been reported elsewhere (Tallowin et
al 2002 Kirkham et al 2002 Kirkham et al 2008) This paper describes botanical and
soil microbial responses over the period 1999-2010 Nomenclature of vascular plants
follows Stace (2010) except for NVC community names which follow Rodwell (1992)
Materials and methods
EXPERIMENT SITES
Experimental plots were established in 1999 in two agriculturally unimproved meadows one at
an upland site at in Cumbria North West England (Raisbeck) and the other at a lowland site in
Monmouthshire South Wales (Pentwyn) The upland meadow is located at 54ordm 27rsquo N and 2ordm 34rsquo
W (National Grid Reference NY635069) near the village of Orton whilst the lowland meadow is
close to Monmouth at 51ordm 46rsquo N and 2ordm 41rsquo W (NGR SO524093)
Annual and seasonal rainfall
Average rainfall differed between the two areas 1999-2009 means (recorded at Meteorological
Office weather stations at Newton Rigg in Cumbria (NGR NY493308) and Ross-on-Wye (NGR
SO598238) in Monmouthshire) were 982 mm and 797 mm for annual rainfall for the upland and
lowland sites respectively and 525 mm and 462 mm respectively for the growing season (April-
October) These values were somewhat higher than the longer term (1980-2009) averages - by
3 and 6 for annual and seasonal rainfall respectively at the upland site and by 8 and 13
respectively at the lowland site
Plant communities
The vegetation of the upland meadow in 1999 (Kirkham et al 2002) corresponded to the MG3b
(Anthoxanthum odoratum-Geranium sylvaticum grassland Briza media sub-community) of the
NVC whilst that at the lowland meadow was close to both MG5a (Cynosurus cristatus-
Centaurea nigra grassland Lathyrus pratensis sub-community) and MG5c (Danthonia
decumbens sub-community) although lacking some of the preferential species differentiating
MG5c from the MG5a (Rodwell 1992)
4
Past management
Both meadows had previously been managed over a long period by cutting for hay with aftermath
grazing Cutting occurred after 1 July at the upland meadow and after the second week in July at
the lowland meadow The upland meadow had received about 12 t FYM ha-1
each year usually in
late April but occasionally in mid summer following the hay harvest and had received periodic
applications of lime (amounts not known) the last one in about 1993 Details of past fertilizer
application at the lowland meadow are uncertain although this site had not received any form of
fertilizer or lime in the preceding 20 years or more there is some evidence to suggest that basic
slag and lime may have been applied at some time before that
Soil properties
Soils at the upland meadow were clay-loam in texture whilst those at the lowland meadow were
a mixture of sandy silt loam to silty clay-loam The upland meadow soils were notably higher in
total N carbon and organic matter (Table 1)
Table 1 Soil chemical properties (top 75 cm) at experimental sites in Cumbria (the upland
meadow) and Wales (the lowland meadow) in 1999
Upland meadow Lowland meadow
Mean Range Mean Range
pH 518 512-527 501 494-511
Total N 065 059-071 034 029-039
C 676 615-776 315 266-351
Organic matter 1718 1577-2069 903 801-973
Olsen extractable P (mg l-1
) 550 444-679 512 410-600
Exchangeable K (mg l-1
) 1971 1629-2479 1747 1323-2205
Exchangeable Mg (mg l-1
) 1548 1368-1957 2014 1606-2389
Exchangeable Ca (mg l-1
) 1480 1334-1723 1047 852-1214
Measured by loss on ignition (LOI)
EXPERIMENTAL DESIGN
All experimental treatments (see Table 2) were applied by hand to individual 7m x 5m plots laid
out in a randomized block design with three replicate blocks at each site Treatments were applied
between March and late April Due to access restrictions resulting from the national outbreak of
Foot and Mouth Disease (FMD) no treatment was applied in 2001 This meant that treatments
requiring annual applications only received a total of 1112 (92) of the intended amounts over
the 12 year period 1999-2010 Triennial treatments were applied in 1999 2002 2005 and 2008
5
Table 2 Treatments applied 1999-2010 at the upland and lowland meadows Values are the mean
amounts applied (kg ha-1
year-1
elemental N P and K) either as FYM (estimated) or in inorganic
form (actual) averaged over 12 yearsa 1999-2010 Treatments 13-15 were limed only in both
1999 and 2005 at the upland site but in 1999 only at the lowland site Treatments 2-12 received
lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Upland meadow Lowland meadow
Treatment N P K N P K
1 Untreated control 00 00 00 00 00 00
2 Limed (in 2005) control 00 00 00 00 00 00
3 FYM at 6 t ha-1
annual 44 50 345 29 38 292
4+ FYM at 12 t ha-1
annual 88 100 689 57 76 585
5 + FYM at 24 t ha
-1 annual 176 199 1378
114 151 1169
6 FYM at 6 t ha-1
triennially 15 22 113 09 13 110
7 + FYM at 12 t ha
-1 triennially 30 45 226
19 26 221
8 + FYM at 24 t ha
-1 triennially 61 90 452
38 52 441
9 + Inorg equivalent to Tr 4 85 84 689 61 63 585
10 + Inorg equivalent to Tr 5 170 168 1378
121 125 1169
11 + Inorg equivalent to Tr 7 30 36 226
24 21 221
12 + Inorg equivalent to Tr 8 61 72 452
48 42 441
13 Lime in years 1 (and 7) 00 00 00 00 00 00
14 Lime as Tr 13 + FYM as Tr 4 88 100 689 57 76 585
15 Lime as tr 13 + FYM as Tr 7 30 45 226 19 26 221 aNote that no fertilizer was applied in 2001 so that for annual treatments the amounts shown
are 1112ths of the mean amount applied in each of the remaining years
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime
in 1999 x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
The FYM used in the experiment was sourced locally from the same farms each year and was
sampled at the time of application for subsequent chemical analysis The ADAS Manure Nitrogen
Evaluation Routine (MANNER Chambers et al 1999) was used between 1999 and 2006 to
predict the amount of plant available N supplied by the FYM after which a refined model
(MANNER-NPK Nicholson et al 2010) was used Refinements to the model included more
detailed N mineralisation functions and predictions of N availability in the year following
application Availabilities of 60 and 90 of total P and K respectively (Anon 2010) were
assumed between 1999 and 2006 and this assumption for K was maintained throughout the 1999-
2010 period From 2007 onwards the assumed availability of P from FYM was increased to 80
of total P in line with information on longer term release of plant-available P from FYM (Smith
et al 1998) For both N and P the mean amounts assumed to have been applied in FYM
treatments averaged over 1999-2010 and shown in Table 2 are based upon revised estimates for
the whole period The inorganic fertilizer N treatments applied each year ie the inorganic
lsquoequivalentsrsquo for specific FYM treatments (see Table 2) were based upon estimates made using
the original MANNER model from 1999-2006 and MANNER-NPK from 2007-2010 Similarly
6
the inorganic fertilizer P applied each year corresponded to 60 of the total P in FYM between
1999 and 2006 and 80 from 2007-2010
Inorganic N P and K were applied as ammonium nitrate triple super-phosphate and muriate of
potash respectively to the relevant treatments The FYM treatments were designed to encompass
the range of inputs traditionally applied to species-rich hay meadows upon which current agri-
environment guidelines are based (Simpson and Jefferson 1996 Crofts and Jefferson 1999)
Lime was applied to treatments 13 14 and 15 only at each site in March-April 1999 and to most
treatments in 2005 (Table 2) The latter included the three previously limed treatments at the
upland meadow but not at the lowland one since soil pH levels had not fallen significantly by
2005 on these plots at the latter site Based on assessments of soil texture and soil pH tests
conducted in February-March the amounts of lime were calculated to be sufficient to raise the
soil pH of the experimental plots to 60 (Anon 2010)
At each site one plot per replicate block received no fertilizer or lime (untreated control)
between 1999 and 2005 whilst another plot per replicate was designated as a continuation of past
fertilizer inputs At the lowland meadow the latter was identical to the untreated control
treatment but at the upland meadow it was identical to Treatment 4 (12 t ha-1
FYM annually) In
2005 a decision was made to treat occasional liming as lsquobackgroundrsquo management for all
fertilizer treatments at both sites to be triggered for each treatment in future years once mean pH
for that treatment had declined to 55 whilst retaining both an untreated control (ie nil fertilizer
nil lime ndash Treatment 1) and a nil fertilizer (limed in 2005) control treatment (Treatment 2) at each
site The rationale behind this decision is addressed in the Discussion section At the lowland
meadow one of the two hitherto untreated control plots per replicate (Treatment 2) was therefore
limed in 2005 along with other treatments Only one nil input plot per replicate was available at
the upland meadow but since the site had a history of liming the existing nil input plots were
limed in 2005 (Treatment 2) and new untreated control plots (Treatment 1) were established
adjacent to each replicate block with the end at which each was located chosen randomly for
each block
The experimental plots were cut for hay after 1 July each year at the upland site and after 15
July at the lowland site the actual cutting date being dependent on weather conditions The hay
aftermath growth was grazed each year with experimental plots being grazed with the remainder
of the field At the upland meadow mature sheep were used normally commencing in September-
October continuing until March or late April The lowland meadow was grazed by beef store
cattle from mid Octoberearly November to late February
Botanical assessments were carried out during May each year except in 2001 at the upland
meadow due to FMD restrictions The percentage cover of each species present was estimated
visually in three 1m2 quadrats positioned at random within each plot in 1999 and then fixed for
the project duration
Soils were sampled in March 1999 2002 2004 2007 and 2010 by taking five 35 mm diameter x
75mm deep soil cores at random from within each treatment plot but outside the fixed positions
of the three 1m2 botanical survey quadrats The five samples per plot were combined dried at
30oC and then ground prior to analysis for organic carbon (C) total nitrogen (N) Olsen
extractable P exchangeable potassium (K) magnesium (Mg) calcium (Ca) sodium (Na) and pH
(in H2O) using standard laboratory methods (Allen 1974 MAFF 1986)
Soils were further sampled in June 1999 2002 2004 2007 and 2010 for soil microbial
assessment by taking five 35 mm diameter x 100 mm deep cores which were subsequently bulked
for each plot sieved (5 mm) and stored at 4oC prior to analysis Microbial community structure
was assessed using phospholipid fatty acid analysis (PLFA) as described by Bardgett et al
(1996) The fatty acids i150 a150 150 i160 170 i170 cy170 cis181ω7 and cy190 were
chosen to represent bacterial PLFAs (Federle 1986 Frostegaringrd Tunlid amp Baringaringth 1993) and
182ω6 was used as an indicator of fungal biomass (Federle 1986) The ratio of 182ω6bacterial
7
PLFAs was taken to represent the ratio of fungal-to-bacterial biomass in soil (Bardgett Hobbs amp
Frostegaringrd 1996 Frostegaringrd amp Baringaringth 1996)
DERIVED BOTANICAL VARIABLES
Estimates of cover for individual species were converted to a percentage of the total live
vegetation cover present in each quadrat in order to minimize the effects of year-to-year variation
and variation between treatments in vegetation density and total cover Several composite
variables were calculated to characterize the vegetation and these were then averaged across the
three quadrats in each plot to give plot mean values for each variable Plot means were then used
as the basic units for subsequent analyses of treatment effects
Key variables
Five key variables were used to investigate the effects of treatment and time on botanical
composition total species-richness (the total number of vascular plant species per m2) the
number per m2 of MG3 and MG5 positive indicator species (the upland and lowland meadows
respectively) the aggregate cover of these species as a percentage of total vegetation cover the
number per m2 of negative indicator species and the aggregate cover of negative indicator
species as a percentage of total vegetation cover Positive mesotrophic indicator species were
identified from lists produced by Robertson and Jefferson (2000) for use in monitoring the
condition of grassland Sites of Special Scientific Interest (SSSIs) in MG3 (Anthoxanthum
odoratum-Geranium sylvaticum grassland) and MG5 (Cynosurus cristatus-Centaurea nigra
grassland) communities of the NVC (Rodwell 1992) A single list of negative indicator species
was compiled from the generic negative indicators for MG3 and MG5 communities (Robertson amp
Jefferson 2000) augmented by species shown in previous work (eg Mountford et al 1993
Kirkham et al 1996) to be favoured by nutrient addition and for which an increase in abundance
would indicate a negative effect on plant community quality Positive and negative indicator
species are listed in Supporting Information Table S3
Additional variables
Among other derived variables was the weighted Ellenberg N score (Smith et al 2003 Kirkham
et al 2008) referred to hereafter as the fertility score The Ellenberg N index represents the
degree of association with soil fertility (not specifically soil N) of a particular species (Ellenberg
1988 Hill et al 1999) Fertility scores were calculated for each quadrat as the average N index
of the component species weighted according to the proportional contribution of each species to
total vegetation cover Plot mean fertility scores were highly correlated at both sites with the
aggregate cover of negative indicator species (NI) (Pearson r=0845 and r=0927 at the upland
and lowland sites respectively in 2010 both Plt0001) The latter variable was preferred to
fertility scores for statistical analyses in this paper because it proved more responsive to fertilizer
treatments although fertility scores are referred to in the Discussion section in relation to results
from elsewhere
DATA ANALYSIS
ANOVAs for treatment effects
For each of the key variables botanical data for all years 1999-2010 except 2001 at the upland
meadow and for all treatments except Treatment 1 at the same meadow were analyzed by
repeated measures analysis of variance (ANOVA - Genstat V Committee 1997) with Year as the
repeated measures factor Separate ANOVAs were carried out for each variable within each site
using three separate ANOVA models in each case In each model variation between replicate
8
blocks was accounted for by treating replicate as a blocking factor The first model included all
the treatments except Treatment 1 at the upland meadow whilst the other two models used
balanced factorial subsets (series) of treatments (see Table 2) the Form x Rate x Frequency
(FRF) series and the Lime x FYM frequency (LFF) series
The FRF series tested the effects of form of fertilizer (ie FYM or inorganic) the rate at which
fertilizers were applied (12 or 24 t FYM ha-1
and inorganic treatments corresponding to these)
the frequency at which treatments were applied (ie annual or triennially) and all two- and three-
way interactions between these factors
The LFF series tested the effects of liming regime ie lime in 1999 (and 2005 at the upland site)
versus lime in 2005 only the frequency of FYM application (ie nil annual or triennially) and the
Lime x FYM frequency interaction All FYM treatments within this series were applied at 12 t ha-
1 At both sites the LFF series consisted of Treatments 2 4 7 13 14 and 15 (see Table 2) The
liming comparison therefore differed slightly between the two sites since liming was repeated in
2005 on plots previously limed in 1999 at the upland meadow but not repeated at the lowland
one
In both the LFF and the FRF models for each variable the first stratum of the ANOVA
consisted of the effects of all factors and interactions averaged over time with the same factors
and interactions repeated in the second stratum with time as an additional factor
In all repeated measures ANOVAs to account for the repeated measures aspect all degrees of
freedom in the Year x Treatment stratum were adjusted to account for departure from
homogeneity of the population covariance matrix using ε-coefficients calculated by the
Greenhouse-Geisser method (Genstat V Committee 1997)
In most years including 2010 all species were recorded individually but at both sites
bryophytes were recorded only as a single group in some years This had only a small influence
on the data since there were seldom more than two bryophyte species present in a quadrat and
more often less Nevertheless for consistency between years bryophytes were treated as a single
group in all years when calculating total species-richness for data for repeated measures
ANOVAs
In addition to the above analyses 2010 data for the key variables were tested alone for treatment
effects by ANOVA and by analysis of covariance (ANCOVA) the latter using 1999 data as the
covariate (Genstat 5 Committee 1997) All three models as described above were tested The
ANCOVAs for the upland meadow excluded Treatment 1 (untreated control plots established in
2005) Where an ANCOVA showed significant treatment covariance with 1999 data the adjusted
results were used In other cases the unadjusted analyses were used Individual bryophyte species
were included in the calculations of species-richness at both sites in these analyses
In all analyses in order to satisfy the conditions for ANOVA (normality of residuals and
homogeneity of residual variation) all percentage data were transformed before analysis by
arcsine(radicp) where p is the percentage value expressed as a proportion All results quoted for
percentage data refer to analyses of arcsine-square root transformed data
Mixed modeling
Mixed model analysis (Residual Estimated Maximum Likelihood ndash REML Genstat V
Committee 1997) was used to identify any effect of form rate or frequency of application on
vegetation composition at each site in 2010 not simply attributable to the total (or mean per year)
amount of fertilizer applied over the study period A variable representing this amount was
included as a continuous explanatory variable in each analysis along with other fixed factors
form rate and frequency of application (Block was included as a random factor) Since the total
(or mean) amounts of N P and K applied over the whole period were completely correlated
across treatments at each site it was not feasible to include all three elements as separate terms in
the model Nor was it feasible to simply use a single notional variable for fertilizer amount (eg t
9
FYM ha-1
year-1
or inorganic equivalent) since there was lack of equivalence of N and P supply
(but not K) between individual FYM treatments and their corresponding inorganic treatments For
each dependent variable tested two separate models were therefore developed using either N or
P ha-1
year-1
as the continuous variable In each case the presence of any non-linearity in response
was tested by including a quadratic term (ie N2 or P
2) or where more appropriate by logn-
transformation of the explanatory variable
For each analysis an initial model was constructed that included all five main effects and all
interactions The importance of each term was evaluated initially by a χ2 test on the Wald statistic
(Genstat V Committee 1997) The number and order of terms in the model were varied but with
N or P amount always included until a model was developed in which each term was significant
The model was then refined by testing the contribution of each term to the model by the more
conservative deviance test ie a χ2 test on the difference in deviance between the model
containing the term and a sub-model from which it was dropped (Welham amp Thompson 1997)
Where there was an interaction between form of fertilizer and the quadratic term but no overall
effect of form - implying that the difference between the two forms of fertilizer lay primarily in a
difference in the curvilinearity of response - a refined model was tested with the P2 (or N
2) x
Form term replaced by Form nested within the quadratic term (eg P2Form) This has the effect
of absorbing the overall effect of form of fertilizer within the interaction term and returns the two
terms P2 and P
2 x Form (Galwey 2006)
These modeling approaches were used with total species-richness and PI species-richness as
(separate) response variables and included data for all treatments limed in 2005 but not in 1999
(ie Trs 2-12)
Results
TEMPORAL CHANGES 1999-2010
Changes over time in total species-richness (number per m2) richness and proportional
aggregate cover of positive indicator (PI) species and proportional aggregate cover of
negative indicator (NI) species at the upland and lowland meadows are represented in
Figs 1 and 2 respectively for ten of the fifteen treatments applied Repeated measures
ANOVA statistics for each of the four variables are given in Supporting Information
Tables S4 S6 S8 and S10 respectively for the upland meadow and Tables S12 S14 S16
and S18 respectively for the lowland meadow Corresponding mean values for each
treatment in each year are given in Tables S5 S7 S9 S11 S13 S15 S17 and S19
respectively Changes in individual species abundance contributing to the temporal trends
in aggregated PI and NI species described below are outlined under Supporting
Information
The upland meadow
The overall effects of both treatment and time were highly significant in the repeated
measures ANOVA for total species-richness at the upland meadow (both P=0001) but
there was no treatment x time interaction (P=0647)
After an initial decline between 1999 and 2000 species-richness remained fairly
constant for most treatments until 2003 after which there was a general increase with the
notable exception of the highest rate annual FYM treatment (Tr 5) (Fig 1a) The latter
treatment declined further between 2005 and 2007 after which it appeared to recover
slightly and averaged over all years this treatment was significantly less species-rich than
all others (Plt001 compared to Tr 14 Plt0001 the remainder) None of the remaining
10
treatments differed significantly from the limed control treatment (Tr2) averaged over all
years However the more species-rich treatments ie nil fertilizer plus lime applied in
both 1999 and 2005 (Tr 13) low rate annual and medium rate triennial FYM (Tr 3 and 7
respectively) and the inorganic equivalent to the latter (Tr 11) were all more species-rich
averaged over all years (Plt005) than annual FYM at the medium rate under either
liming regime (ie Trs 4 and 14 the former representing a continuation of past
management)
The effect of the high rate annual FYM treatment (Tr 5) was largely responsible for
several main effects within the repeated measures ANOVA of treatments within the Form
x Rate x Frequency (FRF) factorial series namely overall effects (averaged over all
years) of rate (mediumgthigh P=0002) and frequency (triennialgtannual P=0001) of
application and a not quite significant effect of form of fertilizer (FYMltinorganic
P=0057) Both the form x rate and the form x frequency interactions were significant
(both Plt005) high rate FYM was significantly less species-rich than all other
combinations averaged over annual and three yearly treatments and all years (Plt0001
compared to medium rate FYM Plt001 compared to both medium and high rate
inorganic fertilizer) and annual FYM was less species-rich than three yearly FYM and
than inorganic fertilizer either annually or triennial (all Plt0001) averaged over the two
application rates and all years The rate x frequency interaction was not significant
(P=0105) but there was a marginally non-significant three-way interaction between
form rate and frequency (P=0070) with a low mean for high rate annual FYM (Tr 5
211 species m-2
) contrasting markedly with all other treatments within the series
particularly with low rate triennial FYM (Tr 6 267 species m-2
)
As with the full treatment ANOVA the overall time factor was highly significant
(Plt0001) but only the effect of application frequency varied significantly with time
(interaction Plt005) Species-richness declined slightly with annual treatments overall
and increased slightly with triennial treatments such that differences between these were
significant in all years from 2005 onwards (Plt001 for 2006-2009 Plt005 in 2005 and
2010)
Both the richness and aggregate cover of PI species (PI) increased generally with time
(Fig 1b and 1c) although in both cases there was a progressive separation between
species The temporal trend was particularly marked for PI although the time factor
was highly significant (Plt0001) in both ANOVAs The overall treatment effect was
marginally non-significant for PI richness (P=0069) with no indication of a time x
treatment interaction (P=0703) whereas for PI there was both a significant treatment
effect (P=0002) and a marginally non-significant treatment x time interaction (P=0075)
Mean PI was significantly lower averaged over all years for annual FYM treatments at
both 12 and 24 t ha-1
(ie Trs 4 14 and 5) compared to several other treatments notably
nil fertilizer treatments limed either in 2005 only (Tr 2 limed control) or in both 1999
and 2005 (Tr 13) (all differences Plt0001 except Tr 2 vs Tr 4 Plt001) and triennial
inorganic treatments (Trs 11 and 12 Plt001 except for Tr 4 vs Tr 11 Plt005) Within
the FRF series ANOVA the increase in PI over time was greater with triennial
treatments compared to annual averaged over fertilizer forms and rates of application
(time x frequency interaction P=0016) with a marked overall difference due to
application frequency averaged over forms rates and all years (P=0005)
11
PI species-richness showed a similar overall effect of application frequency within the
FRF series (triennialgtannual P=0013) and also a significant overall effect of rate of
application (medium rategthigh rate P=0022) The overall effect of form of fertilizer was
marginally non-significant (FYMltinorganic P=0059) and there was no significant
interaction among any of the three factors averaged over all years nor any interaction
with time
16
18
20
22
24
26
28
30
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Specie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime in Year 1+ ann FYM (Tr 14) Nil fert + Lime in Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr1)
a Species richness (total)
3
4
5
6
7
8
9
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
70
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
0
5
10
15
20
25
30
35
40
45
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
Fig 1 Changes 1999-2010 in vegetation composition an upland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control
The mean number of negative indicator (NI) species per m2 averaged over all treatments
varied significantly over time at the upland meadow (P=0002 data not shown) though
with no consistent trend - it was lowest overall in 2002 (499 per m2) and highest in both
1999 and 2008 (548 per m2) The treatment x time interaction was not significant
(P=0469) but the overall treatment effect was highly significant (Plt0001) mean
number of NI species per m2 was higher than the limed control (Tr 1 453 per m
2) with
all fertilizer treatments except Treatments 11 (triennial inorganic fertilizer at the lower
rate) and 13 (nil fertilizer limed in both 1999 and 2005) (Plt0001 for all except Trs 5
and 6 for which Plt001) The highest mean NI m-2
value was for high rate inorganic
12
fertilizer (Tr 10 565 per m2) which differed significantly (Plt005) from its FYM
counterpart Treatment 5 There was however no consistent difference between FYM
treatments and their inorganic counterparts and no overall effect of form of fertilizer
within the FRF series (P=0745) There was a significant form x rate interaction within
this ANOVA (P=0032) but only the difference between the high rate and lower rate
inorganic means was significant (high rategtlower rate Plt001)
The aggregate contribution to vegetation cover of negative indicator species (NI)
showed a very marked divergence between treatments over time (Fig 1d) with NI
either changing little or declining under most treatments again with the notable exception
of high rate annual FYM (Tr 5) under which NI increased over time Time treatment
and the treatment x time interaction were all highly significant (Plt0001) The
predominant temporal trend was downward (from 25 of live cover in 1999 to 14 in
2010 averaged over all treatments) but NI for Treatment 5 had significantly increased
compared to 1999 in 2002 2003 (Plt005) 2005 (Plt0001) 2008 (Plt0001) and 2009
(Plt001) but not in 2010 (Fig 1d) The most marked declines in NI occurred on the
limed control (Tr 2) nil fertilizer plus lime in both 1999 and 2005 (Tr 13) and the lower
rate triennial inorganic fertilizer treatments (Tr 11) differences compared to both 1999
and 2002 were highly significant (Plt0001) in most subsequent years for these
treatments A smaller decline also occurred after 2002 on the treatment equivalent to past
inputs (ie12 t ha-1
year-1
annual FYM Tr 4) In most years this treatment differed little
from the equivalent FYM treatment limed in both 1999 and 2005 (Tr 14)
These trends were reflected in highly significant overall effects on NI within the FRF
series of form (FYMgtinorganic) rate (mediumlthigh) and frequency (triennialltannual)
(all effects Plt0001) Time x rate and time x frequency interactions were both significant
(P=0019 and Plt0001 respectively) with a marginally non-significant rate x frequency
interaction averaged over all years (P=0054) NI declined over time under both
application rates averaged over forms and frequencies of application but the decline was
slower with high rate treatments compared to lower rate Difference between rates were
significant in all years from 2002 onwards (Plt005 in 2002 2006 and 2007 Plt0001 in
other years) Overall differences due to frequency of application were even more marked
Under annual application NI declined initially from 2003-2006 compared to 1999 but
subsequently recovered and did not differ from 1999 levels from 2007 onwards whilst
under triennial application differences were highly significant (Plt0001) compared to
1999 from 2003 onwards with significant differences between years during this same
period (Plt005 in 2003 and 2006 Plt0001 in remaining years)
Repeated-measures ANOVAs within the LFF treatment series at the upland meadow
showed no overall effect of liming regime on temporal trends for any of the above
variables nor any interaction of lime with either time or FYM frequency - note that FYM
was applied at the intermediate rate (12 t ha-1
) in all FYM treatments within this series
The overall effect of FYM frequency was significant for both PI (P=0003) and the
number of NI species m-2
(P=0001) PI was highest with nil FYM and lowest with
annual FYM with triennial intermediate and differing significantly (Plt005) from the
other two means NI species m-2
did not differ between triennial and annual application
within the LFF series but both were significantly higher than nil FYM (Plt001 for
triennial Plt0001 for annual application) There was also a significant time x FYM
frequency interaction for NI within this series (P=0001) reflecting a more rapid
13
decline with nil FYM than with triennial FYM and no significant overall trend with
annual FYM when averaged over both lime treatments
12
14
16
18
20
22
24
26
28
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Spe
cie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime Year 1 + ann FYM (Tr 14) Nil fert + Lime Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr 1)
a Species richness (total)
2
3
4
5
6
7
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
0
10
20
30
40
50
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
Fig 2 Changes 1999-2010 in vegetation composition at a lowland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control treatment and those that had received lime in year 1
Treatments 1 and 2 were identical (nil input) until 2005
The lowland meadow
Both time and treatment were significant within the ANOVA for total species-richness at
the lowland meadow (Plt0001 and P=0009 respectively and in contrast to the upland
site the treatment x time interaction was significant (P=0012) Averaged over all years
high rate annual FYM treatment (Tr 5) was significantly less species-rich than all others
except Treatment 14 (annual FYM at 12 t ha-1
limed in 1999) having declined
progressively between 1999 and 2009 (Fig 2a) with most differences significant at
Plt001 Treatment 14 did not differ significantly overall compared to the corresponding
FYM treatment not limed until 2005 (Tr 4) although these two treatments showed
14
markedly different temporal trends Treatment 14 was less species-rich than others during
the first five years of the experiment but did not change significantly throughout the
whole 12 year period whilst Treatment 4 changed little between 1999 and 2006 but
declined thereafter (Plt001 and Plt0001 for 2009 and 2010 respectively compared to
2006) possibly in response to liming in 2005 However there was no significant overall
effect on total species-richness of liming strategy within the LFF series and no interaction
between liming and either time or FYM application
There were marked differences in temporal trend in species-richness between FYM
treatments and their inorganic counterparts particularly with the high rate annual
treatments (Tr 5 and Tr 10) Whereas species-richness declined more or less continually
throughout under Treatment 5 at least until 2009 (Plt001 for 2004 compared to 1999 and
for both 2009 and 2010 compared to 2004) Treatment 10 declined between 2000 and
2001 (Plt005) and then remained relatively unchanged until 2008 after which it declined
again (2010 Plt005 compared to 2008)
Other treatments within the full treatment series including untreated control (Tr 1) and
limed control (Tr 2) showed no particular temporal trend in the full repeated measures
ANOVA although both the latter treatments showed a dip in species-richness in 2004
(Plt005 compared to both 2003 and 2005)
There was a strong overall effect of form of fertilizer within the FRF series ANOVA
(FYMltinorganic P=0003) a significant effect of frequency of application
(triennialgtannual P=0027) and a not quite significant effect of application rate (high
rateltmedium rate P=0064) but no significant interaction between any of these factors
The time x application frequency interaction was highly significant (Plt0001) species-
richness remained unchanged under triennial application but under annual application
was significantly lower compared to 1999 in each year from 2004 onwards with the
exception of 2006 (Plt005 in 2004 Plt001 in 2005 and 2008 Plt0001 the remainder)
Again with the exception of 2006 differences between triennial and annual application
were significant in each year from 2005 onwards (Plt005 in 2007 Plt001 in 2005 and
2008 Plt0001 in 2009 and 2010)
Both treatment time and their interaction were significant in the full treatment ANOVA
for PI species-richness at the lowland meadow (ie time and treatment both Plt0001
time x treatment P=0006) There was a general decline in PI-richness after 2005 (Fig
2b) with PI species m-2
averaged over all treatments lower from 2007 onwards (Plt0001)
than all years up to 2005 except 2001 Although the initiation of this trend corresponded
with the liming of most treatments in 2005 a similar pattern was shown by treatments
that did not receive this lime application ie the untreated control treatment (Tr 1) the
nil fertilizer treatment limed in 1999 (Tr 13) and the corresponding treatment also
receiving triennial FYM (Tr 15) In all these cases PI-richness showed no significant
difference between years until 2005 but was significantly (Plt001) lower by 2010 than in
2005 Treatment 14 (limed in 1999 plus annual FYM) did not show this pattern but PI
species-richness had been lowest under this treatment in most years between 1999 and
2005 (Fig 2b)
As with total species-richness PI richness declined more or less progressively under
Treatment 5 until 2008 (Plt005 compared to 1999 by 2001 Plt0001 from 2006
onwards) whereas Treatment 4 did not change significantly until 2006 after which it
declined (Plt005 in 2007 compared to 2006 Plt001 thereafter) Both these treatments
15
tended be less rich in PI species than the corresponding inorganic treatment (Trs 9 and
10 respectively) in most years consistently so for Tr 4 compared to Tr 9 but these
differences were not significant in any year nor when averaged over all years There was
however a significant overall effect of form of fertilizer in the FRF series ANOVA
(FYMltinorganic P=0002) and a highly significant effect of frequency of application
(triennialgtannual Plt0001) The time x frequency of application interaction was also
highly significant (Plt0001) Under triennial application PI species per m2 increased
slightly until 2005 (Plt005 compared to 1999) and declined thereafter so that by 2009
levels were significantly lower than in 1999 (Plt001 for 2009 Plt0001 for 2010) By
contrast under annual application PI species m-2
declined progressively until 2009
following a temporary though highly significant (Plt0001) dip in 2000 so that the
difference compared to 1999 was significant in every year from 2002 onwards and highly
significant (Plt0001) from 2005 With the exception of 2003 differences between
triennial and annual application were significant from 2002 onwards and highly
significant (Plt0001) in 2005 and from 2007 to 2010
There was no significant effect other than of time within the LFF treatment series
ANOVA although there was a marginally non-significant time x FYM frequency
interaction (P=0054) This reflected a more rapid decline in PI species-richness under
annual FYM application than with either nil or triennial FYM with the latter two showing
no discernible difference
By contrast with PI species m-2
PI cover percentage (PI) showed no overall treatment
effect within the full treatment ANOVA nor a time x treatment interaction although the
overall effect of time was highly significant (Plt0001) The temporal pattern in PI (Fig
2c) also differed from that shown by PI-richness with PI averaged over all treatments
declining between 1999 and 2005 in three highly significant increments (1999-2000
2000-2002 and 2002-2005 all Plt0001) and increasing again thereafter (2005-2009 and
2009-2010 both Plt0001) There was no significant effect within either the LFF or FRF
treatment series ANOVAs although their was a slight time x frequency of application
effect within the latter (P=0081) PI was slightly higher under annual application than
triennial in each year 1999-2004 after which the reverse was true although differences
were always small
There was no overall effect of treatment on the number of NI species per m2 within the
full treatment range at the lowland meadow (data not shown) but both the overall effect
of time and the time x treatment interaction were significant (Plt0001 and P=0009
respectively) Overall NI species m-2
increased over time from 24 species m-2
in 1999 to
a peak of 42 m-2
in 2008 averaged over all treatments NI species m-2
increased on the
untreated control treatment (Tr 1) from 21 species m-2
in 1999 to 34 species m-2
in 2007
(increase Plt001) then declined to the 1999 value by 2010 The limed (in 2005) control
(Tr 2) showed a somewhat similar pattern though from a slightly higher starting level
(31 species m-2
) and with the increase to 38 species m-2
in 2008 not reaching
significance
These temporal trends in NI species m-2
were much accentuated by annual fertilizer
application particularly by FYM treatments with peak numbers of NI species reaching
52 and 47 species m-2
for Treatments 4 and 5 respectively in 2008 (both Plt0001
compared to 1999) and despite slight declines in both cases remaining significantly
higher in 2010 than in 1999 (both Plt0001) Inorganic counterparts to these treatments
16
showed similar though slightly less marked trends and there was no significant
difference between all four treatments in the peak number reached in 2008 There was no
overall effect of either form of fertilizer or rate of application within the FRF series
ANOVA and only a marginally non-significant effect of frequency of application
(triennialltannual P=0061) Liming in 1999 (Trs 13 14 and 15) also appeared to
accentuate the generally increasing trend although peak numbers in 2008 did not differ
between these treatments and either untreated or limed control in the full treatment range
ANOVA The overall effects of liming regime (1999 or 2005) were not significant within
the LFF treatment series but the faster rate of increase with liming in 1999 was reflected
in a significant time x lime interaction in this analysis (P=0017) The mean number of NI
species m-2
increased very significantly (Plt0001) between 1999 and 2000 following
liming in 1999 with the increment increasing until 2008 whilst on plots un-limed until
2005 NI species m-2
did not increase significantly until 2003 (Plt005) although the
increase compared to 1999 was highly significant (Plt0001) in 2006 2008 and 2010
Nevertheless the differences in rate of increase was at least partly due to a lower 1999
mean for the 1999-limed treatments (170 species m-2
compared to 244 species m-2
difference NS) and although in all years from 2001 onwards mean NI species m-2
was
higher for 1999-limed treatments compared to those not limed until 2005 the difference
was not significant in any year
The contribution of NI species to vegetation cover (NI) showed a very marked
separation between treatments over time at the lowland meadow (Fig 2d) starting from
much lower levels than at the upland meadow (compare with Fig 1d) There were
noticeable differences in temporal trend between annual fertilizer treatments on the one
hand and triennial and nil fertilizer treatments on the other (Fig 2d) The effects of
treatment time and the treatment x time interaction were all highly significant (Plt0001)
in the full treatment range ANOVA The most marked increase in NI occurred with
annual FYM at the high rate (Tr 5) with NI increasing rapidly between 1999 and 2001
(Plt001) and a further highly significant increment between 2004 and 2009 (Plt0001)
(Fig 2d) A similar though less marked trend occurred with annual FYM at the medium
rate (Tr 4) with significant increases between 1999 and 2005 and between 2005 and
2010 (both Plt001) NI also increased markedly with the inorganic treatment
equivalent to Treatment 5 (Tr 10) although the overall rate of increase shown by this
treatment was closer to that shown by Treatment 4 than Treatment 5 There were
significant differences between Treatments 5 and 10 in most years from 2004 onwards
(Plt001 and Plt0001 in 2009 and 2010 respectively)
These effects were reflected in a significant overall effect of form of fertilizer
(FYMgtinorganic P=0008) and highly significant (Plt0001) overall effects of both rate
(high rategtlower rate) and frequency of application (annualgttriennial) within the FRF
series ANOVA and a significant form x rate interaction (P=0043) Mean NI was
highest for high rate annual FYM (Plt0001 compared to all three remaining means) and
lowest for lower rate inorganic (Plt005 compared to high rate inorganic NS compared
to lower rate FYM) Only frequency of application interacted significantly with time
(Plt0001) NI increased moderately under triennial application until 2008 (Plt005 in
2005 compared to 1999 Plt0001 each year 2006-2008) followed by a decline (Plt001
2010 compared to 2008) but with mean NI in 2010 still higher than 1999 (Plt005)
The increasing trend was very much more marked with annual fertilizer application for
17
which there were highly significant (Plt0001) increments between 1999 and 2002 and
between 2002 and 2008 but with no significant increase thereafter Differences between
annual and triennial treatment means were highly significant (Plt0001) from 2003
onwards
There was no significant change in NI over time either under the untreated control
(Tr 1) or the nil fertilizer (Tr 2) treatments in the full treatment ANOVA NI increased
until 2005 on plots limed in 1999 without fertilizer Tr 13 (Plt001 compared to 1999)
and declined slightly but not significantly thereafter Trends were similar with the
treatment limed in 1999 and receiving triennial FYM (Tr 15 ndash not illustrated in Fig 2)
although NI was higher with this treatment than with Tr 13 (Plt005) in both 2005 and
2008 By contrast applying the same rate of FYM annually after liming in 1999 (Tr 14)
caused a more rapid increase with levels by 2005 higher than either Tr 13 (Plt001) or
Tr 4 (Plt005) ndash note that the latter was equivalent to Tr 14 but without lime until 2005
NI declined after 2006 under Tr 14 but continued to increase with Tr 4 with the
difference between these two treatments reversed by 2010 (Plt005)
All main effects and interactions were significant in the LFF treatment series ANOVA
for NI except for lime x FYM frequency and the lime x FYM x time interactions Plots
limed in 1999 supported higher NI levels than those limed in 2005 averaged over the
three FYM frequencies and all years (P=0042) NI increased more rapidly after liming
in 1999 than where liming was delayed until 2005 (P=0033 for time x lime interaction)
with the overall difference due to liming in 1999 highly significant (Plt0001) by 2005
NI declined thereafter on the 1999-limed treatments whilst continuing to increase
following liming in 2005 of previously un-limed treatments both peaking in 2008 at a
very similar level Averaged over lime treatments NI increased much more rapidly
under annual FYM application than under either triennial or nil FYM (P=0002 for the
time x FYM frequency interaction) a highly significant increase had occurred by 2002
with annual FYM (Plt0001 compared to 1999) but increases of similar magnitude were
not reached until 2005 with triennial application and not until 2007 with nil FYM NI
subsequently declined under the latter two FYM regimes but not with annual FYM so
that by 2010 NI was significantly higher with annual FYM than with either triennial or
nil FYM (Plt0001) but with only a small non-significant difference between the latter
two treatments
TREATMENT EFFECTS AFTER 12 YEARS OF APPLICATION
The effects of all treatments on the five key variables at each site in 2010 are shown in
Table S20 with treatments differing significantly (Plt005) from the limed control
treatment (Tr 2) highlighted for each variable Treatment effects in 2010 are not
described in detail here but the modeled responses of total species-richness and of
richness of PI species in 2010 to the mean amounts of N and P applied per year averaged
over 1999-2010 are described below
Relationships with the amounts of fertilizer applied
The effect of neither rate nor frequency of application was significant in any of the
REML models for either total species-richness or PI species-richness at either site when
the amount of N or P applied ha-1
year-1
was included The effect of form of fertilizer was
still significant in most models however either as a main effect or in interaction with the
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
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Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
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Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
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Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
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Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
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Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
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Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
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Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
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Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
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haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
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Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
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Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
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Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
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Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
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studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
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plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
4
Past management
Both meadows had previously been managed over a long period by cutting for hay with aftermath
grazing Cutting occurred after 1 July at the upland meadow and after the second week in July at
the lowland meadow The upland meadow had received about 12 t FYM ha-1
each year usually in
late April but occasionally in mid summer following the hay harvest and had received periodic
applications of lime (amounts not known) the last one in about 1993 Details of past fertilizer
application at the lowland meadow are uncertain although this site had not received any form of
fertilizer or lime in the preceding 20 years or more there is some evidence to suggest that basic
slag and lime may have been applied at some time before that
Soil properties
Soils at the upland meadow were clay-loam in texture whilst those at the lowland meadow were
a mixture of sandy silt loam to silty clay-loam The upland meadow soils were notably higher in
total N carbon and organic matter (Table 1)
Table 1 Soil chemical properties (top 75 cm) at experimental sites in Cumbria (the upland
meadow) and Wales (the lowland meadow) in 1999
Upland meadow Lowland meadow
Mean Range Mean Range
pH 518 512-527 501 494-511
Total N 065 059-071 034 029-039
C 676 615-776 315 266-351
Organic matter 1718 1577-2069 903 801-973
Olsen extractable P (mg l-1
) 550 444-679 512 410-600
Exchangeable K (mg l-1
) 1971 1629-2479 1747 1323-2205
Exchangeable Mg (mg l-1
) 1548 1368-1957 2014 1606-2389
Exchangeable Ca (mg l-1
) 1480 1334-1723 1047 852-1214
Measured by loss on ignition (LOI)
EXPERIMENTAL DESIGN
All experimental treatments (see Table 2) were applied by hand to individual 7m x 5m plots laid
out in a randomized block design with three replicate blocks at each site Treatments were applied
between March and late April Due to access restrictions resulting from the national outbreak of
Foot and Mouth Disease (FMD) no treatment was applied in 2001 This meant that treatments
requiring annual applications only received a total of 1112 (92) of the intended amounts over
the 12 year period 1999-2010 Triennial treatments were applied in 1999 2002 2005 and 2008
5
Table 2 Treatments applied 1999-2010 at the upland and lowland meadows Values are the mean
amounts applied (kg ha-1
year-1
elemental N P and K) either as FYM (estimated) or in inorganic
form (actual) averaged over 12 yearsa 1999-2010 Treatments 13-15 were limed only in both
1999 and 2005 at the upland site but in 1999 only at the lowland site Treatments 2-12 received
lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Upland meadow Lowland meadow
Treatment N P K N P K
1 Untreated control 00 00 00 00 00 00
2 Limed (in 2005) control 00 00 00 00 00 00
3 FYM at 6 t ha-1
annual 44 50 345 29 38 292
4+ FYM at 12 t ha-1
annual 88 100 689 57 76 585
5 + FYM at 24 t ha
-1 annual 176 199 1378
114 151 1169
6 FYM at 6 t ha-1
triennially 15 22 113 09 13 110
7 + FYM at 12 t ha
-1 triennially 30 45 226
19 26 221
8 + FYM at 24 t ha
-1 triennially 61 90 452
38 52 441
9 + Inorg equivalent to Tr 4 85 84 689 61 63 585
10 + Inorg equivalent to Tr 5 170 168 1378
121 125 1169
11 + Inorg equivalent to Tr 7 30 36 226
24 21 221
12 + Inorg equivalent to Tr 8 61 72 452
48 42 441
13 Lime in years 1 (and 7) 00 00 00 00 00 00
14 Lime as Tr 13 + FYM as Tr 4 88 100 689 57 76 585
15 Lime as tr 13 + FYM as Tr 7 30 45 226 19 26 221 aNote that no fertilizer was applied in 2001 so that for annual treatments the amounts shown
are 1112ths of the mean amount applied in each of the remaining years
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime
in 1999 x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
The FYM used in the experiment was sourced locally from the same farms each year and was
sampled at the time of application for subsequent chemical analysis The ADAS Manure Nitrogen
Evaluation Routine (MANNER Chambers et al 1999) was used between 1999 and 2006 to
predict the amount of plant available N supplied by the FYM after which a refined model
(MANNER-NPK Nicholson et al 2010) was used Refinements to the model included more
detailed N mineralisation functions and predictions of N availability in the year following
application Availabilities of 60 and 90 of total P and K respectively (Anon 2010) were
assumed between 1999 and 2006 and this assumption for K was maintained throughout the 1999-
2010 period From 2007 onwards the assumed availability of P from FYM was increased to 80
of total P in line with information on longer term release of plant-available P from FYM (Smith
et al 1998) For both N and P the mean amounts assumed to have been applied in FYM
treatments averaged over 1999-2010 and shown in Table 2 are based upon revised estimates for
the whole period The inorganic fertilizer N treatments applied each year ie the inorganic
lsquoequivalentsrsquo for specific FYM treatments (see Table 2) were based upon estimates made using
the original MANNER model from 1999-2006 and MANNER-NPK from 2007-2010 Similarly
6
the inorganic fertilizer P applied each year corresponded to 60 of the total P in FYM between
1999 and 2006 and 80 from 2007-2010
Inorganic N P and K were applied as ammonium nitrate triple super-phosphate and muriate of
potash respectively to the relevant treatments The FYM treatments were designed to encompass
the range of inputs traditionally applied to species-rich hay meadows upon which current agri-
environment guidelines are based (Simpson and Jefferson 1996 Crofts and Jefferson 1999)
Lime was applied to treatments 13 14 and 15 only at each site in March-April 1999 and to most
treatments in 2005 (Table 2) The latter included the three previously limed treatments at the
upland meadow but not at the lowland one since soil pH levels had not fallen significantly by
2005 on these plots at the latter site Based on assessments of soil texture and soil pH tests
conducted in February-March the amounts of lime were calculated to be sufficient to raise the
soil pH of the experimental plots to 60 (Anon 2010)
At each site one plot per replicate block received no fertilizer or lime (untreated control)
between 1999 and 2005 whilst another plot per replicate was designated as a continuation of past
fertilizer inputs At the lowland meadow the latter was identical to the untreated control
treatment but at the upland meadow it was identical to Treatment 4 (12 t ha-1
FYM annually) In
2005 a decision was made to treat occasional liming as lsquobackgroundrsquo management for all
fertilizer treatments at both sites to be triggered for each treatment in future years once mean pH
for that treatment had declined to 55 whilst retaining both an untreated control (ie nil fertilizer
nil lime ndash Treatment 1) and a nil fertilizer (limed in 2005) control treatment (Treatment 2) at each
site The rationale behind this decision is addressed in the Discussion section At the lowland
meadow one of the two hitherto untreated control plots per replicate (Treatment 2) was therefore
limed in 2005 along with other treatments Only one nil input plot per replicate was available at
the upland meadow but since the site had a history of liming the existing nil input plots were
limed in 2005 (Treatment 2) and new untreated control plots (Treatment 1) were established
adjacent to each replicate block with the end at which each was located chosen randomly for
each block
The experimental plots were cut for hay after 1 July each year at the upland site and after 15
July at the lowland site the actual cutting date being dependent on weather conditions The hay
aftermath growth was grazed each year with experimental plots being grazed with the remainder
of the field At the upland meadow mature sheep were used normally commencing in September-
October continuing until March or late April The lowland meadow was grazed by beef store
cattle from mid Octoberearly November to late February
Botanical assessments were carried out during May each year except in 2001 at the upland
meadow due to FMD restrictions The percentage cover of each species present was estimated
visually in three 1m2 quadrats positioned at random within each plot in 1999 and then fixed for
the project duration
Soils were sampled in March 1999 2002 2004 2007 and 2010 by taking five 35 mm diameter x
75mm deep soil cores at random from within each treatment plot but outside the fixed positions
of the three 1m2 botanical survey quadrats The five samples per plot were combined dried at
30oC and then ground prior to analysis for organic carbon (C) total nitrogen (N) Olsen
extractable P exchangeable potassium (K) magnesium (Mg) calcium (Ca) sodium (Na) and pH
(in H2O) using standard laboratory methods (Allen 1974 MAFF 1986)
Soils were further sampled in June 1999 2002 2004 2007 and 2010 for soil microbial
assessment by taking five 35 mm diameter x 100 mm deep cores which were subsequently bulked
for each plot sieved (5 mm) and stored at 4oC prior to analysis Microbial community structure
was assessed using phospholipid fatty acid analysis (PLFA) as described by Bardgett et al
(1996) The fatty acids i150 a150 150 i160 170 i170 cy170 cis181ω7 and cy190 were
chosen to represent bacterial PLFAs (Federle 1986 Frostegaringrd Tunlid amp Baringaringth 1993) and
182ω6 was used as an indicator of fungal biomass (Federle 1986) The ratio of 182ω6bacterial
7
PLFAs was taken to represent the ratio of fungal-to-bacterial biomass in soil (Bardgett Hobbs amp
Frostegaringrd 1996 Frostegaringrd amp Baringaringth 1996)
DERIVED BOTANICAL VARIABLES
Estimates of cover for individual species were converted to a percentage of the total live
vegetation cover present in each quadrat in order to minimize the effects of year-to-year variation
and variation between treatments in vegetation density and total cover Several composite
variables were calculated to characterize the vegetation and these were then averaged across the
three quadrats in each plot to give plot mean values for each variable Plot means were then used
as the basic units for subsequent analyses of treatment effects
Key variables
Five key variables were used to investigate the effects of treatment and time on botanical
composition total species-richness (the total number of vascular plant species per m2) the
number per m2 of MG3 and MG5 positive indicator species (the upland and lowland meadows
respectively) the aggregate cover of these species as a percentage of total vegetation cover the
number per m2 of negative indicator species and the aggregate cover of negative indicator
species as a percentage of total vegetation cover Positive mesotrophic indicator species were
identified from lists produced by Robertson and Jefferson (2000) for use in monitoring the
condition of grassland Sites of Special Scientific Interest (SSSIs) in MG3 (Anthoxanthum
odoratum-Geranium sylvaticum grassland) and MG5 (Cynosurus cristatus-Centaurea nigra
grassland) communities of the NVC (Rodwell 1992) A single list of negative indicator species
was compiled from the generic negative indicators for MG3 and MG5 communities (Robertson amp
Jefferson 2000) augmented by species shown in previous work (eg Mountford et al 1993
Kirkham et al 1996) to be favoured by nutrient addition and for which an increase in abundance
would indicate a negative effect on plant community quality Positive and negative indicator
species are listed in Supporting Information Table S3
Additional variables
Among other derived variables was the weighted Ellenberg N score (Smith et al 2003 Kirkham
et al 2008) referred to hereafter as the fertility score The Ellenberg N index represents the
degree of association with soil fertility (not specifically soil N) of a particular species (Ellenberg
1988 Hill et al 1999) Fertility scores were calculated for each quadrat as the average N index
of the component species weighted according to the proportional contribution of each species to
total vegetation cover Plot mean fertility scores were highly correlated at both sites with the
aggregate cover of negative indicator species (NI) (Pearson r=0845 and r=0927 at the upland
and lowland sites respectively in 2010 both Plt0001) The latter variable was preferred to
fertility scores for statistical analyses in this paper because it proved more responsive to fertilizer
treatments although fertility scores are referred to in the Discussion section in relation to results
from elsewhere
DATA ANALYSIS
ANOVAs for treatment effects
For each of the key variables botanical data for all years 1999-2010 except 2001 at the upland
meadow and for all treatments except Treatment 1 at the same meadow were analyzed by
repeated measures analysis of variance (ANOVA - Genstat V Committee 1997) with Year as the
repeated measures factor Separate ANOVAs were carried out for each variable within each site
using three separate ANOVA models in each case In each model variation between replicate
8
blocks was accounted for by treating replicate as a blocking factor The first model included all
the treatments except Treatment 1 at the upland meadow whilst the other two models used
balanced factorial subsets (series) of treatments (see Table 2) the Form x Rate x Frequency
(FRF) series and the Lime x FYM frequency (LFF) series
The FRF series tested the effects of form of fertilizer (ie FYM or inorganic) the rate at which
fertilizers were applied (12 or 24 t FYM ha-1
and inorganic treatments corresponding to these)
the frequency at which treatments were applied (ie annual or triennially) and all two- and three-
way interactions between these factors
The LFF series tested the effects of liming regime ie lime in 1999 (and 2005 at the upland site)
versus lime in 2005 only the frequency of FYM application (ie nil annual or triennially) and the
Lime x FYM frequency interaction All FYM treatments within this series were applied at 12 t ha-
1 At both sites the LFF series consisted of Treatments 2 4 7 13 14 and 15 (see Table 2) The
liming comparison therefore differed slightly between the two sites since liming was repeated in
2005 on plots previously limed in 1999 at the upland meadow but not repeated at the lowland
one
In both the LFF and the FRF models for each variable the first stratum of the ANOVA
consisted of the effects of all factors and interactions averaged over time with the same factors
and interactions repeated in the second stratum with time as an additional factor
In all repeated measures ANOVAs to account for the repeated measures aspect all degrees of
freedom in the Year x Treatment stratum were adjusted to account for departure from
homogeneity of the population covariance matrix using ε-coefficients calculated by the
Greenhouse-Geisser method (Genstat V Committee 1997)
In most years including 2010 all species were recorded individually but at both sites
bryophytes were recorded only as a single group in some years This had only a small influence
on the data since there were seldom more than two bryophyte species present in a quadrat and
more often less Nevertheless for consistency between years bryophytes were treated as a single
group in all years when calculating total species-richness for data for repeated measures
ANOVAs
In addition to the above analyses 2010 data for the key variables were tested alone for treatment
effects by ANOVA and by analysis of covariance (ANCOVA) the latter using 1999 data as the
covariate (Genstat 5 Committee 1997) All three models as described above were tested The
ANCOVAs for the upland meadow excluded Treatment 1 (untreated control plots established in
2005) Where an ANCOVA showed significant treatment covariance with 1999 data the adjusted
results were used In other cases the unadjusted analyses were used Individual bryophyte species
were included in the calculations of species-richness at both sites in these analyses
In all analyses in order to satisfy the conditions for ANOVA (normality of residuals and
homogeneity of residual variation) all percentage data were transformed before analysis by
arcsine(radicp) where p is the percentage value expressed as a proportion All results quoted for
percentage data refer to analyses of arcsine-square root transformed data
Mixed modeling
Mixed model analysis (Residual Estimated Maximum Likelihood ndash REML Genstat V
Committee 1997) was used to identify any effect of form rate or frequency of application on
vegetation composition at each site in 2010 not simply attributable to the total (or mean per year)
amount of fertilizer applied over the study period A variable representing this amount was
included as a continuous explanatory variable in each analysis along with other fixed factors
form rate and frequency of application (Block was included as a random factor) Since the total
(or mean) amounts of N P and K applied over the whole period were completely correlated
across treatments at each site it was not feasible to include all three elements as separate terms in
the model Nor was it feasible to simply use a single notional variable for fertilizer amount (eg t
9
FYM ha-1
year-1
or inorganic equivalent) since there was lack of equivalence of N and P supply
(but not K) between individual FYM treatments and their corresponding inorganic treatments For
each dependent variable tested two separate models were therefore developed using either N or
P ha-1
year-1
as the continuous variable In each case the presence of any non-linearity in response
was tested by including a quadratic term (ie N2 or P
2) or where more appropriate by logn-
transformation of the explanatory variable
For each analysis an initial model was constructed that included all five main effects and all
interactions The importance of each term was evaluated initially by a χ2 test on the Wald statistic
(Genstat V Committee 1997) The number and order of terms in the model were varied but with
N or P amount always included until a model was developed in which each term was significant
The model was then refined by testing the contribution of each term to the model by the more
conservative deviance test ie a χ2 test on the difference in deviance between the model
containing the term and a sub-model from which it was dropped (Welham amp Thompson 1997)
Where there was an interaction between form of fertilizer and the quadratic term but no overall
effect of form - implying that the difference between the two forms of fertilizer lay primarily in a
difference in the curvilinearity of response - a refined model was tested with the P2 (or N
2) x
Form term replaced by Form nested within the quadratic term (eg P2Form) This has the effect
of absorbing the overall effect of form of fertilizer within the interaction term and returns the two
terms P2 and P
2 x Form (Galwey 2006)
These modeling approaches were used with total species-richness and PI species-richness as
(separate) response variables and included data for all treatments limed in 2005 but not in 1999
(ie Trs 2-12)
Results
TEMPORAL CHANGES 1999-2010
Changes over time in total species-richness (number per m2) richness and proportional
aggregate cover of positive indicator (PI) species and proportional aggregate cover of
negative indicator (NI) species at the upland and lowland meadows are represented in
Figs 1 and 2 respectively for ten of the fifteen treatments applied Repeated measures
ANOVA statistics for each of the four variables are given in Supporting Information
Tables S4 S6 S8 and S10 respectively for the upland meadow and Tables S12 S14 S16
and S18 respectively for the lowland meadow Corresponding mean values for each
treatment in each year are given in Tables S5 S7 S9 S11 S13 S15 S17 and S19
respectively Changes in individual species abundance contributing to the temporal trends
in aggregated PI and NI species described below are outlined under Supporting
Information
The upland meadow
The overall effects of both treatment and time were highly significant in the repeated
measures ANOVA for total species-richness at the upland meadow (both P=0001) but
there was no treatment x time interaction (P=0647)
After an initial decline between 1999 and 2000 species-richness remained fairly
constant for most treatments until 2003 after which there was a general increase with the
notable exception of the highest rate annual FYM treatment (Tr 5) (Fig 1a) The latter
treatment declined further between 2005 and 2007 after which it appeared to recover
slightly and averaged over all years this treatment was significantly less species-rich than
all others (Plt001 compared to Tr 14 Plt0001 the remainder) None of the remaining
10
treatments differed significantly from the limed control treatment (Tr2) averaged over all
years However the more species-rich treatments ie nil fertilizer plus lime applied in
both 1999 and 2005 (Tr 13) low rate annual and medium rate triennial FYM (Tr 3 and 7
respectively) and the inorganic equivalent to the latter (Tr 11) were all more species-rich
averaged over all years (Plt005) than annual FYM at the medium rate under either
liming regime (ie Trs 4 and 14 the former representing a continuation of past
management)
The effect of the high rate annual FYM treatment (Tr 5) was largely responsible for
several main effects within the repeated measures ANOVA of treatments within the Form
x Rate x Frequency (FRF) factorial series namely overall effects (averaged over all
years) of rate (mediumgthigh P=0002) and frequency (triennialgtannual P=0001) of
application and a not quite significant effect of form of fertilizer (FYMltinorganic
P=0057) Both the form x rate and the form x frequency interactions were significant
(both Plt005) high rate FYM was significantly less species-rich than all other
combinations averaged over annual and three yearly treatments and all years (Plt0001
compared to medium rate FYM Plt001 compared to both medium and high rate
inorganic fertilizer) and annual FYM was less species-rich than three yearly FYM and
than inorganic fertilizer either annually or triennial (all Plt0001) averaged over the two
application rates and all years The rate x frequency interaction was not significant
(P=0105) but there was a marginally non-significant three-way interaction between
form rate and frequency (P=0070) with a low mean for high rate annual FYM (Tr 5
211 species m-2
) contrasting markedly with all other treatments within the series
particularly with low rate triennial FYM (Tr 6 267 species m-2
)
As with the full treatment ANOVA the overall time factor was highly significant
(Plt0001) but only the effect of application frequency varied significantly with time
(interaction Plt005) Species-richness declined slightly with annual treatments overall
and increased slightly with triennial treatments such that differences between these were
significant in all years from 2005 onwards (Plt001 for 2006-2009 Plt005 in 2005 and
2010)
Both the richness and aggregate cover of PI species (PI) increased generally with time
(Fig 1b and 1c) although in both cases there was a progressive separation between
species The temporal trend was particularly marked for PI although the time factor
was highly significant (Plt0001) in both ANOVAs The overall treatment effect was
marginally non-significant for PI richness (P=0069) with no indication of a time x
treatment interaction (P=0703) whereas for PI there was both a significant treatment
effect (P=0002) and a marginally non-significant treatment x time interaction (P=0075)
Mean PI was significantly lower averaged over all years for annual FYM treatments at
both 12 and 24 t ha-1
(ie Trs 4 14 and 5) compared to several other treatments notably
nil fertilizer treatments limed either in 2005 only (Tr 2 limed control) or in both 1999
and 2005 (Tr 13) (all differences Plt0001 except Tr 2 vs Tr 4 Plt001) and triennial
inorganic treatments (Trs 11 and 12 Plt001 except for Tr 4 vs Tr 11 Plt005) Within
the FRF series ANOVA the increase in PI over time was greater with triennial
treatments compared to annual averaged over fertilizer forms and rates of application
(time x frequency interaction P=0016) with a marked overall difference due to
application frequency averaged over forms rates and all years (P=0005)
11
PI species-richness showed a similar overall effect of application frequency within the
FRF series (triennialgtannual P=0013) and also a significant overall effect of rate of
application (medium rategthigh rate P=0022) The overall effect of form of fertilizer was
marginally non-significant (FYMltinorganic P=0059) and there was no significant
interaction among any of the three factors averaged over all years nor any interaction
with time
16
18
20
22
24
26
28
30
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Specie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime in Year 1+ ann FYM (Tr 14) Nil fert + Lime in Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr1)
a Species richness (total)
3
4
5
6
7
8
9
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
70
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
0
5
10
15
20
25
30
35
40
45
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
Fig 1 Changes 1999-2010 in vegetation composition an upland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control
The mean number of negative indicator (NI) species per m2 averaged over all treatments
varied significantly over time at the upland meadow (P=0002 data not shown) though
with no consistent trend - it was lowest overall in 2002 (499 per m2) and highest in both
1999 and 2008 (548 per m2) The treatment x time interaction was not significant
(P=0469) but the overall treatment effect was highly significant (Plt0001) mean
number of NI species per m2 was higher than the limed control (Tr 1 453 per m
2) with
all fertilizer treatments except Treatments 11 (triennial inorganic fertilizer at the lower
rate) and 13 (nil fertilizer limed in both 1999 and 2005) (Plt0001 for all except Trs 5
and 6 for which Plt001) The highest mean NI m-2
value was for high rate inorganic
12
fertilizer (Tr 10 565 per m2) which differed significantly (Plt005) from its FYM
counterpart Treatment 5 There was however no consistent difference between FYM
treatments and their inorganic counterparts and no overall effect of form of fertilizer
within the FRF series (P=0745) There was a significant form x rate interaction within
this ANOVA (P=0032) but only the difference between the high rate and lower rate
inorganic means was significant (high rategtlower rate Plt001)
The aggregate contribution to vegetation cover of negative indicator species (NI)
showed a very marked divergence between treatments over time (Fig 1d) with NI
either changing little or declining under most treatments again with the notable exception
of high rate annual FYM (Tr 5) under which NI increased over time Time treatment
and the treatment x time interaction were all highly significant (Plt0001) The
predominant temporal trend was downward (from 25 of live cover in 1999 to 14 in
2010 averaged over all treatments) but NI for Treatment 5 had significantly increased
compared to 1999 in 2002 2003 (Plt005) 2005 (Plt0001) 2008 (Plt0001) and 2009
(Plt001) but not in 2010 (Fig 1d) The most marked declines in NI occurred on the
limed control (Tr 2) nil fertilizer plus lime in both 1999 and 2005 (Tr 13) and the lower
rate triennial inorganic fertilizer treatments (Tr 11) differences compared to both 1999
and 2002 were highly significant (Plt0001) in most subsequent years for these
treatments A smaller decline also occurred after 2002 on the treatment equivalent to past
inputs (ie12 t ha-1
year-1
annual FYM Tr 4) In most years this treatment differed little
from the equivalent FYM treatment limed in both 1999 and 2005 (Tr 14)
These trends were reflected in highly significant overall effects on NI within the FRF
series of form (FYMgtinorganic) rate (mediumlthigh) and frequency (triennialltannual)
(all effects Plt0001) Time x rate and time x frequency interactions were both significant
(P=0019 and Plt0001 respectively) with a marginally non-significant rate x frequency
interaction averaged over all years (P=0054) NI declined over time under both
application rates averaged over forms and frequencies of application but the decline was
slower with high rate treatments compared to lower rate Difference between rates were
significant in all years from 2002 onwards (Plt005 in 2002 2006 and 2007 Plt0001 in
other years) Overall differences due to frequency of application were even more marked
Under annual application NI declined initially from 2003-2006 compared to 1999 but
subsequently recovered and did not differ from 1999 levels from 2007 onwards whilst
under triennial application differences were highly significant (Plt0001) compared to
1999 from 2003 onwards with significant differences between years during this same
period (Plt005 in 2003 and 2006 Plt0001 in remaining years)
Repeated-measures ANOVAs within the LFF treatment series at the upland meadow
showed no overall effect of liming regime on temporal trends for any of the above
variables nor any interaction of lime with either time or FYM frequency - note that FYM
was applied at the intermediate rate (12 t ha-1
) in all FYM treatments within this series
The overall effect of FYM frequency was significant for both PI (P=0003) and the
number of NI species m-2
(P=0001) PI was highest with nil FYM and lowest with
annual FYM with triennial intermediate and differing significantly (Plt005) from the
other two means NI species m-2
did not differ between triennial and annual application
within the LFF series but both were significantly higher than nil FYM (Plt001 for
triennial Plt0001 for annual application) There was also a significant time x FYM
frequency interaction for NI within this series (P=0001) reflecting a more rapid
13
decline with nil FYM than with triennial FYM and no significant overall trend with
annual FYM when averaged over both lime treatments
12
14
16
18
20
22
24
26
28
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Spe
cie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime Year 1 + ann FYM (Tr 14) Nil fert + Lime Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr 1)
a Species richness (total)
2
3
4
5
6
7
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
0
10
20
30
40
50
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
Fig 2 Changes 1999-2010 in vegetation composition at a lowland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control treatment and those that had received lime in year 1
Treatments 1 and 2 were identical (nil input) until 2005
The lowland meadow
Both time and treatment were significant within the ANOVA for total species-richness at
the lowland meadow (Plt0001 and P=0009 respectively and in contrast to the upland
site the treatment x time interaction was significant (P=0012) Averaged over all years
high rate annual FYM treatment (Tr 5) was significantly less species-rich than all others
except Treatment 14 (annual FYM at 12 t ha-1
limed in 1999) having declined
progressively between 1999 and 2009 (Fig 2a) with most differences significant at
Plt001 Treatment 14 did not differ significantly overall compared to the corresponding
FYM treatment not limed until 2005 (Tr 4) although these two treatments showed
14
markedly different temporal trends Treatment 14 was less species-rich than others during
the first five years of the experiment but did not change significantly throughout the
whole 12 year period whilst Treatment 4 changed little between 1999 and 2006 but
declined thereafter (Plt001 and Plt0001 for 2009 and 2010 respectively compared to
2006) possibly in response to liming in 2005 However there was no significant overall
effect on total species-richness of liming strategy within the LFF series and no interaction
between liming and either time or FYM application
There were marked differences in temporal trend in species-richness between FYM
treatments and their inorganic counterparts particularly with the high rate annual
treatments (Tr 5 and Tr 10) Whereas species-richness declined more or less continually
throughout under Treatment 5 at least until 2009 (Plt001 for 2004 compared to 1999 and
for both 2009 and 2010 compared to 2004) Treatment 10 declined between 2000 and
2001 (Plt005) and then remained relatively unchanged until 2008 after which it declined
again (2010 Plt005 compared to 2008)
Other treatments within the full treatment series including untreated control (Tr 1) and
limed control (Tr 2) showed no particular temporal trend in the full repeated measures
ANOVA although both the latter treatments showed a dip in species-richness in 2004
(Plt005 compared to both 2003 and 2005)
There was a strong overall effect of form of fertilizer within the FRF series ANOVA
(FYMltinorganic P=0003) a significant effect of frequency of application
(triennialgtannual P=0027) and a not quite significant effect of application rate (high
rateltmedium rate P=0064) but no significant interaction between any of these factors
The time x application frequency interaction was highly significant (Plt0001) species-
richness remained unchanged under triennial application but under annual application
was significantly lower compared to 1999 in each year from 2004 onwards with the
exception of 2006 (Plt005 in 2004 Plt001 in 2005 and 2008 Plt0001 the remainder)
Again with the exception of 2006 differences between triennial and annual application
were significant in each year from 2005 onwards (Plt005 in 2007 Plt001 in 2005 and
2008 Plt0001 in 2009 and 2010)
Both treatment time and their interaction were significant in the full treatment ANOVA
for PI species-richness at the lowland meadow (ie time and treatment both Plt0001
time x treatment P=0006) There was a general decline in PI-richness after 2005 (Fig
2b) with PI species m-2
averaged over all treatments lower from 2007 onwards (Plt0001)
than all years up to 2005 except 2001 Although the initiation of this trend corresponded
with the liming of most treatments in 2005 a similar pattern was shown by treatments
that did not receive this lime application ie the untreated control treatment (Tr 1) the
nil fertilizer treatment limed in 1999 (Tr 13) and the corresponding treatment also
receiving triennial FYM (Tr 15) In all these cases PI-richness showed no significant
difference between years until 2005 but was significantly (Plt001) lower by 2010 than in
2005 Treatment 14 (limed in 1999 plus annual FYM) did not show this pattern but PI
species-richness had been lowest under this treatment in most years between 1999 and
2005 (Fig 2b)
As with total species-richness PI richness declined more or less progressively under
Treatment 5 until 2008 (Plt005 compared to 1999 by 2001 Plt0001 from 2006
onwards) whereas Treatment 4 did not change significantly until 2006 after which it
declined (Plt005 in 2007 compared to 2006 Plt001 thereafter) Both these treatments
15
tended be less rich in PI species than the corresponding inorganic treatment (Trs 9 and
10 respectively) in most years consistently so for Tr 4 compared to Tr 9 but these
differences were not significant in any year nor when averaged over all years There was
however a significant overall effect of form of fertilizer in the FRF series ANOVA
(FYMltinorganic P=0002) and a highly significant effect of frequency of application
(triennialgtannual Plt0001) The time x frequency of application interaction was also
highly significant (Plt0001) Under triennial application PI species per m2 increased
slightly until 2005 (Plt005 compared to 1999) and declined thereafter so that by 2009
levels were significantly lower than in 1999 (Plt001 for 2009 Plt0001 for 2010) By
contrast under annual application PI species m-2
declined progressively until 2009
following a temporary though highly significant (Plt0001) dip in 2000 so that the
difference compared to 1999 was significant in every year from 2002 onwards and highly
significant (Plt0001) from 2005 With the exception of 2003 differences between
triennial and annual application were significant from 2002 onwards and highly
significant (Plt0001) in 2005 and from 2007 to 2010
There was no significant effect other than of time within the LFF treatment series
ANOVA although there was a marginally non-significant time x FYM frequency
interaction (P=0054) This reflected a more rapid decline in PI species-richness under
annual FYM application than with either nil or triennial FYM with the latter two showing
no discernible difference
By contrast with PI species m-2
PI cover percentage (PI) showed no overall treatment
effect within the full treatment ANOVA nor a time x treatment interaction although the
overall effect of time was highly significant (Plt0001) The temporal pattern in PI (Fig
2c) also differed from that shown by PI-richness with PI averaged over all treatments
declining between 1999 and 2005 in three highly significant increments (1999-2000
2000-2002 and 2002-2005 all Plt0001) and increasing again thereafter (2005-2009 and
2009-2010 both Plt0001) There was no significant effect within either the LFF or FRF
treatment series ANOVAs although their was a slight time x frequency of application
effect within the latter (P=0081) PI was slightly higher under annual application than
triennial in each year 1999-2004 after which the reverse was true although differences
were always small
There was no overall effect of treatment on the number of NI species per m2 within the
full treatment range at the lowland meadow (data not shown) but both the overall effect
of time and the time x treatment interaction were significant (Plt0001 and P=0009
respectively) Overall NI species m-2
increased over time from 24 species m-2
in 1999 to
a peak of 42 m-2
in 2008 averaged over all treatments NI species m-2
increased on the
untreated control treatment (Tr 1) from 21 species m-2
in 1999 to 34 species m-2
in 2007
(increase Plt001) then declined to the 1999 value by 2010 The limed (in 2005) control
(Tr 2) showed a somewhat similar pattern though from a slightly higher starting level
(31 species m-2
) and with the increase to 38 species m-2
in 2008 not reaching
significance
These temporal trends in NI species m-2
were much accentuated by annual fertilizer
application particularly by FYM treatments with peak numbers of NI species reaching
52 and 47 species m-2
for Treatments 4 and 5 respectively in 2008 (both Plt0001
compared to 1999) and despite slight declines in both cases remaining significantly
higher in 2010 than in 1999 (both Plt0001) Inorganic counterparts to these treatments
16
showed similar though slightly less marked trends and there was no significant
difference between all four treatments in the peak number reached in 2008 There was no
overall effect of either form of fertilizer or rate of application within the FRF series
ANOVA and only a marginally non-significant effect of frequency of application
(triennialltannual P=0061) Liming in 1999 (Trs 13 14 and 15) also appeared to
accentuate the generally increasing trend although peak numbers in 2008 did not differ
between these treatments and either untreated or limed control in the full treatment range
ANOVA The overall effects of liming regime (1999 or 2005) were not significant within
the LFF treatment series but the faster rate of increase with liming in 1999 was reflected
in a significant time x lime interaction in this analysis (P=0017) The mean number of NI
species m-2
increased very significantly (Plt0001) between 1999 and 2000 following
liming in 1999 with the increment increasing until 2008 whilst on plots un-limed until
2005 NI species m-2
did not increase significantly until 2003 (Plt005) although the
increase compared to 1999 was highly significant (Plt0001) in 2006 2008 and 2010
Nevertheless the differences in rate of increase was at least partly due to a lower 1999
mean for the 1999-limed treatments (170 species m-2
compared to 244 species m-2
difference NS) and although in all years from 2001 onwards mean NI species m-2
was
higher for 1999-limed treatments compared to those not limed until 2005 the difference
was not significant in any year
The contribution of NI species to vegetation cover (NI) showed a very marked
separation between treatments over time at the lowland meadow (Fig 2d) starting from
much lower levels than at the upland meadow (compare with Fig 1d) There were
noticeable differences in temporal trend between annual fertilizer treatments on the one
hand and triennial and nil fertilizer treatments on the other (Fig 2d) The effects of
treatment time and the treatment x time interaction were all highly significant (Plt0001)
in the full treatment range ANOVA The most marked increase in NI occurred with
annual FYM at the high rate (Tr 5) with NI increasing rapidly between 1999 and 2001
(Plt001) and a further highly significant increment between 2004 and 2009 (Plt0001)
(Fig 2d) A similar though less marked trend occurred with annual FYM at the medium
rate (Tr 4) with significant increases between 1999 and 2005 and between 2005 and
2010 (both Plt001) NI also increased markedly with the inorganic treatment
equivalent to Treatment 5 (Tr 10) although the overall rate of increase shown by this
treatment was closer to that shown by Treatment 4 than Treatment 5 There were
significant differences between Treatments 5 and 10 in most years from 2004 onwards
(Plt001 and Plt0001 in 2009 and 2010 respectively)
These effects were reflected in a significant overall effect of form of fertilizer
(FYMgtinorganic P=0008) and highly significant (Plt0001) overall effects of both rate
(high rategtlower rate) and frequency of application (annualgttriennial) within the FRF
series ANOVA and a significant form x rate interaction (P=0043) Mean NI was
highest for high rate annual FYM (Plt0001 compared to all three remaining means) and
lowest for lower rate inorganic (Plt005 compared to high rate inorganic NS compared
to lower rate FYM) Only frequency of application interacted significantly with time
(Plt0001) NI increased moderately under triennial application until 2008 (Plt005 in
2005 compared to 1999 Plt0001 each year 2006-2008) followed by a decline (Plt001
2010 compared to 2008) but with mean NI in 2010 still higher than 1999 (Plt005)
The increasing trend was very much more marked with annual fertilizer application for
17
which there were highly significant (Plt0001) increments between 1999 and 2002 and
between 2002 and 2008 but with no significant increase thereafter Differences between
annual and triennial treatment means were highly significant (Plt0001) from 2003
onwards
There was no significant change in NI over time either under the untreated control
(Tr 1) or the nil fertilizer (Tr 2) treatments in the full treatment ANOVA NI increased
until 2005 on plots limed in 1999 without fertilizer Tr 13 (Plt001 compared to 1999)
and declined slightly but not significantly thereafter Trends were similar with the
treatment limed in 1999 and receiving triennial FYM (Tr 15 ndash not illustrated in Fig 2)
although NI was higher with this treatment than with Tr 13 (Plt005) in both 2005 and
2008 By contrast applying the same rate of FYM annually after liming in 1999 (Tr 14)
caused a more rapid increase with levels by 2005 higher than either Tr 13 (Plt001) or
Tr 4 (Plt005) ndash note that the latter was equivalent to Tr 14 but without lime until 2005
NI declined after 2006 under Tr 14 but continued to increase with Tr 4 with the
difference between these two treatments reversed by 2010 (Plt005)
All main effects and interactions were significant in the LFF treatment series ANOVA
for NI except for lime x FYM frequency and the lime x FYM x time interactions Plots
limed in 1999 supported higher NI levels than those limed in 2005 averaged over the
three FYM frequencies and all years (P=0042) NI increased more rapidly after liming
in 1999 than where liming was delayed until 2005 (P=0033 for time x lime interaction)
with the overall difference due to liming in 1999 highly significant (Plt0001) by 2005
NI declined thereafter on the 1999-limed treatments whilst continuing to increase
following liming in 2005 of previously un-limed treatments both peaking in 2008 at a
very similar level Averaged over lime treatments NI increased much more rapidly
under annual FYM application than under either triennial or nil FYM (P=0002 for the
time x FYM frequency interaction) a highly significant increase had occurred by 2002
with annual FYM (Plt0001 compared to 1999) but increases of similar magnitude were
not reached until 2005 with triennial application and not until 2007 with nil FYM NI
subsequently declined under the latter two FYM regimes but not with annual FYM so
that by 2010 NI was significantly higher with annual FYM than with either triennial or
nil FYM (Plt0001) but with only a small non-significant difference between the latter
two treatments
TREATMENT EFFECTS AFTER 12 YEARS OF APPLICATION
The effects of all treatments on the five key variables at each site in 2010 are shown in
Table S20 with treatments differing significantly (Plt005) from the limed control
treatment (Tr 2) highlighted for each variable Treatment effects in 2010 are not
described in detail here but the modeled responses of total species-richness and of
richness of PI species in 2010 to the mean amounts of N and P applied per year averaged
over 1999-2010 are described below
Relationships with the amounts of fertilizer applied
The effect of neither rate nor frequency of application was significant in any of the
REML models for either total species-richness or PI species-richness at either site when
the amount of N or P applied ha-1
year-1
was included The effect of form of fertilizer was
still significant in most models however either as a main effect or in interaction with the
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
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Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
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Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
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Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
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Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
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Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
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Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
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Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
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Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
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Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
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Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
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heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
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Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
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Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
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studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
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plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
5
Table 2 Treatments applied 1999-2010 at the upland and lowland meadows Values are the mean
amounts applied (kg ha-1
year-1
elemental N P and K) either as FYM (estimated) or in inorganic
form (actual) averaged over 12 yearsa 1999-2010 Treatments 13-15 were limed only in both
1999 and 2005 at the upland site but in 1999 only at the lowland site Treatments 2-12 received
lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Upland meadow Lowland meadow
Treatment N P K N P K
1 Untreated control 00 00 00 00 00 00
2 Limed (in 2005) control 00 00 00 00 00 00
3 FYM at 6 t ha-1
annual 44 50 345 29 38 292
4+ FYM at 12 t ha-1
annual 88 100 689 57 76 585
5 + FYM at 24 t ha
-1 annual 176 199 1378
114 151 1169
6 FYM at 6 t ha-1
triennially 15 22 113 09 13 110
7 + FYM at 12 t ha
-1 triennially 30 45 226
19 26 221
8 + FYM at 24 t ha
-1 triennially 61 90 452
38 52 441
9 + Inorg equivalent to Tr 4 85 84 689 61 63 585
10 + Inorg equivalent to Tr 5 170 168 1378
121 125 1169
11 + Inorg equivalent to Tr 7 30 36 226
24 21 221
12 + Inorg equivalent to Tr 8 61 72 452
48 42 441
13 Lime in years 1 (and 7) 00 00 00 00 00 00
14 Lime as Tr 13 + FYM as Tr 4 88 100 689 57 76 585
15 Lime as tr 13 + FYM as Tr 7 30 45 226 19 26 221 aNote that no fertilizer was applied in 2001 so that for annual treatments the amounts shown
are 1112ths of the mean amount applied in each of the remaining years
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime
in 1999 x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
The FYM used in the experiment was sourced locally from the same farms each year and was
sampled at the time of application for subsequent chemical analysis The ADAS Manure Nitrogen
Evaluation Routine (MANNER Chambers et al 1999) was used between 1999 and 2006 to
predict the amount of plant available N supplied by the FYM after which a refined model
(MANNER-NPK Nicholson et al 2010) was used Refinements to the model included more
detailed N mineralisation functions and predictions of N availability in the year following
application Availabilities of 60 and 90 of total P and K respectively (Anon 2010) were
assumed between 1999 and 2006 and this assumption for K was maintained throughout the 1999-
2010 period From 2007 onwards the assumed availability of P from FYM was increased to 80
of total P in line with information on longer term release of plant-available P from FYM (Smith
et al 1998) For both N and P the mean amounts assumed to have been applied in FYM
treatments averaged over 1999-2010 and shown in Table 2 are based upon revised estimates for
the whole period The inorganic fertilizer N treatments applied each year ie the inorganic
lsquoequivalentsrsquo for specific FYM treatments (see Table 2) were based upon estimates made using
the original MANNER model from 1999-2006 and MANNER-NPK from 2007-2010 Similarly
6
the inorganic fertilizer P applied each year corresponded to 60 of the total P in FYM between
1999 and 2006 and 80 from 2007-2010
Inorganic N P and K were applied as ammonium nitrate triple super-phosphate and muriate of
potash respectively to the relevant treatments The FYM treatments were designed to encompass
the range of inputs traditionally applied to species-rich hay meadows upon which current agri-
environment guidelines are based (Simpson and Jefferson 1996 Crofts and Jefferson 1999)
Lime was applied to treatments 13 14 and 15 only at each site in March-April 1999 and to most
treatments in 2005 (Table 2) The latter included the three previously limed treatments at the
upland meadow but not at the lowland one since soil pH levels had not fallen significantly by
2005 on these plots at the latter site Based on assessments of soil texture and soil pH tests
conducted in February-March the amounts of lime were calculated to be sufficient to raise the
soil pH of the experimental plots to 60 (Anon 2010)
At each site one plot per replicate block received no fertilizer or lime (untreated control)
between 1999 and 2005 whilst another plot per replicate was designated as a continuation of past
fertilizer inputs At the lowland meadow the latter was identical to the untreated control
treatment but at the upland meadow it was identical to Treatment 4 (12 t ha-1
FYM annually) In
2005 a decision was made to treat occasional liming as lsquobackgroundrsquo management for all
fertilizer treatments at both sites to be triggered for each treatment in future years once mean pH
for that treatment had declined to 55 whilst retaining both an untreated control (ie nil fertilizer
nil lime ndash Treatment 1) and a nil fertilizer (limed in 2005) control treatment (Treatment 2) at each
site The rationale behind this decision is addressed in the Discussion section At the lowland
meadow one of the two hitherto untreated control plots per replicate (Treatment 2) was therefore
limed in 2005 along with other treatments Only one nil input plot per replicate was available at
the upland meadow but since the site had a history of liming the existing nil input plots were
limed in 2005 (Treatment 2) and new untreated control plots (Treatment 1) were established
adjacent to each replicate block with the end at which each was located chosen randomly for
each block
The experimental plots were cut for hay after 1 July each year at the upland site and after 15
July at the lowland site the actual cutting date being dependent on weather conditions The hay
aftermath growth was grazed each year with experimental plots being grazed with the remainder
of the field At the upland meadow mature sheep were used normally commencing in September-
October continuing until March or late April The lowland meadow was grazed by beef store
cattle from mid Octoberearly November to late February
Botanical assessments were carried out during May each year except in 2001 at the upland
meadow due to FMD restrictions The percentage cover of each species present was estimated
visually in three 1m2 quadrats positioned at random within each plot in 1999 and then fixed for
the project duration
Soils were sampled in March 1999 2002 2004 2007 and 2010 by taking five 35 mm diameter x
75mm deep soil cores at random from within each treatment plot but outside the fixed positions
of the three 1m2 botanical survey quadrats The five samples per plot were combined dried at
30oC and then ground prior to analysis for organic carbon (C) total nitrogen (N) Olsen
extractable P exchangeable potassium (K) magnesium (Mg) calcium (Ca) sodium (Na) and pH
(in H2O) using standard laboratory methods (Allen 1974 MAFF 1986)
Soils were further sampled in June 1999 2002 2004 2007 and 2010 for soil microbial
assessment by taking five 35 mm diameter x 100 mm deep cores which were subsequently bulked
for each plot sieved (5 mm) and stored at 4oC prior to analysis Microbial community structure
was assessed using phospholipid fatty acid analysis (PLFA) as described by Bardgett et al
(1996) The fatty acids i150 a150 150 i160 170 i170 cy170 cis181ω7 and cy190 were
chosen to represent bacterial PLFAs (Federle 1986 Frostegaringrd Tunlid amp Baringaringth 1993) and
182ω6 was used as an indicator of fungal biomass (Federle 1986) The ratio of 182ω6bacterial
7
PLFAs was taken to represent the ratio of fungal-to-bacterial biomass in soil (Bardgett Hobbs amp
Frostegaringrd 1996 Frostegaringrd amp Baringaringth 1996)
DERIVED BOTANICAL VARIABLES
Estimates of cover for individual species were converted to a percentage of the total live
vegetation cover present in each quadrat in order to minimize the effects of year-to-year variation
and variation between treatments in vegetation density and total cover Several composite
variables were calculated to characterize the vegetation and these were then averaged across the
three quadrats in each plot to give plot mean values for each variable Plot means were then used
as the basic units for subsequent analyses of treatment effects
Key variables
Five key variables were used to investigate the effects of treatment and time on botanical
composition total species-richness (the total number of vascular plant species per m2) the
number per m2 of MG3 and MG5 positive indicator species (the upland and lowland meadows
respectively) the aggregate cover of these species as a percentage of total vegetation cover the
number per m2 of negative indicator species and the aggregate cover of negative indicator
species as a percentage of total vegetation cover Positive mesotrophic indicator species were
identified from lists produced by Robertson and Jefferson (2000) for use in monitoring the
condition of grassland Sites of Special Scientific Interest (SSSIs) in MG3 (Anthoxanthum
odoratum-Geranium sylvaticum grassland) and MG5 (Cynosurus cristatus-Centaurea nigra
grassland) communities of the NVC (Rodwell 1992) A single list of negative indicator species
was compiled from the generic negative indicators for MG3 and MG5 communities (Robertson amp
Jefferson 2000) augmented by species shown in previous work (eg Mountford et al 1993
Kirkham et al 1996) to be favoured by nutrient addition and for which an increase in abundance
would indicate a negative effect on plant community quality Positive and negative indicator
species are listed in Supporting Information Table S3
Additional variables
Among other derived variables was the weighted Ellenberg N score (Smith et al 2003 Kirkham
et al 2008) referred to hereafter as the fertility score The Ellenberg N index represents the
degree of association with soil fertility (not specifically soil N) of a particular species (Ellenberg
1988 Hill et al 1999) Fertility scores were calculated for each quadrat as the average N index
of the component species weighted according to the proportional contribution of each species to
total vegetation cover Plot mean fertility scores were highly correlated at both sites with the
aggregate cover of negative indicator species (NI) (Pearson r=0845 and r=0927 at the upland
and lowland sites respectively in 2010 both Plt0001) The latter variable was preferred to
fertility scores for statistical analyses in this paper because it proved more responsive to fertilizer
treatments although fertility scores are referred to in the Discussion section in relation to results
from elsewhere
DATA ANALYSIS
ANOVAs for treatment effects
For each of the key variables botanical data for all years 1999-2010 except 2001 at the upland
meadow and for all treatments except Treatment 1 at the same meadow were analyzed by
repeated measures analysis of variance (ANOVA - Genstat V Committee 1997) with Year as the
repeated measures factor Separate ANOVAs were carried out for each variable within each site
using three separate ANOVA models in each case In each model variation between replicate
8
blocks was accounted for by treating replicate as a blocking factor The first model included all
the treatments except Treatment 1 at the upland meadow whilst the other two models used
balanced factorial subsets (series) of treatments (see Table 2) the Form x Rate x Frequency
(FRF) series and the Lime x FYM frequency (LFF) series
The FRF series tested the effects of form of fertilizer (ie FYM or inorganic) the rate at which
fertilizers were applied (12 or 24 t FYM ha-1
and inorganic treatments corresponding to these)
the frequency at which treatments were applied (ie annual or triennially) and all two- and three-
way interactions between these factors
The LFF series tested the effects of liming regime ie lime in 1999 (and 2005 at the upland site)
versus lime in 2005 only the frequency of FYM application (ie nil annual or triennially) and the
Lime x FYM frequency interaction All FYM treatments within this series were applied at 12 t ha-
1 At both sites the LFF series consisted of Treatments 2 4 7 13 14 and 15 (see Table 2) The
liming comparison therefore differed slightly between the two sites since liming was repeated in
2005 on plots previously limed in 1999 at the upland meadow but not repeated at the lowland
one
In both the LFF and the FRF models for each variable the first stratum of the ANOVA
consisted of the effects of all factors and interactions averaged over time with the same factors
and interactions repeated in the second stratum with time as an additional factor
In all repeated measures ANOVAs to account for the repeated measures aspect all degrees of
freedom in the Year x Treatment stratum were adjusted to account for departure from
homogeneity of the population covariance matrix using ε-coefficients calculated by the
Greenhouse-Geisser method (Genstat V Committee 1997)
In most years including 2010 all species were recorded individually but at both sites
bryophytes were recorded only as a single group in some years This had only a small influence
on the data since there were seldom more than two bryophyte species present in a quadrat and
more often less Nevertheless for consistency between years bryophytes were treated as a single
group in all years when calculating total species-richness for data for repeated measures
ANOVAs
In addition to the above analyses 2010 data for the key variables were tested alone for treatment
effects by ANOVA and by analysis of covariance (ANCOVA) the latter using 1999 data as the
covariate (Genstat 5 Committee 1997) All three models as described above were tested The
ANCOVAs for the upland meadow excluded Treatment 1 (untreated control plots established in
2005) Where an ANCOVA showed significant treatment covariance with 1999 data the adjusted
results were used In other cases the unadjusted analyses were used Individual bryophyte species
were included in the calculations of species-richness at both sites in these analyses
In all analyses in order to satisfy the conditions for ANOVA (normality of residuals and
homogeneity of residual variation) all percentage data were transformed before analysis by
arcsine(radicp) where p is the percentage value expressed as a proportion All results quoted for
percentage data refer to analyses of arcsine-square root transformed data
Mixed modeling
Mixed model analysis (Residual Estimated Maximum Likelihood ndash REML Genstat V
Committee 1997) was used to identify any effect of form rate or frequency of application on
vegetation composition at each site in 2010 not simply attributable to the total (or mean per year)
amount of fertilizer applied over the study period A variable representing this amount was
included as a continuous explanatory variable in each analysis along with other fixed factors
form rate and frequency of application (Block was included as a random factor) Since the total
(or mean) amounts of N P and K applied over the whole period were completely correlated
across treatments at each site it was not feasible to include all three elements as separate terms in
the model Nor was it feasible to simply use a single notional variable for fertilizer amount (eg t
9
FYM ha-1
year-1
or inorganic equivalent) since there was lack of equivalence of N and P supply
(but not K) between individual FYM treatments and their corresponding inorganic treatments For
each dependent variable tested two separate models were therefore developed using either N or
P ha-1
year-1
as the continuous variable In each case the presence of any non-linearity in response
was tested by including a quadratic term (ie N2 or P
2) or where more appropriate by logn-
transformation of the explanatory variable
For each analysis an initial model was constructed that included all five main effects and all
interactions The importance of each term was evaluated initially by a χ2 test on the Wald statistic
(Genstat V Committee 1997) The number and order of terms in the model were varied but with
N or P amount always included until a model was developed in which each term was significant
The model was then refined by testing the contribution of each term to the model by the more
conservative deviance test ie a χ2 test on the difference in deviance between the model
containing the term and a sub-model from which it was dropped (Welham amp Thompson 1997)
Where there was an interaction between form of fertilizer and the quadratic term but no overall
effect of form - implying that the difference between the two forms of fertilizer lay primarily in a
difference in the curvilinearity of response - a refined model was tested with the P2 (or N
2) x
Form term replaced by Form nested within the quadratic term (eg P2Form) This has the effect
of absorbing the overall effect of form of fertilizer within the interaction term and returns the two
terms P2 and P
2 x Form (Galwey 2006)
These modeling approaches were used with total species-richness and PI species-richness as
(separate) response variables and included data for all treatments limed in 2005 but not in 1999
(ie Trs 2-12)
Results
TEMPORAL CHANGES 1999-2010
Changes over time in total species-richness (number per m2) richness and proportional
aggregate cover of positive indicator (PI) species and proportional aggregate cover of
negative indicator (NI) species at the upland and lowland meadows are represented in
Figs 1 and 2 respectively for ten of the fifteen treatments applied Repeated measures
ANOVA statistics for each of the four variables are given in Supporting Information
Tables S4 S6 S8 and S10 respectively for the upland meadow and Tables S12 S14 S16
and S18 respectively for the lowland meadow Corresponding mean values for each
treatment in each year are given in Tables S5 S7 S9 S11 S13 S15 S17 and S19
respectively Changes in individual species abundance contributing to the temporal trends
in aggregated PI and NI species described below are outlined under Supporting
Information
The upland meadow
The overall effects of both treatment and time were highly significant in the repeated
measures ANOVA for total species-richness at the upland meadow (both P=0001) but
there was no treatment x time interaction (P=0647)
After an initial decline between 1999 and 2000 species-richness remained fairly
constant for most treatments until 2003 after which there was a general increase with the
notable exception of the highest rate annual FYM treatment (Tr 5) (Fig 1a) The latter
treatment declined further between 2005 and 2007 after which it appeared to recover
slightly and averaged over all years this treatment was significantly less species-rich than
all others (Plt001 compared to Tr 14 Plt0001 the remainder) None of the remaining
10
treatments differed significantly from the limed control treatment (Tr2) averaged over all
years However the more species-rich treatments ie nil fertilizer plus lime applied in
both 1999 and 2005 (Tr 13) low rate annual and medium rate triennial FYM (Tr 3 and 7
respectively) and the inorganic equivalent to the latter (Tr 11) were all more species-rich
averaged over all years (Plt005) than annual FYM at the medium rate under either
liming regime (ie Trs 4 and 14 the former representing a continuation of past
management)
The effect of the high rate annual FYM treatment (Tr 5) was largely responsible for
several main effects within the repeated measures ANOVA of treatments within the Form
x Rate x Frequency (FRF) factorial series namely overall effects (averaged over all
years) of rate (mediumgthigh P=0002) and frequency (triennialgtannual P=0001) of
application and a not quite significant effect of form of fertilizer (FYMltinorganic
P=0057) Both the form x rate and the form x frequency interactions were significant
(both Plt005) high rate FYM was significantly less species-rich than all other
combinations averaged over annual and three yearly treatments and all years (Plt0001
compared to medium rate FYM Plt001 compared to both medium and high rate
inorganic fertilizer) and annual FYM was less species-rich than three yearly FYM and
than inorganic fertilizer either annually or triennial (all Plt0001) averaged over the two
application rates and all years The rate x frequency interaction was not significant
(P=0105) but there was a marginally non-significant three-way interaction between
form rate and frequency (P=0070) with a low mean for high rate annual FYM (Tr 5
211 species m-2
) contrasting markedly with all other treatments within the series
particularly with low rate triennial FYM (Tr 6 267 species m-2
)
As with the full treatment ANOVA the overall time factor was highly significant
(Plt0001) but only the effect of application frequency varied significantly with time
(interaction Plt005) Species-richness declined slightly with annual treatments overall
and increased slightly with triennial treatments such that differences between these were
significant in all years from 2005 onwards (Plt001 for 2006-2009 Plt005 in 2005 and
2010)
Both the richness and aggregate cover of PI species (PI) increased generally with time
(Fig 1b and 1c) although in both cases there was a progressive separation between
species The temporal trend was particularly marked for PI although the time factor
was highly significant (Plt0001) in both ANOVAs The overall treatment effect was
marginally non-significant for PI richness (P=0069) with no indication of a time x
treatment interaction (P=0703) whereas for PI there was both a significant treatment
effect (P=0002) and a marginally non-significant treatment x time interaction (P=0075)
Mean PI was significantly lower averaged over all years for annual FYM treatments at
both 12 and 24 t ha-1
(ie Trs 4 14 and 5) compared to several other treatments notably
nil fertilizer treatments limed either in 2005 only (Tr 2 limed control) or in both 1999
and 2005 (Tr 13) (all differences Plt0001 except Tr 2 vs Tr 4 Plt001) and triennial
inorganic treatments (Trs 11 and 12 Plt001 except for Tr 4 vs Tr 11 Plt005) Within
the FRF series ANOVA the increase in PI over time was greater with triennial
treatments compared to annual averaged over fertilizer forms and rates of application
(time x frequency interaction P=0016) with a marked overall difference due to
application frequency averaged over forms rates and all years (P=0005)
11
PI species-richness showed a similar overall effect of application frequency within the
FRF series (triennialgtannual P=0013) and also a significant overall effect of rate of
application (medium rategthigh rate P=0022) The overall effect of form of fertilizer was
marginally non-significant (FYMltinorganic P=0059) and there was no significant
interaction among any of the three factors averaged over all years nor any interaction
with time
16
18
20
22
24
26
28
30
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Specie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime in Year 1+ ann FYM (Tr 14) Nil fert + Lime in Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr1)
a Species richness (total)
3
4
5
6
7
8
9
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
70
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
0
5
10
15
20
25
30
35
40
45
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
Fig 1 Changes 1999-2010 in vegetation composition an upland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control
The mean number of negative indicator (NI) species per m2 averaged over all treatments
varied significantly over time at the upland meadow (P=0002 data not shown) though
with no consistent trend - it was lowest overall in 2002 (499 per m2) and highest in both
1999 and 2008 (548 per m2) The treatment x time interaction was not significant
(P=0469) but the overall treatment effect was highly significant (Plt0001) mean
number of NI species per m2 was higher than the limed control (Tr 1 453 per m
2) with
all fertilizer treatments except Treatments 11 (triennial inorganic fertilizer at the lower
rate) and 13 (nil fertilizer limed in both 1999 and 2005) (Plt0001 for all except Trs 5
and 6 for which Plt001) The highest mean NI m-2
value was for high rate inorganic
12
fertilizer (Tr 10 565 per m2) which differed significantly (Plt005) from its FYM
counterpart Treatment 5 There was however no consistent difference between FYM
treatments and their inorganic counterparts and no overall effect of form of fertilizer
within the FRF series (P=0745) There was a significant form x rate interaction within
this ANOVA (P=0032) but only the difference between the high rate and lower rate
inorganic means was significant (high rategtlower rate Plt001)
The aggregate contribution to vegetation cover of negative indicator species (NI)
showed a very marked divergence between treatments over time (Fig 1d) with NI
either changing little or declining under most treatments again with the notable exception
of high rate annual FYM (Tr 5) under which NI increased over time Time treatment
and the treatment x time interaction were all highly significant (Plt0001) The
predominant temporal trend was downward (from 25 of live cover in 1999 to 14 in
2010 averaged over all treatments) but NI for Treatment 5 had significantly increased
compared to 1999 in 2002 2003 (Plt005) 2005 (Plt0001) 2008 (Plt0001) and 2009
(Plt001) but not in 2010 (Fig 1d) The most marked declines in NI occurred on the
limed control (Tr 2) nil fertilizer plus lime in both 1999 and 2005 (Tr 13) and the lower
rate triennial inorganic fertilizer treatments (Tr 11) differences compared to both 1999
and 2002 were highly significant (Plt0001) in most subsequent years for these
treatments A smaller decline also occurred after 2002 on the treatment equivalent to past
inputs (ie12 t ha-1
year-1
annual FYM Tr 4) In most years this treatment differed little
from the equivalent FYM treatment limed in both 1999 and 2005 (Tr 14)
These trends were reflected in highly significant overall effects on NI within the FRF
series of form (FYMgtinorganic) rate (mediumlthigh) and frequency (triennialltannual)
(all effects Plt0001) Time x rate and time x frequency interactions were both significant
(P=0019 and Plt0001 respectively) with a marginally non-significant rate x frequency
interaction averaged over all years (P=0054) NI declined over time under both
application rates averaged over forms and frequencies of application but the decline was
slower with high rate treatments compared to lower rate Difference between rates were
significant in all years from 2002 onwards (Plt005 in 2002 2006 and 2007 Plt0001 in
other years) Overall differences due to frequency of application were even more marked
Under annual application NI declined initially from 2003-2006 compared to 1999 but
subsequently recovered and did not differ from 1999 levels from 2007 onwards whilst
under triennial application differences were highly significant (Plt0001) compared to
1999 from 2003 onwards with significant differences between years during this same
period (Plt005 in 2003 and 2006 Plt0001 in remaining years)
Repeated-measures ANOVAs within the LFF treatment series at the upland meadow
showed no overall effect of liming regime on temporal trends for any of the above
variables nor any interaction of lime with either time or FYM frequency - note that FYM
was applied at the intermediate rate (12 t ha-1
) in all FYM treatments within this series
The overall effect of FYM frequency was significant for both PI (P=0003) and the
number of NI species m-2
(P=0001) PI was highest with nil FYM and lowest with
annual FYM with triennial intermediate and differing significantly (Plt005) from the
other two means NI species m-2
did not differ between triennial and annual application
within the LFF series but both were significantly higher than nil FYM (Plt001 for
triennial Plt0001 for annual application) There was also a significant time x FYM
frequency interaction for NI within this series (P=0001) reflecting a more rapid
13
decline with nil FYM than with triennial FYM and no significant overall trend with
annual FYM when averaged over both lime treatments
12
14
16
18
20
22
24
26
28
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Spe
cie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime Year 1 + ann FYM (Tr 14) Nil fert + Lime Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr 1)
a Species richness (total)
2
3
4
5
6
7
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
0
10
20
30
40
50
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
Fig 2 Changes 1999-2010 in vegetation composition at a lowland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control treatment and those that had received lime in year 1
Treatments 1 and 2 were identical (nil input) until 2005
The lowland meadow
Both time and treatment were significant within the ANOVA for total species-richness at
the lowland meadow (Plt0001 and P=0009 respectively and in contrast to the upland
site the treatment x time interaction was significant (P=0012) Averaged over all years
high rate annual FYM treatment (Tr 5) was significantly less species-rich than all others
except Treatment 14 (annual FYM at 12 t ha-1
limed in 1999) having declined
progressively between 1999 and 2009 (Fig 2a) with most differences significant at
Plt001 Treatment 14 did not differ significantly overall compared to the corresponding
FYM treatment not limed until 2005 (Tr 4) although these two treatments showed
14
markedly different temporal trends Treatment 14 was less species-rich than others during
the first five years of the experiment but did not change significantly throughout the
whole 12 year period whilst Treatment 4 changed little between 1999 and 2006 but
declined thereafter (Plt001 and Plt0001 for 2009 and 2010 respectively compared to
2006) possibly in response to liming in 2005 However there was no significant overall
effect on total species-richness of liming strategy within the LFF series and no interaction
between liming and either time or FYM application
There were marked differences in temporal trend in species-richness between FYM
treatments and their inorganic counterparts particularly with the high rate annual
treatments (Tr 5 and Tr 10) Whereas species-richness declined more or less continually
throughout under Treatment 5 at least until 2009 (Plt001 for 2004 compared to 1999 and
for both 2009 and 2010 compared to 2004) Treatment 10 declined between 2000 and
2001 (Plt005) and then remained relatively unchanged until 2008 after which it declined
again (2010 Plt005 compared to 2008)
Other treatments within the full treatment series including untreated control (Tr 1) and
limed control (Tr 2) showed no particular temporal trend in the full repeated measures
ANOVA although both the latter treatments showed a dip in species-richness in 2004
(Plt005 compared to both 2003 and 2005)
There was a strong overall effect of form of fertilizer within the FRF series ANOVA
(FYMltinorganic P=0003) a significant effect of frequency of application
(triennialgtannual P=0027) and a not quite significant effect of application rate (high
rateltmedium rate P=0064) but no significant interaction between any of these factors
The time x application frequency interaction was highly significant (Plt0001) species-
richness remained unchanged under triennial application but under annual application
was significantly lower compared to 1999 in each year from 2004 onwards with the
exception of 2006 (Plt005 in 2004 Plt001 in 2005 and 2008 Plt0001 the remainder)
Again with the exception of 2006 differences between triennial and annual application
were significant in each year from 2005 onwards (Plt005 in 2007 Plt001 in 2005 and
2008 Plt0001 in 2009 and 2010)
Both treatment time and their interaction were significant in the full treatment ANOVA
for PI species-richness at the lowland meadow (ie time and treatment both Plt0001
time x treatment P=0006) There was a general decline in PI-richness after 2005 (Fig
2b) with PI species m-2
averaged over all treatments lower from 2007 onwards (Plt0001)
than all years up to 2005 except 2001 Although the initiation of this trend corresponded
with the liming of most treatments in 2005 a similar pattern was shown by treatments
that did not receive this lime application ie the untreated control treatment (Tr 1) the
nil fertilizer treatment limed in 1999 (Tr 13) and the corresponding treatment also
receiving triennial FYM (Tr 15) In all these cases PI-richness showed no significant
difference between years until 2005 but was significantly (Plt001) lower by 2010 than in
2005 Treatment 14 (limed in 1999 plus annual FYM) did not show this pattern but PI
species-richness had been lowest under this treatment in most years between 1999 and
2005 (Fig 2b)
As with total species-richness PI richness declined more or less progressively under
Treatment 5 until 2008 (Plt005 compared to 1999 by 2001 Plt0001 from 2006
onwards) whereas Treatment 4 did not change significantly until 2006 after which it
declined (Plt005 in 2007 compared to 2006 Plt001 thereafter) Both these treatments
15
tended be less rich in PI species than the corresponding inorganic treatment (Trs 9 and
10 respectively) in most years consistently so for Tr 4 compared to Tr 9 but these
differences were not significant in any year nor when averaged over all years There was
however a significant overall effect of form of fertilizer in the FRF series ANOVA
(FYMltinorganic P=0002) and a highly significant effect of frequency of application
(triennialgtannual Plt0001) The time x frequency of application interaction was also
highly significant (Plt0001) Under triennial application PI species per m2 increased
slightly until 2005 (Plt005 compared to 1999) and declined thereafter so that by 2009
levels were significantly lower than in 1999 (Plt001 for 2009 Plt0001 for 2010) By
contrast under annual application PI species m-2
declined progressively until 2009
following a temporary though highly significant (Plt0001) dip in 2000 so that the
difference compared to 1999 was significant in every year from 2002 onwards and highly
significant (Plt0001) from 2005 With the exception of 2003 differences between
triennial and annual application were significant from 2002 onwards and highly
significant (Plt0001) in 2005 and from 2007 to 2010
There was no significant effect other than of time within the LFF treatment series
ANOVA although there was a marginally non-significant time x FYM frequency
interaction (P=0054) This reflected a more rapid decline in PI species-richness under
annual FYM application than with either nil or triennial FYM with the latter two showing
no discernible difference
By contrast with PI species m-2
PI cover percentage (PI) showed no overall treatment
effect within the full treatment ANOVA nor a time x treatment interaction although the
overall effect of time was highly significant (Plt0001) The temporal pattern in PI (Fig
2c) also differed from that shown by PI-richness with PI averaged over all treatments
declining between 1999 and 2005 in three highly significant increments (1999-2000
2000-2002 and 2002-2005 all Plt0001) and increasing again thereafter (2005-2009 and
2009-2010 both Plt0001) There was no significant effect within either the LFF or FRF
treatment series ANOVAs although their was a slight time x frequency of application
effect within the latter (P=0081) PI was slightly higher under annual application than
triennial in each year 1999-2004 after which the reverse was true although differences
were always small
There was no overall effect of treatment on the number of NI species per m2 within the
full treatment range at the lowland meadow (data not shown) but both the overall effect
of time and the time x treatment interaction were significant (Plt0001 and P=0009
respectively) Overall NI species m-2
increased over time from 24 species m-2
in 1999 to
a peak of 42 m-2
in 2008 averaged over all treatments NI species m-2
increased on the
untreated control treatment (Tr 1) from 21 species m-2
in 1999 to 34 species m-2
in 2007
(increase Plt001) then declined to the 1999 value by 2010 The limed (in 2005) control
(Tr 2) showed a somewhat similar pattern though from a slightly higher starting level
(31 species m-2
) and with the increase to 38 species m-2
in 2008 not reaching
significance
These temporal trends in NI species m-2
were much accentuated by annual fertilizer
application particularly by FYM treatments with peak numbers of NI species reaching
52 and 47 species m-2
for Treatments 4 and 5 respectively in 2008 (both Plt0001
compared to 1999) and despite slight declines in both cases remaining significantly
higher in 2010 than in 1999 (both Plt0001) Inorganic counterparts to these treatments
16
showed similar though slightly less marked trends and there was no significant
difference between all four treatments in the peak number reached in 2008 There was no
overall effect of either form of fertilizer or rate of application within the FRF series
ANOVA and only a marginally non-significant effect of frequency of application
(triennialltannual P=0061) Liming in 1999 (Trs 13 14 and 15) also appeared to
accentuate the generally increasing trend although peak numbers in 2008 did not differ
between these treatments and either untreated or limed control in the full treatment range
ANOVA The overall effects of liming regime (1999 or 2005) were not significant within
the LFF treatment series but the faster rate of increase with liming in 1999 was reflected
in a significant time x lime interaction in this analysis (P=0017) The mean number of NI
species m-2
increased very significantly (Plt0001) between 1999 and 2000 following
liming in 1999 with the increment increasing until 2008 whilst on plots un-limed until
2005 NI species m-2
did not increase significantly until 2003 (Plt005) although the
increase compared to 1999 was highly significant (Plt0001) in 2006 2008 and 2010
Nevertheless the differences in rate of increase was at least partly due to a lower 1999
mean for the 1999-limed treatments (170 species m-2
compared to 244 species m-2
difference NS) and although in all years from 2001 onwards mean NI species m-2
was
higher for 1999-limed treatments compared to those not limed until 2005 the difference
was not significant in any year
The contribution of NI species to vegetation cover (NI) showed a very marked
separation between treatments over time at the lowland meadow (Fig 2d) starting from
much lower levels than at the upland meadow (compare with Fig 1d) There were
noticeable differences in temporal trend between annual fertilizer treatments on the one
hand and triennial and nil fertilizer treatments on the other (Fig 2d) The effects of
treatment time and the treatment x time interaction were all highly significant (Plt0001)
in the full treatment range ANOVA The most marked increase in NI occurred with
annual FYM at the high rate (Tr 5) with NI increasing rapidly between 1999 and 2001
(Plt001) and a further highly significant increment between 2004 and 2009 (Plt0001)
(Fig 2d) A similar though less marked trend occurred with annual FYM at the medium
rate (Tr 4) with significant increases between 1999 and 2005 and between 2005 and
2010 (both Plt001) NI also increased markedly with the inorganic treatment
equivalent to Treatment 5 (Tr 10) although the overall rate of increase shown by this
treatment was closer to that shown by Treatment 4 than Treatment 5 There were
significant differences between Treatments 5 and 10 in most years from 2004 onwards
(Plt001 and Plt0001 in 2009 and 2010 respectively)
These effects were reflected in a significant overall effect of form of fertilizer
(FYMgtinorganic P=0008) and highly significant (Plt0001) overall effects of both rate
(high rategtlower rate) and frequency of application (annualgttriennial) within the FRF
series ANOVA and a significant form x rate interaction (P=0043) Mean NI was
highest for high rate annual FYM (Plt0001 compared to all three remaining means) and
lowest for lower rate inorganic (Plt005 compared to high rate inorganic NS compared
to lower rate FYM) Only frequency of application interacted significantly with time
(Plt0001) NI increased moderately under triennial application until 2008 (Plt005 in
2005 compared to 1999 Plt0001 each year 2006-2008) followed by a decline (Plt001
2010 compared to 2008) but with mean NI in 2010 still higher than 1999 (Plt005)
The increasing trend was very much more marked with annual fertilizer application for
17
which there were highly significant (Plt0001) increments between 1999 and 2002 and
between 2002 and 2008 but with no significant increase thereafter Differences between
annual and triennial treatment means were highly significant (Plt0001) from 2003
onwards
There was no significant change in NI over time either under the untreated control
(Tr 1) or the nil fertilizer (Tr 2) treatments in the full treatment ANOVA NI increased
until 2005 on plots limed in 1999 without fertilizer Tr 13 (Plt001 compared to 1999)
and declined slightly but not significantly thereafter Trends were similar with the
treatment limed in 1999 and receiving triennial FYM (Tr 15 ndash not illustrated in Fig 2)
although NI was higher with this treatment than with Tr 13 (Plt005) in both 2005 and
2008 By contrast applying the same rate of FYM annually after liming in 1999 (Tr 14)
caused a more rapid increase with levels by 2005 higher than either Tr 13 (Plt001) or
Tr 4 (Plt005) ndash note that the latter was equivalent to Tr 14 but without lime until 2005
NI declined after 2006 under Tr 14 but continued to increase with Tr 4 with the
difference between these two treatments reversed by 2010 (Plt005)
All main effects and interactions were significant in the LFF treatment series ANOVA
for NI except for lime x FYM frequency and the lime x FYM x time interactions Plots
limed in 1999 supported higher NI levels than those limed in 2005 averaged over the
three FYM frequencies and all years (P=0042) NI increased more rapidly after liming
in 1999 than where liming was delayed until 2005 (P=0033 for time x lime interaction)
with the overall difference due to liming in 1999 highly significant (Plt0001) by 2005
NI declined thereafter on the 1999-limed treatments whilst continuing to increase
following liming in 2005 of previously un-limed treatments both peaking in 2008 at a
very similar level Averaged over lime treatments NI increased much more rapidly
under annual FYM application than under either triennial or nil FYM (P=0002 for the
time x FYM frequency interaction) a highly significant increase had occurred by 2002
with annual FYM (Plt0001 compared to 1999) but increases of similar magnitude were
not reached until 2005 with triennial application and not until 2007 with nil FYM NI
subsequently declined under the latter two FYM regimes but not with annual FYM so
that by 2010 NI was significantly higher with annual FYM than with either triennial or
nil FYM (Plt0001) but with only a small non-significant difference between the latter
two treatments
TREATMENT EFFECTS AFTER 12 YEARS OF APPLICATION
The effects of all treatments on the five key variables at each site in 2010 are shown in
Table S20 with treatments differing significantly (Plt005) from the limed control
treatment (Tr 2) highlighted for each variable Treatment effects in 2010 are not
described in detail here but the modeled responses of total species-richness and of
richness of PI species in 2010 to the mean amounts of N and P applied per year averaged
over 1999-2010 are described below
Relationships with the amounts of fertilizer applied
The effect of neither rate nor frequency of application was significant in any of the
REML models for either total species-richness or PI species-richness at either site when
the amount of N or P applied ha-1
year-1
was included The effect of form of fertilizer was
still significant in most models however either as a main effect or in interaction with the
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
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Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
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Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
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Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
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Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
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Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
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Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
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Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
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Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
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Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
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Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
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Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
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Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
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Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
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Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
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Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
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Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
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Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
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studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
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plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
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on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
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Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
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Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
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Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
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Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
6
the inorganic fertilizer P applied each year corresponded to 60 of the total P in FYM between
1999 and 2006 and 80 from 2007-2010
Inorganic N P and K were applied as ammonium nitrate triple super-phosphate and muriate of
potash respectively to the relevant treatments The FYM treatments were designed to encompass
the range of inputs traditionally applied to species-rich hay meadows upon which current agri-
environment guidelines are based (Simpson and Jefferson 1996 Crofts and Jefferson 1999)
Lime was applied to treatments 13 14 and 15 only at each site in March-April 1999 and to most
treatments in 2005 (Table 2) The latter included the three previously limed treatments at the
upland meadow but not at the lowland one since soil pH levels had not fallen significantly by
2005 on these plots at the latter site Based on assessments of soil texture and soil pH tests
conducted in February-March the amounts of lime were calculated to be sufficient to raise the
soil pH of the experimental plots to 60 (Anon 2010)
At each site one plot per replicate block received no fertilizer or lime (untreated control)
between 1999 and 2005 whilst another plot per replicate was designated as a continuation of past
fertilizer inputs At the lowland meadow the latter was identical to the untreated control
treatment but at the upland meadow it was identical to Treatment 4 (12 t ha-1
FYM annually) In
2005 a decision was made to treat occasional liming as lsquobackgroundrsquo management for all
fertilizer treatments at both sites to be triggered for each treatment in future years once mean pH
for that treatment had declined to 55 whilst retaining both an untreated control (ie nil fertilizer
nil lime ndash Treatment 1) and a nil fertilizer (limed in 2005) control treatment (Treatment 2) at each
site The rationale behind this decision is addressed in the Discussion section At the lowland
meadow one of the two hitherto untreated control plots per replicate (Treatment 2) was therefore
limed in 2005 along with other treatments Only one nil input plot per replicate was available at
the upland meadow but since the site had a history of liming the existing nil input plots were
limed in 2005 (Treatment 2) and new untreated control plots (Treatment 1) were established
adjacent to each replicate block with the end at which each was located chosen randomly for
each block
The experimental plots were cut for hay after 1 July each year at the upland site and after 15
July at the lowland site the actual cutting date being dependent on weather conditions The hay
aftermath growth was grazed each year with experimental plots being grazed with the remainder
of the field At the upland meadow mature sheep were used normally commencing in September-
October continuing until March or late April The lowland meadow was grazed by beef store
cattle from mid Octoberearly November to late February
Botanical assessments were carried out during May each year except in 2001 at the upland
meadow due to FMD restrictions The percentage cover of each species present was estimated
visually in three 1m2 quadrats positioned at random within each plot in 1999 and then fixed for
the project duration
Soils were sampled in March 1999 2002 2004 2007 and 2010 by taking five 35 mm diameter x
75mm deep soil cores at random from within each treatment plot but outside the fixed positions
of the three 1m2 botanical survey quadrats The five samples per plot were combined dried at
30oC and then ground prior to analysis for organic carbon (C) total nitrogen (N) Olsen
extractable P exchangeable potassium (K) magnesium (Mg) calcium (Ca) sodium (Na) and pH
(in H2O) using standard laboratory methods (Allen 1974 MAFF 1986)
Soils were further sampled in June 1999 2002 2004 2007 and 2010 for soil microbial
assessment by taking five 35 mm diameter x 100 mm deep cores which were subsequently bulked
for each plot sieved (5 mm) and stored at 4oC prior to analysis Microbial community structure
was assessed using phospholipid fatty acid analysis (PLFA) as described by Bardgett et al
(1996) The fatty acids i150 a150 150 i160 170 i170 cy170 cis181ω7 and cy190 were
chosen to represent bacterial PLFAs (Federle 1986 Frostegaringrd Tunlid amp Baringaringth 1993) and
182ω6 was used as an indicator of fungal biomass (Federle 1986) The ratio of 182ω6bacterial
7
PLFAs was taken to represent the ratio of fungal-to-bacterial biomass in soil (Bardgett Hobbs amp
Frostegaringrd 1996 Frostegaringrd amp Baringaringth 1996)
DERIVED BOTANICAL VARIABLES
Estimates of cover for individual species were converted to a percentage of the total live
vegetation cover present in each quadrat in order to minimize the effects of year-to-year variation
and variation between treatments in vegetation density and total cover Several composite
variables were calculated to characterize the vegetation and these were then averaged across the
three quadrats in each plot to give plot mean values for each variable Plot means were then used
as the basic units for subsequent analyses of treatment effects
Key variables
Five key variables were used to investigate the effects of treatment and time on botanical
composition total species-richness (the total number of vascular plant species per m2) the
number per m2 of MG3 and MG5 positive indicator species (the upland and lowland meadows
respectively) the aggregate cover of these species as a percentage of total vegetation cover the
number per m2 of negative indicator species and the aggregate cover of negative indicator
species as a percentage of total vegetation cover Positive mesotrophic indicator species were
identified from lists produced by Robertson and Jefferson (2000) for use in monitoring the
condition of grassland Sites of Special Scientific Interest (SSSIs) in MG3 (Anthoxanthum
odoratum-Geranium sylvaticum grassland) and MG5 (Cynosurus cristatus-Centaurea nigra
grassland) communities of the NVC (Rodwell 1992) A single list of negative indicator species
was compiled from the generic negative indicators for MG3 and MG5 communities (Robertson amp
Jefferson 2000) augmented by species shown in previous work (eg Mountford et al 1993
Kirkham et al 1996) to be favoured by nutrient addition and for which an increase in abundance
would indicate a negative effect on plant community quality Positive and negative indicator
species are listed in Supporting Information Table S3
Additional variables
Among other derived variables was the weighted Ellenberg N score (Smith et al 2003 Kirkham
et al 2008) referred to hereafter as the fertility score The Ellenberg N index represents the
degree of association with soil fertility (not specifically soil N) of a particular species (Ellenberg
1988 Hill et al 1999) Fertility scores were calculated for each quadrat as the average N index
of the component species weighted according to the proportional contribution of each species to
total vegetation cover Plot mean fertility scores were highly correlated at both sites with the
aggregate cover of negative indicator species (NI) (Pearson r=0845 and r=0927 at the upland
and lowland sites respectively in 2010 both Plt0001) The latter variable was preferred to
fertility scores for statistical analyses in this paper because it proved more responsive to fertilizer
treatments although fertility scores are referred to in the Discussion section in relation to results
from elsewhere
DATA ANALYSIS
ANOVAs for treatment effects
For each of the key variables botanical data for all years 1999-2010 except 2001 at the upland
meadow and for all treatments except Treatment 1 at the same meadow were analyzed by
repeated measures analysis of variance (ANOVA - Genstat V Committee 1997) with Year as the
repeated measures factor Separate ANOVAs were carried out for each variable within each site
using three separate ANOVA models in each case In each model variation between replicate
8
blocks was accounted for by treating replicate as a blocking factor The first model included all
the treatments except Treatment 1 at the upland meadow whilst the other two models used
balanced factorial subsets (series) of treatments (see Table 2) the Form x Rate x Frequency
(FRF) series and the Lime x FYM frequency (LFF) series
The FRF series tested the effects of form of fertilizer (ie FYM or inorganic) the rate at which
fertilizers were applied (12 or 24 t FYM ha-1
and inorganic treatments corresponding to these)
the frequency at which treatments were applied (ie annual or triennially) and all two- and three-
way interactions between these factors
The LFF series tested the effects of liming regime ie lime in 1999 (and 2005 at the upland site)
versus lime in 2005 only the frequency of FYM application (ie nil annual or triennially) and the
Lime x FYM frequency interaction All FYM treatments within this series were applied at 12 t ha-
1 At both sites the LFF series consisted of Treatments 2 4 7 13 14 and 15 (see Table 2) The
liming comparison therefore differed slightly between the two sites since liming was repeated in
2005 on plots previously limed in 1999 at the upland meadow but not repeated at the lowland
one
In both the LFF and the FRF models for each variable the first stratum of the ANOVA
consisted of the effects of all factors and interactions averaged over time with the same factors
and interactions repeated in the second stratum with time as an additional factor
In all repeated measures ANOVAs to account for the repeated measures aspect all degrees of
freedom in the Year x Treatment stratum were adjusted to account for departure from
homogeneity of the population covariance matrix using ε-coefficients calculated by the
Greenhouse-Geisser method (Genstat V Committee 1997)
In most years including 2010 all species were recorded individually but at both sites
bryophytes were recorded only as a single group in some years This had only a small influence
on the data since there were seldom more than two bryophyte species present in a quadrat and
more often less Nevertheless for consistency between years bryophytes were treated as a single
group in all years when calculating total species-richness for data for repeated measures
ANOVAs
In addition to the above analyses 2010 data for the key variables were tested alone for treatment
effects by ANOVA and by analysis of covariance (ANCOVA) the latter using 1999 data as the
covariate (Genstat 5 Committee 1997) All three models as described above were tested The
ANCOVAs for the upland meadow excluded Treatment 1 (untreated control plots established in
2005) Where an ANCOVA showed significant treatment covariance with 1999 data the adjusted
results were used In other cases the unadjusted analyses were used Individual bryophyte species
were included in the calculations of species-richness at both sites in these analyses
In all analyses in order to satisfy the conditions for ANOVA (normality of residuals and
homogeneity of residual variation) all percentage data were transformed before analysis by
arcsine(radicp) where p is the percentage value expressed as a proportion All results quoted for
percentage data refer to analyses of arcsine-square root transformed data
Mixed modeling
Mixed model analysis (Residual Estimated Maximum Likelihood ndash REML Genstat V
Committee 1997) was used to identify any effect of form rate or frequency of application on
vegetation composition at each site in 2010 not simply attributable to the total (or mean per year)
amount of fertilizer applied over the study period A variable representing this amount was
included as a continuous explanatory variable in each analysis along with other fixed factors
form rate and frequency of application (Block was included as a random factor) Since the total
(or mean) amounts of N P and K applied over the whole period were completely correlated
across treatments at each site it was not feasible to include all three elements as separate terms in
the model Nor was it feasible to simply use a single notional variable for fertilizer amount (eg t
9
FYM ha-1
year-1
or inorganic equivalent) since there was lack of equivalence of N and P supply
(but not K) between individual FYM treatments and their corresponding inorganic treatments For
each dependent variable tested two separate models were therefore developed using either N or
P ha-1
year-1
as the continuous variable In each case the presence of any non-linearity in response
was tested by including a quadratic term (ie N2 or P
2) or where more appropriate by logn-
transformation of the explanatory variable
For each analysis an initial model was constructed that included all five main effects and all
interactions The importance of each term was evaluated initially by a χ2 test on the Wald statistic
(Genstat V Committee 1997) The number and order of terms in the model were varied but with
N or P amount always included until a model was developed in which each term was significant
The model was then refined by testing the contribution of each term to the model by the more
conservative deviance test ie a χ2 test on the difference in deviance between the model
containing the term and a sub-model from which it was dropped (Welham amp Thompson 1997)
Where there was an interaction between form of fertilizer and the quadratic term but no overall
effect of form - implying that the difference between the two forms of fertilizer lay primarily in a
difference in the curvilinearity of response - a refined model was tested with the P2 (or N
2) x
Form term replaced by Form nested within the quadratic term (eg P2Form) This has the effect
of absorbing the overall effect of form of fertilizer within the interaction term and returns the two
terms P2 and P
2 x Form (Galwey 2006)
These modeling approaches were used with total species-richness and PI species-richness as
(separate) response variables and included data for all treatments limed in 2005 but not in 1999
(ie Trs 2-12)
Results
TEMPORAL CHANGES 1999-2010
Changes over time in total species-richness (number per m2) richness and proportional
aggregate cover of positive indicator (PI) species and proportional aggregate cover of
negative indicator (NI) species at the upland and lowland meadows are represented in
Figs 1 and 2 respectively for ten of the fifteen treatments applied Repeated measures
ANOVA statistics for each of the four variables are given in Supporting Information
Tables S4 S6 S8 and S10 respectively for the upland meadow and Tables S12 S14 S16
and S18 respectively for the lowland meadow Corresponding mean values for each
treatment in each year are given in Tables S5 S7 S9 S11 S13 S15 S17 and S19
respectively Changes in individual species abundance contributing to the temporal trends
in aggregated PI and NI species described below are outlined under Supporting
Information
The upland meadow
The overall effects of both treatment and time were highly significant in the repeated
measures ANOVA for total species-richness at the upland meadow (both P=0001) but
there was no treatment x time interaction (P=0647)
After an initial decline between 1999 and 2000 species-richness remained fairly
constant for most treatments until 2003 after which there was a general increase with the
notable exception of the highest rate annual FYM treatment (Tr 5) (Fig 1a) The latter
treatment declined further between 2005 and 2007 after which it appeared to recover
slightly and averaged over all years this treatment was significantly less species-rich than
all others (Plt001 compared to Tr 14 Plt0001 the remainder) None of the remaining
10
treatments differed significantly from the limed control treatment (Tr2) averaged over all
years However the more species-rich treatments ie nil fertilizer plus lime applied in
both 1999 and 2005 (Tr 13) low rate annual and medium rate triennial FYM (Tr 3 and 7
respectively) and the inorganic equivalent to the latter (Tr 11) were all more species-rich
averaged over all years (Plt005) than annual FYM at the medium rate under either
liming regime (ie Trs 4 and 14 the former representing a continuation of past
management)
The effect of the high rate annual FYM treatment (Tr 5) was largely responsible for
several main effects within the repeated measures ANOVA of treatments within the Form
x Rate x Frequency (FRF) factorial series namely overall effects (averaged over all
years) of rate (mediumgthigh P=0002) and frequency (triennialgtannual P=0001) of
application and a not quite significant effect of form of fertilizer (FYMltinorganic
P=0057) Both the form x rate and the form x frequency interactions were significant
(both Plt005) high rate FYM was significantly less species-rich than all other
combinations averaged over annual and three yearly treatments and all years (Plt0001
compared to medium rate FYM Plt001 compared to both medium and high rate
inorganic fertilizer) and annual FYM was less species-rich than three yearly FYM and
than inorganic fertilizer either annually or triennial (all Plt0001) averaged over the two
application rates and all years The rate x frequency interaction was not significant
(P=0105) but there was a marginally non-significant three-way interaction between
form rate and frequency (P=0070) with a low mean for high rate annual FYM (Tr 5
211 species m-2
) contrasting markedly with all other treatments within the series
particularly with low rate triennial FYM (Tr 6 267 species m-2
)
As with the full treatment ANOVA the overall time factor was highly significant
(Plt0001) but only the effect of application frequency varied significantly with time
(interaction Plt005) Species-richness declined slightly with annual treatments overall
and increased slightly with triennial treatments such that differences between these were
significant in all years from 2005 onwards (Plt001 for 2006-2009 Plt005 in 2005 and
2010)
Both the richness and aggregate cover of PI species (PI) increased generally with time
(Fig 1b and 1c) although in both cases there was a progressive separation between
species The temporal trend was particularly marked for PI although the time factor
was highly significant (Plt0001) in both ANOVAs The overall treatment effect was
marginally non-significant for PI richness (P=0069) with no indication of a time x
treatment interaction (P=0703) whereas for PI there was both a significant treatment
effect (P=0002) and a marginally non-significant treatment x time interaction (P=0075)
Mean PI was significantly lower averaged over all years for annual FYM treatments at
both 12 and 24 t ha-1
(ie Trs 4 14 and 5) compared to several other treatments notably
nil fertilizer treatments limed either in 2005 only (Tr 2 limed control) or in both 1999
and 2005 (Tr 13) (all differences Plt0001 except Tr 2 vs Tr 4 Plt001) and triennial
inorganic treatments (Trs 11 and 12 Plt001 except for Tr 4 vs Tr 11 Plt005) Within
the FRF series ANOVA the increase in PI over time was greater with triennial
treatments compared to annual averaged over fertilizer forms and rates of application
(time x frequency interaction P=0016) with a marked overall difference due to
application frequency averaged over forms rates and all years (P=0005)
11
PI species-richness showed a similar overall effect of application frequency within the
FRF series (triennialgtannual P=0013) and also a significant overall effect of rate of
application (medium rategthigh rate P=0022) The overall effect of form of fertilizer was
marginally non-significant (FYMltinorganic P=0059) and there was no significant
interaction among any of the three factors averaged over all years nor any interaction
with time
16
18
20
22
24
26
28
30
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Specie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime in Year 1+ ann FYM (Tr 14) Nil fert + Lime in Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr1)
a Species richness (total)
3
4
5
6
7
8
9
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
70
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
0
5
10
15
20
25
30
35
40
45
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
Fig 1 Changes 1999-2010 in vegetation composition an upland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control
The mean number of negative indicator (NI) species per m2 averaged over all treatments
varied significantly over time at the upland meadow (P=0002 data not shown) though
with no consistent trend - it was lowest overall in 2002 (499 per m2) and highest in both
1999 and 2008 (548 per m2) The treatment x time interaction was not significant
(P=0469) but the overall treatment effect was highly significant (Plt0001) mean
number of NI species per m2 was higher than the limed control (Tr 1 453 per m
2) with
all fertilizer treatments except Treatments 11 (triennial inorganic fertilizer at the lower
rate) and 13 (nil fertilizer limed in both 1999 and 2005) (Plt0001 for all except Trs 5
and 6 for which Plt001) The highest mean NI m-2
value was for high rate inorganic
12
fertilizer (Tr 10 565 per m2) which differed significantly (Plt005) from its FYM
counterpart Treatment 5 There was however no consistent difference between FYM
treatments and their inorganic counterparts and no overall effect of form of fertilizer
within the FRF series (P=0745) There was a significant form x rate interaction within
this ANOVA (P=0032) but only the difference between the high rate and lower rate
inorganic means was significant (high rategtlower rate Plt001)
The aggregate contribution to vegetation cover of negative indicator species (NI)
showed a very marked divergence between treatments over time (Fig 1d) with NI
either changing little or declining under most treatments again with the notable exception
of high rate annual FYM (Tr 5) under which NI increased over time Time treatment
and the treatment x time interaction were all highly significant (Plt0001) The
predominant temporal trend was downward (from 25 of live cover in 1999 to 14 in
2010 averaged over all treatments) but NI for Treatment 5 had significantly increased
compared to 1999 in 2002 2003 (Plt005) 2005 (Plt0001) 2008 (Plt0001) and 2009
(Plt001) but not in 2010 (Fig 1d) The most marked declines in NI occurred on the
limed control (Tr 2) nil fertilizer plus lime in both 1999 and 2005 (Tr 13) and the lower
rate triennial inorganic fertilizer treatments (Tr 11) differences compared to both 1999
and 2002 were highly significant (Plt0001) in most subsequent years for these
treatments A smaller decline also occurred after 2002 on the treatment equivalent to past
inputs (ie12 t ha-1
year-1
annual FYM Tr 4) In most years this treatment differed little
from the equivalent FYM treatment limed in both 1999 and 2005 (Tr 14)
These trends were reflected in highly significant overall effects on NI within the FRF
series of form (FYMgtinorganic) rate (mediumlthigh) and frequency (triennialltannual)
(all effects Plt0001) Time x rate and time x frequency interactions were both significant
(P=0019 and Plt0001 respectively) with a marginally non-significant rate x frequency
interaction averaged over all years (P=0054) NI declined over time under both
application rates averaged over forms and frequencies of application but the decline was
slower with high rate treatments compared to lower rate Difference between rates were
significant in all years from 2002 onwards (Plt005 in 2002 2006 and 2007 Plt0001 in
other years) Overall differences due to frequency of application were even more marked
Under annual application NI declined initially from 2003-2006 compared to 1999 but
subsequently recovered and did not differ from 1999 levels from 2007 onwards whilst
under triennial application differences were highly significant (Plt0001) compared to
1999 from 2003 onwards with significant differences between years during this same
period (Plt005 in 2003 and 2006 Plt0001 in remaining years)
Repeated-measures ANOVAs within the LFF treatment series at the upland meadow
showed no overall effect of liming regime on temporal trends for any of the above
variables nor any interaction of lime with either time or FYM frequency - note that FYM
was applied at the intermediate rate (12 t ha-1
) in all FYM treatments within this series
The overall effect of FYM frequency was significant for both PI (P=0003) and the
number of NI species m-2
(P=0001) PI was highest with nil FYM and lowest with
annual FYM with triennial intermediate and differing significantly (Plt005) from the
other two means NI species m-2
did not differ between triennial and annual application
within the LFF series but both were significantly higher than nil FYM (Plt001 for
triennial Plt0001 for annual application) There was also a significant time x FYM
frequency interaction for NI within this series (P=0001) reflecting a more rapid
13
decline with nil FYM than with triennial FYM and no significant overall trend with
annual FYM when averaged over both lime treatments
12
14
16
18
20
22
24
26
28
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Spe
cie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime Year 1 + ann FYM (Tr 14) Nil fert + Lime Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr 1)
a Species richness (total)
2
3
4
5
6
7
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
0
10
20
30
40
50
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
Fig 2 Changes 1999-2010 in vegetation composition at a lowland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control treatment and those that had received lime in year 1
Treatments 1 and 2 were identical (nil input) until 2005
The lowland meadow
Both time and treatment were significant within the ANOVA for total species-richness at
the lowland meadow (Plt0001 and P=0009 respectively and in contrast to the upland
site the treatment x time interaction was significant (P=0012) Averaged over all years
high rate annual FYM treatment (Tr 5) was significantly less species-rich than all others
except Treatment 14 (annual FYM at 12 t ha-1
limed in 1999) having declined
progressively between 1999 and 2009 (Fig 2a) with most differences significant at
Plt001 Treatment 14 did not differ significantly overall compared to the corresponding
FYM treatment not limed until 2005 (Tr 4) although these two treatments showed
14
markedly different temporal trends Treatment 14 was less species-rich than others during
the first five years of the experiment but did not change significantly throughout the
whole 12 year period whilst Treatment 4 changed little between 1999 and 2006 but
declined thereafter (Plt001 and Plt0001 for 2009 and 2010 respectively compared to
2006) possibly in response to liming in 2005 However there was no significant overall
effect on total species-richness of liming strategy within the LFF series and no interaction
between liming and either time or FYM application
There were marked differences in temporal trend in species-richness between FYM
treatments and their inorganic counterparts particularly with the high rate annual
treatments (Tr 5 and Tr 10) Whereas species-richness declined more or less continually
throughout under Treatment 5 at least until 2009 (Plt001 for 2004 compared to 1999 and
for both 2009 and 2010 compared to 2004) Treatment 10 declined between 2000 and
2001 (Plt005) and then remained relatively unchanged until 2008 after which it declined
again (2010 Plt005 compared to 2008)
Other treatments within the full treatment series including untreated control (Tr 1) and
limed control (Tr 2) showed no particular temporal trend in the full repeated measures
ANOVA although both the latter treatments showed a dip in species-richness in 2004
(Plt005 compared to both 2003 and 2005)
There was a strong overall effect of form of fertilizer within the FRF series ANOVA
(FYMltinorganic P=0003) a significant effect of frequency of application
(triennialgtannual P=0027) and a not quite significant effect of application rate (high
rateltmedium rate P=0064) but no significant interaction between any of these factors
The time x application frequency interaction was highly significant (Plt0001) species-
richness remained unchanged under triennial application but under annual application
was significantly lower compared to 1999 in each year from 2004 onwards with the
exception of 2006 (Plt005 in 2004 Plt001 in 2005 and 2008 Plt0001 the remainder)
Again with the exception of 2006 differences between triennial and annual application
were significant in each year from 2005 onwards (Plt005 in 2007 Plt001 in 2005 and
2008 Plt0001 in 2009 and 2010)
Both treatment time and their interaction were significant in the full treatment ANOVA
for PI species-richness at the lowland meadow (ie time and treatment both Plt0001
time x treatment P=0006) There was a general decline in PI-richness after 2005 (Fig
2b) with PI species m-2
averaged over all treatments lower from 2007 onwards (Plt0001)
than all years up to 2005 except 2001 Although the initiation of this trend corresponded
with the liming of most treatments in 2005 a similar pattern was shown by treatments
that did not receive this lime application ie the untreated control treatment (Tr 1) the
nil fertilizer treatment limed in 1999 (Tr 13) and the corresponding treatment also
receiving triennial FYM (Tr 15) In all these cases PI-richness showed no significant
difference between years until 2005 but was significantly (Plt001) lower by 2010 than in
2005 Treatment 14 (limed in 1999 plus annual FYM) did not show this pattern but PI
species-richness had been lowest under this treatment in most years between 1999 and
2005 (Fig 2b)
As with total species-richness PI richness declined more or less progressively under
Treatment 5 until 2008 (Plt005 compared to 1999 by 2001 Plt0001 from 2006
onwards) whereas Treatment 4 did not change significantly until 2006 after which it
declined (Plt005 in 2007 compared to 2006 Plt001 thereafter) Both these treatments
15
tended be less rich in PI species than the corresponding inorganic treatment (Trs 9 and
10 respectively) in most years consistently so for Tr 4 compared to Tr 9 but these
differences were not significant in any year nor when averaged over all years There was
however a significant overall effect of form of fertilizer in the FRF series ANOVA
(FYMltinorganic P=0002) and a highly significant effect of frequency of application
(triennialgtannual Plt0001) The time x frequency of application interaction was also
highly significant (Plt0001) Under triennial application PI species per m2 increased
slightly until 2005 (Plt005 compared to 1999) and declined thereafter so that by 2009
levels were significantly lower than in 1999 (Plt001 for 2009 Plt0001 for 2010) By
contrast under annual application PI species m-2
declined progressively until 2009
following a temporary though highly significant (Plt0001) dip in 2000 so that the
difference compared to 1999 was significant in every year from 2002 onwards and highly
significant (Plt0001) from 2005 With the exception of 2003 differences between
triennial and annual application were significant from 2002 onwards and highly
significant (Plt0001) in 2005 and from 2007 to 2010
There was no significant effect other than of time within the LFF treatment series
ANOVA although there was a marginally non-significant time x FYM frequency
interaction (P=0054) This reflected a more rapid decline in PI species-richness under
annual FYM application than with either nil or triennial FYM with the latter two showing
no discernible difference
By contrast with PI species m-2
PI cover percentage (PI) showed no overall treatment
effect within the full treatment ANOVA nor a time x treatment interaction although the
overall effect of time was highly significant (Plt0001) The temporal pattern in PI (Fig
2c) also differed from that shown by PI-richness with PI averaged over all treatments
declining between 1999 and 2005 in three highly significant increments (1999-2000
2000-2002 and 2002-2005 all Plt0001) and increasing again thereafter (2005-2009 and
2009-2010 both Plt0001) There was no significant effect within either the LFF or FRF
treatment series ANOVAs although their was a slight time x frequency of application
effect within the latter (P=0081) PI was slightly higher under annual application than
triennial in each year 1999-2004 after which the reverse was true although differences
were always small
There was no overall effect of treatment on the number of NI species per m2 within the
full treatment range at the lowland meadow (data not shown) but both the overall effect
of time and the time x treatment interaction were significant (Plt0001 and P=0009
respectively) Overall NI species m-2
increased over time from 24 species m-2
in 1999 to
a peak of 42 m-2
in 2008 averaged over all treatments NI species m-2
increased on the
untreated control treatment (Tr 1) from 21 species m-2
in 1999 to 34 species m-2
in 2007
(increase Plt001) then declined to the 1999 value by 2010 The limed (in 2005) control
(Tr 2) showed a somewhat similar pattern though from a slightly higher starting level
(31 species m-2
) and with the increase to 38 species m-2
in 2008 not reaching
significance
These temporal trends in NI species m-2
were much accentuated by annual fertilizer
application particularly by FYM treatments with peak numbers of NI species reaching
52 and 47 species m-2
for Treatments 4 and 5 respectively in 2008 (both Plt0001
compared to 1999) and despite slight declines in both cases remaining significantly
higher in 2010 than in 1999 (both Plt0001) Inorganic counterparts to these treatments
16
showed similar though slightly less marked trends and there was no significant
difference between all four treatments in the peak number reached in 2008 There was no
overall effect of either form of fertilizer or rate of application within the FRF series
ANOVA and only a marginally non-significant effect of frequency of application
(triennialltannual P=0061) Liming in 1999 (Trs 13 14 and 15) also appeared to
accentuate the generally increasing trend although peak numbers in 2008 did not differ
between these treatments and either untreated or limed control in the full treatment range
ANOVA The overall effects of liming regime (1999 or 2005) were not significant within
the LFF treatment series but the faster rate of increase with liming in 1999 was reflected
in a significant time x lime interaction in this analysis (P=0017) The mean number of NI
species m-2
increased very significantly (Plt0001) between 1999 and 2000 following
liming in 1999 with the increment increasing until 2008 whilst on plots un-limed until
2005 NI species m-2
did not increase significantly until 2003 (Plt005) although the
increase compared to 1999 was highly significant (Plt0001) in 2006 2008 and 2010
Nevertheless the differences in rate of increase was at least partly due to a lower 1999
mean for the 1999-limed treatments (170 species m-2
compared to 244 species m-2
difference NS) and although in all years from 2001 onwards mean NI species m-2
was
higher for 1999-limed treatments compared to those not limed until 2005 the difference
was not significant in any year
The contribution of NI species to vegetation cover (NI) showed a very marked
separation between treatments over time at the lowland meadow (Fig 2d) starting from
much lower levels than at the upland meadow (compare with Fig 1d) There were
noticeable differences in temporal trend between annual fertilizer treatments on the one
hand and triennial and nil fertilizer treatments on the other (Fig 2d) The effects of
treatment time and the treatment x time interaction were all highly significant (Plt0001)
in the full treatment range ANOVA The most marked increase in NI occurred with
annual FYM at the high rate (Tr 5) with NI increasing rapidly between 1999 and 2001
(Plt001) and a further highly significant increment between 2004 and 2009 (Plt0001)
(Fig 2d) A similar though less marked trend occurred with annual FYM at the medium
rate (Tr 4) with significant increases between 1999 and 2005 and between 2005 and
2010 (both Plt001) NI also increased markedly with the inorganic treatment
equivalent to Treatment 5 (Tr 10) although the overall rate of increase shown by this
treatment was closer to that shown by Treatment 4 than Treatment 5 There were
significant differences between Treatments 5 and 10 in most years from 2004 onwards
(Plt001 and Plt0001 in 2009 and 2010 respectively)
These effects were reflected in a significant overall effect of form of fertilizer
(FYMgtinorganic P=0008) and highly significant (Plt0001) overall effects of both rate
(high rategtlower rate) and frequency of application (annualgttriennial) within the FRF
series ANOVA and a significant form x rate interaction (P=0043) Mean NI was
highest for high rate annual FYM (Plt0001 compared to all three remaining means) and
lowest for lower rate inorganic (Plt005 compared to high rate inorganic NS compared
to lower rate FYM) Only frequency of application interacted significantly with time
(Plt0001) NI increased moderately under triennial application until 2008 (Plt005 in
2005 compared to 1999 Plt0001 each year 2006-2008) followed by a decline (Plt001
2010 compared to 2008) but with mean NI in 2010 still higher than 1999 (Plt005)
The increasing trend was very much more marked with annual fertilizer application for
17
which there were highly significant (Plt0001) increments between 1999 and 2002 and
between 2002 and 2008 but with no significant increase thereafter Differences between
annual and triennial treatment means were highly significant (Plt0001) from 2003
onwards
There was no significant change in NI over time either under the untreated control
(Tr 1) or the nil fertilizer (Tr 2) treatments in the full treatment ANOVA NI increased
until 2005 on plots limed in 1999 without fertilizer Tr 13 (Plt001 compared to 1999)
and declined slightly but not significantly thereafter Trends were similar with the
treatment limed in 1999 and receiving triennial FYM (Tr 15 ndash not illustrated in Fig 2)
although NI was higher with this treatment than with Tr 13 (Plt005) in both 2005 and
2008 By contrast applying the same rate of FYM annually after liming in 1999 (Tr 14)
caused a more rapid increase with levels by 2005 higher than either Tr 13 (Plt001) or
Tr 4 (Plt005) ndash note that the latter was equivalent to Tr 14 but without lime until 2005
NI declined after 2006 under Tr 14 but continued to increase with Tr 4 with the
difference between these two treatments reversed by 2010 (Plt005)
All main effects and interactions were significant in the LFF treatment series ANOVA
for NI except for lime x FYM frequency and the lime x FYM x time interactions Plots
limed in 1999 supported higher NI levels than those limed in 2005 averaged over the
three FYM frequencies and all years (P=0042) NI increased more rapidly after liming
in 1999 than where liming was delayed until 2005 (P=0033 for time x lime interaction)
with the overall difference due to liming in 1999 highly significant (Plt0001) by 2005
NI declined thereafter on the 1999-limed treatments whilst continuing to increase
following liming in 2005 of previously un-limed treatments both peaking in 2008 at a
very similar level Averaged over lime treatments NI increased much more rapidly
under annual FYM application than under either triennial or nil FYM (P=0002 for the
time x FYM frequency interaction) a highly significant increase had occurred by 2002
with annual FYM (Plt0001 compared to 1999) but increases of similar magnitude were
not reached until 2005 with triennial application and not until 2007 with nil FYM NI
subsequently declined under the latter two FYM regimes but not with annual FYM so
that by 2010 NI was significantly higher with annual FYM than with either triennial or
nil FYM (Plt0001) but with only a small non-significant difference between the latter
two treatments
TREATMENT EFFECTS AFTER 12 YEARS OF APPLICATION
The effects of all treatments on the five key variables at each site in 2010 are shown in
Table S20 with treatments differing significantly (Plt005) from the limed control
treatment (Tr 2) highlighted for each variable Treatment effects in 2010 are not
described in detail here but the modeled responses of total species-richness and of
richness of PI species in 2010 to the mean amounts of N and P applied per year averaged
over 1999-2010 are described below
Relationships with the amounts of fertilizer applied
The effect of neither rate nor frequency of application was significant in any of the
REML models for either total species-richness or PI species-richness at either site when
the amount of N or P applied ha-1
year-1
was included The effect of form of fertilizer was
still significant in most models however either as a main effect or in interaction with the
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
References
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Anon (2010) Fertilizer Manual (RB 209) 8th Edition Her Majestyrsquos Stationery Office London
Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
Kluwer Academic Publishers Dordrecht
Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
restoration of species-rich meadows in the Netherlands Biological Conservation 62 59-65
Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
as an indicator of ecosystem self-regulation in temperate meadow grasslands Biology and
Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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and Fertility of Soils 22 261-264
Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
Hopkins JJ (1999) The extent of semi-natural grassland communities in lowland England
and Wales a review of conservation surveys 1978-96 Grass and Forage Science 54 1-18
Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
phosphorous vs nitrogen enrichment in plant species loss across North-west Europen
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Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
and losses following application of manures to arable land MANNER Soil Use and
Management 15 137-143
Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
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vegetation in English Environmentally Sensitive Areas Grass Forage Science 57 82-92
Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
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Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
activity in botanically diverse haymeadows effects of litter type and fertilizer additions Soil
Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
7
PLFAs was taken to represent the ratio of fungal-to-bacterial biomass in soil (Bardgett Hobbs amp
Frostegaringrd 1996 Frostegaringrd amp Baringaringth 1996)
DERIVED BOTANICAL VARIABLES
Estimates of cover for individual species were converted to a percentage of the total live
vegetation cover present in each quadrat in order to minimize the effects of year-to-year variation
and variation between treatments in vegetation density and total cover Several composite
variables were calculated to characterize the vegetation and these were then averaged across the
three quadrats in each plot to give plot mean values for each variable Plot means were then used
as the basic units for subsequent analyses of treatment effects
Key variables
Five key variables were used to investigate the effects of treatment and time on botanical
composition total species-richness (the total number of vascular plant species per m2) the
number per m2 of MG3 and MG5 positive indicator species (the upland and lowland meadows
respectively) the aggregate cover of these species as a percentage of total vegetation cover the
number per m2 of negative indicator species and the aggregate cover of negative indicator
species as a percentage of total vegetation cover Positive mesotrophic indicator species were
identified from lists produced by Robertson and Jefferson (2000) for use in monitoring the
condition of grassland Sites of Special Scientific Interest (SSSIs) in MG3 (Anthoxanthum
odoratum-Geranium sylvaticum grassland) and MG5 (Cynosurus cristatus-Centaurea nigra
grassland) communities of the NVC (Rodwell 1992) A single list of negative indicator species
was compiled from the generic negative indicators for MG3 and MG5 communities (Robertson amp
Jefferson 2000) augmented by species shown in previous work (eg Mountford et al 1993
Kirkham et al 1996) to be favoured by nutrient addition and for which an increase in abundance
would indicate a negative effect on plant community quality Positive and negative indicator
species are listed in Supporting Information Table S3
Additional variables
Among other derived variables was the weighted Ellenberg N score (Smith et al 2003 Kirkham
et al 2008) referred to hereafter as the fertility score The Ellenberg N index represents the
degree of association with soil fertility (not specifically soil N) of a particular species (Ellenberg
1988 Hill et al 1999) Fertility scores were calculated for each quadrat as the average N index
of the component species weighted according to the proportional contribution of each species to
total vegetation cover Plot mean fertility scores were highly correlated at both sites with the
aggregate cover of negative indicator species (NI) (Pearson r=0845 and r=0927 at the upland
and lowland sites respectively in 2010 both Plt0001) The latter variable was preferred to
fertility scores for statistical analyses in this paper because it proved more responsive to fertilizer
treatments although fertility scores are referred to in the Discussion section in relation to results
from elsewhere
DATA ANALYSIS
ANOVAs for treatment effects
For each of the key variables botanical data for all years 1999-2010 except 2001 at the upland
meadow and for all treatments except Treatment 1 at the same meadow were analyzed by
repeated measures analysis of variance (ANOVA - Genstat V Committee 1997) with Year as the
repeated measures factor Separate ANOVAs were carried out for each variable within each site
using three separate ANOVA models in each case In each model variation between replicate
8
blocks was accounted for by treating replicate as a blocking factor The first model included all
the treatments except Treatment 1 at the upland meadow whilst the other two models used
balanced factorial subsets (series) of treatments (see Table 2) the Form x Rate x Frequency
(FRF) series and the Lime x FYM frequency (LFF) series
The FRF series tested the effects of form of fertilizer (ie FYM or inorganic) the rate at which
fertilizers were applied (12 or 24 t FYM ha-1
and inorganic treatments corresponding to these)
the frequency at which treatments were applied (ie annual or triennially) and all two- and three-
way interactions between these factors
The LFF series tested the effects of liming regime ie lime in 1999 (and 2005 at the upland site)
versus lime in 2005 only the frequency of FYM application (ie nil annual or triennially) and the
Lime x FYM frequency interaction All FYM treatments within this series were applied at 12 t ha-
1 At both sites the LFF series consisted of Treatments 2 4 7 13 14 and 15 (see Table 2) The
liming comparison therefore differed slightly between the two sites since liming was repeated in
2005 on plots previously limed in 1999 at the upland meadow but not repeated at the lowland
one
In both the LFF and the FRF models for each variable the first stratum of the ANOVA
consisted of the effects of all factors and interactions averaged over time with the same factors
and interactions repeated in the second stratum with time as an additional factor
In all repeated measures ANOVAs to account for the repeated measures aspect all degrees of
freedom in the Year x Treatment stratum were adjusted to account for departure from
homogeneity of the population covariance matrix using ε-coefficients calculated by the
Greenhouse-Geisser method (Genstat V Committee 1997)
In most years including 2010 all species were recorded individually but at both sites
bryophytes were recorded only as a single group in some years This had only a small influence
on the data since there were seldom more than two bryophyte species present in a quadrat and
more often less Nevertheless for consistency between years bryophytes were treated as a single
group in all years when calculating total species-richness for data for repeated measures
ANOVAs
In addition to the above analyses 2010 data for the key variables were tested alone for treatment
effects by ANOVA and by analysis of covariance (ANCOVA) the latter using 1999 data as the
covariate (Genstat 5 Committee 1997) All three models as described above were tested The
ANCOVAs for the upland meadow excluded Treatment 1 (untreated control plots established in
2005) Where an ANCOVA showed significant treatment covariance with 1999 data the adjusted
results were used In other cases the unadjusted analyses were used Individual bryophyte species
were included in the calculations of species-richness at both sites in these analyses
In all analyses in order to satisfy the conditions for ANOVA (normality of residuals and
homogeneity of residual variation) all percentage data were transformed before analysis by
arcsine(radicp) where p is the percentage value expressed as a proportion All results quoted for
percentage data refer to analyses of arcsine-square root transformed data
Mixed modeling
Mixed model analysis (Residual Estimated Maximum Likelihood ndash REML Genstat V
Committee 1997) was used to identify any effect of form rate or frequency of application on
vegetation composition at each site in 2010 not simply attributable to the total (or mean per year)
amount of fertilizer applied over the study period A variable representing this amount was
included as a continuous explanatory variable in each analysis along with other fixed factors
form rate and frequency of application (Block was included as a random factor) Since the total
(or mean) amounts of N P and K applied over the whole period were completely correlated
across treatments at each site it was not feasible to include all three elements as separate terms in
the model Nor was it feasible to simply use a single notional variable for fertilizer amount (eg t
9
FYM ha-1
year-1
or inorganic equivalent) since there was lack of equivalence of N and P supply
(but not K) between individual FYM treatments and their corresponding inorganic treatments For
each dependent variable tested two separate models were therefore developed using either N or
P ha-1
year-1
as the continuous variable In each case the presence of any non-linearity in response
was tested by including a quadratic term (ie N2 or P
2) or where more appropriate by logn-
transformation of the explanatory variable
For each analysis an initial model was constructed that included all five main effects and all
interactions The importance of each term was evaluated initially by a χ2 test on the Wald statistic
(Genstat V Committee 1997) The number and order of terms in the model were varied but with
N or P amount always included until a model was developed in which each term was significant
The model was then refined by testing the contribution of each term to the model by the more
conservative deviance test ie a χ2 test on the difference in deviance between the model
containing the term and a sub-model from which it was dropped (Welham amp Thompson 1997)
Where there was an interaction between form of fertilizer and the quadratic term but no overall
effect of form - implying that the difference between the two forms of fertilizer lay primarily in a
difference in the curvilinearity of response - a refined model was tested with the P2 (or N
2) x
Form term replaced by Form nested within the quadratic term (eg P2Form) This has the effect
of absorbing the overall effect of form of fertilizer within the interaction term and returns the two
terms P2 and P
2 x Form (Galwey 2006)
These modeling approaches were used with total species-richness and PI species-richness as
(separate) response variables and included data for all treatments limed in 2005 but not in 1999
(ie Trs 2-12)
Results
TEMPORAL CHANGES 1999-2010
Changes over time in total species-richness (number per m2) richness and proportional
aggregate cover of positive indicator (PI) species and proportional aggregate cover of
negative indicator (NI) species at the upland and lowland meadows are represented in
Figs 1 and 2 respectively for ten of the fifteen treatments applied Repeated measures
ANOVA statistics for each of the four variables are given in Supporting Information
Tables S4 S6 S8 and S10 respectively for the upland meadow and Tables S12 S14 S16
and S18 respectively for the lowland meadow Corresponding mean values for each
treatment in each year are given in Tables S5 S7 S9 S11 S13 S15 S17 and S19
respectively Changes in individual species abundance contributing to the temporal trends
in aggregated PI and NI species described below are outlined under Supporting
Information
The upland meadow
The overall effects of both treatment and time were highly significant in the repeated
measures ANOVA for total species-richness at the upland meadow (both P=0001) but
there was no treatment x time interaction (P=0647)
After an initial decline between 1999 and 2000 species-richness remained fairly
constant for most treatments until 2003 after which there was a general increase with the
notable exception of the highest rate annual FYM treatment (Tr 5) (Fig 1a) The latter
treatment declined further between 2005 and 2007 after which it appeared to recover
slightly and averaged over all years this treatment was significantly less species-rich than
all others (Plt001 compared to Tr 14 Plt0001 the remainder) None of the remaining
10
treatments differed significantly from the limed control treatment (Tr2) averaged over all
years However the more species-rich treatments ie nil fertilizer plus lime applied in
both 1999 and 2005 (Tr 13) low rate annual and medium rate triennial FYM (Tr 3 and 7
respectively) and the inorganic equivalent to the latter (Tr 11) were all more species-rich
averaged over all years (Plt005) than annual FYM at the medium rate under either
liming regime (ie Trs 4 and 14 the former representing a continuation of past
management)
The effect of the high rate annual FYM treatment (Tr 5) was largely responsible for
several main effects within the repeated measures ANOVA of treatments within the Form
x Rate x Frequency (FRF) factorial series namely overall effects (averaged over all
years) of rate (mediumgthigh P=0002) and frequency (triennialgtannual P=0001) of
application and a not quite significant effect of form of fertilizer (FYMltinorganic
P=0057) Both the form x rate and the form x frequency interactions were significant
(both Plt005) high rate FYM was significantly less species-rich than all other
combinations averaged over annual and three yearly treatments and all years (Plt0001
compared to medium rate FYM Plt001 compared to both medium and high rate
inorganic fertilizer) and annual FYM was less species-rich than three yearly FYM and
than inorganic fertilizer either annually or triennial (all Plt0001) averaged over the two
application rates and all years The rate x frequency interaction was not significant
(P=0105) but there was a marginally non-significant three-way interaction between
form rate and frequency (P=0070) with a low mean for high rate annual FYM (Tr 5
211 species m-2
) contrasting markedly with all other treatments within the series
particularly with low rate triennial FYM (Tr 6 267 species m-2
)
As with the full treatment ANOVA the overall time factor was highly significant
(Plt0001) but only the effect of application frequency varied significantly with time
(interaction Plt005) Species-richness declined slightly with annual treatments overall
and increased slightly with triennial treatments such that differences between these were
significant in all years from 2005 onwards (Plt001 for 2006-2009 Plt005 in 2005 and
2010)
Both the richness and aggregate cover of PI species (PI) increased generally with time
(Fig 1b and 1c) although in both cases there was a progressive separation between
species The temporal trend was particularly marked for PI although the time factor
was highly significant (Plt0001) in both ANOVAs The overall treatment effect was
marginally non-significant for PI richness (P=0069) with no indication of a time x
treatment interaction (P=0703) whereas for PI there was both a significant treatment
effect (P=0002) and a marginally non-significant treatment x time interaction (P=0075)
Mean PI was significantly lower averaged over all years for annual FYM treatments at
both 12 and 24 t ha-1
(ie Trs 4 14 and 5) compared to several other treatments notably
nil fertilizer treatments limed either in 2005 only (Tr 2 limed control) or in both 1999
and 2005 (Tr 13) (all differences Plt0001 except Tr 2 vs Tr 4 Plt001) and triennial
inorganic treatments (Trs 11 and 12 Plt001 except for Tr 4 vs Tr 11 Plt005) Within
the FRF series ANOVA the increase in PI over time was greater with triennial
treatments compared to annual averaged over fertilizer forms and rates of application
(time x frequency interaction P=0016) with a marked overall difference due to
application frequency averaged over forms rates and all years (P=0005)
11
PI species-richness showed a similar overall effect of application frequency within the
FRF series (triennialgtannual P=0013) and also a significant overall effect of rate of
application (medium rategthigh rate P=0022) The overall effect of form of fertilizer was
marginally non-significant (FYMltinorganic P=0059) and there was no significant
interaction among any of the three factors averaged over all years nor any interaction
with time
16
18
20
22
24
26
28
30
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Specie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime in Year 1+ ann FYM (Tr 14) Nil fert + Lime in Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr1)
a Species richness (total)
3
4
5
6
7
8
9
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
70
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
0
5
10
15
20
25
30
35
40
45
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
Fig 1 Changes 1999-2010 in vegetation composition an upland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control
The mean number of negative indicator (NI) species per m2 averaged over all treatments
varied significantly over time at the upland meadow (P=0002 data not shown) though
with no consistent trend - it was lowest overall in 2002 (499 per m2) and highest in both
1999 and 2008 (548 per m2) The treatment x time interaction was not significant
(P=0469) but the overall treatment effect was highly significant (Plt0001) mean
number of NI species per m2 was higher than the limed control (Tr 1 453 per m
2) with
all fertilizer treatments except Treatments 11 (triennial inorganic fertilizer at the lower
rate) and 13 (nil fertilizer limed in both 1999 and 2005) (Plt0001 for all except Trs 5
and 6 for which Plt001) The highest mean NI m-2
value was for high rate inorganic
12
fertilizer (Tr 10 565 per m2) which differed significantly (Plt005) from its FYM
counterpart Treatment 5 There was however no consistent difference between FYM
treatments and their inorganic counterparts and no overall effect of form of fertilizer
within the FRF series (P=0745) There was a significant form x rate interaction within
this ANOVA (P=0032) but only the difference between the high rate and lower rate
inorganic means was significant (high rategtlower rate Plt001)
The aggregate contribution to vegetation cover of negative indicator species (NI)
showed a very marked divergence between treatments over time (Fig 1d) with NI
either changing little or declining under most treatments again with the notable exception
of high rate annual FYM (Tr 5) under which NI increased over time Time treatment
and the treatment x time interaction were all highly significant (Plt0001) The
predominant temporal trend was downward (from 25 of live cover in 1999 to 14 in
2010 averaged over all treatments) but NI for Treatment 5 had significantly increased
compared to 1999 in 2002 2003 (Plt005) 2005 (Plt0001) 2008 (Plt0001) and 2009
(Plt001) but not in 2010 (Fig 1d) The most marked declines in NI occurred on the
limed control (Tr 2) nil fertilizer plus lime in both 1999 and 2005 (Tr 13) and the lower
rate triennial inorganic fertilizer treatments (Tr 11) differences compared to both 1999
and 2002 were highly significant (Plt0001) in most subsequent years for these
treatments A smaller decline also occurred after 2002 on the treatment equivalent to past
inputs (ie12 t ha-1
year-1
annual FYM Tr 4) In most years this treatment differed little
from the equivalent FYM treatment limed in both 1999 and 2005 (Tr 14)
These trends were reflected in highly significant overall effects on NI within the FRF
series of form (FYMgtinorganic) rate (mediumlthigh) and frequency (triennialltannual)
(all effects Plt0001) Time x rate and time x frequency interactions were both significant
(P=0019 and Plt0001 respectively) with a marginally non-significant rate x frequency
interaction averaged over all years (P=0054) NI declined over time under both
application rates averaged over forms and frequencies of application but the decline was
slower with high rate treatments compared to lower rate Difference between rates were
significant in all years from 2002 onwards (Plt005 in 2002 2006 and 2007 Plt0001 in
other years) Overall differences due to frequency of application were even more marked
Under annual application NI declined initially from 2003-2006 compared to 1999 but
subsequently recovered and did not differ from 1999 levels from 2007 onwards whilst
under triennial application differences were highly significant (Plt0001) compared to
1999 from 2003 onwards with significant differences between years during this same
period (Plt005 in 2003 and 2006 Plt0001 in remaining years)
Repeated-measures ANOVAs within the LFF treatment series at the upland meadow
showed no overall effect of liming regime on temporal trends for any of the above
variables nor any interaction of lime with either time or FYM frequency - note that FYM
was applied at the intermediate rate (12 t ha-1
) in all FYM treatments within this series
The overall effect of FYM frequency was significant for both PI (P=0003) and the
number of NI species m-2
(P=0001) PI was highest with nil FYM and lowest with
annual FYM with triennial intermediate and differing significantly (Plt005) from the
other two means NI species m-2
did not differ between triennial and annual application
within the LFF series but both were significantly higher than nil FYM (Plt001 for
triennial Plt0001 for annual application) There was also a significant time x FYM
frequency interaction for NI within this series (P=0001) reflecting a more rapid
13
decline with nil FYM than with triennial FYM and no significant overall trend with
annual FYM when averaged over both lime treatments
12
14
16
18
20
22
24
26
28
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Spe
cie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime Year 1 + ann FYM (Tr 14) Nil fert + Lime Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr 1)
a Species richness (total)
2
3
4
5
6
7
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
0
10
20
30
40
50
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
Fig 2 Changes 1999-2010 in vegetation composition at a lowland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control treatment and those that had received lime in year 1
Treatments 1 and 2 were identical (nil input) until 2005
The lowland meadow
Both time and treatment were significant within the ANOVA for total species-richness at
the lowland meadow (Plt0001 and P=0009 respectively and in contrast to the upland
site the treatment x time interaction was significant (P=0012) Averaged over all years
high rate annual FYM treatment (Tr 5) was significantly less species-rich than all others
except Treatment 14 (annual FYM at 12 t ha-1
limed in 1999) having declined
progressively between 1999 and 2009 (Fig 2a) with most differences significant at
Plt001 Treatment 14 did not differ significantly overall compared to the corresponding
FYM treatment not limed until 2005 (Tr 4) although these two treatments showed
14
markedly different temporal trends Treatment 14 was less species-rich than others during
the first five years of the experiment but did not change significantly throughout the
whole 12 year period whilst Treatment 4 changed little between 1999 and 2006 but
declined thereafter (Plt001 and Plt0001 for 2009 and 2010 respectively compared to
2006) possibly in response to liming in 2005 However there was no significant overall
effect on total species-richness of liming strategy within the LFF series and no interaction
between liming and either time or FYM application
There were marked differences in temporal trend in species-richness between FYM
treatments and their inorganic counterparts particularly with the high rate annual
treatments (Tr 5 and Tr 10) Whereas species-richness declined more or less continually
throughout under Treatment 5 at least until 2009 (Plt001 for 2004 compared to 1999 and
for both 2009 and 2010 compared to 2004) Treatment 10 declined between 2000 and
2001 (Plt005) and then remained relatively unchanged until 2008 after which it declined
again (2010 Plt005 compared to 2008)
Other treatments within the full treatment series including untreated control (Tr 1) and
limed control (Tr 2) showed no particular temporal trend in the full repeated measures
ANOVA although both the latter treatments showed a dip in species-richness in 2004
(Plt005 compared to both 2003 and 2005)
There was a strong overall effect of form of fertilizer within the FRF series ANOVA
(FYMltinorganic P=0003) a significant effect of frequency of application
(triennialgtannual P=0027) and a not quite significant effect of application rate (high
rateltmedium rate P=0064) but no significant interaction between any of these factors
The time x application frequency interaction was highly significant (Plt0001) species-
richness remained unchanged under triennial application but under annual application
was significantly lower compared to 1999 in each year from 2004 onwards with the
exception of 2006 (Plt005 in 2004 Plt001 in 2005 and 2008 Plt0001 the remainder)
Again with the exception of 2006 differences between triennial and annual application
were significant in each year from 2005 onwards (Plt005 in 2007 Plt001 in 2005 and
2008 Plt0001 in 2009 and 2010)
Both treatment time and their interaction were significant in the full treatment ANOVA
for PI species-richness at the lowland meadow (ie time and treatment both Plt0001
time x treatment P=0006) There was a general decline in PI-richness after 2005 (Fig
2b) with PI species m-2
averaged over all treatments lower from 2007 onwards (Plt0001)
than all years up to 2005 except 2001 Although the initiation of this trend corresponded
with the liming of most treatments in 2005 a similar pattern was shown by treatments
that did not receive this lime application ie the untreated control treatment (Tr 1) the
nil fertilizer treatment limed in 1999 (Tr 13) and the corresponding treatment also
receiving triennial FYM (Tr 15) In all these cases PI-richness showed no significant
difference between years until 2005 but was significantly (Plt001) lower by 2010 than in
2005 Treatment 14 (limed in 1999 plus annual FYM) did not show this pattern but PI
species-richness had been lowest under this treatment in most years between 1999 and
2005 (Fig 2b)
As with total species-richness PI richness declined more or less progressively under
Treatment 5 until 2008 (Plt005 compared to 1999 by 2001 Plt0001 from 2006
onwards) whereas Treatment 4 did not change significantly until 2006 after which it
declined (Plt005 in 2007 compared to 2006 Plt001 thereafter) Both these treatments
15
tended be less rich in PI species than the corresponding inorganic treatment (Trs 9 and
10 respectively) in most years consistently so for Tr 4 compared to Tr 9 but these
differences were not significant in any year nor when averaged over all years There was
however a significant overall effect of form of fertilizer in the FRF series ANOVA
(FYMltinorganic P=0002) and a highly significant effect of frequency of application
(triennialgtannual Plt0001) The time x frequency of application interaction was also
highly significant (Plt0001) Under triennial application PI species per m2 increased
slightly until 2005 (Plt005 compared to 1999) and declined thereafter so that by 2009
levels were significantly lower than in 1999 (Plt001 for 2009 Plt0001 for 2010) By
contrast under annual application PI species m-2
declined progressively until 2009
following a temporary though highly significant (Plt0001) dip in 2000 so that the
difference compared to 1999 was significant in every year from 2002 onwards and highly
significant (Plt0001) from 2005 With the exception of 2003 differences between
triennial and annual application were significant from 2002 onwards and highly
significant (Plt0001) in 2005 and from 2007 to 2010
There was no significant effect other than of time within the LFF treatment series
ANOVA although there was a marginally non-significant time x FYM frequency
interaction (P=0054) This reflected a more rapid decline in PI species-richness under
annual FYM application than with either nil or triennial FYM with the latter two showing
no discernible difference
By contrast with PI species m-2
PI cover percentage (PI) showed no overall treatment
effect within the full treatment ANOVA nor a time x treatment interaction although the
overall effect of time was highly significant (Plt0001) The temporal pattern in PI (Fig
2c) also differed from that shown by PI-richness with PI averaged over all treatments
declining between 1999 and 2005 in three highly significant increments (1999-2000
2000-2002 and 2002-2005 all Plt0001) and increasing again thereafter (2005-2009 and
2009-2010 both Plt0001) There was no significant effect within either the LFF or FRF
treatment series ANOVAs although their was a slight time x frequency of application
effect within the latter (P=0081) PI was slightly higher under annual application than
triennial in each year 1999-2004 after which the reverse was true although differences
were always small
There was no overall effect of treatment on the number of NI species per m2 within the
full treatment range at the lowland meadow (data not shown) but both the overall effect
of time and the time x treatment interaction were significant (Plt0001 and P=0009
respectively) Overall NI species m-2
increased over time from 24 species m-2
in 1999 to
a peak of 42 m-2
in 2008 averaged over all treatments NI species m-2
increased on the
untreated control treatment (Tr 1) from 21 species m-2
in 1999 to 34 species m-2
in 2007
(increase Plt001) then declined to the 1999 value by 2010 The limed (in 2005) control
(Tr 2) showed a somewhat similar pattern though from a slightly higher starting level
(31 species m-2
) and with the increase to 38 species m-2
in 2008 not reaching
significance
These temporal trends in NI species m-2
were much accentuated by annual fertilizer
application particularly by FYM treatments with peak numbers of NI species reaching
52 and 47 species m-2
for Treatments 4 and 5 respectively in 2008 (both Plt0001
compared to 1999) and despite slight declines in both cases remaining significantly
higher in 2010 than in 1999 (both Plt0001) Inorganic counterparts to these treatments
16
showed similar though slightly less marked trends and there was no significant
difference between all four treatments in the peak number reached in 2008 There was no
overall effect of either form of fertilizer or rate of application within the FRF series
ANOVA and only a marginally non-significant effect of frequency of application
(triennialltannual P=0061) Liming in 1999 (Trs 13 14 and 15) also appeared to
accentuate the generally increasing trend although peak numbers in 2008 did not differ
between these treatments and either untreated or limed control in the full treatment range
ANOVA The overall effects of liming regime (1999 or 2005) were not significant within
the LFF treatment series but the faster rate of increase with liming in 1999 was reflected
in a significant time x lime interaction in this analysis (P=0017) The mean number of NI
species m-2
increased very significantly (Plt0001) between 1999 and 2000 following
liming in 1999 with the increment increasing until 2008 whilst on plots un-limed until
2005 NI species m-2
did not increase significantly until 2003 (Plt005) although the
increase compared to 1999 was highly significant (Plt0001) in 2006 2008 and 2010
Nevertheless the differences in rate of increase was at least partly due to a lower 1999
mean for the 1999-limed treatments (170 species m-2
compared to 244 species m-2
difference NS) and although in all years from 2001 onwards mean NI species m-2
was
higher for 1999-limed treatments compared to those not limed until 2005 the difference
was not significant in any year
The contribution of NI species to vegetation cover (NI) showed a very marked
separation between treatments over time at the lowland meadow (Fig 2d) starting from
much lower levels than at the upland meadow (compare with Fig 1d) There were
noticeable differences in temporal trend between annual fertilizer treatments on the one
hand and triennial and nil fertilizer treatments on the other (Fig 2d) The effects of
treatment time and the treatment x time interaction were all highly significant (Plt0001)
in the full treatment range ANOVA The most marked increase in NI occurred with
annual FYM at the high rate (Tr 5) with NI increasing rapidly between 1999 and 2001
(Plt001) and a further highly significant increment between 2004 and 2009 (Plt0001)
(Fig 2d) A similar though less marked trend occurred with annual FYM at the medium
rate (Tr 4) with significant increases between 1999 and 2005 and between 2005 and
2010 (both Plt001) NI also increased markedly with the inorganic treatment
equivalent to Treatment 5 (Tr 10) although the overall rate of increase shown by this
treatment was closer to that shown by Treatment 4 than Treatment 5 There were
significant differences between Treatments 5 and 10 in most years from 2004 onwards
(Plt001 and Plt0001 in 2009 and 2010 respectively)
These effects were reflected in a significant overall effect of form of fertilizer
(FYMgtinorganic P=0008) and highly significant (Plt0001) overall effects of both rate
(high rategtlower rate) and frequency of application (annualgttriennial) within the FRF
series ANOVA and a significant form x rate interaction (P=0043) Mean NI was
highest for high rate annual FYM (Plt0001 compared to all three remaining means) and
lowest for lower rate inorganic (Plt005 compared to high rate inorganic NS compared
to lower rate FYM) Only frequency of application interacted significantly with time
(Plt0001) NI increased moderately under triennial application until 2008 (Plt005 in
2005 compared to 1999 Plt0001 each year 2006-2008) followed by a decline (Plt001
2010 compared to 2008) but with mean NI in 2010 still higher than 1999 (Plt005)
The increasing trend was very much more marked with annual fertilizer application for
17
which there were highly significant (Plt0001) increments between 1999 and 2002 and
between 2002 and 2008 but with no significant increase thereafter Differences between
annual and triennial treatment means were highly significant (Plt0001) from 2003
onwards
There was no significant change in NI over time either under the untreated control
(Tr 1) or the nil fertilizer (Tr 2) treatments in the full treatment ANOVA NI increased
until 2005 on plots limed in 1999 without fertilizer Tr 13 (Plt001 compared to 1999)
and declined slightly but not significantly thereafter Trends were similar with the
treatment limed in 1999 and receiving triennial FYM (Tr 15 ndash not illustrated in Fig 2)
although NI was higher with this treatment than with Tr 13 (Plt005) in both 2005 and
2008 By contrast applying the same rate of FYM annually after liming in 1999 (Tr 14)
caused a more rapid increase with levels by 2005 higher than either Tr 13 (Plt001) or
Tr 4 (Plt005) ndash note that the latter was equivalent to Tr 14 but without lime until 2005
NI declined after 2006 under Tr 14 but continued to increase with Tr 4 with the
difference between these two treatments reversed by 2010 (Plt005)
All main effects and interactions were significant in the LFF treatment series ANOVA
for NI except for lime x FYM frequency and the lime x FYM x time interactions Plots
limed in 1999 supported higher NI levels than those limed in 2005 averaged over the
three FYM frequencies and all years (P=0042) NI increased more rapidly after liming
in 1999 than where liming was delayed until 2005 (P=0033 for time x lime interaction)
with the overall difference due to liming in 1999 highly significant (Plt0001) by 2005
NI declined thereafter on the 1999-limed treatments whilst continuing to increase
following liming in 2005 of previously un-limed treatments both peaking in 2008 at a
very similar level Averaged over lime treatments NI increased much more rapidly
under annual FYM application than under either triennial or nil FYM (P=0002 for the
time x FYM frequency interaction) a highly significant increase had occurred by 2002
with annual FYM (Plt0001 compared to 1999) but increases of similar magnitude were
not reached until 2005 with triennial application and not until 2007 with nil FYM NI
subsequently declined under the latter two FYM regimes but not with annual FYM so
that by 2010 NI was significantly higher with annual FYM than with either triennial or
nil FYM (Plt0001) but with only a small non-significant difference between the latter
two treatments
TREATMENT EFFECTS AFTER 12 YEARS OF APPLICATION
The effects of all treatments on the five key variables at each site in 2010 are shown in
Table S20 with treatments differing significantly (Plt005) from the limed control
treatment (Tr 2) highlighted for each variable Treatment effects in 2010 are not
described in detail here but the modeled responses of total species-richness and of
richness of PI species in 2010 to the mean amounts of N and P applied per year averaged
over 1999-2010 are described below
Relationships with the amounts of fertilizer applied
The effect of neither rate nor frequency of application was significant in any of the
REML models for either total species-richness or PI species-richness at either site when
the amount of N or P applied ha-1
year-1
was included The effect of form of fertilizer was
still significant in most models however either as a main effect or in interaction with the
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
References
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Anon (2010) Fertilizer Manual (RB 209) 8th Edition Her Majestyrsquos Stationery Office London
Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
Kluwer Academic Publishers Dordrecht
Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
restoration of species-rich meadows in the Netherlands Biological Conservation 62 59-65
Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
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Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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and Fertility of Soils 22 261-264
Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
Hopkins JJ (1999) The extent of semi-natural grassland communities in lowland England
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Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
phosphorous vs nitrogen enrichment in plant species loss across North-west Europen
grasslands Journal of Applied Ecology 48 1155-1163
Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
and losses following application of manures to arable land MANNER Soil Use and
Management 15 137-143
Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
(2002a) Plant species-richness functional type and soil properties in grasslands and allied
vegetation in English Environmentally Sensitive Areas Grass Forage Science 57 82-92
Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
2002b) Association between lowland grassland plant communities and soil properties
Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
activity in botanically diverse haymeadows effects of litter type and fertilizer additions Soil
Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
8
blocks was accounted for by treating replicate as a blocking factor The first model included all
the treatments except Treatment 1 at the upland meadow whilst the other two models used
balanced factorial subsets (series) of treatments (see Table 2) the Form x Rate x Frequency
(FRF) series and the Lime x FYM frequency (LFF) series
The FRF series tested the effects of form of fertilizer (ie FYM or inorganic) the rate at which
fertilizers were applied (12 or 24 t FYM ha-1
and inorganic treatments corresponding to these)
the frequency at which treatments were applied (ie annual or triennially) and all two- and three-
way interactions between these factors
The LFF series tested the effects of liming regime ie lime in 1999 (and 2005 at the upland site)
versus lime in 2005 only the frequency of FYM application (ie nil annual or triennially) and the
Lime x FYM frequency interaction All FYM treatments within this series were applied at 12 t ha-
1 At both sites the LFF series consisted of Treatments 2 4 7 13 14 and 15 (see Table 2) The
liming comparison therefore differed slightly between the two sites since liming was repeated in
2005 on plots previously limed in 1999 at the upland meadow but not repeated at the lowland
one
In both the LFF and the FRF models for each variable the first stratum of the ANOVA
consisted of the effects of all factors and interactions averaged over time with the same factors
and interactions repeated in the second stratum with time as an additional factor
In all repeated measures ANOVAs to account for the repeated measures aspect all degrees of
freedom in the Year x Treatment stratum were adjusted to account for departure from
homogeneity of the population covariance matrix using ε-coefficients calculated by the
Greenhouse-Geisser method (Genstat V Committee 1997)
In most years including 2010 all species were recorded individually but at both sites
bryophytes were recorded only as a single group in some years This had only a small influence
on the data since there were seldom more than two bryophyte species present in a quadrat and
more often less Nevertheless for consistency between years bryophytes were treated as a single
group in all years when calculating total species-richness for data for repeated measures
ANOVAs
In addition to the above analyses 2010 data for the key variables were tested alone for treatment
effects by ANOVA and by analysis of covariance (ANCOVA) the latter using 1999 data as the
covariate (Genstat 5 Committee 1997) All three models as described above were tested The
ANCOVAs for the upland meadow excluded Treatment 1 (untreated control plots established in
2005) Where an ANCOVA showed significant treatment covariance with 1999 data the adjusted
results were used In other cases the unadjusted analyses were used Individual bryophyte species
were included in the calculations of species-richness at both sites in these analyses
In all analyses in order to satisfy the conditions for ANOVA (normality of residuals and
homogeneity of residual variation) all percentage data were transformed before analysis by
arcsine(radicp) where p is the percentage value expressed as a proportion All results quoted for
percentage data refer to analyses of arcsine-square root transformed data
Mixed modeling
Mixed model analysis (Residual Estimated Maximum Likelihood ndash REML Genstat V
Committee 1997) was used to identify any effect of form rate or frequency of application on
vegetation composition at each site in 2010 not simply attributable to the total (or mean per year)
amount of fertilizer applied over the study period A variable representing this amount was
included as a continuous explanatory variable in each analysis along with other fixed factors
form rate and frequency of application (Block was included as a random factor) Since the total
(or mean) amounts of N P and K applied over the whole period were completely correlated
across treatments at each site it was not feasible to include all three elements as separate terms in
the model Nor was it feasible to simply use a single notional variable for fertilizer amount (eg t
9
FYM ha-1
year-1
or inorganic equivalent) since there was lack of equivalence of N and P supply
(but not K) between individual FYM treatments and their corresponding inorganic treatments For
each dependent variable tested two separate models were therefore developed using either N or
P ha-1
year-1
as the continuous variable In each case the presence of any non-linearity in response
was tested by including a quadratic term (ie N2 or P
2) or where more appropriate by logn-
transformation of the explanatory variable
For each analysis an initial model was constructed that included all five main effects and all
interactions The importance of each term was evaluated initially by a χ2 test on the Wald statistic
(Genstat V Committee 1997) The number and order of terms in the model were varied but with
N or P amount always included until a model was developed in which each term was significant
The model was then refined by testing the contribution of each term to the model by the more
conservative deviance test ie a χ2 test on the difference in deviance between the model
containing the term and a sub-model from which it was dropped (Welham amp Thompson 1997)
Where there was an interaction between form of fertilizer and the quadratic term but no overall
effect of form - implying that the difference between the two forms of fertilizer lay primarily in a
difference in the curvilinearity of response - a refined model was tested with the P2 (or N
2) x
Form term replaced by Form nested within the quadratic term (eg P2Form) This has the effect
of absorbing the overall effect of form of fertilizer within the interaction term and returns the two
terms P2 and P
2 x Form (Galwey 2006)
These modeling approaches were used with total species-richness and PI species-richness as
(separate) response variables and included data for all treatments limed in 2005 but not in 1999
(ie Trs 2-12)
Results
TEMPORAL CHANGES 1999-2010
Changes over time in total species-richness (number per m2) richness and proportional
aggregate cover of positive indicator (PI) species and proportional aggregate cover of
negative indicator (NI) species at the upland and lowland meadows are represented in
Figs 1 and 2 respectively for ten of the fifteen treatments applied Repeated measures
ANOVA statistics for each of the four variables are given in Supporting Information
Tables S4 S6 S8 and S10 respectively for the upland meadow and Tables S12 S14 S16
and S18 respectively for the lowland meadow Corresponding mean values for each
treatment in each year are given in Tables S5 S7 S9 S11 S13 S15 S17 and S19
respectively Changes in individual species abundance contributing to the temporal trends
in aggregated PI and NI species described below are outlined under Supporting
Information
The upland meadow
The overall effects of both treatment and time were highly significant in the repeated
measures ANOVA for total species-richness at the upland meadow (both P=0001) but
there was no treatment x time interaction (P=0647)
After an initial decline between 1999 and 2000 species-richness remained fairly
constant for most treatments until 2003 after which there was a general increase with the
notable exception of the highest rate annual FYM treatment (Tr 5) (Fig 1a) The latter
treatment declined further between 2005 and 2007 after which it appeared to recover
slightly and averaged over all years this treatment was significantly less species-rich than
all others (Plt001 compared to Tr 14 Plt0001 the remainder) None of the remaining
10
treatments differed significantly from the limed control treatment (Tr2) averaged over all
years However the more species-rich treatments ie nil fertilizer plus lime applied in
both 1999 and 2005 (Tr 13) low rate annual and medium rate triennial FYM (Tr 3 and 7
respectively) and the inorganic equivalent to the latter (Tr 11) were all more species-rich
averaged over all years (Plt005) than annual FYM at the medium rate under either
liming regime (ie Trs 4 and 14 the former representing a continuation of past
management)
The effect of the high rate annual FYM treatment (Tr 5) was largely responsible for
several main effects within the repeated measures ANOVA of treatments within the Form
x Rate x Frequency (FRF) factorial series namely overall effects (averaged over all
years) of rate (mediumgthigh P=0002) and frequency (triennialgtannual P=0001) of
application and a not quite significant effect of form of fertilizer (FYMltinorganic
P=0057) Both the form x rate and the form x frequency interactions were significant
(both Plt005) high rate FYM was significantly less species-rich than all other
combinations averaged over annual and three yearly treatments and all years (Plt0001
compared to medium rate FYM Plt001 compared to both medium and high rate
inorganic fertilizer) and annual FYM was less species-rich than three yearly FYM and
than inorganic fertilizer either annually or triennial (all Plt0001) averaged over the two
application rates and all years The rate x frequency interaction was not significant
(P=0105) but there was a marginally non-significant three-way interaction between
form rate and frequency (P=0070) with a low mean for high rate annual FYM (Tr 5
211 species m-2
) contrasting markedly with all other treatments within the series
particularly with low rate triennial FYM (Tr 6 267 species m-2
)
As with the full treatment ANOVA the overall time factor was highly significant
(Plt0001) but only the effect of application frequency varied significantly with time
(interaction Plt005) Species-richness declined slightly with annual treatments overall
and increased slightly with triennial treatments such that differences between these were
significant in all years from 2005 onwards (Plt001 for 2006-2009 Plt005 in 2005 and
2010)
Both the richness and aggregate cover of PI species (PI) increased generally with time
(Fig 1b and 1c) although in both cases there was a progressive separation between
species The temporal trend was particularly marked for PI although the time factor
was highly significant (Plt0001) in both ANOVAs The overall treatment effect was
marginally non-significant for PI richness (P=0069) with no indication of a time x
treatment interaction (P=0703) whereas for PI there was both a significant treatment
effect (P=0002) and a marginally non-significant treatment x time interaction (P=0075)
Mean PI was significantly lower averaged over all years for annual FYM treatments at
both 12 and 24 t ha-1
(ie Trs 4 14 and 5) compared to several other treatments notably
nil fertilizer treatments limed either in 2005 only (Tr 2 limed control) or in both 1999
and 2005 (Tr 13) (all differences Plt0001 except Tr 2 vs Tr 4 Plt001) and triennial
inorganic treatments (Trs 11 and 12 Plt001 except for Tr 4 vs Tr 11 Plt005) Within
the FRF series ANOVA the increase in PI over time was greater with triennial
treatments compared to annual averaged over fertilizer forms and rates of application
(time x frequency interaction P=0016) with a marked overall difference due to
application frequency averaged over forms rates and all years (P=0005)
11
PI species-richness showed a similar overall effect of application frequency within the
FRF series (triennialgtannual P=0013) and also a significant overall effect of rate of
application (medium rategthigh rate P=0022) The overall effect of form of fertilizer was
marginally non-significant (FYMltinorganic P=0059) and there was no significant
interaction among any of the three factors averaged over all years nor any interaction
with time
16
18
20
22
24
26
28
30
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Specie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime in Year 1+ ann FYM (Tr 14) Nil fert + Lime in Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr1)
a Species richness (total)
3
4
5
6
7
8
9
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
70
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
0
5
10
15
20
25
30
35
40
45
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
Fig 1 Changes 1999-2010 in vegetation composition an upland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control
The mean number of negative indicator (NI) species per m2 averaged over all treatments
varied significantly over time at the upland meadow (P=0002 data not shown) though
with no consistent trend - it was lowest overall in 2002 (499 per m2) and highest in both
1999 and 2008 (548 per m2) The treatment x time interaction was not significant
(P=0469) but the overall treatment effect was highly significant (Plt0001) mean
number of NI species per m2 was higher than the limed control (Tr 1 453 per m
2) with
all fertilizer treatments except Treatments 11 (triennial inorganic fertilizer at the lower
rate) and 13 (nil fertilizer limed in both 1999 and 2005) (Plt0001 for all except Trs 5
and 6 for which Plt001) The highest mean NI m-2
value was for high rate inorganic
12
fertilizer (Tr 10 565 per m2) which differed significantly (Plt005) from its FYM
counterpart Treatment 5 There was however no consistent difference between FYM
treatments and their inorganic counterparts and no overall effect of form of fertilizer
within the FRF series (P=0745) There was a significant form x rate interaction within
this ANOVA (P=0032) but only the difference between the high rate and lower rate
inorganic means was significant (high rategtlower rate Plt001)
The aggregate contribution to vegetation cover of negative indicator species (NI)
showed a very marked divergence between treatments over time (Fig 1d) with NI
either changing little or declining under most treatments again with the notable exception
of high rate annual FYM (Tr 5) under which NI increased over time Time treatment
and the treatment x time interaction were all highly significant (Plt0001) The
predominant temporal trend was downward (from 25 of live cover in 1999 to 14 in
2010 averaged over all treatments) but NI for Treatment 5 had significantly increased
compared to 1999 in 2002 2003 (Plt005) 2005 (Plt0001) 2008 (Plt0001) and 2009
(Plt001) but not in 2010 (Fig 1d) The most marked declines in NI occurred on the
limed control (Tr 2) nil fertilizer plus lime in both 1999 and 2005 (Tr 13) and the lower
rate triennial inorganic fertilizer treatments (Tr 11) differences compared to both 1999
and 2002 were highly significant (Plt0001) in most subsequent years for these
treatments A smaller decline also occurred after 2002 on the treatment equivalent to past
inputs (ie12 t ha-1
year-1
annual FYM Tr 4) In most years this treatment differed little
from the equivalent FYM treatment limed in both 1999 and 2005 (Tr 14)
These trends were reflected in highly significant overall effects on NI within the FRF
series of form (FYMgtinorganic) rate (mediumlthigh) and frequency (triennialltannual)
(all effects Plt0001) Time x rate and time x frequency interactions were both significant
(P=0019 and Plt0001 respectively) with a marginally non-significant rate x frequency
interaction averaged over all years (P=0054) NI declined over time under both
application rates averaged over forms and frequencies of application but the decline was
slower with high rate treatments compared to lower rate Difference between rates were
significant in all years from 2002 onwards (Plt005 in 2002 2006 and 2007 Plt0001 in
other years) Overall differences due to frequency of application were even more marked
Under annual application NI declined initially from 2003-2006 compared to 1999 but
subsequently recovered and did not differ from 1999 levels from 2007 onwards whilst
under triennial application differences were highly significant (Plt0001) compared to
1999 from 2003 onwards with significant differences between years during this same
period (Plt005 in 2003 and 2006 Plt0001 in remaining years)
Repeated-measures ANOVAs within the LFF treatment series at the upland meadow
showed no overall effect of liming regime on temporal trends for any of the above
variables nor any interaction of lime with either time or FYM frequency - note that FYM
was applied at the intermediate rate (12 t ha-1
) in all FYM treatments within this series
The overall effect of FYM frequency was significant for both PI (P=0003) and the
number of NI species m-2
(P=0001) PI was highest with nil FYM and lowest with
annual FYM with triennial intermediate and differing significantly (Plt005) from the
other two means NI species m-2
did not differ between triennial and annual application
within the LFF series but both were significantly higher than nil FYM (Plt001 for
triennial Plt0001 for annual application) There was also a significant time x FYM
frequency interaction for NI within this series (P=0001) reflecting a more rapid
13
decline with nil FYM than with triennial FYM and no significant overall trend with
annual FYM when averaged over both lime treatments
12
14
16
18
20
22
24
26
28
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Spe
cie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime Year 1 + ann FYM (Tr 14) Nil fert + Lime Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr 1)
a Species richness (total)
2
3
4
5
6
7
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
0
10
20
30
40
50
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
Fig 2 Changes 1999-2010 in vegetation composition at a lowland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control treatment and those that had received lime in year 1
Treatments 1 and 2 were identical (nil input) until 2005
The lowland meadow
Both time and treatment were significant within the ANOVA for total species-richness at
the lowland meadow (Plt0001 and P=0009 respectively and in contrast to the upland
site the treatment x time interaction was significant (P=0012) Averaged over all years
high rate annual FYM treatment (Tr 5) was significantly less species-rich than all others
except Treatment 14 (annual FYM at 12 t ha-1
limed in 1999) having declined
progressively between 1999 and 2009 (Fig 2a) with most differences significant at
Plt001 Treatment 14 did not differ significantly overall compared to the corresponding
FYM treatment not limed until 2005 (Tr 4) although these two treatments showed
14
markedly different temporal trends Treatment 14 was less species-rich than others during
the first five years of the experiment but did not change significantly throughout the
whole 12 year period whilst Treatment 4 changed little between 1999 and 2006 but
declined thereafter (Plt001 and Plt0001 for 2009 and 2010 respectively compared to
2006) possibly in response to liming in 2005 However there was no significant overall
effect on total species-richness of liming strategy within the LFF series and no interaction
between liming and either time or FYM application
There were marked differences in temporal trend in species-richness between FYM
treatments and their inorganic counterparts particularly with the high rate annual
treatments (Tr 5 and Tr 10) Whereas species-richness declined more or less continually
throughout under Treatment 5 at least until 2009 (Plt001 for 2004 compared to 1999 and
for both 2009 and 2010 compared to 2004) Treatment 10 declined between 2000 and
2001 (Plt005) and then remained relatively unchanged until 2008 after which it declined
again (2010 Plt005 compared to 2008)
Other treatments within the full treatment series including untreated control (Tr 1) and
limed control (Tr 2) showed no particular temporal trend in the full repeated measures
ANOVA although both the latter treatments showed a dip in species-richness in 2004
(Plt005 compared to both 2003 and 2005)
There was a strong overall effect of form of fertilizer within the FRF series ANOVA
(FYMltinorganic P=0003) a significant effect of frequency of application
(triennialgtannual P=0027) and a not quite significant effect of application rate (high
rateltmedium rate P=0064) but no significant interaction between any of these factors
The time x application frequency interaction was highly significant (Plt0001) species-
richness remained unchanged under triennial application but under annual application
was significantly lower compared to 1999 in each year from 2004 onwards with the
exception of 2006 (Plt005 in 2004 Plt001 in 2005 and 2008 Plt0001 the remainder)
Again with the exception of 2006 differences between triennial and annual application
were significant in each year from 2005 onwards (Plt005 in 2007 Plt001 in 2005 and
2008 Plt0001 in 2009 and 2010)
Both treatment time and their interaction were significant in the full treatment ANOVA
for PI species-richness at the lowland meadow (ie time and treatment both Plt0001
time x treatment P=0006) There was a general decline in PI-richness after 2005 (Fig
2b) with PI species m-2
averaged over all treatments lower from 2007 onwards (Plt0001)
than all years up to 2005 except 2001 Although the initiation of this trend corresponded
with the liming of most treatments in 2005 a similar pattern was shown by treatments
that did not receive this lime application ie the untreated control treatment (Tr 1) the
nil fertilizer treatment limed in 1999 (Tr 13) and the corresponding treatment also
receiving triennial FYM (Tr 15) In all these cases PI-richness showed no significant
difference between years until 2005 but was significantly (Plt001) lower by 2010 than in
2005 Treatment 14 (limed in 1999 plus annual FYM) did not show this pattern but PI
species-richness had been lowest under this treatment in most years between 1999 and
2005 (Fig 2b)
As with total species-richness PI richness declined more or less progressively under
Treatment 5 until 2008 (Plt005 compared to 1999 by 2001 Plt0001 from 2006
onwards) whereas Treatment 4 did not change significantly until 2006 after which it
declined (Plt005 in 2007 compared to 2006 Plt001 thereafter) Both these treatments
15
tended be less rich in PI species than the corresponding inorganic treatment (Trs 9 and
10 respectively) in most years consistently so for Tr 4 compared to Tr 9 but these
differences were not significant in any year nor when averaged over all years There was
however a significant overall effect of form of fertilizer in the FRF series ANOVA
(FYMltinorganic P=0002) and a highly significant effect of frequency of application
(triennialgtannual Plt0001) The time x frequency of application interaction was also
highly significant (Plt0001) Under triennial application PI species per m2 increased
slightly until 2005 (Plt005 compared to 1999) and declined thereafter so that by 2009
levels were significantly lower than in 1999 (Plt001 for 2009 Plt0001 for 2010) By
contrast under annual application PI species m-2
declined progressively until 2009
following a temporary though highly significant (Plt0001) dip in 2000 so that the
difference compared to 1999 was significant in every year from 2002 onwards and highly
significant (Plt0001) from 2005 With the exception of 2003 differences between
triennial and annual application were significant from 2002 onwards and highly
significant (Plt0001) in 2005 and from 2007 to 2010
There was no significant effect other than of time within the LFF treatment series
ANOVA although there was a marginally non-significant time x FYM frequency
interaction (P=0054) This reflected a more rapid decline in PI species-richness under
annual FYM application than with either nil or triennial FYM with the latter two showing
no discernible difference
By contrast with PI species m-2
PI cover percentage (PI) showed no overall treatment
effect within the full treatment ANOVA nor a time x treatment interaction although the
overall effect of time was highly significant (Plt0001) The temporal pattern in PI (Fig
2c) also differed from that shown by PI-richness with PI averaged over all treatments
declining between 1999 and 2005 in three highly significant increments (1999-2000
2000-2002 and 2002-2005 all Plt0001) and increasing again thereafter (2005-2009 and
2009-2010 both Plt0001) There was no significant effect within either the LFF or FRF
treatment series ANOVAs although their was a slight time x frequency of application
effect within the latter (P=0081) PI was slightly higher under annual application than
triennial in each year 1999-2004 after which the reverse was true although differences
were always small
There was no overall effect of treatment on the number of NI species per m2 within the
full treatment range at the lowland meadow (data not shown) but both the overall effect
of time and the time x treatment interaction were significant (Plt0001 and P=0009
respectively) Overall NI species m-2
increased over time from 24 species m-2
in 1999 to
a peak of 42 m-2
in 2008 averaged over all treatments NI species m-2
increased on the
untreated control treatment (Tr 1) from 21 species m-2
in 1999 to 34 species m-2
in 2007
(increase Plt001) then declined to the 1999 value by 2010 The limed (in 2005) control
(Tr 2) showed a somewhat similar pattern though from a slightly higher starting level
(31 species m-2
) and with the increase to 38 species m-2
in 2008 not reaching
significance
These temporal trends in NI species m-2
were much accentuated by annual fertilizer
application particularly by FYM treatments with peak numbers of NI species reaching
52 and 47 species m-2
for Treatments 4 and 5 respectively in 2008 (both Plt0001
compared to 1999) and despite slight declines in both cases remaining significantly
higher in 2010 than in 1999 (both Plt0001) Inorganic counterparts to these treatments
16
showed similar though slightly less marked trends and there was no significant
difference between all four treatments in the peak number reached in 2008 There was no
overall effect of either form of fertilizer or rate of application within the FRF series
ANOVA and only a marginally non-significant effect of frequency of application
(triennialltannual P=0061) Liming in 1999 (Trs 13 14 and 15) also appeared to
accentuate the generally increasing trend although peak numbers in 2008 did not differ
between these treatments and either untreated or limed control in the full treatment range
ANOVA The overall effects of liming regime (1999 or 2005) were not significant within
the LFF treatment series but the faster rate of increase with liming in 1999 was reflected
in a significant time x lime interaction in this analysis (P=0017) The mean number of NI
species m-2
increased very significantly (Plt0001) between 1999 and 2000 following
liming in 1999 with the increment increasing until 2008 whilst on plots un-limed until
2005 NI species m-2
did not increase significantly until 2003 (Plt005) although the
increase compared to 1999 was highly significant (Plt0001) in 2006 2008 and 2010
Nevertheless the differences in rate of increase was at least partly due to a lower 1999
mean for the 1999-limed treatments (170 species m-2
compared to 244 species m-2
difference NS) and although in all years from 2001 onwards mean NI species m-2
was
higher for 1999-limed treatments compared to those not limed until 2005 the difference
was not significant in any year
The contribution of NI species to vegetation cover (NI) showed a very marked
separation between treatments over time at the lowland meadow (Fig 2d) starting from
much lower levels than at the upland meadow (compare with Fig 1d) There were
noticeable differences in temporal trend between annual fertilizer treatments on the one
hand and triennial and nil fertilizer treatments on the other (Fig 2d) The effects of
treatment time and the treatment x time interaction were all highly significant (Plt0001)
in the full treatment range ANOVA The most marked increase in NI occurred with
annual FYM at the high rate (Tr 5) with NI increasing rapidly between 1999 and 2001
(Plt001) and a further highly significant increment between 2004 and 2009 (Plt0001)
(Fig 2d) A similar though less marked trend occurred with annual FYM at the medium
rate (Tr 4) with significant increases between 1999 and 2005 and between 2005 and
2010 (both Plt001) NI also increased markedly with the inorganic treatment
equivalent to Treatment 5 (Tr 10) although the overall rate of increase shown by this
treatment was closer to that shown by Treatment 4 than Treatment 5 There were
significant differences between Treatments 5 and 10 in most years from 2004 onwards
(Plt001 and Plt0001 in 2009 and 2010 respectively)
These effects were reflected in a significant overall effect of form of fertilizer
(FYMgtinorganic P=0008) and highly significant (Plt0001) overall effects of both rate
(high rategtlower rate) and frequency of application (annualgttriennial) within the FRF
series ANOVA and a significant form x rate interaction (P=0043) Mean NI was
highest for high rate annual FYM (Plt0001 compared to all three remaining means) and
lowest for lower rate inorganic (Plt005 compared to high rate inorganic NS compared
to lower rate FYM) Only frequency of application interacted significantly with time
(Plt0001) NI increased moderately under triennial application until 2008 (Plt005 in
2005 compared to 1999 Plt0001 each year 2006-2008) followed by a decline (Plt001
2010 compared to 2008) but with mean NI in 2010 still higher than 1999 (Plt005)
The increasing trend was very much more marked with annual fertilizer application for
17
which there were highly significant (Plt0001) increments between 1999 and 2002 and
between 2002 and 2008 but with no significant increase thereafter Differences between
annual and triennial treatment means were highly significant (Plt0001) from 2003
onwards
There was no significant change in NI over time either under the untreated control
(Tr 1) or the nil fertilizer (Tr 2) treatments in the full treatment ANOVA NI increased
until 2005 on plots limed in 1999 without fertilizer Tr 13 (Plt001 compared to 1999)
and declined slightly but not significantly thereafter Trends were similar with the
treatment limed in 1999 and receiving triennial FYM (Tr 15 ndash not illustrated in Fig 2)
although NI was higher with this treatment than with Tr 13 (Plt005) in both 2005 and
2008 By contrast applying the same rate of FYM annually after liming in 1999 (Tr 14)
caused a more rapid increase with levels by 2005 higher than either Tr 13 (Plt001) or
Tr 4 (Plt005) ndash note that the latter was equivalent to Tr 14 but without lime until 2005
NI declined after 2006 under Tr 14 but continued to increase with Tr 4 with the
difference between these two treatments reversed by 2010 (Plt005)
All main effects and interactions were significant in the LFF treatment series ANOVA
for NI except for lime x FYM frequency and the lime x FYM x time interactions Plots
limed in 1999 supported higher NI levels than those limed in 2005 averaged over the
three FYM frequencies and all years (P=0042) NI increased more rapidly after liming
in 1999 than where liming was delayed until 2005 (P=0033 for time x lime interaction)
with the overall difference due to liming in 1999 highly significant (Plt0001) by 2005
NI declined thereafter on the 1999-limed treatments whilst continuing to increase
following liming in 2005 of previously un-limed treatments both peaking in 2008 at a
very similar level Averaged over lime treatments NI increased much more rapidly
under annual FYM application than under either triennial or nil FYM (P=0002 for the
time x FYM frequency interaction) a highly significant increase had occurred by 2002
with annual FYM (Plt0001 compared to 1999) but increases of similar magnitude were
not reached until 2005 with triennial application and not until 2007 with nil FYM NI
subsequently declined under the latter two FYM regimes but not with annual FYM so
that by 2010 NI was significantly higher with annual FYM than with either triennial or
nil FYM (Plt0001) but with only a small non-significant difference between the latter
two treatments
TREATMENT EFFECTS AFTER 12 YEARS OF APPLICATION
The effects of all treatments on the five key variables at each site in 2010 are shown in
Table S20 with treatments differing significantly (Plt005) from the limed control
treatment (Tr 2) highlighted for each variable Treatment effects in 2010 are not
described in detail here but the modeled responses of total species-richness and of
richness of PI species in 2010 to the mean amounts of N and P applied per year averaged
over 1999-2010 are described below
Relationships with the amounts of fertilizer applied
The effect of neither rate nor frequency of application was significant in any of the
REML models for either total species-richness or PI species-richness at either site when
the amount of N or P applied ha-1
year-1
was included The effect of form of fertilizer was
still significant in most models however either as a main effect or in interaction with the
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
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Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
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Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
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Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
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Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
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Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
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Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
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Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
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Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
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haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
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Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
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Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
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Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
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Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
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Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
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plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
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on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
9
FYM ha-1
year-1
or inorganic equivalent) since there was lack of equivalence of N and P supply
(but not K) between individual FYM treatments and their corresponding inorganic treatments For
each dependent variable tested two separate models were therefore developed using either N or
P ha-1
year-1
as the continuous variable In each case the presence of any non-linearity in response
was tested by including a quadratic term (ie N2 or P
2) or where more appropriate by logn-
transformation of the explanatory variable
For each analysis an initial model was constructed that included all five main effects and all
interactions The importance of each term was evaluated initially by a χ2 test on the Wald statistic
(Genstat V Committee 1997) The number and order of terms in the model were varied but with
N or P amount always included until a model was developed in which each term was significant
The model was then refined by testing the contribution of each term to the model by the more
conservative deviance test ie a χ2 test on the difference in deviance between the model
containing the term and a sub-model from which it was dropped (Welham amp Thompson 1997)
Where there was an interaction between form of fertilizer and the quadratic term but no overall
effect of form - implying that the difference between the two forms of fertilizer lay primarily in a
difference in the curvilinearity of response - a refined model was tested with the P2 (or N
2) x
Form term replaced by Form nested within the quadratic term (eg P2Form) This has the effect
of absorbing the overall effect of form of fertilizer within the interaction term and returns the two
terms P2 and P
2 x Form (Galwey 2006)
These modeling approaches were used with total species-richness and PI species-richness as
(separate) response variables and included data for all treatments limed in 2005 but not in 1999
(ie Trs 2-12)
Results
TEMPORAL CHANGES 1999-2010
Changes over time in total species-richness (number per m2) richness and proportional
aggregate cover of positive indicator (PI) species and proportional aggregate cover of
negative indicator (NI) species at the upland and lowland meadows are represented in
Figs 1 and 2 respectively for ten of the fifteen treatments applied Repeated measures
ANOVA statistics for each of the four variables are given in Supporting Information
Tables S4 S6 S8 and S10 respectively for the upland meadow and Tables S12 S14 S16
and S18 respectively for the lowland meadow Corresponding mean values for each
treatment in each year are given in Tables S5 S7 S9 S11 S13 S15 S17 and S19
respectively Changes in individual species abundance contributing to the temporal trends
in aggregated PI and NI species described below are outlined under Supporting
Information
The upland meadow
The overall effects of both treatment and time were highly significant in the repeated
measures ANOVA for total species-richness at the upland meadow (both P=0001) but
there was no treatment x time interaction (P=0647)
After an initial decline between 1999 and 2000 species-richness remained fairly
constant for most treatments until 2003 after which there was a general increase with the
notable exception of the highest rate annual FYM treatment (Tr 5) (Fig 1a) The latter
treatment declined further between 2005 and 2007 after which it appeared to recover
slightly and averaged over all years this treatment was significantly less species-rich than
all others (Plt001 compared to Tr 14 Plt0001 the remainder) None of the remaining
10
treatments differed significantly from the limed control treatment (Tr2) averaged over all
years However the more species-rich treatments ie nil fertilizer plus lime applied in
both 1999 and 2005 (Tr 13) low rate annual and medium rate triennial FYM (Tr 3 and 7
respectively) and the inorganic equivalent to the latter (Tr 11) were all more species-rich
averaged over all years (Plt005) than annual FYM at the medium rate under either
liming regime (ie Trs 4 and 14 the former representing a continuation of past
management)
The effect of the high rate annual FYM treatment (Tr 5) was largely responsible for
several main effects within the repeated measures ANOVA of treatments within the Form
x Rate x Frequency (FRF) factorial series namely overall effects (averaged over all
years) of rate (mediumgthigh P=0002) and frequency (triennialgtannual P=0001) of
application and a not quite significant effect of form of fertilizer (FYMltinorganic
P=0057) Both the form x rate and the form x frequency interactions were significant
(both Plt005) high rate FYM was significantly less species-rich than all other
combinations averaged over annual and three yearly treatments and all years (Plt0001
compared to medium rate FYM Plt001 compared to both medium and high rate
inorganic fertilizer) and annual FYM was less species-rich than three yearly FYM and
than inorganic fertilizer either annually or triennial (all Plt0001) averaged over the two
application rates and all years The rate x frequency interaction was not significant
(P=0105) but there was a marginally non-significant three-way interaction between
form rate and frequency (P=0070) with a low mean for high rate annual FYM (Tr 5
211 species m-2
) contrasting markedly with all other treatments within the series
particularly with low rate triennial FYM (Tr 6 267 species m-2
)
As with the full treatment ANOVA the overall time factor was highly significant
(Plt0001) but only the effect of application frequency varied significantly with time
(interaction Plt005) Species-richness declined slightly with annual treatments overall
and increased slightly with triennial treatments such that differences between these were
significant in all years from 2005 onwards (Plt001 for 2006-2009 Plt005 in 2005 and
2010)
Both the richness and aggregate cover of PI species (PI) increased generally with time
(Fig 1b and 1c) although in both cases there was a progressive separation between
species The temporal trend was particularly marked for PI although the time factor
was highly significant (Plt0001) in both ANOVAs The overall treatment effect was
marginally non-significant for PI richness (P=0069) with no indication of a time x
treatment interaction (P=0703) whereas for PI there was both a significant treatment
effect (P=0002) and a marginally non-significant treatment x time interaction (P=0075)
Mean PI was significantly lower averaged over all years for annual FYM treatments at
both 12 and 24 t ha-1
(ie Trs 4 14 and 5) compared to several other treatments notably
nil fertilizer treatments limed either in 2005 only (Tr 2 limed control) or in both 1999
and 2005 (Tr 13) (all differences Plt0001 except Tr 2 vs Tr 4 Plt001) and triennial
inorganic treatments (Trs 11 and 12 Plt001 except for Tr 4 vs Tr 11 Plt005) Within
the FRF series ANOVA the increase in PI over time was greater with triennial
treatments compared to annual averaged over fertilizer forms and rates of application
(time x frequency interaction P=0016) with a marked overall difference due to
application frequency averaged over forms rates and all years (P=0005)
11
PI species-richness showed a similar overall effect of application frequency within the
FRF series (triennialgtannual P=0013) and also a significant overall effect of rate of
application (medium rategthigh rate P=0022) The overall effect of form of fertilizer was
marginally non-significant (FYMltinorganic P=0059) and there was no significant
interaction among any of the three factors averaged over all years nor any interaction
with time
16
18
20
22
24
26
28
30
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Specie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime in Year 1+ ann FYM (Tr 14) Nil fert + Lime in Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr1)
a Species richness (total)
3
4
5
6
7
8
9
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
70
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
0
5
10
15
20
25
30
35
40
45
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
Fig 1 Changes 1999-2010 in vegetation composition an upland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control
The mean number of negative indicator (NI) species per m2 averaged over all treatments
varied significantly over time at the upland meadow (P=0002 data not shown) though
with no consistent trend - it was lowest overall in 2002 (499 per m2) and highest in both
1999 and 2008 (548 per m2) The treatment x time interaction was not significant
(P=0469) but the overall treatment effect was highly significant (Plt0001) mean
number of NI species per m2 was higher than the limed control (Tr 1 453 per m
2) with
all fertilizer treatments except Treatments 11 (triennial inorganic fertilizer at the lower
rate) and 13 (nil fertilizer limed in both 1999 and 2005) (Plt0001 for all except Trs 5
and 6 for which Plt001) The highest mean NI m-2
value was for high rate inorganic
12
fertilizer (Tr 10 565 per m2) which differed significantly (Plt005) from its FYM
counterpart Treatment 5 There was however no consistent difference between FYM
treatments and their inorganic counterparts and no overall effect of form of fertilizer
within the FRF series (P=0745) There was a significant form x rate interaction within
this ANOVA (P=0032) but only the difference between the high rate and lower rate
inorganic means was significant (high rategtlower rate Plt001)
The aggregate contribution to vegetation cover of negative indicator species (NI)
showed a very marked divergence between treatments over time (Fig 1d) with NI
either changing little or declining under most treatments again with the notable exception
of high rate annual FYM (Tr 5) under which NI increased over time Time treatment
and the treatment x time interaction were all highly significant (Plt0001) The
predominant temporal trend was downward (from 25 of live cover in 1999 to 14 in
2010 averaged over all treatments) but NI for Treatment 5 had significantly increased
compared to 1999 in 2002 2003 (Plt005) 2005 (Plt0001) 2008 (Plt0001) and 2009
(Plt001) but not in 2010 (Fig 1d) The most marked declines in NI occurred on the
limed control (Tr 2) nil fertilizer plus lime in both 1999 and 2005 (Tr 13) and the lower
rate triennial inorganic fertilizer treatments (Tr 11) differences compared to both 1999
and 2002 were highly significant (Plt0001) in most subsequent years for these
treatments A smaller decline also occurred after 2002 on the treatment equivalent to past
inputs (ie12 t ha-1
year-1
annual FYM Tr 4) In most years this treatment differed little
from the equivalent FYM treatment limed in both 1999 and 2005 (Tr 14)
These trends were reflected in highly significant overall effects on NI within the FRF
series of form (FYMgtinorganic) rate (mediumlthigh) and frequency (triennialltannual)
(all effects Plt0001) Time x rate and time x frequency interactions were both significant
(P=0019 and Plt0001 respectively) with a marginally non-significant rate x frequency
interaction averaged over all years (P=0054) NI declined over time under both
application rates averaged over forms and frequencies of application but the decline was
slower with high rate treatments compared to lower rate Difference between rates were
significant in all years from 2002 onwards (Plt005 in 2002 2006 and 2007 Plt0001 in
other years) Overall differences due to frequency of application were even more marked
Under annual application NI declined initially from 2003-2006 compared to 1999 but
subsequently recovered and did not differ from 1999 levels from 2007 onwards whilst
under triennial application differences were highly significant (Plt0001) compared to
1999 from 2003 onwards with significant differences between years during this same
period (Plt005 in 2003 and 2006 Plt0001 in remaining years)
Repeated-measures ANOVAs within the LFF treatment series at the upland meadow
showed no overall effect of liming regime on temporal trends for any of the above
variables nor any interaction of lime with either time or FYM frequency - note that FYM
was applied at the intermediate rate (12 t ha-1
) in all FYM treatments within this series
The overall effect of FYM frequency was significant for both PI (P=0003) and the
number of NI species m-2
(P=0001) PI was highest with nil FYM and lowest with
annual FYM with triennial intermediate and differing significantly (Plt005) from the
other two means NI species m-2
did not differ between triennial and annual application
within the LFF series but both were significantly higher than nil FYM (Plt001 for
triennial Plt0001 for annual application) There was also a significant time x FYM
frequency interaction for NI within this series (P=0001) reflecting a more rapid
13
decline with nil FYM than with triennial FYM and no significant overall trend with
annual FYM when averaged over both lime treatments
12
14
16
18
20
22
24
26
28
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Spe
cie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime Year 1 + ann FYM (Tr 14) Nil fert + Lime Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr 1)
a Species richness (total)
2
3
4
5
6
7
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
0
10
20
30
40
50
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
Fig 2 Changes 1999-2010 in vegetation composition at a lowland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control treatment and those that had received lime in year 1
Treatments 1 and 2 were identical (nil input) until 2005
The lowland meadow
Both time and treatment were significant within the ANOVA for total species-richness at
the lowland meadow (Plt0001 and P=0009 respectively and in contrast to the upland
site the treatment x time interaction was significant (P=0012) Averaged over all years
high rate annual FYM treatment (Tr 5) was significantly less species-rich than all others
except Treatment 14 (annual FYM at 12 t ha-1
limed in 1999) having declined
progressively between 1999 and 2009 (Fig 2a) with most differences significant at
Plt001 Treatment 14 did not differ significantly overall compared to the corresponding
FYM treatment not limed until 2005 (Tr 4) although these two treatments showed
14
markedly different temporal trends Treatment 14 was less species-rich than others during
the first five years of the experiment but did not change significantly throughout the
whole 12 year period whilst Treatment 4 changed little between 1999 and 2006 but
declined thereafter (Plt001 and Plt0001 for 2009 and 2010 respectively compared to
2006) possibly in response to liming in 2005 However there was no significant overall
effect on total species-richness of liming strategy within the LFF series and no interaction
between liming and either time or FYM application
There were marked differences in temporal trend in species-richness between FYM
treatments and their inorganic counterparts particularly with the high rate annual
treatments (Tr 5 and Tr 10) Whereas species-richness declined more or less continually
throughout under Treatment 5 at least until 2009 (Plt001 for 2004 compared to 1999 and
for both 2009 and 2010 compared to 2004) Treatment 10 declined between 2000 and
2001 (Plt005) and then remained relatively unchanged until 2008 after which it declined
again (2010 Plt005 compared to 2008)
Other treatments within the full treatment series including untreated control (Tr 1) and
limed control (Tr 2) showed no particular temporal trend in the full repeated measures
ANOVA although both the latter treatments showed a dip in species-richness in 2004
(Plt005 compared to both 2003 and 2005)
There was a strong overall effect of form of fertilizer within the FRF series ANOVA
(FYMltinorganic P=0003) a significant effect of frequency of application
(triennialgtannual P=0027) and a not quite significant effect of application rate (high
rateltmedium rate P=0064) but no significant interaction between any of these factors
The time x application frequency interaction was highly significant (Plt0001) species-
richness remained unchanged under triennial application but under annual application
was significantly lower compared to 1999 in each year from 2004 onwards with the
exception of 2006 (Plt005 in 2004 Plt001 in 2005 and 2008 Plt0001 the remainder)
Again with the exception of 2006 differences between triennial and annual application
were significant in each year from 2005 onwards (Plt005 in 2007 Plt001 in 2005 and
2008 Plt0001 in 2009 and 2010)
Both treatment time and their interaction were significant in the full treatment ANOVA
for PI species-richness at the lowland meadow (ie time and treatment both Plt0001
time x treatment P=0006) There was a general decline in PI-richness after 2005 (Fig
2b) with PI species m-2
averaged over all treatments lower from 2007 onwards (Plt0001)
than all years up to 2005 except 2001 Although the initiation of this trend corresponded
with the liming of most treatments in 2005 a similar pattern was shown by treatments
that did not receive this lime application ie the untreated control treatment (Tr 1) the
nil fertilizer treatment limed in 1999 (Tr 13) and the corresponding treatment also
receiving triennial FYM (Tr 15) In all these cases PI-richness showed no significant
difference between years until 2005 but was significantly (Plt001) lower by 2010 than in
2005 Treatment 14 (limed in 1999 plus annual FYM) did not show this pattern but PI
species-richness had been lowest under this treatment in most years between 1999 and
2005 (Fig 2b)
As with total species-richness PI richness declined more or less progressively under
Treatment 5 until 2008 (Plt005 compared to 1999 by 2001 Plt0001 from 2006
onwards) whereas Treatment 4 did not change significantly until 2006 after which it
declined (Plt005 in 2007 compared to 2006 Plt001 thereafter) Both these treatments
15
tended be less rich in PI species than the corresponding inorganic treatment (Trs 9 and
10 respectively) in most years consistently so for Tr 4 compared to Tr 9 but these
differences were not significant in any year nor when averaged over all years There was
however a significant overall effect of form of fertilizer in the FRF series ANOVA
(FYMltinorganic P=0002) and a highly significant effect of frequency of application
(triennialgtannual Plt0001) The time x frequency of application interaction was also
highly significant (Plt0001) Under triennial application PI species per m2 increased
slightly until 2005 (Plt005 compared to 1999) and declined thereafter so that by 2009
levels were significantly lower than in 1999 (Plt001 for 2009 Plt0001 for 2010) By
contrast under annual application PI species m-2
declined progressively until 2009
following a temporary though highly significant (Plt0001) dip in 2000 so that the
difference compared to 1999 was significant in every year from 2002 onwards and highly
significant (Plt0001) from 2005 With the exception of 2003 differences between
triennial and annual application were significant from 2002 onwards and highly
significant (Plt0001) in 2005 and from 2007 to 2010
There was no significant effect other than of time within the LFF treatment series
ANOVA although there was a marginally non-significant time x FYM frequency
interaction (P=0054) This reflected a more rapid decline in PI species-richness under
annual FYM application than with either nil or triennial FYM with the latter two showing
no discernible difference
By contrast with PI species m-2
PI cover percentage (PI) showed no overall treatment
effect within the full treatment ANOVA nor a time x treatment interaction although the
overall effect of time was highly significant (Plt0001) The temporal pattern in PI (Fig
2c) also differed from that shown by PI-richness with PI averaged over all treatments
declining between 1999 and 2005 in three highly significant increments (1999-2000
2000-2002 and 2002-2005 all Plt0001) and increasing again thereafter (2005-2009 and
2009-2010 both Plt0001) There was no significant effect within either the LFF or FRF
treatment series ANOVAs although their was a slight time x frequency of application
effect within the latter (P=0081) PI was slightly higher under annual application than
triennial in each year 1999-2004 after which the reverse was true although differences
were always small
There was no overall effect of treatment on the number of NI species per m2 within the
full treatment range at the lowland meadow (data not shown) but both the overall effect
of time and the time x treatment interaction were significant (Plt0001 and P=0009
respectively) Overall NI species m-2
increased over time from 24 species m-2
in 1999 to
a peak of 42 m-2
in 2008 averaged over all treatments NI species m-2
increased on the
untreated control treatment (Tr 1) from 21 species m-2
in 1999 to 34 species m-2
in 2007
(increase Plt001) then declined to the 1999 value by 2010 The limed (in 2005) control
(Tr 2) showed a somewhat similar pattern though from a slightly higher starting level
(31 species m-2
) and with the increase to 38 species m-2
in 2008 not reaching
significance
These temporal trends in NI species m-2
were much accentuated by annual fertilizer
application particularly by FYM treatments with peak numbers of NI species reaching
52 and 47 species m-2
for Treatments 4 and 5 respectively in 2008 (both Plt0001
compared to 1999) and despite slight declines in both cases remaining significantly
higher in 2010 than in 1999 (both Plt0001) Inorganic counterparts to these treatments
16
showed similar though slightly less marked trends and there was no significant
difference between all four treatments in the peak number reached in 2008 There was no
overall effect of either form of fertilizer or rate of application within the FRF series
ANOVA and only a marginally non-significant effect of frequency of application
(triennialltannual P=0061) Liming in 1999 (Trs 13 14 and 15) also appeared to
accentuate the generally increasing trend although peak numbers in 2008 did not differ
between these treatments and either untreated or limed control in the full treatment range
ANOVA The overall effects of liming regime (1999 or 2005) were not significant within
the LFF treatment series but the faster rate of increase with liming in 1999 was reflected
in a significant time x lime interaction in this analysis (P=0017) The mean number of NI
species m-2
increased very significantly (Plt0001) between 1999 and 2000 following
liming in 1999 with the increment increasing until 2008 whilst on plots un-limed until
2005 NI species m-2
did not increase significantly until 2003 (Plt005) although the
increase compared to 1999 was highly significant (Plt0001) in 2006 2008 and 2010
Nevertheless the differences in rate of increase was at least partly due to a lower 1999
mean for the 1999-limed treatments (170 species m-2
compared to 244 species m-2
difference NS) and although in all years from 2001 onwards mean NI species m-2
was
higher for 1999-limed treatments compared to those not limed until 2005 the difference
was not significant in any year
The contribution of NI species to vegetation cover (NI) showed a very marked
separation between treatments over time at the lowland meadow (Fig 2d) starting from
much lower levels than at the upland meadow (compare with Fig 1d) There were
noticeable differences in temporal trend between annual fertilizer treatments on the one
hand and triennial and nil fertilizer treatments on the other (Fig 2d) The effects of
treatment time and the treatment x time interaction were all highly significant (Plt0001)
in the full treatment range ANOVA The most marked increase in NI occurred with
annual FYM at the high rate (Tr 5) with NI increasing rapidly between 1999 and 2001
(Plt001) and a further highly significant increment between 2004 and 2009 (Plt0001)
(Fig 2d) A similar though less marked trend occurred with annual FYM at the medium
rate (Tr 4) with significant increases between 1999 and 2005 and between 2005 and
2010 (both Plt001) NI also increased markedly with the inorganic treatment
equivalent to Treatment 5 (Tr 10) although the overall rate of increase shown by this
treatment was closer to that shown by Treatment 4 than Treatment 5 There were
significant differences between Treatments 5 and 10 in most years from 2004 onwards
(Plt001 and Plt0001 in 2009 and 2010 respectively)
These effects were reflected in a significant overall effect of form of fertilizer
(FYMgtinorganic P=0008) and highly significant (Plt0001) overall effects of both rate
(high rategtlower rate) and frequency of application (annualgttriennial) within the FRF
series ANOVA and a significant form x rate interaction (P=0043) Mean NI was
highest for high rate annual FYM (Plt0001 compared to all three remaining means) and
lowest for lower rate inorganic (Plt005 compared to high rate inorganic NS compared
to lower rate FYM) Only frequency of application interacted significantly with time
(Plt0001) NI increased moderately under triennial application until 2008 (Plt005 in
2005 compared to 1999 Plt0001 each year 2006-2008) followed by a decline (Plt001
2010 compared to 2008) but with mean NI in 2010 still higher than 1999 (Plt005)
The increasing trend was very much more marked with annual fertilizer application for
17
which there were highly significant (Plt0001) increments between 1999 and 2002 and
between 2002 and 2008 but with no significant increase thereafter Differences between
annual and triennial treatment means were highly significant (Plt0001) from 2003
onwards
There was no significant change in NI over time either under the untreated control
(Tr 1) or the nil fertilizer (Tr 2) treatments in the full treatment ANOVA NI increased
until 2005 on plots limed in 1999 without fertilizer Tr 13 (Plt001 compared to 1999)
and declined slightly but not significantly thereafter Trends were similar with the
treatment limed in 1999 and receiving triennial FYM (Tr 15 ndash not illustrated in Fig 2)
although NI was higher with this treatment than with Tr 13 (Plt005) in both 2005 and
2008 By contrast applying the same rate of FYM annually after liming in 1999 (Tr 14)
caused a more rapid increase with levels by 2005 higher than either Tr 13 (Plt001) or
Tr 4 (Plt005) ndash note that the latter was equivalent to Tr 14 but without lime until 2005
NI declined after 2006 under Tr 14 but continued to increase with Tr 4 with the
difference between these two treatments reversed by 2010 (Plt005)
All main effects and interactions were significant in the LFF treatment series ANOVA
for NI except for lime x FYM frequency and the lime x FYM x time interactions Plots
limed in 1999 supported higher NI levels than those limed in 2005 averaged over the
three FYM frequencies and all years (P=0042) NI increased more rapidly after liming
in 1999 than where liming was delayed until 2005 (P=0033 for time x lime interaction)
with the overall difference due to liming in 1999 highly significant (Plt0001) by 2005
NI declined thereafter on the 1999-limed treatments whilst continuing to increase
following liming in 2005 of previously un-limed treatments both peaking in 2008 at a
very similar level Averaged over lime treatments NI increased much more rapidly
under annual FYM application than under either triennial or nil FYM (P=0002 for the
time x FYM frequency interaction) a highly significant increase had occurred by 2002
with annual FYM (Plt0001 compared to 1999) but increases of similar magnitude were
not reached until 2005 with triennial application and not until 2007 with nil FYM NI
subsequently declined under the latter two FYM regimes but not with annual FYM so
that by 2010 NI was significantly higher with annual FYM than with either triennial or
nil FYM (Plt0001) but with only a small non-significant difference between the latter
two treatments
TREATMENT EFFECTS AFTER 12 YEARS OF APPLICATION
The effects of all treatments on the five key variables at each site in 2010 are shown in
Table S20 with treatments differing significantly (Plt005) from the limed control
treatment (Tr 2) highlighted for each variable Treatment effects in 2010 are not
described in detail here but the modeled responses of total species-richness and of
richness of PI species in 2010 to the mean amounts of N and P applied per year averaged
over 1999-2010 are described below
Relationships with the amounts of fertilizer applied
The effect of neither rate nor frequency of application was significant in any of the
REML models for either total species-richness or PI species-richness at either site when
the amount of N or P applied ha-1
year-1
was included The effect of form of fertilizer was
still significant in most models however either as a main effect or in interaction with the
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
References
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Anon (2010) Fertilizer Manual (RB 209) 8th Edition Her Majestyrsquos Stationery Office London
Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
Kluwer Academic Publishers Dordrecht
Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
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Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
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Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
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Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
phosphorous vs nitrogen enrichment in plant species loss across North-west Europen
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Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
and losses following application of manures to arable land MANNER Soil Use and
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Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
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Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
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Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
activity in botanically diverse haymeadows effects of litter type and fertilizer additions Soil
Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
10
treatments differed significantly from the limed control treatment (Tr2) averaged over all
years However the more species-rich treatments ie nil fertilizer plus lime applied in
both 1999 and 2005 (Tr 13) low rate annual and medium rate triennial FYM (Tr 3 and 7
respectively) and the inorganic equivalent to the latter (Tr 11) were all more species-rich
averaged over all years (Plt005) than annual FYM at the medium rate under either
liming regime (ie Trs 4 and 14 the former representing a continuation of past
management)
The effect of the high rate annual FYM treatment (Tr 5) was largely responsible for
several main effects within the repeated measures ANOVA of treatments within the Form
x Rate x Frequency (FRF) factorial series namely overall effects (averaged over all
years) of rate (mediumgthigh P=0002) and frequency (triennialgtannual P=0001) of
application and a not quite significant effect of form of fertilizer (FYMltinorganic
P=0057) Both the form x rate and the form x frequency interactions were significant
(both Plt005) high rate FYM was significantly less species-rich than all other
combinations averaged over annual and three yearly treatments and all years (Plt0001
compared to medium rate FYM Plt001 compared to both medium and high rate
inorganic fertilizer) and annual FYM was less species-rich than three yearly FYM and
than inorganic fertilizer either annually or triennial (all Plt0001) averaged over the two
application rates and all years The rate x frequency interaction was not significant
(P=0105) but there was a marginally non-significant three-way interaction between
form rate and frequency (P=0070) with a low mean for high rate annual FYM (Tr 5
211 species m-2
) contrasting markedly with all other treatments within the series
particularly with low rate triennial FYM (Tr 6 267 species m-2
)
As with the full treatment ANOVA the overall time factor was highly significant
(Plt0001) but only the effect of application frequency varied significantly with time
(interaction Plt005) Species-richness declined slightly with annual treatments overall
and increased slightly with triennial treatments such that differences between these were
significant in all years from 2005 onwards (Plt001 for 2006-2009 Plt005 in 2005 and
2010)
Both the richness and aggregate cover of PI species (PI) increased generally with time
(Fig 1b and 1c) although in both cases there was a progressive separation between
species The temporal trend was particularly marked for PI although the time factor
was highly significant (Plt0001) in both ANOVAs The overall treatment effect was
marginally non-significant for PI richness (P=0069) with no indication of a time x
treatment interaction (P=0703) whereas for PI there was both a significant treatment
effect (P=0002) and a marginally non-significant treatment x time interaction (P=0075)
Mean PI was significantly lower averaged over all years for annual FYM treatments at
both 12 and 24 t ha-1
(ie Trs 4 14 and 5) compared to several other treatments notably
nil fertilizer treatments limed either in 2005 only (Tr 2 limed control) or in both 1999
and 2005 (Tr 13) (all differences Plt0001 except Tr 2 vs Tr 4 Plt001) and triennial
inorganic treatments (Trs 11 and 12 Plt001 except for Tr 4 vs Tr 11 Plt005) Within
the FRF series ANOVA the increase in PI over time was greater with triennial
treatments compared to annual averaged over fertilizer forms and rates of application
(time x frequency interaction P=0016) with a marked overall difference due to
application frequency averaged over forms rates and all years (P=0005)
11
PI species-richness showed a similar overall effect of application frequency within the
FRF series (triennialgtannual P=0013) and also a significant overall effect of rate of
application (medium rategthigh rate P=0022) The overall effect of form of fertilizer was
marginally non-significant (FYMltinorganic P=0059) and there was no significant
interaction among any of the three factors averaged over all years nor any interaction
with time
16
18
20
22
24
26
28
30
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Specie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime in Year 1+ ann FYM (Tr 14) Nil fert + Lime in Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr1)
a Species richness (total)
3
4
5
6
7
8
9
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
70
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
0
5
10
15
20
25
30
35
40
45
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
Fig 1 Changes 1999-2010 in vegetation composition an upland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control
The mean number of negative indicator (NI) species per m2 averaged over all treatments
varied significantly over time at the upland meadow (P=0002 data not shown) though
with no consistent trend - it was lowest overall in 2002 (499 per m2) and highest in both
1999 and 2008 (548 per m2) The treatment x time interaction was not significant
(P=0469) but the overall treatment effect was highly significant (Plt0001) mean
number of NI species per m2 was higher than the limed control (Tr 1 453 per m
2) with
all fertilizer treatments except Treatments 11 (triennial inorganic fertilizer at the lower
rate) and 13 (nil fertilizer limed in both 1999 and 2005) (Plt0001 for all except Trs 5
and 6 for which Plt001) The highest mean NI m-2
value was for high rate inorganic
12
fertilizer (Tr 10 565 per m2) which differed significantly (Plt005) from its FYM
counterpart Treatment 5 There was however no consistent difference between FYM
treatments and their inorganic counterparts and no overall effect of form of fertilizer
within the FRF series (P=0745) There was a significant form x rate interaction within
this ANOVA (P=0032) but only the difference between the high rate and lower rate
inorganic means was significant (high rategtlower rate Plt001)
The aggregate contribution to vegetation cover of negative indicator species (NI)
showed a very marked divergence between treatments over time (Fig 1d) with NI
either changing little or declining under most treatments again with the notable exception
of high rate annual FYM (Tr 5) under which NI increased over time Time treatment
and the treatment x time interaction were all highly significant (Plt0001) The
predominant temporal trend was downward (from 25 of live cover in 1999 to 14 in
2010 averaged over all treatments) but NI for Treatment 5 had significantly increased
compared to 1999 in 2002 2003 (Plt005) 2005 (Plt0001) 2008 (Plt0001) and 2009
(Plt001) but not in 2010 (Fig 1d) The most marked declines in NI occurred on the
limed control (Tr 2) nil fertilizer plus lime in both 1999 and 2005 (Tr 13) and the lower
rate triennial inorganic fertilizer treatments (Tr 11) differences compared to both 1999
and 2002 were highly significant (Plt0001) in most subsequent years for these
treatments A smaller decline also occurred after 2002 on the treatment equivalent to past
inputs (ie12 t ha-1
year-1
annual FYM Tr 4) In most years this treatment differed little
from the equivalent FYM treatment limed in both 1999 and 2005 (Tr 14)
These trends were reflected in highly significant overall effects on NI within the FRF
series of form (FYMgtinorganic) rate (mediumlthigh) and frequency (triennialltannual)
(all effects Plt0001) Time x rate and time x frequency interactions were both significant
(P=0019 and Plt0001 respectively) with a marginally non-significant rate x frequency
interaction averaged over all years (P=0054) NI declined over time under both
application rates averaged over forms and frequencies of application but the decline was
slower with high rate treatments compared to lower rate Difference between rates were
significant in all years from 2002 onwards (Plt005 in 2002 2006 and 2007 Plt0001 in
other years) Overall differences due to frequency of application were even more marked
Under annual application NI declined initially from 2003-2006 compared to 1999 but
subsequently recovered and did not differ from 1999 levels from 2007 onwards whilst
under triennial application differences were highly significant (Plt0001) compared to
1999 from 2003 onwards with significant differences between years during this same
period (Plt005 in 2003 and 2006 Plt0001 in remaining years)
Repeated-measures ANOVAs within the LFF treatment series at the upland meadow
showed no overall effect of liming regime on temporal trends for any of the above
variables nor any interaction of lime with either time or FYM frequency - note that FYM
was applied at the intermediate rate (12 t ha-1
) in all FYM treatments within this series
The overall effect of FYM frequency was significant for both PI (P=0003) and the
number of NI species m-2
(P=0001) PI was highest with nil FYM and lowest with
annual FYM with triennial intermediate and differing significantly (Plt005) from the
other two means NI species m-2
did not differ between triennial and annual application
within the LFF series but both were significantly higher than nil FYM (Plt001 for
triennial Plt0001 for annual application) There was also a significant time x FYM
frequency interaction for NI within this series (P=0001) reflecting a more rapid
13
decline with nil FYM than with triennial FYM and no significant overall trend with
annual FYM when averaged over both lime treatments
12
14
16
18
20
22
24
26
28
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Spe
cie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime Year 1 + ann FYM (Tr 14) Nil fert + Lime Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr 1)
a Species richness (total)
2
3
4
5
6
7
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
0
10
20
30
40
50
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
Fig 2 Changes 1999-2010 in vegetation composition at a lowland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control treatment and those that had received lime in year 1
Treatments 1 and 2 were identical (nil input) until 2005
The lowland meadow
Both time and treatment were significant within the ANOVA for total species-richness at
the lowland meadow (Plt0001 and P=0009 respectively and in contrast to the upland
site the treatment x time interaction was significant (P=0012) Averaged over all years
high rate annual FYM treatment (Tr 5) was significantly less species-rich than all others
except Treatment 14 (annual FYM at 12 t ha-1
limed in 1999) having declined
progressively between 1999 and 2009 (Fig 2a) with most differences significant at
Plt001 Treatment 14 did not differ significantly overall compared to the corresponding
FYM treatment not limed until 2005 (Tr 4) although these two treatments showed
14
markedly different temporal trends Treatment 14 was less species-rich than others during
the first five years of the experiment but did not change significantly throughout the
whole 12 year period whilst Treatment 4 changed little between 1999 and 2006 but
declined thereafter (Plt001 and Plt0001 for 2009 and 2010 respectively compared to
2006) possibly in response to liming in 2005 However there was no significant overall
effect on total species-richness of liming strategy within the LFF series and no interaction
between liming and either time or FYM application
There were marked differences in temporal trend in species-richness between FYM
treatments and their inorganic counterparts particularly with the high rate annual
treatments (Tr 5 and Tr 10) Whereas species-richness declined more or less continually
throughout under Treatment 5 at least until 2009 (Plt001 for 2004 compared to 1999 and
for both 2009 and 2010 compared to 2004) Treatment 10 declined between 2000 and
2001 (Plt005) and then remained relatively unchanged until 2008 after which it declined
again (2010 Plt005 compared to 2008)
Other treatments within the full treatment series including untreated control (Tr 1) and
limed control (Tr 2) showed no particular temporal trend in the full repeated measures
ANOVA although both the latter treatments showed a dip in species-richness in 2004
(Plt005 compared to both 2003 and 2005)
There was a strong overall effect of form of fertilizer within the FRF series ANOVA
(FYMltinorganic P=0003) a significant effect of frequency of application
(triennialgtannual P=0027) and a not quite significant effect of application rate (high
rateltmedium rate P=0064) but no significant interaction between any of these factors
The time x application frequency interaction was highly significant (Plt0001) species-
richness remained unchanged under triennial application but under annual application
was significantly lower compared to 1999 in each year from 2004 onwards with the
exception of 2006 (Plt005 in 2004 Plt001 in 2005 and 2008 Plt0001 the remainder)
Again with the exception of 2006 differences between triennial and annual application
were significant in each year from 2005 onwards (Plt005 in 2007 Plt001 in 2005 and
2008 Plt0001 in 2009 and 2010)
Both treatment time and their interaction were significant in the full treatment ANOVA
for PI species-richness at the lowland meadow (ie time and treatment both Plt0001
time x treatment P=0006) There was a general decline in PI-richness after 2005 (Fig
2b) with PI species m-2
averaged over all treatments lower from 2007 onwards (Plt0001)
than all years up to 2005 except 2001 Although the initiation of this trend corresponded
with the liming of most treatments in 2005 a similar pattern was shown by treatments
that did not receive this lime application ie the untreated control treatment (Tr 1) the
nil fertilizer treatment limed in 1999 (Tr 13) and the corresponding treatment also
receiving triennial FYM (Tr 15) In all these cases PI-richness showed no significant
difference between years until 2005 but was significantly (Plt001) lower by 2010 than in
2005 Treatment 14 (limed in 1999 plus annual FYM) did not show this pattern but PI
species-richness had been lowest under this treatment in most years between 1999 and
2005 (Fig 2b)
As with total species-richness PI richness declined more or less progressively under
Treatment 5 until 2008 (Plt005 compared to 1999 by 2001 Plt0001 from 2006
onwards) whereas Treatment 4 did not change significantly until 2006 after which it
declined (Plt005 in 2007 compared to 2006 Plt001 thereafter) Both these treatments
15
tended be less rich in PI species than the corresponding inorganic treatment (Trs 9 and
10 respectively) in most years consistently so for Tr 4 compared to Tr 9 but these
differences were not significant in any year nor when averaged over all years There was
however a significant overall effect of form of fertilizer in the FRF series ANOVA
(FYMltinorganic P=0002) and a highly significant effect of frequency of application
(triennialgtannual Plt0001) The time x frequency of application interaction was also
highly significant (Plt0001) Under triennial application PI species per m2 increased
slightly until 2005 (Plt005 compared to 1999) and declined thereafter so that by 2009
levels were significantly lower than in 1999 (Plt001 for 2009 Plt0001 for 2010) By
contrast under annual application PI species m-2
declined progressively until 2009
following a temporary though highly significant (Plt0001) dip in 2000 so that the
difference compared to 1999 was significant in every year from 2002 onwards and highly
significant (Plt0001) from 2005 With the exception of 2003 differences between
triennial and annual application were significant from 2002 onwards and highly
significant (Plt0001) in 2005 and from 2007 to 2010
There was no significant effect other than of time within the LFF treatment series
ANOVA although there was a marginally non-significant time x FYM frequency
interaction (P=0054) This reflected a more rapid decline in PI species-richness under
annual FYM application than with either nil or triennial FYM with the latter two showing
no discernible difference
By contrast with PI species m-2
PI cover percentage (PI) showed no overall treatment
effect within the full treatment ANOVA nor a time x treatment interaction although the
overall effect of time was highly significant (Plt0001) The temporal pattern in PI (Fig
2c) also differed from that shown by PI-richness with PI averaged over all treatments
declining between 1999 and 2005 in three highly significant increments (1999-2000
2000-2002 and 2002-2005 all Plt0001) and increasing again thereafter (2005-2009 and
2009-2010 both Plt0001) There was no significant effect within either the LFF or FRF
treatment series ANOVAs although their was a slight time x frequency of application
effect within the latter (P=0081) PI was slightly higher under annual application than
triennial in each year 1999-2004 after which the reverse was true although differences
were always small
There was no overall effect of treatment on the number of NI species per m2 within the
full treatment range at the lowland meadow (data not shown) but both the overall effect
of time and the time x treatment interaction were significant (Plt0001 and P=0009
respectively) Overall NI species m-2
increased over time from 24 species m-2
in 1999 to
a peak of 42 m-2
in 2008 averaged over all treatments NI species m-2
increased on the
untreated control treatment (Tr 1) from 21 species m-2
in 1999 to 34 species m-2
in 2007
(increase Plt001) then declined to the 1999 value by 2010 The limed (in 2005) control
(Tr 2) showed a somewhat similar pattern though from a slightly higher starting level
(31 species m-2
) and with the increase to 38 species m-2
in 2008 not reaching
significance
These temporal trends in NI species m-2
were much accentuated by annual fertilizer
application particularly by FYM treatments with peak numbers of NI species reaching
52 and 47 species m-2
for Treatments 4 and 5 respectively in 2008 (both Plt0001
compared to 1999) and despite slight declines in both cases remaining significantly
higher in 2010 than in 1999 (both Plt0001) Inorganic counterparts to these treatments
16
showed similar though slightly less marked trends and there was no significant
difference between all four treatments in the peak number reached in 2008 There was no
overall effect of either form of fertilizer or rate of application within the FRF series
ANOVA and only a marginally non-significant effect of frequency of application
(triennialltannual P=0061) Liming in 1999 (Trs 13 14 and 15) also appeared to
accentuate the generally increasing trend although peak numbers in 2008 did not differ
between these treatments and either untreated or limed control in the full treatment range
ANOVA The overall effects of liming regime (1999 or 2005) were not significant within
the LFF treatment series but the faster rate of increase with liming in 1999 was reflected
in a significant time x lime interaction in this analysis (P=0017) The mean number of NI
species m-2
increased very significantly (Plt0001) between 1999 and 2000 following
liming in 1999 with the increment increasing until 2008 whilst on plots un-limed until
2005 NI species m-2
did not increase significantly until 2003 (Plt005) although the
increase compared to 1999 was highly significant (Plt0001) in 2006 2008 and 2010
Nevertheless the differences in rate of increase was at least partly due to a lower 1999
mean for the 1999-limed treatments (170 species m-2
compared to 244 species m-2
difference NS) and although in all years from 2001 onwards mean NI species m-2
was
higher for 1999-limed treatments compared to those not limed until 2005 the difference
was not significant in any year
The contribution of NI species to vegetation cover (NI) showed a very marked
separation between treatments over time at the lowland meadow (Fig 2d) starting from
much lower levels than at the upland meadow (compare with Fig 1d) There were
noticeable differences in temporal trend between annual fertilizer treatments on the one
hand and triennial and nil fertilizer treatments on the other (Fig 2d) The effects of
treatment time and the treatment x time interaction were all highly significant (Plt0001)
in the full treatment range ANOVA The most marked increase in NI occurred with
annual FYM at the high rate (Tr 5) with NI increasing rapidly between 1999 and 2001
(Plt001) and a further highly significant increment between 2004 and 2009 (Plt0001)
(Fig 2d) A similar though less marked trend occurred with annual FYM at the medium
rate (Tr 4) with significant increases between 1999 and 2005 and between 2005 and
2010 (both Plt001) NI also increased markedly with the inorganic treatment
equivalent to Treatment 5 (Tr 10) although the overall rate of increase shown by this
treatment was closer to that shown by Treatment 4 than Treatment 5 There were
significant differences between Treatments 5 and 10 in most years from 2004 onwards
(Plt001 and Plt0001 in 2009 and 2010 respectively)
These effects were reflected in a significant overall effect of form of fertilizer
(FYMgtinorganic P=0008) and highly significant (Plt0001) overall effects of both rate
(high rategtlower rate) and frequency of application (annualgttriennial) within the FRF
series ANOVA and a significant form x rate interaction (P=0043) Mean NI was
highest for high rate annual FYM (Plt0001 compared to all three remaining means) and
lowest for lower rate inorganic (Plt005 compared to high rate inorganic NS compared
to lower rate FYM) Only frequency of application interacted significantly with time
(Plt0001) NI increased moderately under triennial application until 2008 (Plt005 in
2005 compared to 1999 Plt0001 each year 2006-2008) followed by a decline (Plt001
2010 compared to 2008) but with mean NI in 2010 still higher than 1999 (Plt005)
The increasing trend was very much more marked with annual fertilizer application for
17
which there were highly significant (Plt0001) increments between 1999 and 2002 and
between 2002 and 2008 but with no significant increase thereafter Differences between
annual and triennial treatment means were highly significant (Plt0001) from 2003
onwards
There was no significant change in NI over time either under the untreated control
(Tr 1) or the nil fertilizer (Tr 2) treatments in the full treatment ANOVA NI increased
until 2005 on plots limed in 1999 without fertilizer Tr 13 (Plt001 compared to 1999)
and declined slightly but not significantly thereafter Trends were similar with the
treatment limed in 1999 and receiving triennial FYM (Tr 15 ndash not illustrated in Fig 2)
although NI was higher with this treatment than with Tr 13 (Plt005) in both 2005 and
2008 By contrast applying the same rate of FYM annually after liming in 1999 (Tr 14)
caused a more rapid increase with levels by 2005 higher than either Tr 13 (Plt001) or
Tr 4 (Plt005) ndash note that the latter was equivalent to Tr 14 but without lime until 2005
NI declined after 2006 under Tr 14 but continued to increase with Tr 4 with the
difference between these two treatments reversed by 2010 (Plt005)
All main effects and interactions were significant in the LFF treatment series ANOVA
for NI except for lime x FYM frequency and the lime x FYM x time interactions Plots
limed in 1999 supported higher NI levels than those limed in 2005 averaged over the
three FYM frequencies and all years (P=0042) NI increased more rapidly after liming
in 1999 than where liming was delayed until 2005 (P=0033 for time x lime interaction)
with the overall difference due to liming in 1999 highly significant (Plt0001) by 2005
NI declined thereafter on the 1999-limed treatments whilst continuing to increase
following liming in 2005 of previously un-limed treatments both peaking in 2008 at a
very similar level Averaged over lime treatments NI increased much more rapidly
under annual FYM application than under either triennial or nil FYM (P=0002 for the
time x FYM frequency interaction) a highly significant increase had occurred by 2002
with annual FYM (Plt0001 compared to 1999) but increases of similar magnitude were
not reached until 2005 with triennial application and not until 2007 with nil FYM NI
subsequently declined under the latter two FYM regimes but not with annual FYM so
that by 2010 NI was significantly higher with annual FYM than with either triennial or
nil FYM (Plt0001) but with only a small non-significant difference between the latter
two treatments
TREATMENT EFFECTS AFTER 12 YEARS OF APPLICATION
The effects of all treatments on the five key variables at each site in 2010 are shown in
Table S20 with treatments differing significantly (Plt005) from the limed control
treatment (Tr 2) highlighted for each variable Treatment effects in 2010 are not
described in detail here but the modeled responses of total species-richness and of
richness of PI species in 2010 to the mean amounts of N and P applied per year averaged
over 1999-2010 are described below
Relationships with the amounts of fertilizer applied
The effect of neither rate nor frequency of application was significant in any of the
REML models for either total species-richness or PI species-richness at either site when
the amount of N or P applied ha-1
year-1
was included The effect of form of fertilizer was
still significant in most models however either as a main effect or in interaction with the
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
References
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Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
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Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
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Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
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Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
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Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
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Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
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Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
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Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
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haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
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28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
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maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
11
PI species-richness showed a similar overall effect of application frequency within the
FRF series (triennialgtannual P=0013) and also a significant overall effect of rate of
application (medium rategthigh rate P=0022) The overall effect of form of fertilizer was
marginally non-significant (FYMltinorganic P=0059) and there was no significant
interaction among any of the three factors averaged over all years nor any interaction
with time
16
18
20
22
24
26
28
30
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Specie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime in Year 1+ ann FYM (Tr 14) Nil fert + Lime in Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr1)
a Species richness (total)
3
4
5
6
7
8
9
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
70
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
0
5
10
15
20
25
30
35
40
45
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
Fig 1 Changes 1999-2010 in vegetation composition an upland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control
The mean number of negative indicator (NI) species per m2 averaged over all treatments
varied significantly over time at the upland meadow (P=0002 data not shown) though
with no consistent trend - it was lowest overall in 2002 (499 per m2) and highest in both
1999 and 2008 (548 per m2) The treatment x time interaction was not significant
(P=0469) but the overall treatment effect was highly significant (Plt0001) mean
number of NI species per m2 was higher than the limed control (Tr 1 453 per m
2) with
all fertilizer treatments except Treatments 11 (triennial inorganic fertilizer at the lower
rate) and 13 (nil fertilizer limed in both 1999 and 2005) (Plt0001 for all except Trs 5
and 6 for which Plt001) The highest mean NI m-2
value was for high rate inorganic
12
fertilizer (Tr 10 565 per m2) which differed significantly (Plt005) from its FYM
counterpart Treatment 5 There was however no consistent difference between FYM
treatments and their inorganic counterparts and no overall effect of form of fertilizer
within the FRF series (P=0745) There was a significant form x rate interaction within
this ANOVA (P=0032) but only the difference between the high rate and lower rate
inorganic means was significant (high rategtlower rate Plt001)
The aggregate contribution to vegetation cover of negative indicator species (NI)
showed a very marked divergence between treatments over time (Fig 1d) with NI
either changing little or declining under most treatments again with the notable exception
of high rate annual FYM (Tr 5) under which NI increased over time Time treatment
and the treatment x time interaction were all highly significant (Plt0001) The
predominant temporal trend was downward (from 25 of live cover in 1999 to 14 in
2010 averaged over all treatments) but NI for Treatment 5 had significantly increased
compared to 1999 in 2002 2003 (Plt005) 2005 (Plt0001) 2008 (Plt0001) and 2009
(Plt001) but not in 2010 (Fig 1d) The most marked declines in NI occurred on the
limed control (Tr 2) nil fertilizer plus lime in both 1999 and 2005 (Tr 13) and the lower
rate triennial inorganic fertilizer treatments (Tr 11) differences compared to both 1999
and 2002 were highly significant (Plt0001) in most subsequent years for these
treatments A smaller decline also occurred after 2002 on the treatment equivalent to past
inputs (ie12 t ha-1
year-1
annual FYM Tr 4) In most years this treatment differed little
from the equivalent FYM treatment limed in both 1999 and 2005 (Tr 14)
These trends were reflected in highly significant overall effects on NI within the FRF
series of form (FYMgtinorganic) rate (mediumlthigh) and frequency (triennialltannual)
(all effects Plt0001) Time x rate and time x frequency interactions were both significant
(P=0019 and Plt0001 respectively) with a marginally non-significant rate x frequency
interaction averaged over all years (P=0054) NI declined over time under both
application rates averaged over forms and frequencies of application but the decline was
slower with high rate treatments compared to lower rate Difference between rates were
significant in all years from 2002 onwards (Plt005 in 2002 2006 and 2007 Plt0001 in
other years) Overall differences due to frequency of application were even more marked
Under annual application NI declined initially from 2003-2006 compared to 1999 but
subsequently recovered and did not differ from 1999 levels from 2007 onwards whilst
under triennial application differences were highly significant (Plt0001) compared to
1999 from 2003 onwards with significant differences between years during this same
period (Plt005 in 2003 and 2006 Plt0001 in remaining years)
Repeated-measures ANOVAs within the LFF treatment series at the upland meadow
showed no overall effect of liming regime on temporal trends for any of the above
variables nor any interaction of lime with either time or FYM frequency - note that FYM
was applied at the intermediate rate (12 t ha-1
) in all FYM treatments within this series
The overall effect of FYM frequency was significant for both PI (P=0003) and the
number of NI species m-2
(P=0001) PI was highest with nil FYM and lowest with
annual FYM with triennial intermediate and differing significantly (Plt005) from the
other two means NI species m-2
did not differ between triennial and annual application
within the LFF series but both were significantly higher than nil FYM (Plt001 for
triennial Plt0001 for annual application) There was also a significant time x FYM
frequency interaction for NI within this series (P=0001) reflecting a more rapid
13
decline with nil FYM than with triennial FYM and no significant overall trend with
annual FYM when averaged over both lime treatments
12
14
16
18
20
22
24
26
28
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Spe
cie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime Year 1 + ann FYM (Tr 14) Nil fert + Lime Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr 1)
a Species richness (total)
2
3
4
5
6
7
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
0
10
20
30
40
50
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
Fig 2 Changes 1999-2010 in vegetation composition at a lowland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control treatment and those that had received lime in year 1
Treatments 1 and 2 were identical (nil input) until 2005
The lowland meadow
Both time and treatment were significant within the ANOVA for total species-richness at
the lowland meadow (Plt0001 and P=0009 respectively and in contrast to the upland
site the treatment x time interaction was significant (P=0012) Averaged over all years
high rate annual FYM treatment (Tr 5) was significantly less species-rich than all others
except Treatment 14 (annual FYM at 12 t ha-1
limed in 1999) having declined
progressively between 1999 and 2009 (Fig 2a) with most differences significant at
Plt001 Treatment 14 did not differ significantly overall compared to the corresponding
FYM treatment not limed until 2005 (Tr 4) although these two treatments showed
14
markedly different temporal trends Treatment 14 was less species-rich than others during
the first five years of the experiment but did not change significantly throughout the
whole 12 year period whilst Treatment 4 changed little between 1999 and 2006 but
declined thereafter (Plt001 and Plt0001 for 2009 and 2010 respectively compared to
2006) possibly in response to liming in 2005 However there was no significant overall
effect on total species-richness of liming strategy within the LFF series and no interaction
between liming and either time or FYM application
There were marked differences in temporal trend in species-richness between FYM
treatments and their inorganic counterparts particularly with the high rate annual
treatments (Tr 5 and Tr 10) Whereas species-richness declined more or less continually
throughout under Treatment 5 at least until 2009 (Plt001 for 2004 compared to 1999 and
for both 2009 and 2010 compared to 2004) Treatment 10 declined between 2000 and
2001 (Plt005) and then remained relatively unchanged until 2008 after which it declined
again (2010 Plt005 compared to 2008)
Other treatments within the full treatment series including untreated control (Tr 1) and
limed control (Tr 2) showed no particular temporal trend in the full repeated measures
ANOVA although both the latter treatments showed a dip in species-richness in 2004
(Plt005 compared to both 2003 and 2005)
There was a strong overall effect of form of fertilizer within the FRF series ANOVA
(FYMltinorganic P=0003) a significant effect of frequency of application
(triennialgtannual P=0027) and a not quite significant effect of application rate (high
rateltmedium rate P=0064) but no significant interaction between any of these factors
The time x application frequency interaction was highly significant (Plt0001) species-
richness remained unchanged under triennial application but under annual application
was significantly lower compared to 1999 in each year from 2004 onwards with the
exception of 2006 (Plt005 in 2004 Plt001 in 2005 and 2008 Plt0001 the remainder)
Again with the exception of 2006 differences between triennial and annual application
were significant in each year from 2005 onwards (Plt005 in 2007 Plt001 in 2005 and
2008 Plt0001 in 2009 and 2010)
Both treatment time and their interaction were significant in the full treatment ANOVA
for PI species-richness at the lowland meadow (ie time and treatment both Plt0001
time x treatment P=0006) There was a general decline in PI-richness after 2005 (Fig
2b) with PI species m-2
averaged over all treatments lower from 2007 onwards (Plt0001)
than all years up to 2005 except 2001 Although the initiation of this trend corresponded
with the liming of most treatments in 2005 a similar pattern was shown by treatments
that did not receive this lime application ie the untreated control treatment (Tr 1) the
nil fertilizer treatment limed in 1999 (Tr 13) and the corresponding treatment also
receiving triennial FYM (Tr 15) In all these cases PI-richness showed no significant
difference between years until 2005 but was significantly (Plt001) lower by 2010 than in
2005 Treatment 14 (limed in 1999 plus annual FYM) did not show this pattern but PI
species-richness had been lowest under this treatment in most years between 1999 and
2005 (Fig 2b)
As with total species-richness PI richness declined more or less progressively under
Treatment 5 until 2008 (Plt005 compared to 1999 by 2001 Plt0001 from 2006
onwards) whereas Treatment 4 did not change significantly until 2006 after which it
declined (Plt005 in 2007 compared to 2006 Plt001 thereafter) Both these treatments
15
tended be less rich in PI species than the corresponding inorganic treatment (Trs 9 and
10 respectively) in most years consistently so for Tr 4 compared to Tr 9 but these
differences were not significant in any year nor when averaged over all years There was
however a significant overall effect of form of fertilizer in the FRF series ANOVA
(FYMltinorganic P=0002) and a highly significant effect of frequency of application
(triennialgtannual Plt0001) The time x frequency of application interaction was also
highly significant (Plt0001) Under triennial application PI species per m2 increased
slightly until 2005 (Plt005 compared to 1999) and declined thereafter so that by 2009
levels were significantly lower than in 1999 (Plt001 for 2009 Plt0001 for 2010) By
contrast under annual application PI species m-2
declined progressively until 2009
following a temporary though highly significant (Plt0001) dip in 2000 so that the
difference compared to 1999 was significant in every year from 2002 onwards and highly
significant (Plt0001) from 2005 With the exception of 2003 differences between
triennial and annual application were significant from 2002 onwards and highly
significant (Plt0001) in 2005 and from 2007 to 2010
There was no significant effect other than of time within the LFF treatment series
ANOVA although there was a marginally non-significant time x FYM frequency
interaction (P=0054) This reflected a more rapid decline in PI species-richness under
annual FYM application than with either nil or triennial FYM with the latter two showing
no discernible difference
By contrast with PI species m-2
PI cover percentage (PI) showed no overall treatment
effect within the full treatment ANOVA nor a time x treatment interaction although the
overall effect of time was highly significant (Plt0001) The temporal pattern in PI (Fig
2c) also differed from that shown by PI-richness with PI averaged over all treatments
declining between 1999 and 2005 in three highly significant increments (1999-2000
2000-2002 and 2002-2005 all Plt0001) and increasing again thereafter (2005-2009 and
2009-2010 both Plt0001) There was no significant effect within either the LFF or FRF
treatment series ANOVAs although their was a slight time x frequency of application
effect within the latter (P=0081) PI was slightly higher under annual application than
triennial in each year 1999-2004 after which the reverse was true although differences
were always small
There was no overall effect of treatment on the number of NI species per m2 within the
full treatment range at the lowland meadow (data not shown) but both the overall effect
of time and the time x treatment interaction were significant (Plt0001 and P=0009
respectively) Overall NI species m-2
increased over time from 24 species m-2
in 1999 to
a peak of 42 m-2
in 2008 averaged over all treatments NI species m-2
increased on the
untreated control treatment (Tr 1) from 21 species m-2
in 1999 to 34 species m-2
in 2007
(increase Plt001) then declined to the 1999 value by 2010 The limed (in 2005) control
(Tr 2) showed a somewhat similar pattern though from a slightly higher starting level
(31 species m-2
) and with the increase to 38 species m-2
in 2008 not reaching
significance
These temporal trends in NI species m-2
were much accentuated by annual fertilizer
application particularly by FYM treatments with peak numbers of NI species reaching
52 and 47 species m-2
for Treatments 4 and 5 respectively in 2008 (both Plt0001
compared to 1999) and despite slight declines in both cases remaining significantly
higher in 2010 than in 1999 (both Plt0001) Inorganic counterparts to these treatments
16
showed similar though slightly less marked trends and there was no significant
difference between all four treatments in the peak number reached in 2008 There was no
overall effect of either form of fertilizer or rate of application within the FRF series
ANOVA and only a marginally non-significant effect of frequency of application
(triennialltannual P=0061) Liming in 1999 (Trs 13 14 and 15) also appeared to
accentuate the generally increasing trend although peak numbers in 2008 did not differ
between these treatments and either untreated or limed control in the full treatment range
ANOVA The overall effects of liming regime (1999 or 2005) were not significant within
the LFF treatment series but the faster rate of increase with liming in 1999 was reflected
in a significant time x lime interaction in this analysis (P=0017) The mean number of NI
species m-2
increased very significantly (Plt0001) between 1999 and 2000 following
liming in 1999 with the increment increasing until 2008 whilst on plots un-limed until
2005 NI species m-2
did not increase significantly until 2003 (Plt005) although the
increase compared to 1999 was highly significant (Plt0001) in 2006 2008 and 2010
Nevertheless the differences in rate of increase was at least partly due to a lower 1999
mean for the 1999-limed treatments (170 species m-2
compared to 244 species m-2
difference NS) and although in all years from 2001 onwards mean NI species m-2
was
higher for 1999-limed treatments compared to those not limed until 2005 the difference
was not significant in any year
The contribution of NI species to vegetation cover (NI) showed a very marked
separation between treatments over time at the lowland meadow (Fig 2d) starting from
much lower levels than at the upland meadow (compare with Fig 1d) There were
noticeable differences in temporal trend between annual fertilizer treatments on the one
hand and triennial and nil fertilizer treatments on the other (Fig 2d) The effects of
treatment time and the treatment x time interaction were all highly significant (Plt0001)
in the full treatment range ANOVA The most marked increase in NI occurred with
annual FYM at the high rate (Tr 5) with NI increasing rapidly between 1999 and 2001
(Plt001) and a further highly significant increment between 2004 and 2009 (Plt0001)
(Fig 2d) A similar though less marked trend occurred with annual FYM at the medium
rate (Tr 4) with significant increases between 1999 and 2005 and between 2005 and
2010 (both Plt001) NI also increased markedly with the inorganic treatment
equivalent to Treatment 5 (Tr 10) although the overall rate of increase shown by this
treatment was closer to that shown by Treatment 4 than Treatment 5 There were
significant differences between Treatments 5 and 10 in most years from 2004 onwards
(Plt001 and Plt0001 in 2009 and 2010 respectively)
These effects were reflected in a significant overall effect of form of fertilizer
(FYMgtinorganic P=0008) and highly significant (Plt0001) overall effects of both rate
(high rategtlower rate) and frequency of application (annualgttriennial) within the FRF
series ANOVA and a significant form x rate interaction (P=0043) Mean NI was
highest for high rate annual FYM (Plt0001 compared to all three remaining means) and
lowest for lower rate inorganic (Plt005 compared to high rate inorganic NS compared
to lower rate FYM) Only frequency of application interacted significantly with time
(Plt0001) NI increased moderately under triennial application until 2008 (Plt005 in
2005 compared to 1999 Plt0001 each year 2006-2008) followed by a decline (Plt001
2010 compared to 2008) but with mean NI in 2010 still higher than 1999 (Plt005)
The increasing trend was very much more marked with annual fertilizer application for
17
which there were highly significant (Plt0001) increments between 1999 and 2002 and
between 2002 and 2008 but with no significant increase thereafter Differences between
annual and triennial treatment means were highly significant (Plt0001) from 2003
onwards
There was no significant change in NI over time either under the untreated control
(Tr 1) or the nil fertilizer (Tr 2) treatments in the full treatment ANOVA NI increased
until 2005 on plots limed in 1999 without fertilizer Tr 13 (Plt001 compared to 1999)
and declined slightly but not significantly thereafter Trends were similar with the
treatment limed in 1999 and receiving triennial FYM (Tr 15 ndash not illustrated in Fig 2)
although NI was higher with this treatment than with Tr 13 (Plt005) in both 2005 and
2008 By contrast applying the same rate of FYM annually after liming in 1999 (Tr 14)
caused a more rapid increase with levels by 2005 higher than either Tr 13 (Plt001) or
Tr 4 (Plt005) ndash note that the latter was equivalent to Tr 14 but without lime until 2005
NI declined after 2006 under Tr 14 but continued to increase with Tr 4 with the
difference between these two treatments reversed by 2010 (Plt005)
All main effects and interactions were significant in the LFF treatment series ANOVA
for NI except for lime x FYM frequency and the lime x FYM x time interactions Plots
limed in 1999 supported higher NI levels than those limed in 2005 averaged over the
three FYM frequencies and all years (P=0042) NI increased more rapidly after liming
in 1999 than where liming was delayed until 2005 (P=0033 for time x lime interaction)
with the overall difference due to liming in 1999 highly significant (Plt0001) by 2005
NI declined thereafter on the 1999-limed treatments whilst continuing to increase
following liming in 2005 of previously un-limed treatments both peaking in 2008 at a
very similar level Averaged over lime treatments NI increased much more rapidly
under annual FYM application than under either triennial or nil FYM (P=0002 for the
time x FYM frequency interaction) a highly significant increase had occurred by 2002
with annual FYM (Plt0001 compared to 1999) but increases of similar magnitude were
not reached until 2005 with triennial application and not until 2007 with nil FYM NI
subsequently declined under the latter two FYM regimes but not with annual FYM so
that by 2010 NI was significantly higher with annual FYM than with either triennial or
nil FYM (Plt0001) but with only a small non-significant difference between the latter
two treatments
TREATMENT EFFECTS AFTER 12 YEARS OF APPLICATION
The effects of all treatments on the five key variables at each site in 2010 are shown in
Table S20 with treatments differing significantly (Plt005) from the limed control
treatment (Tr 2) highlighted for each variable Treatment effects in 2010 are not
described in detail here but the modeled responses of total species-richness and of
richness of PI species in 2010 to the mean amounts of N and P applied per year averaged
over 1999-2010 are described below
Relationships with the amounts of fertilizer applied
The effect of neither rate nor frequency of application was significant in any of the
REML models for either total species-richness or PI species-richness at either site when
the amount of N or P applied ha-1
year-1
was included The effect of form of fertilizer was
still significant in most models however either as a main effect or in interaction with the
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
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Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
Kluwer Academic Publishers Dordrecht
Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
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Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
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Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
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Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
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Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
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Management 15 137-143
Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
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Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
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Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
activity in botanically diverse haymeadows effects of litter type and fertilizer additions Soil
Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
12
fertilizer (Tr 10 565 per m2) which differed significantly (Plt005) from its FYM
counterpart Treatment 5 There was however no consistent difference between FYM
treatments and their inorganic counterparts and no overall effect of form of fertilizer
within the FRF series (P=0745) There was a significant form x rate interaction within
this ANOVA (P=0032) but only the difference between the high rate and lower rate
inorganic means was significant (high rategtlower rate Plt001)
The aggregate contribution to vegetation cover of negative indicator species (NI)
showed a very marked divergence between treatments over time (Fig 1d) with NI
either changing little or declining under most treatments again with the notable exception
of high rate annual FYM (Tr 5) under which NI increased over time Time treatment
and the treatment x time interaction were all highly significant (Plt0001) The
predominant temporal trend was downward (from 25 of live cover in 1999 to 14 in
2010 averaged over all treatments) but NI for Treatment 5 had significantly increased
compared to 1999 in 2002 2003 (Plt005) 2005 (Plt0001) 2008 (Plt0001) and 2009
(Plt001) but not in 2010 (Fig 1d) The most marked declines in NI occurred on the
limed control (Tr 2) nil fertilizer plus lime in both 1999 and 2005 (Tr 13) and the lower
rate triennial inorganic fertilizer treatments (Tr 11) differences compared to both 1999
and 2002 were highly significant (Plt0001) in most subsequent years for these
treatments A smaller decline also occurred after 2002 on the treatment equivalent to past
inputs (ie12 t ha-1
year-1
annual FYM Tr 4) In most years this treatment differed little
from the equivalent FYM treatment limed in both 1999 and 2005 (Tr 14)
These trends were reflected in highly significant overall effects on NI within the FRF
series of form (FYMgtinorganic) rate (mediumlthigh) and frequency (triennialltannual)
(all effects Plt0001) Time x rate and time x frequency interactions were both significant
(P=0019 and Plt0001 respectively) with a marginally non-significant rate x frequency
interaction averaged over all years (P=0054) NI declined over time under both
application rates averaged over forms and frequencies of application but the decline was
slower with high rate treatments compared to lower rate Difference between rates were
significant in all years from 2002 onwards (Plt005 in 2002 2006 and 2007 Plt0001 in
other years) Overall differences due to frequency of application were even more marked
Under annual application NI declined initially from 2003-2006 compared to 1999 but
subsequently recovered and did not differ from 1999 levels from 2007 onwards whilst
under triennial application differences were highly significant (Plt0001) compared to
1999 from 2003 onwards with significant differences between years during this same
period (Plt005 in 2003 and 2006 Plt0001 in remaining years)
Repeated-measures ANOVAs within the LFF treatment series at the upland meadow
showed no overall effect of liming regime on temporal trends for any of the above
variables nor any interaction of lime with either time or FYM frequency - note that FYM
was applied at the intermediate rate (12 t ha-1
) in all FYM treatments within this series
The overall effect of FYM frequency was significant for both PI (P=0003) and the
number of NI species m-2
(P=0001) PI was highest with nil FYM and lowest with
annual FYM with triennial intermediate and differing significantly (Plt005) from the
other two means NI species m-2
did not differ between triennial and annual application
within the LFF series but both were significantly higher than nil FYM (Plt001 for
triennial Plt0001 for annual application) There was also a significant time x FYM
frequency interaction for NI within this series (P=0001) reflecting a more rapid
13
decline with nil FYM than with triennial FYM and no significant overall trend with
annual FYM when averaged over both lime treatments
12
14
16
18
20
22
24
26
28
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Spe
cie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime Year 1 + ann FYM (Tr 14) Nil fert + Lime Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr 1)
a Species richness (total)
2
3
4
5
6
7
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
0
10
20
30
40
50
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
Fig 2 Changes 1999-2010 in vegetation composition at a lowland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control treatment and those that had received lime in year 1
Treatments 1 and 2 were identical (nil input) until 2005
The lowland meadow
Both time and treatment were significant within the ANOVA for total species-richness at
the lowland meadow (Plt0001 and P=0009 respectively and in contrast to the upland
site the treatment x time interaction was significant (P=0012) Averaged over all years
high rate annual FYM treatment (Tr 5) was significantly less species-rich than all others
except Treatment 14 (annual FYM at 12 t ha-1
limed in 1999) having declined
progressively between 1999 and 2009 (Fig 2a) with most differences significant at
Plt001 Treatment 14 did not differ significantly overall compared to the corresponding
FYM treatment not limed until 2005 (Tr 4) although these two treatments showed
14
markedly different temporal trends Treatment 14 was less species-rich than others during
the first five years of the experiment but did not change significantly throughout the
whole 12 year period whilst Treatment 4 changed little between 1999 and 2006 but
declined thereafter (Plt001 and Plt0001 for 2009 and 2010 respectively compared to
2006) possibly in response to liming in 2005 However there was no significant overall
effect on total species-richness of liming strategy within the LFF series and no interaction
between liming and either time or FYM application
There were marked differences in temporal trend in species-richness between FYM
treatments and their inorganic counterparts particularly with the high rate annual
treatments (Tr 5 and Tr 10) Whereas species-richness declined more or less continually
throughout under Treatment 5 at least until 2009 (Plt001 for 2004 compared to 1999 and
for both 2009 and 2010 compared to 2004) Treatment 10 declined between 2000 and
2001 (Plt005) and then remained relatively unchanged until 2008 after which it declined
again (2010 Plt005 compared to 2008)
Other treatments within the full treatment series including untreated control (Tr 1) and
limed control (Tr 2) showed no particular temporal trend in the full repeated measures
ANOVA although both the latter treatments showed a dip in species-richness in 2004
(Plt005 compared to both 2003 and 2005)
There was a strong overall effect of form of fertilizer within the FRF series ANOVA
(FYMltinorganic P=0003) a significant effect of frequency of application
(triennialgtannual P=0027) and a not quite significant effect of application rate (high
rateltmedium rate P=0064) but no significant interaction between any of these factors
The time x application frequency interaction was highly significant (Plt0001) species-
richness remained unchanged under triennial application but under annual application
was significantly lower compared to 1999 in each year from 2004 onwards with the
exception of 2006 (Plt005 in 2004 Plt001 in 2005 and 2008 Plt0001 the remainder)
Again with the exception of 2006 differences between triennial and annual application
were significant in each year from 2005 onwards (Plt005 in 2007 Plt001 in 2005 and
2008 Plt0001 in 2009 and 2010)
Both treatment time and their interaction were significant in the full treatment ANOVA
for PI species-richness at the lowland meadow (ie time and treatment both Plt0001
time x treatment P=0006) There was a general decline in PI-richness after 2005 (Fig
2b) with PI species m-2
averaged over all treatments lower from 2007 onwards (Plt0001)
than all years up to 2005 except 2001 Although the initiation of this trend corresponded
with the liming of most treatments in 2005 a similar pattern was shown by treatments
that did not receive this lime application ie the untreated control treatment (Tr 1) the
nil fertilizer treatment limed in 1999 (Tr 13) and the corresponding treatment also
receiving triennial FYM (Tr 15) In all these cases PI-richness showed no significant
difference between years until 2005 but was significantly (Plt001) lower by 2010 than in
2005 Treatment 14 (limed in 1999 plus annual FYM) did not show this pattern but PI
species-richness had been lowest under this treatment in most years between 1999 and
2005 (Fig 2b)
As with total species-richness PI richness declined more or less progressively under
Treatment 5 until 2008 (Plt005 compared to 1999 by 2001 Plt0001 from 2006
onwards) whereas Treatment 4 did not change significantly until 2006 after which it
declined (Plt005 in 2007 compared to 2006 Plt001 thereafter) Both these treatments
15
tended be less rich in PI species than the corresponding inorganic treatment (Trs 9 and
10 respectively) in most years consistently so for Tr 4 compared to Tr 9 but these
differences were not significant in any year nor when averaged over all years There was
however a significant overall effect of form of fertilizer in the FRF series ANOVA
(FYMltinorganic P=0002) and a highly significant effect of frequency of application
(triennialgtannual Plt0001) The time x frequency of application interaction was also
highly significant (Plt0001) Under triennial application PI species per m2 increased
slightly until 2005 (Plt005 compared to 1999) and declined thereafter so that by 2009
levels were significantly lower than in 1999 (Plt001 for 2009 Plt0001 for 2010) By
contrast under annual application PI species m-2
declined progressively until 2009
following a temporary though highly significant (Plt0001) dip in 2000 so that the
difference compared to 1999 was significant in every year from 2002 onwards and highly
significant (Plt0001) from 2005 With the exception of 2003 differences between
triennial and annual application were significant from 2002 onwards and highly
significant (Plt0001) in 2005 and from 2007 to 2010
There was no significant effect other than of time within the LFF treatment series
ANOVA although there was a marginally non-significant time x FYM frequency
interaction (P=0054) This reflected a more rapid decline in PI species-richness under
annual FYM application than with either nil or triennial FYM with the latter two showing
no discernible difference
By contrast with PI species m-2
PI cover percentage (PI) showed no overall treatment
effect within the full treatment ANOVA nor a time x treatment interaction although the
overall effect of time was highly significant (Plt0001) The temporal pattern in PI (Fig
2c) also differed from that shown by PI-richness with PI averaged over all treatments
declining between 1999 and 2005 in three highly significant increments (1999-2000
2000-2002 and 2002-2005 all Plt0001) and increasing again thereafter (2005-2009 and
2009-2010 both Plt0001) There was no significant effect within either the LFF or FRF
treatment series ANOVAs although their was a slight time x frequency of application
effect within the latter (P=0081) PI was slightly higher under annual application than
triennial in each year 1999-2004 after which the reverse was true although differences
were always small
There was no overall effect of treatment on the number of NI species per m2 within the
full treatment range at the lowland meadow (data not shown) but both the overall effect
of time and the time x treatment interaction were significant (Plt0001 and P=0009
respectively) Overall NI species m-2
increased over time from 24 species m-2
in 1999 to
a peak of 42 m-2
in 2008 averaged over all treatments NI species m-2
increased on the
untreated control treatment (Tr 1) from 21 species m-2
in 1999 to 34 species m-2
in 2007
(increase Plt001) then declined to the 1999 value by 2010 The limed (in 2005) control
(Tr 2) showed a somewhat similar pattern though from a slightly higher starting level
(31 species m-2
) and with the increase to 38 species m-2
in 2008 not reaching
significance
These temporal trends in NI species m-2
were much accentuated by annual fertilizer
application particularly by FYM treatments with peak numbers of NI species reaching
52 and 47 species m-2
for Treatments 4 and 5 respectively in 2008 (both Plt0001
compared to 1999) and despite slight declines in both cases remaining significantly
higher in 2010 than in 1999 (both Plt0001) Inorganic counterparts to these treatments
16
showed similar though slightly less marked trends and there was no significant
difference between all four treatments in the peak number reached in 2008 There was no
overall effect of either form of fertilizer or rate of application within the FRF series
ANOVA and only a marginally non-significant effect of frequency of application
(triennialltannual P=0061) Liming in 1999 (Trs 13 14 and 15) also appeared to
accentuate the generally increasing trend although peak numbers in 2008 did not differ
between these treatments and either untreated or limed control in the full treatment range
ANOVA The overall effects of liming regime (1999 or 2005) were not significant within
the LFF treatment series but the faster rate of increase with liming in 1999 was reflected
in a significant time x lime interaction in this analysis (P=0017) The mean number of NI
species m-2
increased very significantly (Plt0001) between 1999 and 2000 following
liming in 1999 with the increment increasing until 2008 whilst on plots un-limed until
2005 NI species m-2
did not increase significantly until 2003 (Plt005) although the
increase compared to 1999 was highly significant (Plt0001) in 2006 2008 and 2010
Nevertheless the differences in rate of increase was at least partly due to a lower 1999
mean for the 1999-limed treatments (170 species m-2
compared to 244 species m-2
difference NS) and although in all years from 2001 onwards mean NI species m-2
was
higher for 1999-limed treatments compared to those not limed until 2005 the difference
was not significant in any year
The contribution of NI species to vegetation cover (NI) showed a very marked
separation between treatments over time at the lowland meadow (Fig 2d) starting from
much lower levels than at the upland meadow (compare with Fig 1d) There were
noticeable differences in temporal trend between annual fertilizer treatments on the one
hand and triennial and nil fertilizer treatments on the other (Fig 2d) The effects of
treatment time and the treatment x time interaction were all highly significant (Plt0001)
in the full treatment range ANOVA The most marked increase in NI occurred with
annual FYM at the high rate (Tr 5) with NI increasing rapidly between 1999 and 2001
(Plt001) and a further highly significant increment between 2004 and 2009 (Plt0001)
(Fig 2d) A similar though less marked trend occurred with annual FYM at the medium
rate (Tr 4) with significant increases between 1999 and 2005 and between 2005 and
2010 (both Plt001) NI also increased markedly with the inorganic treatment
equivalent to Treatment 5 (Tr 10) although the overall rate of increase shown by this
treatment was closer to that shown by Treatment 4 than Treatment 5 There were
significant differences between Treatments 5 and 10 in most years from 2004 onwards
(Plt001 and Plt0001 in 2009 and 2010 respectively)
These effects were reflected in a significant overall effect of form of fertilizer
(FYMgtinorganic P=0008) and highly significant (Plt0001) overall effects of both rate
(high rategtlower rate) and frequency of application (annualgttriennial) within the FRF
series ANOVA and a significant form x rate interaction (P=0043) Mean NI was
highest for high rate annual FYM (Plt0001 compared to all three remaining means) and
lowest for lower rate inorganic (Plt005 compared to high rate inorganic NS compared
to lower rate FYM) Only frequency of application interacted significantly with time
(Plt0001) NI increased moderately under triennial application until 2008 (Plt005 in
2005 compared to 1999 Plt0001 each year 2006-2008) followed by a decline (Plt001
2010 compared to 2008) but with mean NI in 2010 still higher than 1999 (Plt005)
The increasing trend was very much more marked with annual fertilizer application for
17
which there were highly significant (Plt0001) increments between 1999 and 2002 and
between 2002 and 2008 but with no significant increase thereafter Differences between
annual and triennial treatment means were highly significant (Plt0001) from 2003
onwards
There was no significant change in NI over time either under the untreated control
(Tr 1) or the nil fertilizer (Tr 2) treatments in the full treatment ANOVA NI increased
until 2005 on plots limed in 1999 without fertilizer Tr 13 (Plt001 compared to 1999)
and declined slightly but not significantly thereafter Trends were similar with the
treatment limed in 1999 and receiving triennial FYM (Tr 15 ndash not illustrated in Fig 2)
although NI was higher with this treatment than with Tr 13 (Plt005) in both 2005 and
2008 By contrast applying the same rate of FYM annually after liming in 1999 (Tr 14)
caused a more rapid increase with levels by 2005 higher than either Tr 13 (Plt001) or
Tr 4 (Plt005) ndash note that the latter was equivalent to Tr 14 but without lime until 2005
NI declined after 2006 under Tr 14 but continued to increase with Tr 4 with the
difference between these two treatments reversed by 2010 (Plt005)
All main effects and interactions were significant in the LFF treatment series ANOVA
for NI except for lime x FYM frequency and the lime x FYM x time interactions Plots
limed in 1999 supported higher NI levels than those limed in 2005 averaged over the
three FYM frequencies and all years (P=0042) NI increased more rapidly after liming
in 1999 than where liming was delayed until 2005 (P=0033 for time x lime interaction)
with the overall difference due to liming in 1999 highly significant (Plt0001) by 2005
NI declined thereafter on the 1999-limed treatments whilst continuing to increase
following liming in 2005 of previously un-limed treatments both peaking in 2008 at a
very similar level Averaged over lime treatments NI increased much more rapidly
under annual FYM application than under either triennial or nil FYM (P=0002 for the
time x FYM frequency interaction) a highly significant increase had occurred by 2002
with annual FYM (Plt0001 compared to 1999) but increases of similar magnitude were
not reached until 2005 with triennial application and not until 2007 with nil FYM NI
subsequently declined under the latter two FYM regimes but not with annual FYM so
that by 2010 NI was significantly higher with annual FYM than with either triennial or
nil FYM (Plt0001) but with only a small non-significant difference between the latter
two treatments
TREATMENT EFFECTS AFTER 12 YEARS OF APPLICATION
The effects of all treatments on the five key variables at each site in 2010 are shown in
Table S20 with treatments differing significantly (Plt005) from the limed control
treatment (Tr 2) highlighted for each variable Treatment effects in 2010 are not
described in detail here but the modeled responses of total species-richness and of
richness of PI species in 2010 to the mean amounts of N and P applied per year averaged
over 1999-2010 are described below
Relationships with the amounts of fertilizer applied
The effect of neither rate nor frequency of application was significant in any of the
REML models for either total species-richness or PI species-richness at either site when
the amount of N or P applied ha-1
year-1
was included The effect of form of fertilizer was
still significant in most models however either as a main effect or in interaction with the
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
References
Allen SE (1974) Chemical Analysis of Ecological Materials Blackwell Oxford UK
Anon (2010) Fertilizer Manual (RB 209) 8th Edition Her Majestyrsquos Stationery Office London
Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
Kluwer Academic Publishers Dordrecht
Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
restoration of species-rich meadows in the Netherlands Biological Conservation 62 59-65
Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
as an indicator of ecosystem self-regulation in temperate meadow grasslands Biology and
Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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and Fertility of Soils 22 261-264
Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
Hopkins JJ (1999) The extent of semi-natural grassland communities in lowland England
and Wales a review of conservation surveys 1978-96 Grass and Forage Science 54 1-18
Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
phosphorous vs nitrogen enrichment in plant species loss across North-west Europen
grasslands Journal of Applied Ecology 48 1155-1163
Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
and losses following application of manures to arable land MANNER Soil Use and
Management 15 137-143
Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
(2002a) Plant species-richness functional type and soil properties in grasslands and allied
vegetation in English Environmentally Sensitive Areas Grass Forage Science 57 82-92
Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
2002b) Association between lowland grassland plant communities and soil properties
Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
activity in botanically diverse haymeadows effects of litter type and fertilizer additions Soil
Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
13
decline with nil FYM than with triennial FYM and no significant overall trend with
annual FYM when averaged over both lime treatments
12
14
16
18
20
22
24
26
28
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
Spe
cie
s p
er
m2
12 t FYMha annual (Tr 4) 24 t FYMha annual (Tr 5) Inor equiv 12 t annual (Tr 9)
Inor equiv 24 t annual (Tr 10) 12 t FYMha 3-yearly (Tr 7) Inor equiv 12 t 3-yearly (Tr 11)
Lime Year 1 + ann FYM (Tr 14) Nil fert + Lime Year 1 (Tr 13) Limed control (Tr 2)
Untreated control (Tr 1)
a Species richness (total)
2
3
4
5
6
7
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s p
er
m2
b Positive indicator species richness
0
10
20
30
40
50
60
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
NI
sp
ecie
s
of liv
e c
over
d Negative indicator species cover
0
10
20
30
40
50
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year
PI sp
ecie
s
of liv
e c
ove
r
c Positive indicator species cover
Fig 2 Changes 1999-2010 in vegetation composition at a lowland meadow as influenced by
fertilizer and lime treatments (selected treatments) Note that all treatments received lime in year
7 (2005) except for the untreated control treatment and those that had received lime in year 1
Treatments 1 and 2 were identical (nil input) until 2005
The lowland meadow
Both time and treatment were significant within the ANOVA for total species-richness at
the lowland meadow (Plt0001 and P=0009 respectively and in contrast to the upland
site the treatment x time interaction was significant (P=0012) Averaged over all years
high rate annual FYM treatment (Tr 5) was significantly less species-rich than all others
except Treatment 14 (annual FYM at 12 t ha-1
limed in 1999) having declined
progressively between 1999 and 2009 (Fig 2a) with most differences significant at
Plt001 Treatment 14 did not differ significantly overall compared to the corresponding
FYM treatment not limed until 2005 (Tr 4) although these two treatments showed
14
markedly different temporal trends Treatment 14 was less species-rich than others during
the first five years of the experiment but did not change significantly throughout the
whole 12 year period whilst Treatment 4 changed little between 1999 and 2006 but
declined thereafter (Plt001 and Plt0001 for 2009 and 2010 respectively compared to
2006) possibly in response to liming in 2005 However there was no significant overall
effect on total species-richness of liming strategy within the LFF series and no interaction
between liming and either time or FYM application
There were marked differences in temporal trend in species-richness between FYM
treatments and their inorganic counterparts particularly with the high rate annual
treatments (Tr 5 and Tr 10) Whereas species-richness declined more or less continually
throughout under Treatment 5 at least until 2009 (Plt001 for 2004 compared to 1999 and
for both 2009 and 2010 compared to 2004) Treatment 10 declined between 2000 and
2001 (Plt005) and then remained relatively unchanged until 2008 after which it declined
again (2010 Plt005 compared to 2008)
Other treatments within the full treatment series including untreated control (Tr 1) and
limed control (Tr 2) showed no particular temporal trend in the full repeated measures
ANOVA although both the latter treatments showed a dip in species-richness in 2004
(Plt005 compared to both 2003 and 2005)
There was a strong overall effect of form of fertilizer within the FRF series ANOVA
(FYMltinorganic P=0003) a significant effect of frequency of application
(triennialgtannual P=0027) and a not quite significant effect of application rate (high
rateltmedium rate P=0064) but no significant interaction between any of these factors
The time x application frequency interaction was highly significant (Plt0001) species-
richness remained unchanged under triennial application but under annual application
was significantly lower compared to 1999 in each year from 2004 onwards with the
exception of 2006 (Plt005 in 2004 Plt001 in 2005 and 2008 Plt0001 the remainder)
Again with the exception of 2006 differences between triennial and annual application
were significant in each year from 2005 onwards (Plt005 in 2007 Plt001 in 2005 and
2008 Plt0001 in 2009 and 2010)
Both treatment time and their interaction were significant in the full treatment ANOVA
for PI species-richness at the lowland meadow (ie time and treatment both Plt0001
time x treatment P=0006) There was a general decline in PI-richness after 2005 (Fig
2b) with PI species m-2
averaged over all treatments lower from 2007 onwards (Plt0001)
than all years up to 2005 except 2001 Although the initiation of this trend corresponded
with the liming of most treatments in 2005 a similar pattern was shown by treatments
that did not receive this lime application ie the untreated control treatment (Tr 1) the
nil fertilizer treatment limed in 1999 (Tr 13) and the corresponding treatment also
receiving triennial FYM (Tr 15) In all these cases PI-richness showed no significant
difference between years until 2005 but was significantly (Plt001) lower by 2010 than in
2005 Treatment 14 (limed in 1999 plus annual FYM) did not show this pattern but PI
species-richness had been lowest under this treatment in most years between 1999 and
2005 (Fig 2b)
As with total species-richness PI richness declined more or less progressively under
Treatment 5 until 2008 (Plt005 compared to 1999 by 2001 Plt0001 from 2006
onwards) whereas Treatment 4 did not change significantly until 2006 after which it
declined (Plt005 in 2007 compared to 2006 Plt001 thereafter) Both these treatments
15
tended be less rich in PI species than the corresponding inorganic treatment (Trs 9 and
10 respectively) in most years consistently so for Tr 4 compared to Tr 9 but these
differences were not significant in any year nor when averaged over all years There was
however a significant overall effect of form of fertilizer in the FRF series ANOVA
(FYMltinorganic P=0002) and a highly significant effect of frequency of application
(triennialgtannual Plt0001) The time x frequency of application interaction was also
highly significant (Plt0001) Under triennial application PI species per m2 increased
slightly until 2005 (Plt005 compared to 1999) and declined thereafter so that by 2009
levels were significantly lower than in 1999 (Plt001 for 2009 Plt0001 for 2010) By
contrast under annual application PI species m-2
declined progressively until 2009
following a temporary though highly significant (Plt0001) dip in 2000 so that the
difference compared to 1999 was significant in every year from 2002 onwards and highly
significant (Plt0001) from 2005 With the exception of 2003 differences between
triennial and annual application were significant from 2002 onwards and highly
significant (Plt0001) in 2005 and from 2007 to 2010
There was no significant effect other than of time within the LFF treatment series
ANOVA although there was a marginally non-significant time x FYM frequency
interaction (P=0054) This reflected a more rapid decline in PI species-richness under
annual FYM application than with either nil or triennial FYM with the latter two showing
no discernible difference
By contrast with PI species m-2
PI cover percentage (PI) showed no overall treatment
effect within the full treatment ANOVA nor a time x treatment interaction although the
overall effect of time was highly significant (Plt0001) The temporal pattern in PI (Fig
2c) also differed from that shown by PI-richness with PI averaged over all treatments
declining between 1999 and 2005 in three highly significant increments (1999-2000
2000-2002 and 2002-2005 all Plt0001) and increasing again thereafter (2005-2009 and
2009-2010 both Plt0001) There was no significant effect within either the LFF or FRF
treatment series ANOVAs although their was a slight time x frequency of application
effect within the latter (P=0081) PI was slightly higher under annual application than
triennial in each year 1999-2004 after which the reverse was true although differences
were always small
There was no overall effect of treatment on the number of NI species per m2 within the
full treatment range at the lowland meadow (data not shown) but both the overall effect
of time and the time x treatment interaction were significant (Plt0001 and P=0009
respectively) Overall NI species m-2
increased over time from 24 species m-2
in 1999 to
a peak of 42 m-2
in 2008 averaged over all treatments NI species m-2
increased on the
untreated control treatment (Tr 1) from 21 species m-2
in 1999 to 34 species m-2
in 2007
(increase Plt001) then declined to the 1999 value by 2010 The limed (in 2005) control
(Tr 2) showed a somewhat similar pattern though from a slightly higher starting level
(31 species m-2
) and with the increase to 38 species m-2
in 2008 not reaching
significance
These temporal trends in NI species m-2
were much accentuated by annual fertilizer
application particularly by FYM treatments with peak numbers of NI species reaching
52 and 47 species m-2
for Treatments 4 and 5 respectively in 2008 (both Plt0001
compared to 1999) and despite slight declines in both cases remaining significantly
higher in 2010 than in 1999 (both Plt0001) Inorganic counterparts to these treatments
16
showed similar though slightly less marked trends and there was no significant
difference between all four treatments in the peak number reached in 2008 There was no
overall effect of either form of fertilizer or rate of application within the FRF series
ANOVA and only a marginally non-significant effect of frequency of application
(triennialltannual P=0061) Liming in 1999 (Trs 13 14 and 15) also appeared to
accentuate the generally increasing trend although peak numbers in 2008 did not differ
between these treatments and either untreated or limed control in the full treatment range
ANOVA The overall effects of liming regime (1999 or 2005) were not significant within
the LFF treatment series but the faster rate of increase with liming in 1999 was reflected
in a significant time x lime interaction in this analysis (P=0017) The mean number of NI
species m-2
increased very significantly (Plt0001) between 1999 and 2000 following
liming in 1999 with the increment increasing until 2008 whilst on plots un-limed until
2005 NI species m-2
did not increase significantly until 2003 (Plt005) although the
increase compared to 1999 was highly significant (Plt0001) in 2006 2008 and 2010
Nevertheless the differences in rate of increase was at least partly due to a lower 1999
mean for the 1999-limed treatments (170 species m-2
compared to 244 species m-2
difference NS) and although in all years from 2001 onwards mean NI species m-2
was
higher for 1999-limed treatments compared to those not limed until 2005 the difference
was not significant in any year
The contribution of NI species to vegetation cover (NI) showed a very marked
separation between treatments over time at the lowland meadow (Fig 2d) starting from
much lower levels than at the upland meadow (compare with Fig 1d) There were
noticeable differences in temporal trend between annual fertilizer treatments on the one
hand and triennial and nil fertilizer treatments on the other (Fig 2d) The effects of
treatment time and the treatment x time interaction were all highly significant (Plt0001)
in the full treatment range ANOVA The most marked increase in NI occurred with
annual FYM at the high rate (Tr 5) with NI increasing rapidly between 1999 and 2001
(Plt001) and a further highly significant increment between 2004 and 2009 (Plt0001)
(Fig 2d) A similar though less marked trend occurred with annual FYM at the medium
rate (Tr 4) with significant increases between 1999 and 2005 and between 2005 and
2010 (both Plt001) NI also increased markedly with the inorganic treatment
equivalent to Treatment 5 (Tr 10) although the overall rate of increase shown by this
treatment was closer to that shown by Treatment 4 than Treatment 5 There were
significant differences between Treatments 5 and 10 in most years from 2004 onwards
(Plt001 and Plt0001 in 2009 and 2010 respectively)
These effects were reflected in a significant overall effect of form of fertilizer
(FYMgtinorganic P=0008) and highly significant (Plt0001) overall effects of both rate
(high rategtlower rate) and frequency of application (annualgttriennial) within the FRF
series ANOVA and a significant form x rate interaction (P=0043) Mean NI was
highest for high rate annual FYM (Plt0001 compared to all three remaining means) and
lowest for lower rate inorganic (Plt005 compared to high rate inorganic NS compared
to lower rate FYM) Only frequency of application interacted significantly with time
(Plt0001) NI increased moderately under triennial application until 2008 (Plt005 in
2005 compared to 1999 Plt0001 each year 2006-2008) followed by a decline (Plt001
2010 compared to 2008) but with mean NI in 2010 still higher than 1999 (Plt005)
The increasing trend was very much more marked with annual fertilizer application for
17
which there were highly significant (Plt0001) increments between 1999 and 2002 and
between 2002 and 2008 but with no significant increase thereafter Differences between
annual and triennial treatment means were highly significant (Plt0001) from 2003
onwards
There was no significant change in NI over time either under the untreated control
(Tr 1) or the nil fertilizer (Tr 2) treatments in the full treatment ANOVA NI increased
until 2005 on plots limed in 1999 without fertilizer Tr 13 (Plt001 compared to 1999)
and declined slightly but not significantly thereafter Trends were similar with the
treatment limed in 1999 and receiving triennial FYM (Tr 15 ndash not illustrated in Fig 2)
although NI was higher with this treatment than with Tr 13 (Plt005) in both 2005 and
2008 By contrast applying the same rate of FYM annually after liming in 1999 (Tr 14)
caused a more rapid increase with levels by 2005 higher than either Tr 13 (Plt001) or
Tr 4 (Plt005) ndash note that the latter was equivalent to Tr 14 but without lime until 2005
NI declined after 2006 under Tr 14 but continued to increase with Tr 4 with the
difference between these two treatments reversed by 2010 (Plt005)
All main effects and interactions were significant in the LFF treatment series ANOVA
for NI except for lime x FYM frequency and the lime x FYM x time interactions Plots
limed in 1999 supported higher NI levels than those limed in 2005 averaged over the
three FYM frequencies and all years (P=0042) NI increased more rapidly after liming
in 1999 than where liming was delayed until 2005 (P=0033 for time x lime interaction)
with the overall difference due to liming in 1999 highly significant (Plt0001) by 2005
NI declined thereafter on the 1999-limed treatments whilst continuing to increase
following liming in 2005 of previously un-limed treatments both peaking in 2008 at a
very similar level Averaged over lime treatments NI increased much more rapidly
under annual FYM application than under either triennial or nil FYM (P=0002 for the
time x FYM frequency interaction) a highly significant increase had occurred by 2002
with annual FYM (Plt0001 compared to 1999) but increases of similar magnitude were
not reached until 2005 with triennial application and not until 2007 with nil FYM NI
subsequently declined under the latter two FYM regimes but not with annual FYM so
that by 2010 NI was significantly higher with annual FYM than with either triennial or
nil FYM (Plt0001) but with only a small non-significant difference between the latter
two treatments
TREATMENT EFFECTS AFTER 12 YEARS OF APPLICATION
The effects of all treatments on the five key variables at each site in 2010 are shown in
Table S20 with treatments differing significantly (Plt005) from the limed control
treatment (Tr 2) highlighted for each variable Treatment effects in 2010 are not
described in detail here but the modeled responses of total species-richness and of
richness of PI species in 2010 to the mean amounts of N and P applied per year averaged
over 1999-2010 are described below
Relationships with the amounts of fertilizer applied
The effect of neither rate nor frequency of application was significant in any of the
REML models for either total species-richness or PI species-richness at either site when
the amount of N or P applied ha-1
year-1
was included The effect of form of fertilizer was
still significant in most models however either as a main effect or in interaction with the
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
References
Allen SE (1974) Chemical Analysis of Ecological Materials Blackwell Oxford UK
Anon (2010) Fertilizer Manual (RB 209) 8th Edition Her Majestyrsquos Stationery Office London
Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
Kluwer Academic Publishers Dordrecht
Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
restoration of species-rich meadows in the Netherlands Biological Conservation 62 59-65
Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
as an indicator of ecosystem self-regulation in temperate meadow grasslands Biology and
Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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and Fertility of Soils 22 261-264
Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
Hopkins JJ (1999) The extent of semi-natural grassland communities in lowland England
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Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
phosphorous vs nitrogen enrichment in plant species loss across North-west Europen
grasslands Journal of Applied Ecology 48 1155-1163
Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
and losses following application of manures to arable land MANNER Soil Use and
Management 15 137-143
Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
(2002a) Plant species-richness functional type and soil properties in grasslands and allied
vegetation in English Environmentally Sensitive Areas Grass Forage Science 57 82-92
Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
2002b) Association between lowland grassland plant communities and soil properties
Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
activity in botanically diverse haymeadows effects of litter type and fertilizer additions Soil
Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
14
markedly different temporal trends Treatment 14 was less species-rich than others during
the first five years of the experiment but did not change significantly throughout the
whole 12 year period whilst Treatment 4 changed little between 1999 and 2006 but
declined thereafter (Plt001 and Plt0001 for 2009 and 2010 respectively compared to
2006) possibly in response to liming in 2005 However there was no significant overall
effect on total species-richness of liming strategy within the LFF series and no interaction
between liming and either time or FYM application
There were marked differences in temporal trend in species-richness between FYM
treatments and their inorganic counterparts particularly with the high rate annual
treatments (Tr 5 and Tr 10) Whereas species-richness declined more or less continually
throughout under Treatment 5 at least until 2009 (Plt001 for 2004 compared to 1999 and
for both 2009 and 2010 compared to 2004) Treatment 10 declined between 2000 and
2001 (Plt005) and then remained relatively unchanged until 2008 after which it declined
again (2010 Plt005 compared to 2008)
Other treatments within the full treatment series including untreated control (Tr 1) and
limed control (Tr 2) showed no particular temporal trend in the full repeated measures
ANOVA although both the latter treatments showed a dip in species-richness in 2004
(Plt005 compared to both 2003 and 2005)
There was a strong overall effect of form of fertilizer within the FRF series ANOVA
(FYMltinorganic P=0003) a significant effect of frequency of application
(triennialgtannual P=0027) and a not quite significant effect of application rate (high
rateltmedium rate P=0064) but no significant interaction between any of these factors
The time x application frequency interaction was highly significant (Plt0001) species-
richness remained unchanged under triennial application but under annual application
was significantly lower compared to 1999 in each year from 2004 onwards with the
exception of 2006 (Plt005 in 2004 Plt001 in 2005 and 2008 Plt0001 the remainder)
Again with the exception of 2006 differences between triennial and annual application
were significant in each year from 2005 onwards (Plt005 in 2007 Plt001 in 2005 and
2008 Plt0001 in 2009 and 2010)
Both treatment time and their interaction were significant in the full treatment ANOVA
for PI species-richness at the lowland meadow (ie time and treatment both Plt0001
time x treatment P=0006) There was a general decline in PI-richness after 2005 (Fig
2b) with PI species m-2
averaged over all treatments lower from 2007 onwards (Plt0001)
than all years up to 2005 except 2001 Although the initiation of this trend corresponded
with the liming of most treatments in 2005 a similar pattern was shown by treatments
that did not receive this lime application ie the untreated control treatment (Tr 1) the
nil fertilizer treatment limed in 1999 (Tr 13) and the corresponding treatment also
receiving triennial FYM (Tr 15) In all these cases PI-richness showed no significant
difference between years until 2005 but was significantly (Plt001) lower by 2010 than in
2005 Treatment 14 (limed in 1999 plus annual FYM) did not show this pattern but PI
species-richness had been lowest under this treatment in most years between 1999 and
2005 (Fig 2b)
As with total species-richness PI richness declined more or less progressively under
Treatment 5 until 2008 (Plt005 compared to 1999 by 2001 Plt0001 from 2006
onwards) whereas Treatment 4 did not change significantly until 2006 after which it
declined (Plt005 in 2007 compared to 2006 Plt001 thereafter) Both these treatments
15
tended be less rich in PI species than the corresponding inorganic treatment (Trs 9 and
10 respectively) in most years consistently so for Tr 4 compared to Tr 9 but these
differences were not significant in any year nor when averaged over all years There was
however a significant overall effect of form of fertilizer in the FRF series ANOVA
(FYMltinorganic P=0002) and a highly significant effect of frequency of application
(triennialgtannual Plt0001) The time x frequency of application interaction was also
highly significant (Plt0001) Under triennial application PI species per m2 increased
slightly until 2005 (Plt005 compared to 1999) and declined thereafter so that by 2009
levels were significantly lower than in 1999 (Plt001 for 2009 Plt0001 for 2010) By
contrast under annual application PI species m-2
declined progressively until 2009
following a temporary though highly significant (Plt0001) dip in 2000 so that the
difference compared to 1999 was significant in every year from 2002 onwards and highly
significant (Plt0001) from 2005 With the exception of 2003 differences between
triennial and annual application were significant from 2002 onwards and highly
significant (Plt0001) in 2005 and from 2007 to 2010
There was no significant effect other than of time within the LFF treatment series
ANOVA although there was a marginally non-significant time x FYM frequency
interaction (P=0054) This reflected a more rapid decline in PI species-richness under
annual FYM application than with either nil or triennial FYM with the latter two showing
no discernible difference
By contrast with PI species m-2
PI cover percentage (PI) showed no overall treatment
effect within the full treatment ANOVA nor a time x treatment interaction although the
overall effect of time was highly significant (Plt0001) The temporal pattern in PI (Fig
2c) also differed from that shown by PI-richness with PI averaged over all treatments
declining between 1999 and 2005 in three highly significant increments (1999-2000
2000-2002 and 2002-2005 all Plt0001) and increasing again thereafter (2005-2009 and
2009-2010 both Plt0001) There was no significant effect within either the LFF or FRF
treatment series ANOVAs although their was a slight time x frequency of application
effect within the latter (P=0081) PI was slightly higher under annual application than
triennial in each year 1999-2004 after which the reverse was true although differences
were always small
There was no overall effect of treatment on the number of NI species per m2 within the
full treatment range at the lowland meadow (data not shown) but both the overall effect
of time and the time x treatment interaction were significant (Plt0001 and P=0009
respectively) Overall NI species m-2
increased over time from 24 species m-2
in 1999 to
a peak of 42 m-2
in 2008 averaged over all treatments NI species m-2
increased on the
untreated control treatment (Tr 1) from 21 species m-2
in 1999 to 34 species m-2
in 2007
(increase Plt001) then declined to the 1999 value by 2010 The limed (in 2005) control
(Tr 2) showed a somewhat similar pattern though from a slightly higher starting level
(31 species m-2
) and with the increase to 38 species m-2
in 2008 not reaching
significance
These temporal trends in NI species m-2
were much accentuated by annual fertilizer
application particularly by FYM treatments with peak numbers of NI species reaching
52 and 47 species m-2
for Treatments 4 and 5 respectively in 2008 (both Plt0001
compared to 1999) and despite slight declines in both cases remaining significantly
higher in 2010 than in 1999 (both Plt0001) Inorganic counterparts to these treatments
16
showed similar though slightly less marked trends and there was no significant
difference between all four treatments in the peak number reached in 2008 There was no
overall effect of either form of fertilizer or rate of application within the FRF series
ANOVA and only a marginally non-significant effect of frequency of application
(triennialltannual P=0061) Liming in 1999 (Trs 13 14 and 15) also appeared to
accentuate the generally increasing trend although peak numbers in 2008 did not differ
between these treatments and either untreated or limed control in the full treatment range
ANOVA The overall effects of liming regime (1999 or 2005) were not significant within
the LFF treatment series but the faster rate of increase with liming in 1999 was reflected
in a significant time x lime interaction in this analysis (P=0017) The mean number of NI
species m-2
increased very significantly (Plt0001) between 1999 and 2000 following
liming in 1999 with the increment increasing until 2008 whilst on plots un-limed until
2005 NI species m-2
did not increase significantly until 2003 (Plt005) although the
increase compared to 1999 was highly significant (Plt0001) in 2006 2008 and 2010
Nevertheless the differences in rate of increase was at least partly due to a lower 1999
mean for the 1999-limed treatments (170 species m-2
compared to 244 species m-2
difference NS) and although in all years from 2001 onwards mean NI species m-2
was
higher for 1999-limed treatments compared to those not limed until 2005 the difference
was not significant in any year
The contribution of NI species to vegetation cover (NI) showed a very marked
separation between treatments over time at the lowland meadow (Fig 2d) starting from
much lower levels than at the upland meadow (compare with Fig 1d) There were
noticeable differences in temporal trend between annual fertilizer treatments on the one
hand and triennial and nil fertilizer treatments on the other (Fig 2d) The effects of
treatment time and the treatment x time interaction were all highly significant (Plt0001)
in the full treatment range ANOVA The most marked increase in NI occurred with
annual FYM at the high rate (Tr 5) with NI increasing rapidly between 1999 and 2001
(Plt001) and a further highly significant increment between 2004 and 2009 (Plt0001)
(Fig 2d) A similar though less marked trend occurred with annual FYM at the medium
rate (Tr 4) with significant increases between 1999 and 2005 and between 2005 and
2010 (both Plt001) NI also increased markedly with the inorganic treatment
equivalent to Treatment 5 (Tr 10) although the overall rate of increase shown by this
treatment was closer to that shown by Treatment 4 than Treatment 5 There were
significant differences between Treatments 5 and 10 in most years from 2004 onwards
(Plt001 and Plt0001 in 2009 and 2010 respectively)
These effects were reflected in a significant overall effect of form of fertilizer
(FYMgtinorganic P=0008) and highly significant (Plt0001) overall effects of both rate
(high rategtlower rate) and frequency of application (annualgttriennial) within the FRF
series ANOVA and a significant form x rate interaction (P=0043) Mean NI was
highest for high rate annual FYM (Plt0001 compared to all three remaining means) and
lowest for lower rate inorganic (Plt005 compared to high rate inorganic NS compared
to lower rate FYM) Only frequency of application interacted significantly with time
(Plt0001) NI increased moderately under triennial application until 2008 (Plt005 in
2005 compared to 1999 Plt0001 each year 2006-2008) followed by a decline (Plt001
2010 compared to 2008) but with mean NI in 2010 still higher than 1999 (Plt005)
The increasing trend was very much more marked with annual fertilizer application for
17
which there were highly significant (Plt0001) increments between 1999 and 2002 and
between 2002 and 2008 but with no significant increase thereafter Differences between
annual and triennial treatment means were highly significant (Plt0001) from 2003
onwards
There was no significant change in NI over time either under the untreated control
(Tr 1) or the nil fertilizer (Tr 2) treatments in the full treatment ANOVA NI increased
until 2005 on plots limed in 1999 without fertilizer Tr 13 (Plt001 compared to 1999)
and declined slightly but not significantly thereafter Trends were similar with the
treatment limed in 1999 and receiving triennial FYM (Tr 15 ndash not illustrated in Fig 2)
although NI was higher with this treatment than with Tr 13 (Plt005) in both 2005 and
2008 By contrast applying the same rate of FYM annually after liming in 1999 (Tr 14)
caused a more rapid increase with levels by 2005 higher than either Tr 13 (Plt001) or
Tr 4 (Plt005) ndash note that the latter was equivalent to Tr 14 but without lime until 2005
NI declined after 2006 under Tr 14 but continued to increase with Tr 4 with the
difference between these two treatments reversed by 2010 (Plt005)
All main effects and interactions were significant in the LFF treatment series ANOVA
for NI except for lime x FYM frequency and the lime x FYM x time interactions Plots
limed in 1999 supported higher NI levels than those limed in 2005 averaged over the
three FYM frequencies and all years (P=0042) NI increased more rapidly after liming
in 1999 than where liming was delayed until 2005 (P=0033 for time x lime interaction)
with the overall difference due to liming in 1999 highly significant (Plt0001) by 2005
NI declined thereafter on the 1999-limed treatments whilst continuing to increase
following liming in 2005 of previously un-limed treatments both peaking in 2008 at a
very similar level Averaged over lime treatments NI increased much more rapidly
under annual FYM application than under either triennial or nil FYM (P=0002 for the
time x FYM frequency interaction) a highly significant increase had occurred by 2002
with annual FYM (Plt0001 compared to 1999) but increases of similar magnitude were
not reached until 2005 with triennial application and not until 2007 with nil FYM NI
subsequently declined under the latter two FYM regimes but not with annual FYM so
that by 2010 NI was significantly higher with annual FYM than with either triennial or
nil FYM (Plt0001) but with only a small non-significant difference between the latter
two treatments
TREATMENT EFFECTS AFTER 12 YEARS OF APPLICATION
The effects of all treatments on the five key variables at each site in 2010 are shown in
Table S20 with treatments differing significantly (Plt005) from the limed control
treatment (Tr 2) highlighted for each variable Treatment effects in 2010 are not
described in detail here but the modeled responses of total species-richness and of
richness of PI species in 2010 to the mean amounts of N and P applied per year averaged
over 1999-2010 are described below
Relationships with the amounts of fertilizer applied
The effect of neither rate nor frequency of application was significant in any of the
REML models for either total species-richness or PI species-richness at either site when
the amount of N or P applied ha-1
year-1
was included The effect of form of fertilizer was
still significant in most models however either as a main effect or in interaction with the
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
References
Allen SE (1974) Chemical Analysis of Ecological Materials Blackwell Oxford UK
Anon (2010) Fertilizer Manual (RB 209) 8th Edition Her Majestyrsquos Stationery Office London
Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
Kluwer Academic Publishers Dordrecht
Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
restoration of species-rich meadows in the Netherlands Biological Conservation 62 59-65
Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
as an indicator of ecosystem self-regulation in temperate meadow grasslands Biology and
Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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and Fertility of Soils 22 261-264
Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
Hopkins JJ (1999) The extent of semi-natural grassland communities in lowland England
and Wales a review of conservation surveys 1978-96 Grass and Forage Science 54 1-18
Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
phosphorous vs nitrogen enrichment in plant species loss across North-west Europen
grasslands Journal of Applied Ecology 48 1155-1163
Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
and losses following application of manures to arable land MANNER Soil Use and
Management 15 137-143
Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
(2002a) Plant species-richness functional type and soil properties in grasslands and allied
vegetation in English Environmentally Sensitive Areas Grass Forage Science 57 82-92
Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
2002b) Association between lowland grassland plant communities and soil properties
Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
activity in botanically diverse haymeadows effects of litter type and fertilizer additions Soil
Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
15
tended be less rich in PI species than the corresponding inorganic treatment (Trs 9 and
10 respectively) in most years consistently so for Tr 4 compared to Tr 9 but these
differences were not significant in any year nor when averaged over all years There was
however a significant overall effect of form of fertilizer in the FRF series ANOVA
(FYMltinorganic P=0002) and a highly significant effect of frequency of application
(triennialgtannual Plt0001) The time x frequency of application interaction was also
highly significant (Plt0001) Under triennial application PI species per m2 increased
slightly until 2005 (Plt005 compared to 1999) and declined thereafter so that by 2009
levels were significantly lower than in 1999 (Plt001 for 2009 Plt0001 for 2010) By
contrast under annual application PI species m-2
declined progressively until 2009
following a temporary though highly significant (Plt0001) dip in 2000 so that the
difference compared to 1999 was significant in every year from 2002 onwards and highly
significant (Plt0001) from 2005 With the exception of 2003 differences between
triennial and annual application were significant from 2002 onwards and highly
significant (Plt0001) in 2005 and from 2007 to 2010
There was no significant effect other than of time within the LFF treatment series
ANOVA although there was a marginally non-significant time x FYM frequency
interaction (P=0054) This reflected a more rapid decline in PI species-richness under
annual FYM application than with either nil or triennial FYM with the latter two showing
no discernible difference
By contrast with PI species m-2
PI cover percentage (PI) showed no overall treatment
effect within the full treatment ANOVA nor a time x treatment interaction although the
overall effect of time was highly significant (Plt0001) The temporal pattern in PI (Fig
2c) also differed from that shown by PI-richness with PI averaged over all treatments
declining between 1999 and 2005 in three highly significant increments (1999-2000
2000-2002 and 2002-2005 all Plt0001) and increasing again thereafter (2005-2009 and
2009-2010 both Plt0001) There was no significant effect within either the LFF or FRF
treatment series ANOVAs although their was a slight time x frequency of application
effect within the latter (P=0081) PI was slightly higher under annual application than
triennial in each year 1999-2004 after which the reverse was true although differences
were always small
There was no overall effect of treatment on the number of NI species per m2 within the
full treatment range at the lowland meadow (data not shown) but both the overall effect
of time and the time x treatment interaction were significant (Plt0001 and P=0009
respectively) Overall NI species m-2
increased over time from 24 species m-2
in 1999 to
a peak of 42 m-2
in 2008 averaged over all treatments NI species m-2
increased on the
untreated control treatment (Tr 1) from 21 species m-2
in 1999 to 34 species m-2
in 2007
(increase Plt001) then declined to the 1999 value by 2010 The limed (in 2005) control
(Tr 2) showed a somewhat similar pattern though from a slightly higher starting level
(31 species m-2
) and with the increase to 38 species m-2
in 2008 not reaching
significance
These temporal trends in NI species m-2
were much accentuated by annual fertilizer
application particularly by FYM treatments with peak numbers of NI species reaching
52 and 47 species m-2
for Treatments 4 and 5 respectively in 2008 (both Plt0001
compared to 1999) and despite slight declines in both cases remaining significantly
higher in 2010 than in 1999 (both Plt0001) Inorganic counterparts to these treatments
16
showed similar though slightly less marked trends and there was no significant
difference between all four treatments in the peak number reached in 2008 There was no
overall effect of either form of fertilizer or rate of application within the FRF series
ANOVA and only a marginally non-significant effect of frequency of application
(triennialltannual P=0061) Liming in 1999 (Trs 13 14 and 15) also appeared to
accentuate the generally increasing trend although peak numbers in 2008 did not differ
between these treatments and either untreated or limed control in the full treatment range
ANOVA The overall effects of liming regime (1999 or 2005) were not significant within
the LFF treatment series but the faster rate of increase with liming in 1999 was reflected
in a significant time x lime interaction in this analysis (P=0017) The mean number of NI
species m-2
increased very significantly (Plt0001) between 1999 and 2000 following
liming in 1999 with the increment increasing until 2008 whilst on plots un-limed until
2005 NI species m-2
did not increase significantly until 2003 (Plt005) although the
increase compared to 1999 was highly significant (Plt0001) in 2006 2008 and 2010
Nevertheless the differences in rate of increase was at least partly due to a lower 1999
mean for the 1999-limed treatments (170 species m-2
compared to 244 species m-2
difference NS) and although in all years from 2001 onwards mean NI species m-2
was
higher for 1999-limed treatments compared to those not limed until 2005 the difference
was not significant in any year
The contribution of NI species to vegetation cover (NI) showed a very marked
separation between treatments over time at the lowland meadow (Fig 2d) starting from
much lower levels than at the upland meadow (compare with Fig 1d) There were
noticeable differences in temporal trend between annual fertilizer treatments on the one
hand and triennial and nil fertilizer treatments on the other (Fig 2d) The effects of
treatment time and the treatment x time interaction were all highly significant (Plt0001)
in the full treatment range ANOVA The most marked increase in NI occurred with
annual FYM at the high rate (Tr 5) with NI increasing rapidly between 1999 and 2001
(Plt001) and a further highly significant increment between 2004 and 2009 (Plt0001)
(Fig 2d) A similar though less marked trend occurred with annual FYM at the medium
rate (Tr 4) with significant increases between 1999 and 2005 and between 2005 and
2010 (both Plt001) NI also increased markedly with the inorganic treatment
equivalent to Treatment 5 (Tr 10) although the overall rate of increase shown by this
treatment was closer to that shown by Treatment 4 than Treatment 5 There were
significant differences between Treatments 5 and 10 in most years from 2004 onwards
(Plt001 and Plt0001 in 2009 and 2010 respectively)
These effects were reflected in a significant overall effect of form of fertilizer
(FYMgtinorganic P=0008) and highly significant (Plt0001) overall effects of both rate
(high rategtlower rate) and frequency of application (annualgttriennial) within the FRF
series ANOVA and a significant form x rate interaction (P=0043) Mean NI was
highest for high rate annual FYM (Plt0001 compared to all three remaining means) and
lowest for lower rate inorganic (Plt005 compared to high rate inorganic NS compared
to lower rate FYM) Only frequency of application interacted significantly with time
(Plt0001) NI increased moderately under triennial application until 2008 (Plt005 in
2005 compared to 1999 Plt0001 each year 2006-2008) followed by a decline (Plt001
2010 compared to 2008) but with mean NI in 2010 still higher than 1999 (Plt005)
The increasing trend was very much more marked with annual fertilizer application for
17
which there were highly significant (Plt0001) increments between 1999 and 2002 and
between 2002 and 2008 but with no significant increase thereafter Differences between
annual and triennial treatment means were highly significant (Plt0001) from 2003
onwards
There was no significant change in NI over time either under the untreated control
(Tr 1) or the nil fertilizer (Tr 2) treatments in the full treatment ANOVA NI increased
until 2005 on plots limed in 1999 without fertilizer Tr 13 (Plt001 compared to 1999)
and declined slightly but not significantly thereafter Trends were similar with the
treatment limed in 1999 and receiving triennial FYM (Tr 15 ndash not illustrated in Fig 2)
although NI was higher with this treatment than with Tr 13 (Plt005) in both 2005 and
2008 By contrast applying the same rate of FYM annually after liming in 1999 (Tr 14)
caused a more rapid increase with levels by 2005 higher than either Tr 13 (Plt001) or
Tr 4 (Plt005) ndash note that the latter was equivalent to Tr 14 but without lime until 2005
NI declined after 2006 under Tr 14 but continued to increase with Tr 4 with the
difference between these two treatments reversed by 2010 (Plt005)
All main effects and interactions were significant in the LFF treatment series ANOVA
for NI except for lime x FYM frequency and the lime x FYM x time interactions Plots
limed in 1999 supported higher NI levels than those limed in 2005 averaged over the
three FYM frequencies and all years (P=0042) NI increased more rapidly after liming
in 1999 than where liming was delayed until 2005 (P=0033 for time x lime interaction)
with the overall difference due to liming in 1999 highly significant (Plt0001) by 2005
NI declined thereafter on the 1999-limed treatments whilst continuing to increase
following liming in 2005 of previously un-limed treatments both peaking in 2008 at a
very similar level Averaged over lime treatments NI increased much more rapidly
under annual FYM application than under either triennial or nil FYM (P=0002 for the
time x FYM frequency interaction) a highly significant increase had occurred by 2002
with annual FYM (Plt0001 compared to 1999) but increases of similar magnitude were
not reached until 2005 with triennial application and not until 2007 with nil FYM NI
subsequently declined under the latter two FYM regimes but not with annual FYM so
that by 2010 NI was significantly higher with annual FYM than with either triennial or
nil FYM (Plt0001) but with only a small non-significant difference between the latter
two treatments
TREATMENT EFFECTS AFTER 12 YEARS OF APPLICATION
The effects of all treatments on the five key variables at each site in 2010 are shown in
Table S20 with treatments differing significantly (Plt005) from the limed control
treatment (Tr 2) highlighted for each variable Treatment effects in 2010 are not
described in detail here but the modeled responses of total species-richness and of
richness of PI species in 2010 to the mean amounts of N and P applied per year averaged
over 1999-2010 are described below
Relationships with the amounts of fertilizer applied
The effect of neither rate nor frequency of application was significant in any of the
REML models for either total species-richness or PI species-richness at either site when
the amount of N or P applied ha-1
year-1
was included The effect of form of fertilizer was
still significant in most models however either as a main effect or in interaction with the
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
References
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Anon (2010) Fertilizer Manual (RB 209) 8th Edition Her Majestyrsquos Stationery Office London
Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
Kluwer Academic Publishers Dordrecht
Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
restoration of species-rich meadows in the Netherlands Biological Conservation 62 59-65
Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
as an indicator of ecosystem self-regulation in temperate meadow grasslands Biology and
Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
ratios following reductions in the intensity of management of an upland grassland Biology
and Fertility of Soils 22 261-264
Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
Hopkins JJ (1999) The extent of semi-natural grassland communities in lowland England
and Wales a review of conservation surveys 1978-96 Grass and Forage Science 54 1-18
Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
phosphorous vs nitrogen enrichment in plant species loss across North-west Europen
grasslands Journal of Applied Ecology 48 1155-1163
Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
and losses following application of manures to arable land MANNER Soil Use and
Management 15 137-143
Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
(2002a) Plant species-richness functional type and soil properties in grasslands and allied
vegetation in English Environmentally Sensitive Areas Grass Forage Science 57 82-92
Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
2002b) Association between lowland grassland plant communities and soil properties
Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
activity in botanically diverse haymeadows effects of litter type and fertilizer additions Soil
Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
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Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
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Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
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DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
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Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
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Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
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Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
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maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
16
showed similar though slightly less marked trends and there was no significant
difference between all four treatments in the peak number reached in 2008 There was no
overall effect of either form of fertilizer or rate of application within the FRF series
ANOVA and only a marginally non-significant effect of frequency of application
(triennialltannual P=0061) Liming in 1999 (Trs 13 14 and 15) also appeared to
accentuate the generally increasing trend although peak numbers in 2008 did not differ
between these treatments and either untreated or limed control in the full treatment range
ANOVA The overall effects of liming regime (1999 or 2005) were not significant within
the LFF treatment series but the faster rate of increase with liming in 1999 was reflected
in a significant time x lime interaction in this analysis (P=0017) The mean number of NI
species m-2
increased very significantly (Plt0001) between 1999 and 2000 following
liming in 1999 with the increment increasing until 2008 whilst on plots un-limed until
2005 NI species m-2
did not increase significantly until 2003 (Plt005) although the
increase compared to 1999 was highly significant (Plt0001) in 2006 2008 and 2010
Nevertheless the differences in rate of increase was at least partly due to a lower 1999
mean for the 1999-limed treatments (170 species m-2
compared to 244 species m-2
difference NS) and although in all years from 2001 onwards mean NI species m-2
was
higher for 1999-limed treatments compared to those not limed until 2005 the difference
was not significant in any year
The contribution of NI species to vegetation cover (NI) showed a very marked
separation between treatments over time at the lowland meadow (Fig 2d) starting from
much lower levels than at the upland meadow (compare with Fig 1d) There were
noticeable differences in temporal trend between annual fertilizer treatments on the one
hand and triennial and nil fertilizer treatments on the other (Fig 2d) The effects of
treatment time and the treatment x time interaction were all highly significant (Plt0001)
in the full treatment range ANOVA The most marked increase in NI occurred with
annual FYM at the high rate (Tr 5) with NI increasing rapidly between 1999 and 2001
(Plt001) and a further highly significant increment between 2004 and 2009 (Plt0001)
(Fig 2d) A similar though less marked trend occurred with annual FYM at the medium
rate (Tr 4) with significant increases between 1999 and 2005 and between 2005 and
2010 (both Plt001) NI also increased markedly with the inorganic treatment
equivalent to Treatment 5 (Tr 10) although the overall rate of increase shown by this
treatment was closer to that shown by Treatment 4 than Treatment 5 There were
significant differences between Treatments 5 and 10 in most years from 2004 onwards
(Plt001 and Plt0001 in 2009 and 2010 respectively)
These effects were reflected in a significant overall effect of form of fertilizer
(FYMgtinorganic P=0008) and highly significant (Plt0001) overall effects of both rate
(high rategtlower rate) and frequency of application (annualgttriennial) within the FRF
series ANOVA and a significant form x rate interaction (P=0043) Mean NI was
highest for high rate annual FYM (Plt0001 compared to all three remaining means) and
lowest for lower rate inorganic (Plt005 compared to high rate inorganic NS compared
to lower rate FYM) Only frequency of application interacted significantly with time
(Plt0001) NI increased moderately under triennial application until 2008 (Plt005 in
2005 compared to 1999 Plt0001 each year 2006-2008) followed by a decline (Plt001
2010 compared to 2008) but with mean NI in 2010 still higher than 1999 (Plt005)
The increasing trend was very much more marked with annual fertilizer application for
17
which there were highly significant (Plt0001) increments between 1999 and 2002 and
between 2002 and 2008 but with no significant increase thereafter Differences between
annual and triennial treatment means were highly significant (Plt0001) from 2003
onwards
There was no significant change in NI over time either under the untreated control
(Tr 1) or the nil fertilizer (Tr 2) treatments in the full treatment ANOVA NI increased
until 2005 on plots limed in 1999 without fertilizer Tr 13 (Plt001 compared to 1999)
and declined slightly but not significantly thereafter Trends were similar with the
treatment limed in 1999 and receiving triennial FYM (Tr 15 ndash not illustrated in Fig 2)
although NI was higher with this treatment than with Tr 13 (Plt005) in both 2005 and
2008 By contrast applying the same rate of FYM annually after liming in 1999 (Tr 14)
caused a more rapid increase with levels by 2005 higher than either Tr 13 (Plt001) or
Tr 4 (Plt005) ndash note that the latter was equivalent to Tr 14 but without lime until 2005
NI declined after 2006 under Tr 14 but continued to increase with Tr 4 with the
difference between these two treatments reversed by 2010 (Plt005)
All main effects and interactions were significant in the LFF treatment series ANOVA
for NI except for lime x FYM frequency and the lime x FYM x time interactions Plots
limed in 1999 supported higher NI levels than those limed in 2005 averaged over the
three FYM frequencies and all years (P=0042) NI increased more rapidly after liming
in 1999 than where liming was delayed until 2005 (P=0033 for time x lime interaction)
with the overall difference due to liming in 1999 highly significant (Plt0001) by 2005
NI declined thereafter on the 1999-limed treatments whilst continuing to increase
following liming in 2005 of previously un-limed treatments both peaking in 2008 at a
very similar level Averaged over lime treatments NI increased much more rapidly
under annual FYM application than under either triennial or nil FYM (P=0002 for the
time x FYM frequency interaction) a highly significant increase had occurred by 2002
with annual FYM (Plt0001 compared to 1999) but increases of similar magnitude were
not reached until 2005 with triennial application and not until 2007 with nil FYM NI
subsequently declined under the latter two FYM regimes but not with annual FYM so
that by 2010 NI was significantly higher with annual FYM than with either triennial or
nil FYM (Plt0001) but with only a small non-significant difference between the latter
two treatments
TREATMENT EFFECTS AFTER 12 YEARS OF APPLICATION
The effects of all treatments on the five key variables at each site in 2010 are shown in
Table S20 with treatments differing significantly (Plt005) from the limed control
treatment (Tr 2) highlighted for each variable Treatment effects in 2010 are not
described in detail here but the modeled responses of total species-richness and of
richness of PI species in 2010 to the mean amounts of N and P applied per year averaged
over 1999-2010 are described below
Relationships with the amounts of fertilizer applied
The effect of neither rate nor frequency of application was significant in any of the
REML models for either total species-richness or PI species-richness at either site when
the amount of N or P applied ha-1
year-1
was included The effect of form of fertilizer was
still significant in most models however either as a main effect or in interaction with the
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
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Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
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Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
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Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
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Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
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Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
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Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
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Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
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Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
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Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
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Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
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Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
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Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
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Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
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studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
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plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
17
which there were highly significant (Plt0001) increments between 1999 and 2002 and
between 2002 and 2008 but with no significant increase thereafter Differences between
annual and triennial treatment means were highly significant (Plt0001) from 2003
onwards
There was no significant change in NI over time either under the untreated control
(Tr 1) or the nil fertilizer (Tr 2) treatments in the full treatment ANOVA NI increased
until 2005 on plots limed in 1999 without fertilizer Tr 13 (Plt001 compared to 1999)
and declined slightly but not significantly thereafter Trends were similar with the
treatment limed in 1999 and receiving triennial FYM (Tr 15 ndash not illustrated in Fig 2)
although NI was higher with this treatment than with Tr 13 (Plt005) in both 2005 and
2008 By contrast applying the same rate of FYM annually after liming in 1999 (Tr 14)
caused a more rapid increase with levels by 2005 higher than either Tr 13 (Plt001) or
Tr 4 (Plt005) ndash note that the latter was equivalent to Tr 14 but without lime until 2005
NI declined after 2006 under Tr 14 but continued to increase with Tr 4 with the
difference between these two treatments reversed by 2010 (Plt005)
All main effects and interactions were significant in the LFF treatment series ANOVA
for NI except for lime x FYM frequency and the lime x FYM x time interactions Plots
limed in 1999 supported higher NI levels than those limed in 2005 averaged over the
three FYM frequencies and all years (P=0042) NI increased more rapidly after liming
in 1999 than where liming was delayed until 2005 (P=0033 for time x lime interaction)
with the overall difference due to liming in 1999 highly significant (Plt0001) by 2005
NI declined thereafter on the 1999-limed treatments whilst continuing to increase
following liming in 2005 of previously un-limed treatments both peaking in 2008 at a
very similar level Averaged over lime treatments NI increased much more rapidly
under annual FYM application than under either triennial or nil FYM (P=0002 for the
time x FYM frequency interaction) a highly significant increase had occurred by 2002
with annual FYM (Plt0001 compared to 1999) but increases of similar magnitude were
not reached until 2005 with triennial application and not until 2007 with nil FYM NI
subsequently declined under the latter two FYM regimes but not with annual FYM so
that by 2010 NI was significantly higher with annual FYM than with either triennial or
nil FYM (Plt0001) but with only a small non-significant difference between the latter
two treatments
TREATMENT EFFECTS AFTER 12 YEARS OF APPLICATION
The effects of all treatments on the five key variables at each site in 2010 are shown in
Table S20 with treatments differing significantly (Plt005) from the limed control
treatment (Tr 2) highlighted for each variable Treatment effects in 2010 are not
described in detail here but the modeled responses of total species-richness and of
richness of PI species in 2010 to the mean amounts of N and P applied per year averaged
over 1999-2010 are described below
Relationships with the amounts of fertilizer applied
The effect of neither rate nor frequency of application was significant in any of the
REML models for either total species-richness or PI species-richness at either site when
the amount of N or P applied ha-1
year-1
was included The effect of form of fertilizer was
still significant in most models however either as a main effect or in interaction with the
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
References
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Anon (2010) Fertilizer Manual (RB 209) 8th Edition Her Majestyrsquos Stationery Office London
Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
Kluwer Academic Publishers Dordrecht
Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
restoration of species-rich meadows in the Netherlands Biological Conservation 62 59-65
Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
as an indicator of ecosystem self-regulation in temperate meadow grasslands Biology and
Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
ratios following reductions in the intensity of management of an upland grassland Biology
and Fertility of Soils 22 261-264
Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
Hopkins JJ (1999) The extent of semi-natural grassland communities in lowland England
and Wales a review of conservation surveys 1978-96 Grass and Forage Science 54 1-18
Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
phosphorous vs nitrogen enrichment in plant species loss across North-west Europen
grasslands Journal of Applied Ecology 48 1155-1163
Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
and losses following application of manures to arable land MANNER Soil Use and
Management 15 137-143
Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
(2002a) Plant species-richness functional type and soil properties in grasslands and allied
vegetation in English Environmentally Sensitive Areas Grass Forage Science 57 82-92
Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
2002b) Association between lowland grassland plant communities and soil properties
Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
activity in botanically diverse haymeadows effects of litter type and fertilizer additions Soil
Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
18
squared term for the relevant nutrient (ie N2 or P
2) Since the mean amounts of N and P
applied were completely correlated across treatment N and P models usually showed
similar main effects although the Form term was usually more significant in N than P
models due to the smaller discrepancies between FYM and inorganic lsquoequivalentrsquo
treatments in the amount of N applied compared to P (see Table 2) Attention is focussed
primarily on P models here since P availability was considered to be the main limiting
element at both sites (see Discussion)
The best fit model for species-richness in the upland meadow showed both a marked
non-linear response to fertilizer application (particularly FYM) and a marked difference
in response between FYM and inorganic treatments (Fig 3a)The main effect of form of
fertilizer was not significant but a model that included Form nested within the squared
term (P2) was highly significant (Plt0001) with each term significant in the model
(P=0010 Plt0001 and P=0024 for P P2 and P
2 x Form respectively and Plt0001 for
the constant) (see Fig 3a for formula)
10
15
20
25
30
0 2 4 6 8 10 12 14 16
kg P ha-1
year-1
Sp
ecie
s p
er
m2
Annual FYM Annual Inorganic Triennial FYM
Triennial inorganic Nil fertilizer
c Total species-richness
SR = 216 - 440 lognP + x
x = 77 for inorganic x = 51 for FYM
Lowland meadowUpland meadow
16
18
20
22
24
26
28
30
32
0 5 10 15 20
kg P ha-1
year-1
Sp
ecie
s p
er
m2
2
3
4
5
6
0 2 4 6 8 10 12 14 16
kg P ha-1 year-1
PI specie
s p
er
m2
PIn = 475 - 0107P
d Positive indicator species richness
a Total species-richness
SR = 257 + 0483 P - 0027 P2 ndash x P2
x = 0 for inorganic x = 0011 for FYM
2
3
4
5
6
7
8
9
0 5 10 15 20
kg P ha-1
year-1
PI sp
ecie
s p
er
m2
b Positive indicator species richness
PIn = 733 - 0113 P +x
x = 085 for inorganic x = 0 23 for FYM
Fig 3 Relationships between the amount of P applied per year (mean of 1999-2010) as either
inorganic fertilizer or as FYM and species-richness (a and c) and the number of positive
indicator species per m2 (b and d) The lines or curves on each graph were fitted by REML with P
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
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Anon (2010) Fertilizer Manual (RB 209) 8th Edition Her Majestyrsquos Stationery Office London
Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
Kluwer Academic Publishers Dordrecht
Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
restoration of species-rich meadows in the Netherlands Biological Conservation 62 59-65
Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
as an indicator of ecosystem self-regulation in temperate meadow grasslands Biology and
Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
ratios following reductions in the intensity of management of an upland grassland Biology
and Fertility of Soils 22 261-264
Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
Hopkins JJ (1999) The extent of semi-natural grassland communities in lowland England
and Wales a review of conservation surveys 1978-96 Grass and Forage Science 54 1-18
Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
phosphorous vs nitrogen enrichment in plant species loss across North-west Europen
grasslands Journal of Applied Ecology 48 1155-1163
Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
and losses following application of manures to arable land MANNER Soil Use and
Management 15 137-143
Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
(2002a) Plant species-richness functional type and soil properties in grasslands and allied
vegetation in English Environmentally Sensitive Areas Grass Forage Science 57 82-92
Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
2002b) Association between lowland grassland plant communities and soil properties
Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
activity in botanically diverse haymeadows effects of litter type and fertilizer additions Soil
Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
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Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
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Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
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DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
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Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
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Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
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maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
19
as a continuous explanatory variable and with form of fertilizer as a factor Error bars are
plusmnstandard errors of treatment means
The overall relationship between PI species-richness and nutrient input was linear at the
upland meadow (Fig 3b) The addition of the Form term gave a marginally non-
significant improvement to the P model (P=0081) but this term was significant in the N
model (P=0039) The influence of nutrient amount (N or P) was highly significant in
both models (both Plt0001) but with no significant interaction with form of fertilizer
Inorganic treatments were slightly more rich in PI species at a given level of N or P input
than FYM (by 075 and 062 PI species per m2 according to the N and P models
respectively) In both models the predicted intercept values for inorganic fertilizers were
somewhat higher than the actual limed control mean reflecting a lack of response to
inorganic fertilizer levels equivalent to up to about 8 -10 kg P ha-1
year-1
relative to nil
fertilizer
Species-richness at the lowland meadow (Fig 3c) was reduced both by FYM and
inorganic treatments in a non-linear fashion best described by a linear relationship against
lognP (or lognN) over the range of fertilizer treatments (but not nil fertilizer) The effect
of form of fertilizer was very strong in each model (Plt0001) but there was no significant
interaction so that both models predicted a constant difference between inorganic and
FYM treatments (+26 and +39 species m-2
for inorganic fertilizer compared to FYM in
P and N models respectively) However in contrast to the polynomial response at the
upland meadow the logarithmic relationship at the lowland site did not adequately
describe the response at the lowest input levels compared to nil fertilizer which
suggested a humped pattern with the lowest fertilizer values at its peak (see Fig 3c) A
simple second or third order polynomial response was inadequate to describe this overall
relationship but no further attempt was made to find a more complex model
The effect of form of fertiliser was not significant (P=0111) in the P model for PI
species m-2
at the lowland meadow but was significant in the N model (P=0014) with
the overall linear response to the amount applied highly significant (Plt0001) in both
models (Fig 3d) The N model predicted higher PI-richness with inorganic treatments
than with FYM with an overall difference of 062 PI species m-2
These results suggest that treatments delivering up to about 10 kg P ha-1
year-1
(equivalent to 11-12 t FYM ha-1
year-1
) were consistent with maintenance of species
diversity at the upland meadow with some indication that lower rates were beneficial
Only about 3 kg P ha-1
year-1
applied as FYM (equivalent to about 4 t FYM ha-1
year-1
) or
5-6 kg P ha-1
year-1
as inorganic fertilizer were sustainable at the lowland site
Soil microbial community responses
Fertilizer treatments had no effect on total fungal and bacterial PLFArsquos or on the ratio of
fungalbacterial PLFA at either site in 2010 (Tables S21 and S22) Likewise no
treatment effects on these variables were detected in previous years (data not presented)
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
References
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Anon (2010) Fertilizer Manual (RB 209) 8th Edition Her Majestyrsquos Stationery Office London
Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
Kluwer Academic Publishers Dordrecht
Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
restoration of species-rich meadows in the Netherlands Biological Conservation 62 59-65
Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
as an indicator of ecosystem self-regulation in temperate meadow grasslands Biology and
Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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and Fertility of Soils 22 261-264
Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
Hopkins JJ (1999) The extent of semi-natural grassland communities in lowland England
and Wales a review of conservation surveys 1978-96 Grass and Forage Science 54 1-18
Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
phosphorous vs nitrogen enrichment in plant species loss across North-west Europen
grasslands Journal of Applied Ecology 48 1155-1163
Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
and losses following application of manures to arable land MANNER Soil Use and
Management 15 137-143
Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
(2002a) Plant species-richness functional type and soil properties in grasslands and allied
vegetation in English Environmentally Sensitive Areas Grass Forage Science 57 82-92
Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
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Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
activity in botanically diverse haymeadows effects of litter type and fertilizer additions Soil
Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
20
Discussion
PLANT COMMUNITY AND SOIL CHARACTERISTICS OF THE EXPERIMENTAL SITES
Several other sources of data from within the UK (eg Rodwell 1992 Gibson 1996
1997 Critchley et al 2002ab Smith et al 2003) allow the meadows studied here and
the treatment effects occurring at them to be put into a national context in terms of plant
community and soil characteristics The similarities between these meadows and lowland
oceanic and upland meadows in Europe (Rodwell et al 2007) mean that these results
should have wide applicability outside the UK
Plant communities
Ordination of the vegetation composition in 2007 in the context of the NVC (RA
Sanderson unpublished) showed most treatments at the upland meadow to have retained
a close affinity to the MG3b (Briza media) NVC sub-community with the notable
exception of the high rate (24 t ha-1
year-1
) annual FYM which had moved to a position
intermediate between MG3a and MG3b the generally less species-rich Bromus
hordeaceus ssp hordeaceus sub-community (Rodwell 1992) Species-richness on the
latter treatment had declined to 18 species per m2 by 2007 a level more typical of MG6
(Lolium perenne-Cynosurus cristatus grassland) (Critchley et al 2002a) Although the
vegetation had recovered to a small extent to 20 species per m2 by 2010 under this
treatment it was still markedly less species-rich than others and with a substantially
higher aggregate cover of negative indicator species By contrast the quality of
vegetation under other fertilizer treatments - particularly those incorporating lower
nutrient inputs than used in the past (12 t ha-1
applied annually) - had improved in terms
of increased number and cover of positive indicator species and a decline in the aggregate
cover of negative indicator species
Unfertilized treatments at the lowland meadow contained about 22-24 species per m2
between 1999 and 2007 the higher value being fairly typical of MG5 grassland (Gibson
1996 1997 Critchley et al 2002a Species-richness declined thereafter under most
treatments including limed control and untreated control treatments with the declines
most noticeable in terms of positive indicator species The reasons for this general decline
are not clear although a series of later hay harvests during this period is the probable
cause (Kirkham amp Tallowin 1995) The decline in total species-richness under the high
rate annual FYM treatment (to 133 species m-2
in 2009) was even more marked than at
the upland meadow with this level approaching the average for MG7 (Lolium perenne-
dominated) grasslands (118 species m-2
) in the ESA survey (Critchley et al 2002a)
The weighted Ellenberg fertility score is useful for characterizing vegetation in relation
to variations in soil fertility and nutrient inputs (Smith et al 2003 Kirkham et al 2008
The mean scores for the upland and lowland meadows in 1999 were 447 and 400
respectively with the upland meadow mean falling within the 433-467 range of target
values for the MG3b community identified by Smith et al (2003) Medium and high rate
(12 and 24 t ha-1
) annual FYM treatments at the upland meadow exceeded this target
range in 2010 although the former only marginally so at 472 (509 for the high rate)
whereas both these treatments increased the fertility scores markedly at the lowland
meadow (486 and 520 respectively in 2010) All other treatments at the upland meadow
fell within the 433-467 MG3 target range in 2010 with the exception of the inorganic
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
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Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
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Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
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Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
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Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
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Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
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Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
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Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
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Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
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Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
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Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
21
equivalent to high rate FYM (480) Values for fertilizer treatments were generally higher
at the lowland meadow than at the upland one although the opposite was true for
unfertilized vegetation These differences closely reflect the corresponding differences
between sites in the percentage contribution to vegetation cover of negative indicator
species (NI) with which fertility scores were closely correlated
Soil chemical properties
Soil pH was low at both sites in 1999 (518 and 501 at upland and lowland meadows
respectively) compared to typical mean values for ESA MG3 and MG5 grasslands of 64
SDplusmn089 and 62 SDplusmn100 respectively (Critchley et al 2002b) in each case falling
outside the range represented by plusmn one standard deviation (SD) about the ESA mean All
treatments except untreated controls fell within this range following liming in 1999
andor 2005 most exceeding the target of 60 This target corresponds to the mean pH in
3260 English grassland sites currently candidate for an agri-environment agreement or
within existing agreements (Natural England (NE) unpublished) although higher than
the average of 54 for MG5a grasslands in Wales (Stevens et al 2010)
Oslashlsen extractable soil P at both sites in 1999 was within the range associated with
maximal species-richness in ESA grasslands of 4-15 mg l-1
(Critchley et al 2002a) and
the mean value at the upland meadow (550 mg l-1
) was within the SD range typical of
MG3 grasslands though somewhat below the mean (77 plusmn449 mg l-1
) (Critchley et al
2002b) Soil P levels were considerably lower at the lowland meadow (512 mg l-1
) than
typical values for ESA MG5 grasslands (96 plusmn449 mg l-1
) and low at both sites
compared to the mean for the NE agri-environment scheme database of 135 mg l-1
(although it should be noted that this database includes candidate sites for biodiversity
restoration as well as unimproved grasslands)
The significance of plant available soil P for vegetation response to fertilizers is
discussed later
Soil organic matter (measured by loss on ignition) was lower at the lowland meadow
(90) than at the upland one (172) although within typical ranges for MG5 (106
plusmn427) and MG3 (146 plusmn422) communities respectively (Critchley et al 2002b) Total
soil nitrogen values differed between sites correspondingly (034 and 065 at lowland
and upland sites respectively) with these values also low compared to values of 067
plusmn0299 for MG5 and 090 plusmn0425 for MG3 ESA grasslands
Exchangeable soil K values were high at both sites (at 197 and 175 mg l-1
for upland
and lowland meadows respectively) compared to MG3 (128 plusmn640) and MG5 (135 plusmn390
mg l-1
) ESA grasslands and compared to sites within the NE database (135 mg l-1
) and
were more typical of MG6 grasslands (164 plusmn820 mg l-1
) (Critchley et al 2002b)
Soil K levels in 2010 were noticeably raised at both sites by annual FYM treatments at
the medium and high rates and by the inorganic counterparts to these treatments (see
Supporting Information Tables S1 and S2) A similar effect was noticeable with Oslashlsen P
levels though mainly at the higher annual FYM rate but no such effect was noticeable
for soil organic matter (LOI) soil carbon or total soil N Nevertheless even with the
high rate FYM treatment extractable soil P levels were still lower at the lowland meadow
in 2010 than the mean for MG5 grasslands and still well within the range for MG3 at the
upland meadow whilst exchangeable K at both sites was within the range more typical of
improved (MG7) grasslands (213 plusmn1197 mg l-1
Critchley et al 2002b)
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
References
Allen SE (1974) Chemical Analysis of Ecological Materials Blackwell Oxford UK
Anon (2010) Fertilizer Manual (RB 209) 8th Edition Her Majestyrsquos Stationery Office London
Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
Kluwer Academic Publishers Dordrecht
Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
restoration of species-rich meadows in the Netherlands Biological Conservation 62 59-65
Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
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Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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and Fertility of Soils 22 261-264
Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
Hopkins JJ (1999) The extent of semi-natural grassland communities in lowland England
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Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
phosphorous vs nitrogen enrichment in plant species loss across North-west Europen
grasslands Journal of Applied Ecology 48 1155-1163
Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
and losses following application of manures to arable land MANNER Soil Use and
Management 15 137-143
Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
(2002a) Plant species-richness functional type and soil properties in grasslands and allied
vegetation in English Environmentally Sensitive Areas Grass Forage Science 57 82-92
Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
2002b) Association between lowland grassland plant communities and soil properties
Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
activity in botanically diverse haymeadows effects of litter type and fertilizer additions Soil
Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
22
Even at the upland meadow K availability in 2010 would be considered at least
moderate in agronomic terms on all treatments ie soil index 1 (61-120 mg K l-1
)
according to the classification system used in the UK (Anon 2010) and satisfactory on
some (ie index 2 and 3 121-240 and 241-400 mg K l-1
respectively whilst soil K
indices were 2 or 3 under all treatments at the lowland meadow in 2010 including
untreated controls By contrast soil P was still well within the deficient category (index
0 0-9 mg P l-1
) under all treatments at both sites in 2010 with the exception of the high
rate FYM treatment at the upland meadow which fell just within the index 1 (moderate)
range (10-15 mg P l-1
)
Soil microbial communities
The fungal-to-bacterial PLFA ratio (FBR) values which ranged from 006 to 011
suggest that the microbial communities of both sites were fungal-dominated which is
typical of traditionally managed species-rich grasslands Other studies have confirmed
that grassland microbial soil communities are strongly affected by inorganic nitrogen
enrichment with fertilizer addition often reducing the FBR and resulting in a shift to
bacterial dominance (Bardgett amp McAlister 1999 Donnison et al 2000ab Smith et al
2008) The lack of response in this study however most likely reflects the low amounts
of nutrients added to soil in fertilizers but also potentially the high resistance of fungal-
dominated microbial communities of species-rich grasslands to environmental changes
(Gordon Haygarth amp Bardgett 2008)
SIGNIFICANCE OF BETWEEN-SITE DIFFERENCES FOR RESILIENCE TO FERTILITY INPUTS
Whilst the lighter texture and lower organic matter content of soils at the lowland site
might imply a higher potential for nutrient leaching this would probably be outweighed
by the higher rainfall at the upland site and in terms of N availability by the much higher
levels of N deposition ie c 33 kg N ha-1
year-1
compared to c 21 kg N ha-1
year-1
for
the lowland site (source the Air Pollution Information System at wwwapisacuk)
Probably of more significance the lowland meadow - with lower rainfall lower
atmospheric N input and lower soil organic matter (and thus lower soil water holding
capacity) - was a more stressed environment than the upland meadow This combined
with a history of no fertilizer or lime inputs for at least 20 years was apparently reflected
in a grassland system less resilient to fertilizer inputs than the upland meadow
FREQUENCY AND RATE OF APPLICATION
Not surprisingly perhaps the influence of rate of application of fertilizers on both
species-richness and the richness of positive indicator species were shown to be entirely a
function of the mean amount of nutrients added per year The same was true of frequency
of application An earlier analysis of 2005 data from these sites suggested a positive
benefit of triennial application compared to annual application of correspondingly lower
amounts (Kirkham et al 2008) However in contrast to the period reported here which
encompassed a further two completed three-year cycles (including that started in 2005)
in the earlier analysis the last instalment of the triennial treatment had been applied only
about a month before the 2005 botanical assessment and will have contributed little to the
effects of the treatment in that year More subtle effects of application frequency may
have occurred however at the plant community or individual plant species level that
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
References
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Anon (2010) Fertilizer Manual (RB 209) 8th Edition Her Majestyrsquos Stationery Office London
Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
Kluwer Academic Publishers Dordrecht
Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
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Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
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Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
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Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
phosphorous vs nitrogen enrichment in plant species loss across North-west Europen
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Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
and losses following application of manures to arable land MANNER Soil Use and
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Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
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Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
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Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
activity in botanically diverse haymeadows effects of litter type and fertilizer additions Soil
Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
23
were not shown by the composite variables analysed and there might also be agronomic
or logistical advantages of intermittent fertilizer application beyond the scope of
discussion here
DIFFERENCES BETWEEN FYM AND INORGANIC FERTILIZERS
The lesser effect of inorganic fertilizer on vegetation compared to FYM was reported
previously (Kirkham et al 2008) However the subsequent revision made in 2007 of
estimates of N and P availability from FYM inorganic lsquoequivalentrsquo treatments were now
considered to have been supplying substantially less P in particular than was estimated
for their FYM counterparts These discrepancies were taken into account in the REML
modelling approach but although differences in response between the two forms of
fertilizer were much reduced a difference remained in most cases These differentials
might have narrowed further after a longer time period had elapsed following the
revisions to the amounts of N and P applied in inorganic treatments particularly as
discrepancies in the amount of P applied were both larger and most likely of more
significance than those in N (see discussion below) Furthermore although some residual
availability from FYM of both N and P were allowed for in the revised estimates this
may not have fully accounted for cumulative effects of these residues and for differences
between FYM and inorganic fertilizers in this respect
THE SIGNIFICANCE OF PHOSPHORUS AVAILABILITY
The very low underlying availability of soil P at both sites relative to N and K makes it
very likely that P was limiting relative to the other two macro-nutrients at most if not all
levels of fertilizer application This hypothesis is supported by analyses of 2005 data
from the lowland meadow and from an adjacent agriculturally-improved meadow (Bush)
incorporating a wider range of treatments than those reported here and which provided
some variation in the ratios between the amounts of N P and K applied (Kirkham et al
2007) Proprietary organic fertilizers delivering similar amounts of N to the high rate
FYM treatment but containing less P and K had relatively little effect on vegetation and
regression analyses of data across all treatments suggested a much stronger influence of P
than of N at both unimproved and improved lowland meadows the latter also having a
low soil P status in 1999 (56 mg Oslashlsen extractable P l-1
) Other experimental and survey
evidence shows that P enrichment is generally a more important driver of species-loss
and reductions in species-diversity from semi-natural grasslands than N enrichment
(Kirkham et al 1996 Ceulemans et al 2011) and that maximum species-richness in
grasslands is confined to a very narrow band of low soil P availability (Janssens et al
1998 Critchley et al 2002a) Many more endangered species persist under P-limited
than under N-limited conditions (Wassen et al 2005)
These studies do not demonstrate a causal link but species that are particularly adapted
to low P availability are characteristically deep-rooted herbs and other species with low
relative growth rates (Grime amp Hunt 1975) Higher susceptibility to elevated P is
associated with stress-tolerance low stature and association with arbuscular mycorrhizae
(Ceulemans et al 2011) the latter enhancing their access to soil P resources (van der
Heijden et al 1998) These properties apparently allow them to co-exist in relatively
large numbers under low P availability along with smaller numbers of potentially more
competitive species whose dominance is restrained by the nutrient-poor conditions
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
References
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Anon (2010) Fertilizer Manual (RB 209) 8th Edition Her Majestyrsquos Stationery Office London
Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
Kluwer Academic Publishers Dordrecht
Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
restoration of species-rich meadows in the Netherlands Biological Conservation 62 59-65
Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
as an indicator of ecosystem self-regulation in temperate meadow grasslands Biology and
Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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and Fertility of Soils 22 261-264
Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
Hopkins JJ (1999) The extent of semi-natural grassland communities in lowland England
and Wales a review of conservation surveys 1978-96 Grass and Forage Science 54 1-18
Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
phosphorous vs nitrogen enrichment in plant species loss across North-west Europen
grasslands Journal of Applied Ecology 48 1155-1163
Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
and losses following application of manures to arable land MANNER Soil Use and
Management 15 137-143
Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
(2002a) Plant species-richness functional type and soil properties in grasslands and allied
vegetation in English Environmentally Sensitive Areas Grass Forage Science 57 82-92
Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
2002b) Association between lowland grassland plant communities and soil properties
Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
activity in botanically diverse haymeadows effects of litter type and fertilizer additions Soil
Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
24
It is important to note that the amounts of N applied in FYM were relatively small
compared to amounts used in other fertilizer experiments (eg Mountford et al 1993
Kirkham et al 1996 Silvertown et al 2006) but the proportions of P and K relative to
N in FYM were much higher - both in comparison with treatments applied in these earlier
experiments and with most commercial compound fertilizers The evidence suggests that
in this context at least as much attention should be focussed upon the amount of P
applied in relation to the initial P status of the soil as on the amount of N being applied
RESPONSE TO LIME
Liming alone had little or no detrimental effect on vegetation at either site but apparently
reduced vegetation quality when applied in conjunction with annual FYM at the lowland
meadow The latter effect was not evident at the upland meadow and probably reflects
differences between sites in past management It is debateable whether the plant
community at the lowland meadow would eventually acclimatise to this regime and even
a 12-year period is insufficient to gauge this partly because soil pH did not decline
rapidly enough at the lowland meadow after liming in 1999 to justify repetition of the
treatment Further evidence of the damaging effect of liming in conjunction with annual
FYM application was provided by a decline in vegetation quality on the corresponding
previously un-limed FYM treatment following liming in 2005 although this effect was
masked somewhat by a general trend of decline after 2005 which included the untreated
control treatment By contrast there was no evidence of any detrimental effect of lime in
conjunction with triennial FYM application
Rationale behind the liming policy
A decision was made in 2005 to treat liming as a background management for all
fertilizer treatments at both sites whilst retaining (or in the case of the upland meadow
establishing) a non-limed unfertilised treatment This policy was adopted on the basis that
use of lime is widespread in hay meadows and occasional liming can be considered
traditional management (Tallowin 1998 Walsh Peel amp Jefferson 2011) It is unlikely
that either inorganic fertilizers or FYM would be used in practice without occasional
liming to maintain soil pH at levels which would allow sufficient response to these inputs
to justify their use on agronomic grounds To forego this strategy at the upland meadow
would have been doubly unjustifiable in view of the high quality of the plant community
there and the known history of occasional liming
IMPLICATIONS FOR THE ECOLOGICALLY SUSTAINABLE MANAGEMENT OF SEMI-NATURAL
MEADOWS
These results show that levels of annual FYM application equivalent to those already in
use at the upland meadow (ie 12 tonnes of FYM per hectare) are likely to be
ecologically sustainable in the long term within similar MG3 meadows but that higher
rates may not be and amounts as high as 24 t ha-1
per year almost certainly are not
sustainable within meadows comparable to either of those in this study Moreover whilst
past inputs appear to be sustainable at the upland meadow reductions in the level of
inputs were beneficial
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
References
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Anon (2010) Fertilizer Manual (RB 209) 8th Edition Her Majestyrsquos Stationery Office London
Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
Kluwer Academic Publishers Dordrecht
Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
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Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
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Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
Hopkins JJ (1999) The extent of semi-natural grassland communities in lowland England
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Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
phosphorous vs nitrogen enrichment in plant species loss across North-west Europen
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Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
and losses following application of manures to arable land MANNER Soil Use and
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Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
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Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
2002b) Association between lowland grassland plant communities and soil properties
Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
activity in botanically diverse haymeadows effects of litter type and fertilizer additions Soil
Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
25
Occasional liming to raise soil pH to 60 appeared to be consistent with maintaining
vegetation quality within MG3 plant communities of which the upland meadow is
typical
It was less easy to define with any certainty safe levels of fertilizer input at the lowland
meadow than at the upland one However FYM inputs equivalent to up 12 tonnes ha-1
triennialy or about 4 tonnes annually or levels of inorganic fertilizer equivalent in N P
and K content to these levels appeared to be sustainable for the maintenance of a high
nature value meadow at the lowland site These levels are slightly lower than the le6
tonnes FYM ha-1
year-1
suggested by 1999-2005 results (Kirkham et al 2008) ie for a
period five years less than that reported here and it is possible that even these lower
levels may turn out to be unsustainable in the longer term However as noted above
defining such trends has been hampered somewhat by an apparent small decline in
vegetation quality over recent years apparently independent of treatments but possibly
due to a succession of late harvests in later years
This study shows no evidence to suggest that inorganic fertilizers supplying equivalent
amounts of N P and K to ecologically-sustainable levels of FYM could not be substituted
for these FYM treatments at either site
Liming either alone or in conjunction with intermittent FYM application equivalent to
the levels quoted above appeared to be sustainable at the lowland meadow and liming
showed no adverse effect at the upland site either alone or in interaction with fertilizers
The negative effects of fairly moderate annual inputs of FYM to the lowland meadow
and the apparent interaction with liming may reflect historic adaptation of vegetation to
the absence of recent inputs of lime or fertilizer and the consequent low levels of soil pH
phosphorus and total nitrogen or may more closely reflect site-specific factors such as
soil texture and annual rainfall It would be unwise therefore to extrapolate directly the
results of this study to MG5 meadows with a more recent history of either FYM or lime
use or conversely to sites where this history is unknown Nevertheless at sites
comparable in botanical composition and soil status to the lowland meadow the low
fertilizer inputs quoted above are likely to be ecologically sustainable and may also bring
about a gradual adaptation of the vegetation to slightly higher fertility levels in the longer
term
Acknowledgements
We would like to thank Helen Adamson Deborah Beaumont John Fowbert Jo Goodyear Anna
Gundrey Emma Pilgrim Ken Milner Anne Moon Steven Shepherd Roger Smith Julia
Tallowin James Towers Jan Winder and Barry Wright for carrying out botanical assessments
and Helen Adamson Gail Bennett Jo Goodyear and other staff from IGER (now Rothamsted
Research North Wyke) and ADAS for applying treatments Mike Burke and Alison Mole were
responsible for data management The project was conceived supervised and funded by the
Department for Environment Food and Rural Affairs English Nature (now Natural England) and
the Countryside Council for Wales We would like to acknowledge the contributions made by
Tim Blackstock Val Brown Richard Brand-Hardy Andrew Cooke Richard Jefferson Steve
Peel Carrie Rimes Stuart Smith and the late David Stevens We are grateful to Mr and Mrs Joe
Winder Tim Winder and the Gwent Wildlife Trust for provision of sites and for cooperation and
assistance with field operations
26
References
Allen SE (1974) Chemical Analysis of Ecological Materials Blackwell Oxford UK
Anon (2010) Fertilizer Manual (RB 209) 8th Edition Her Majestyrsquos Stationery Office London
Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
Kluwer Academic Publishers Dordrecht
Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
restoration of species-rich meadows in the Netherlands Biological Conservation 62 59-65
Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
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Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
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and Fertility of Soils 22 261-264
Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
Hopkins JJ (1999) The extent of semi-natural grassland communities in lowland England
and Wales a review of conservation surveys 1978-96 Grass and Forage Science 54 1-18
Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
phosphorous vs nitrogen enrichment in plant species loss across North-west Europen
grasslands Journal of Applied Ecology 48 1155-1163
Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
and losses following application of manures to arable land MANNER Soil Use and
Management 15 137-143
Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
(2002a) Plant species-richness functional type and soil properties in grasslands and allied
vegetation in English Environmentally Sensitive Areas Grass Forage Science 57 82-92
Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
2002b) Association between lowland grassland plant communities and soil properties
Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
activity in botanically diverse haymeadows effects of litter type and fertilizer additions Soil
Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
26
References
Allen SE (1974) Chemical Analysis of Ecological Materials Blackwell Oxford UK
Anon (2010) Fertilizer Manual (RB 209) 8th Edition Her Majestyrsquos Stationery Office London
Bakker JP (1989) Nature Management by Grazing and Cutting Geobotany Series No 14
Kluwer Academic Publishers Dordrecht
Berendse F Oomes MJM Altena HJ amp Elberse W Th (1992) Experiments on the
restoration of species-rich meadows in the Netherlands Biological Conservation 62 59-65
Bardgett R D amp McAlister E (1999) The measurement of soil fungalbacterial biomass ratios
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Fertility of Soils 29 282-290
Bardgett R D Hobbs P J amp Frostegaringrd A (1996) Changes in soil fungalbacterial biomass
ratios following reductions in the intensity of management of an upland grassland Biology
and Fertility of Soils 22 261-264
Blackstock TH Rimes CA Stevens DP Jefferson RG Robertson HJ Mackintosh J amp
Hopkins JJ (1999) The extent of semi-natural grassland communities in lowland England
and Wales a review of conservation surveys 1978-96 Grass and Forage Science 54 1-18
Ceulemans T Merckx R Hens M amp Honnay O (2011) A trat-based analysis of the role of
phosphorous vs nitrogen enrichment in plant species loss across North-west Europen
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Chambers BJ Lord EI Nicholson FA amp Smith KA (1999) Predicting nitrogen availability
and losses following application of manures to arable land MANNER Soil Use and
Management 15 137-143
Critchley C N R Chambers B J Fowbert JA Bhogal A Rose S C amp Sanderson R A
(2002a) Plant species-richness functional type and soil properties in grasslands and allied
vegetation in English Environmentally Sensitive Areas Grass Forage Science 57 82-92
Critchley C N R Chambers B J Fowbert JA Sanderson R A Bhogal A amp Rose S C (
2002b) Association between lowland grassland plant communities and soil properties
Biological Conservation 105 199-215
Crofts A amp Jefferson RG (1999) The Lowland Grassland Management Handbook 2nd edn
English NatureThe Wildlife Trusts Peterborough
Donnison L M Griffith G S amp Bardgett R D (2000a) Determinants of fungal growth and
activity in botanically diverse haymeadows effects of litter type and fertilizer additions Soil
Biology and Biochemistry 32 289-294
Donnison L M Griffith G S Hedger J Hobbs P J amp Bardgett R D (2000b) Management
influences on soil microbial communities and their function in botanically diverse
haymeadows of northern England and Wales Soil Biology and Biochemistry 32 253-263
Ellenberg H (1988) Vegetation Ecology of Central Europe Cambridge University Press
Cambridge
Federle T W (1986) Microbial distribution in soil ndash new techniques Perspectives in microbial
ecology pp 493-498 (eds F Megusar amp M Ganter) Slovene Society for Microbiology
Ljubljana Slovenia
Frostegaringrd A amp Baringaringth E (1996) The use of phospholipid fatty analysis to estimate bacterial
and fungal biomass in soil Biology and Fertility of Soils 22 59-65
Frostegaringrd A Tunlid A amp Baringaringth E (1993) Phospholipid fatty acid composition biomass and
activity of microbial communities from two soil types experimentally exposed to different
heavy metals Applied Environmental Microbiology 59 3605-3617
Fuller RM (1987) The changing extent and conservation interest of lowland grasslands in
England and Wales a review of grassland surveys 1930-1984 Biological Conservation 40
281-300
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
27
Galwey NW (2006) Introduction to Mixed Modelling Beyond Regression and Analysis of
Variance John Wiley amp Sons Ltd Chichester
Genstat 5 Committee (1997) Genstat for Windows Command Language Manual 3rd edn
Numerical Algorithms Group Oxford
Gibson CWD (1996) The effects of horse grazing on species-rich grasslands English Nature
Research Report No 164 English Nature Peterborough
Gibson CWD (1997) The effects of horse and cattle grazing on English species-rich grasslands
English Nature Research Report No 210 English Nature Peterborough
Gordon H Haygarth PM amp Bardgett RD (2008) Drying and rewetting effects on soil
microbial community composition and nutrient leaching Soil Biology and Biochemistry 40
302-311
Goulson D Hanley ME Darvill B Ellis JS amp Knight ME (2005) Causes of rarity in
bumblebees Biological Conservation 122 1-8
Grime JP amp Hunt R (1975) Relative growth rate its range and adaptive significance in a local
flora Journal of Ecology 63 393-422
Hill MO Mountford JO Roy DB Bunce RGH 1999 Ellenbergs indicator values for
British plants ECOFACT Volume 2 Technical Annexe HMSO Norwich
Janssens F Peeters A Tallowin JRB Bakker JP Bekker RM Verweij GL Fillat F amp
Oomes MJM (1998) Relationship between soil chemical factors and grassland diversity
Plant and Soil 202 68-78
Jefferson RG (2005) The conservation management of upland hay meadows in Britain a
review Grass and Forage Science 60 322-331
Kirkham FW amp Tallowin JRB (1995) The influence of cutting date and previous fertilizer
treatment on the productivity and botanical composition of species-rich hay meadows on the
Somerset Levels Grass and Forage Science 50 365-377
Kirkham FW Mountford JO amp Wilkins RJ (1996) The effects of nitrogen potassium and
phosphorus addition on the vegetation of a Somerset peat moor under cutting management
Journal of Applied Ecology 33 1013-1029
Kirkham FW Tallowin JRB Sanderson RA Hoppeacute GM amp Goodyear J (2002) Role of
organic fertilizers in the sustainable management of semi-natural grasslands II ndash Botanical
studies Conservation Pays (ed J Frame) pp 153-157 Proceedings of British Grassland
Society Occasional Symposium No 36 British Grassland Society Reading UK
Kirkham FW Tallowin JRB Sanderson RA Bhogal A Chambers BJ amp Stevens DP
(2008) The impact of organic and inorganic fertilizers and lime on the species-richness and
plant functional characteristics of hay meadow communities Biological Conservation 141
1411-1427
MAFF (1986) Analysis of Agricultural Materials (RB427) HMSO London UK
Mountford JO Lakhani K amp Kirkham FW (1993) Experimental assessment of the effects of
nitrogen addition under hay cutting and aftermath grazing on the vegetation of hay meadows
on a Somerset peat moor Journal of Applied Ecology 30 321-332
Myklestad Aring amp Saeligtersdal M (2004) The importance of traditional meadow management
techniques for conservation of vascular plant species richness in Norway Biological
Conservation 118 133-139
Nicholson F Rollett AJ Bhogal A Lord E Thorman RE Williams JR Smith KA
Misselbrook TH Chadwick DR amp Chambers BJ (2010) MANNER-NPK Climate Water
and Soil Science Policy and Practice (eds K Crichton amp R Audsley) pp 328-333
Proceedings of the SA and SEPA biennial conference Macaulay Land Use Research Institute
Aberdeen UK
Ormerod SJ 2003 Restoration in applied ecology Journal of Ecology 40 44-50
Ratcliffe DA (ed) (1977) A Nature Conservation Review (Two volumes) Cambridge University
Press Cambridge
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
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Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
28
Ratcliffe DA (1984) Post-mediaeval and recent changes in British vegetation the culmination
of human influence New Phytologist 98 73-100
Rich TCG amp Woodruff ER (1996) Changes in the vascular plant floras of England and
Scotland between 1930-60 and 1987-88 the BSBI monitoring scheme Biological
Conservation 75 217-229
Robertson HJamp Jefferson RG (2000) Monitoring the condition of lowland grassland SSSIs
I English Naturersquos rapid assessment method English Nature Research Report No315
English Nature Peterborough
Rodwell JS (ed) (1992) British Plant Communities Volume 3 Grasslands and montane
Communities Cambridge University Press Cambridge
Rodwell JS Morgan V Jefferson RG amp Moss D (2007) The European context of British
lowland grasslands Joint Nature Conservation Committee Report No 394 JNCC
Peterborough
Silvertown J Poulton P Johnston E Edwards G Heard M amp Biss PM (2006) The Park
Grass Experiment 1856-2006 Its contribution to ecology Journal of Ecology 94 801-814
Simpson NA amp Jefferson RG (1996) Use of Farmyard Manure on Semi-natural (Meadow)
Grassland English Nature Research Report No 150 English Nature Peterborough
Smith KA Chalmers AG Chambers BJ amp Christie P (1998) Organic manure phosphorus
accumulation mobility and management Soil Use and Management 14 154-159
Smith RS (1988) Farming and conservation of traditional meadowland in the Pennine Dales
Environmentally Sensitive Area Ecological Change in the Uplands (eds) MB Usher amp
DBA Thompson British Ecological Society Special Publication No 7 Blackwell Scientific
Publications Oxford pp 183-199
Smith RS amp Jones L (1991) The phenology of mesotrophic grassland in the Pennine Dales
Northern England historic hay cutting dates vegetation variation and plant species
phenologies Journal of Applied Ecology 28 42-59
Smith RS amp Rushton SP (1994) The effects of grazing management on the vegetation of
mesotrophic (meadow) grassland in Northern England Journal of Applied Ecology 31 13-24
Smith RS Buckingham H Bullard MJ Shiel RS amp Younger A (1996) The conservation
management of mesotrophic (meadow) grassland in northern England I Effects of grazing
cutting date and fertilizer on the vegetation of a traditionally managed sward Grass and
Forage Science 51 278-291
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Rolph G Hobbs PJ
Peacock S 2003 Soil microbial community fertility vegetation and diversity as targets in
the restoration management of a meadow grassland Journal of Applied Ecology 40 51-64
Smith RS Shiel RS Bardgett RD Millward D Corkhill P Evans P Quirk H Hobbs
PJ amp Kometa ST (2008) Long-term change in vegetation and soil microbial communities
during the phased restoration of traditional meadow grassland Journal of Applied Ecology
45 670-679
Stace CA (2010) New Flora of the British Isles 3rd edn Cambridge University Press
Cambridge
Stevens DP Smith SLN Blackstock TH Bosanquet SDS amp Stevens JP (2010)
Grasslands of Wales a survey of lowland species- rich grasslands 1987ndash2004 University of
Wales Cardiff Press Cardiff
Tallowin JRB (1998) Use and effects of lime application on semi-natural grasslands in Britain
Countryside Council for Wales Contract Science Report No 261 Countryside Council for
Wales Bangor
Tallowin JRB Kirkham FW Goodyear J amp Hoppeacute GM (2002) Role of organic fertilizers
in the sustainable management of semi-natural grasslands I ndash Agronomic studies
Conservation Pays (ed J Frame) pp 149-152 BGS Occasional Symposium No 36 British
Grassland Society Reading
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
29
Van der Heijden MGA Klironomos JN Ursic M Moutoglis P Streitwolfe-Engel R
Boller T Wiemken A amp Saunders IR (1998) Mycorrhizal fungal diversity determines plant
diversity ecosystem variability and productivity Nature 396 69-72
Walsh G Peel S amp Jefferson R (2011) The use of lime on semi-natural grassland in agri-
environment schemes Natural England Technical Information Note TIN 045 Natural
England Available at httpnaturalenglandetraderstorescom NaturalEnglandShopTIN045
Wassen MJ Venterink HO Lapshina ED Tannenberger F (2005) Endangered species
persist under phosphorus limitation Nature 437 547-550
Welham SJ amp Thompson R (1997) Likelihood ratio tests for fixed model terms using residual
maximum likelihood Journal of the Royal Statistical Society Series B 59 701-714
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
30
Supporting Information
SPECIES RESPONSIBLE FOR TEMPORAL TRENDS IN PI AND NI SPECIES
The upland meadow
The small but significant (Plt005) overall decline in PI species-richness between 1999
and 2000 (Fig 1b) was largely attributable to small declines in the abundance of
Alchemilla glabra and Euphrasia officinalis agg both of which subsequently increased
to levels equivalent to or higher than 1999 levels (data for individual species not
presented) This initial decline was not reflected in PI which increased significantly
between most years including 1999 and 2000 (Plt0001) The marked progressive overall
increase in PI (from 14 of live cover in 1999 to 51 in 2010 back-transformed
means) was mainly attributable to increases in Conopodium majus across the experiment
as a whole and to increases in Leontodon hispidus in nil and low input treatments in
particular with the latter species declining under annual high rate FYM (Tr 5) Both
Centaurea nigra and Geranium sylvaticum increased in general including under Tr 5
although G sylvaticum was rather unevenly distributed among treatments Some of the
overall increase in PI under Tr 5 was attributable to Filipendula ulmaria which was
recorded on only one plot throughout the whole period but increased on this plot from
less than 2 of live cover in 1999 to 18 in 2010
Declines in NI were largely attributable to diminishing cover of Holcus lanatus -
which accounted for 10-15 of vegetation cover in 1999 - and to a lesser extent Rumex
acetosa which occupied 3-5 of cover initially Holcus also declined under Tr 5 but this
was at least matched by increases in Poa trivialis with R acetosa with Heracleum
sphondylium also increasing and a noticeable increase in Lolium perenne in 2007-2009
Similar trends occurred with both Trs 4 and 14 but with smaller increases in the latter
four species and no increase in P trivialis with Tr 14 this species remaining at low
levels with all treatments where lime was applied in 1999 (Trs 13-15)
The lowland meadow
Temporal trends in PI were not attributable to any one species but reflect the combined
effect of differing patterns in several species Centaurea nigra cover followed a
somewhat similar trend to the overall one though less marked Leontodon hispidus
declined progressively over the whole period with most treatments from initial levels of
3-20 of vegetation cover though not with the untreated control treatment (Tr 1) and
only to a small extent on the limed control (Tr 2) Rhinanthus minor declined steeply
from 2-8 in 1999 to lt15 in 2001 but recovered to initial levels between 2008 and
2010
Lathyrus pratensis remained at very low levels overall (lt05) until 2005 then rose to
levels of up to 15 of the vegetation on fertilized plots by 2010 Lotus corniculatus
accounted for 4-15 of vegetation cover initially declining to levels averaging about 2
in 2001-2002 and increasing slightly to 2-8 from 2005 onwards
Most initial increases in NI were attributable to increases in H lanatus but under both
FYM and inorganic treatments this species was progressively replaced by P trivialis
mainly after 2005 The interchange between these two species was not clearly related to
liming in 2005 since it also occurred on Tr 14 plots (limed in 1999) although this was
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
31
not evident until 2004 Trifolium repens was prominent on most plots between 2001 and
2007 particularly with Tr 13 (nil fertilizer limed in 1999) though not with Tr5 This
species increased under Tr 4 initially but was replaced between 2005 and 2007 by
increases in Holcus before the latter declined in favour of P trivialis
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
32
Table S1 Soil chemical properties in 2010 at the upland meadow Treatments 13-15 were limed in 1999 and 2005 and Treatments 2-12 received
lime in 2005 only Treatment 1 received no fertilizer lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 540 046 510 1487 57 950 1000 1112
2 Limed (in 2005) control 590 049 500 1333 34 997 830 1500
3 FYM at 6 t ha-1
annual 630 060 500 1500 47 1167 897 1774
4+ FYM at 12 t ha-1
annual 617 060 522 1392 50 1432 1040 1657
5 + FYM at 24 t ha
-1 annual 633 061 543 1523 103 1933 1050 1778
6 FYM at 6 t ha-1
triennially 623 060 527 1480 31 1117 867 1718
7 + FYM at 12 t ha
-1 triennially 607 044 453 1353 57 830 840 1570
8 + FYM at 24 t ha
-1 triennially 623 052 500 1357 47 923 837 1649
9 + Inorg equivalent to Tr 4 593 059 507 1463 63 1470 780 1453
10 + Inorg equivalent to Tr 5 600 059 497 1340 67 2603 730 1513
11 + Inorg equivalent to Tr 7 620 054 487 1440 57 1043 717 1668
12 + Inorg equivalent to Tr 8 620 050 517 1390 31 1087 793 1678
13 Lime in years 1 and 7 587 066 567 1653 43 1210 1307 1554
14 Lime as Tr 13 + FYM as Tr 4 610 057 513 1515 43 1413 1160 1692
15 Lime as tr 13 + FYM as Tr 7 623 061 530 1567 37 1050 1073 1662 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (andor
2005) x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Soil chemical properties relate to air-dried soil from samples taken to a depth of 75 cm N C and LOI
refer to total N total C and loss on ignition respectively values for P (Oslashlsen extractable) and for K Mg and
Ca (extractable) are Mg l-1
of dried soil
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
33
Table S2 Soil chemical properties in 2010 at the lowland meadow At this site Treatments 13-15 were limed in 1999 only and Treatments 2-12
received lime in 2005 only Treatment 1 received no fertilizer or lime in any year
Treatment pH N C LOI P K Mg Ca
1 Untreated control 530 031 364 742 46 2677 2119 1404
2 Limed (in 2005) control 613 029 341 681 41 2049 1494 2133
3 FYM at 6 t ha-1
annual 610 032 345 756 47 2769 2033 2247
4+ FYM at 12 t ha-1
annual 624 032 364 745 51 2861 1946 2378
5 + FYM at 24 t ha
-1 annual 629 032 362 716 65 2979 2335 2261
6 FYM at 6 t ha-1
triennially 619 032 374 767 40 2142 1791 2319
7 + FYM at 12 t ha
-1 triennially 604 031 367 721 42 2164 1761 2138
8 + FYM at 24 t ha
-1 triennially 622 034 389 754 47 2758 1928 2509
9 + Inorg equivalent to Tr 4 603 031 352 714 47 2726 1491 2035
10 + Inorg equivalent to Tr 5 602 032 350 678 54 3288 1439 2237
11 + Inorg equivalent to Tr 7 591 031 355 700 42 2204 1689 2064
12 + Inorg equivalent to Tr 8 601 032 353 699 43 2469 1563 2179
13 Lime in years 1 602 031 387 734 36 1903 1569 2462
14 Lime as Tr 13 + FYM as Tr 4 621 032 367 717 50 2484 1760 2291
15 Lime as tr 13 + FYM as Tr 7 609 031 344 708 46 2055 1544 2349 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 (or 2005)
x FYM frequency (LFF) are indicated by lsquo+rsquo and lsquorsquo respectively
Other notes as Table S1
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
34
Table S3 Positive and negative indicator species Positive indicator species for MG3 and MG5 communities are taken from
Robertson amp Jefferson 2000 Negative indicator species include species taken from the same source supplemented by others
associated with loss of conservation value when nutrients are added to semi-natural meadows in previous work (see main paper text) Positive indicator species Negative indicator species all communities) MG3 MG5 MG5 (contd) Alchemilla glabra Agrimonia eupatoria Polygala vulgaris Acer campestre Rumex acetosa
Alchemilla spp Alchemilla glabra Polygala spp Acer pseudoplatanus Rumex crispus Anemone nemorosa Alchemilla spp Potentilla erecta Arrhenatherum elatius Rumex obtusifolius Centaurea nigra Anemone nemorosa Primula veris Cirsium arvense Salix alba Cirsium heterophyllum Carex flacca Rhinanthus minor Cirsium vulgare Salix caprea Conopodium majus Carex nigra Poterium sanguisorba Crataegus monogyna Salix cinerea Euphrasia officinalis agg Carex panicea Sanguisorba officinalis Dactylis glomerata Salix fragilis Euphrasia spp Centaurea nigra Serratula tinctoria Fagus sylvatica Sambucus nigra Filipendula ulmaria Euphrasia officinalis agg Silaum silaus Fraxinus excelsior Senecio jacobaea Geranium sylvaticum Euphrasia spp Betonica officinalis Galium aparine Sorbus sp Geum rivale Filipendula ulmaria Succisa pratensis Heracleum sphondylium Stellaria media Lathyrus pratensis Filipendula vulgaris Tragopogon pratensis Holcus lanatus Trifolium repens
Scorzoneroides autumnalis Galium verum Ilex aquifolium Ulex europaeus Leontodon hispidus Genista tinctoria Lolium perenne Ulmus sp Leontodon spp Lathyrus linifolius Poa trivialis Tree seedling unid Lotus corniculatus Lathyrus pratensis Prunus spinosa Urtica dioica Persicaria bistorta Leontodon hispidus Quercus sp Viburnum lantana Rhinanthus minor Leontodon saxatilis Ranunculus repens Sanguisorba officinalis Leucanthemum vulgare Rosa arvensis Succisa pratensis Lotus corniculatus Rosa spp Trollius europaeus Pimpinella saxifraga Rubus fruticosus Species highlighted in bold in the MG3 and MG5 lists were recorded in at least one year at upland and lowland meadows respectively
Bolded negative indicator species were those recorded at either site
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
35
Table S4 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the upland meadow 1999-2010 Degrees of freedom for Time and interactions with
Time are adjusted to account for departure from homogeneity of the population
covariance matrix using ε-coefficients calculated by the Greenhouse-Geisser method
(Genstat V Committee 1997 ndash see main paper for references) Significance levels
Plt005 Plt001 Plt0001 NS not significant (ie P gt005)
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 395 0001
Time 7 211 842 lt0001
Treat x Time 88 211 093 0647 NS Form x Rate Form (Fo) 1 14 434 0057 NS
x Frequency (FRF) Rate (R) 1 14 1392 0002
Frequency (Fr) 1 14 1571 0001
Fo x R 1 14 477 0047
Fo x Fr 1 14 584 0030
R x Fr 1 14 300 1050 NS
Fo x R x Fr 1 14 385 0070 NS
Time (T) 5 88 522 lt0001
T xFo 5 88 067 0647 NS
T xR 5 88 136 0247 NS
T xFr 5 88 238 0050
T x Fo x R 5 88 063 0677 NS
T x Fo x Fr 5 88 014 0983 NS
T x R x Fr 5 88 121 0311 NS
T x Fo x R x Fr 5 88 134 0255 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 088 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
36
Table S5 Totala species-richness (species m
-2) at the upland meadow 1999-2010 Treatments 13-15 were limed in 1999 and 2005 and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 260 269 252 263 278 278
2 Limed (in 2005) control 256 252 240 231 251 250 254 241 263 258 252
3 FYM at 6 t ha-1
annual 267 262 259 253 261 261 278 259 277 268 277
4 +FYM at 12 t ha-1
annual 266 248 226 233 244 258 238 226 252 246 265
5 +FYM at 24 t ha-1
annual 261 221 223 224 220 216 198 179 191 186 202
6 FYM at 6 t ha-1
triennially 257 243 238 247 261 258 250 259 287 271 270
7 +FYM at 12 t ha-1
triennially 267 262 250 263 270 280 283 256 272 261 276
8 +FYM at 24 t ha-1
triennially 248 254 238 243 261 271 267 250 260 260 278
9 +Inorg equivalent to Tr 4 259 251 260 252 263 266 254 229 264 258 266
10 +Inorg equivalent to Tr 5 273 243 240 247 249 262 248 232 261 242 260
11 +Inorg equivalent to Tr 7 250 256 256 254 260 269 276 260 289 276 269
12 +Inorg equivalent to Tr 8 263 254 233 240 261 269 257 241 273 260 270
13 Lime in years 1 and 7 268 248 261 258 273 273 276 274 281 268 264
14 Lime in year 1 + FYM as Tr 4 256 224 242 229 239 251 238 239 242 227 262
15 Lime in year 1 + FYM as Tr 7 242 244 243 241 264 264 269 233 264 252 263 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
37
Table S6 Repeated measures ANOVA statistics for PI species m-2
at the upland meadow
1999-2010 Other nots as in caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 193 0069 NS
Time 6 194 6668 lt0001
Treat x Time 81 194 090 0703 NS Form x Rate Form (Fo) 1 14 422 0059 NS
x Frequency (FRF) Rate (R) 1 14 665 0022
Frequency (Fr) 1 14 818 0013
Fo x R 1 14 081 0383 NS
Fo x Fr 1 14 018 0678 NS
R x Fr 1 14 000 1000 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 5 83 3585 lt0001
T xFo 5 83 098 0435 NS
T xR 5 83 195 0096 NS
T xFr 5 83 060 0700 NS
T x Fo x R 5 83 095 0453 NS
T x Fo x Fr 5 83 088 0498 NS
T x R x Fr 5 83 084 0525 NS
T x Fo x R x Fr 5 83 023 0948 NS Lime99 x Lime99 (L) 1 10 025 0628 NS
FYM frequency (LFF) FYMfreq (F) 2 10 113 0361 NS
L x F 2 10 220 0162 NS
Time (T) 5 58 386 0004
T x L 5 58 035 0880 NS
T x F 10 58 068 0738 NS
T x L x F 10 58 043 0926 NS
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
38
Table S7 PI species m-2
at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 733 733 700 711 767 789
2 Limed (in 2005) control 522 511 633 667 622 622 633 611 622 678 733
3 FYM at 6 t ha-1
annual 500 456 633 667 689 711 700 678 656 656 667
4 +FYM at 12 t ha-1
annual 522 450 600 594 606 656 622 628 628 667 644
5 +FYM at 24 t ha-1
annual 478 389 556 589 522 533 533 522 500 522 533
6 FYM at 6 t ha-1
triennially 467 411 600 667 644 644 589 656 633 667 689
7 +FYM at 12 t ha-1
triennially 600 522 633 678 722 722 711 733 722 733 778
8 +FYM at 24 t ha-1
triennially 456 456 578 611 633 644 578 656 589 656 656
9 +Inorg equivalent to Tr 4 444 456 600 633 678 689 678 667 700 733 744
10 +Inorg equivalent to Tr 5 511 433 600 611 622 689 633 611 589 611 622
11 +Inorg equivalent to Tr 7 533 600 733 756 733 789 778 789 811 789 800
12 +Inorg equivalent to Tr 8 544 533 678 689 722 778 733 711 733 711 700
13 Lime in years 1 and 7 500 489 600 667 667 689 656 689 678 722 711
14 Lime in year 1 + FYM as Tr 4 467 389 589 533 600 611 578 667 533 544 600
15 Lime in year 1 + FYM as Tr 7 389 456 500 578 578 556 589 578 567 600 622 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are
indicated by lsquo+rsquo and lsquorsquo respectively
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
39
Table S8 Repeated measures ANOVA statistics for PI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 367 0002
Time 5 161 281 lt0001
Treat x Time 67 161 133 0075 NS
Form x Rate Form (Fo) 1 14 269 0123 NS
x Frequency (FRF) Rate (R) 1 14 122 0288 NS
Frequency (Fr) 1 14 1089 0005
Fo x R 1 14 060 0452 NS
Fo x Fr 1 14 010 0757 NS
R x Fr 1 14 001 0922 NS
Fo x R x Fr 1 14 038 0548 NS
Time (T) 4 70 13996 lt0001
T xFo 4 70 142 0236 NS
T xR 4 70 151 0209 NS
T xFr 4 70 329 0016
T x Fo x R 4 70 106 0383 NS
T x Fo x Fr 4 70 124 0302 NS
T x R x Fr 4 70 051 0729 NS
T x Fo x R x Fr 4 70 027 0896 NS
Lime99 x Lime99 (L) 1 10 063 0446 NS
FYM frequency (LFF) FYMfreq (F) 2 10 1126 0003
L x F 2 10 061 0562 NS
Time (T) 4 44 11331 lt0001
T x L 4 44 041 0801 NS
T x F 7 44 198 0080 NS
T x L x F 7 44 052 0815 NS
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
40
Table S9 PI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 380 436 397 454 501 482
2 Limed (in 2005) control 167 236 307 358 425 476 502 510 582 562 636
3 FYM at 6 t ha-1
annual 183 179 312 396 366 396 443 437 478 531 518
4 +FYM at 12 t ha-1
annual 127 163 228 298 280 320 420 334 371 422 436
5 +FYM at 24 t ha-1
annual 136 133 190 254 191 286 436 311 335 405 434
6 FYM at 6 t ha-1
triennially 91 185 241 285 353 384 428 453 414 475 520
7 +FYM at 12 t ha-1
triennially 183 203 257 370 400 383 422 469 459 527 584
8 +FYM at 24 t ha-1
triennially 142 158 225 298 308 331 396 400 425 458 524
9 +Inorg equivalent to Tr 4 137 221 275 330 322 336 336 322 424 474 486
10 +Inorg equivalent to Tr 5 142 152 232 352 272 327 412 338 403 406 385
11 +Inorg equivalent to Tr 7 166 231 307 404 382 383 417 484 451 587 585
12 +Inorg equivalent to Tr 8 171 235 269 381 409 396 513 512 486 542 582
13 Lime in years 1 and 7 162 252 355 452 438 506 505 534 557 588 593
14 Lime in year 1 + FYM as Tr 4 146 153 221 243 231 299 377 301 352 384 422
15 Lime in year 1 + FYM as Tr 7 112 185 217 295 314 356 389 403 412 478 551
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated
by lsquo+rsquo and lsquorsquo respectively
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
41
Table S10 Repeated measures ANOVA statistics for NI of live cover (PI) arcsine-
square root transformed data at the upland meadow 1999-2010 Other notes as in
caption to Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 13 29 1810 lt0001
Time 6 194 3774 lt0001
Treat x Time 81 194 202 lt0001
Form x Rate Form (Fo) 1 14 1767 lt0001
x Frequency (FRF) Rate (R) 1 14 2554 lt0001
Frequency (Fr) 1 14 2866 lt0001
Fo x R 1 14 041 0532 NS
Fo x Fr 1 14 105 0323 NS
R x Fr 1 14 442 0054 NS
Fo x R x Fr 1 14 026 0618 NS
Time (T) 6 99 1564 lt0001
T xFo 6 99 119 0318 NS
T xR 6 99 267 0019
T xFr 6 99 661 lt0001
T x Fo x R 6 99 082 0557 NS
T x Fo x Fr 6 99 067 0674 NS
T x R x Fr 6 99 153 0176 NS
T x Fo x R x Fr 6 99 119 0318 NS
Lime99 x Lime99 (L) 1 10 027 0615 NS
FYM frequency (LFF) FYMfreq (F) 2 10 2613 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 53 2225 lt0001
T x L 4 53 074 0569 NS
T x F 9 53 372 0010
T x L x F 9 53 075 0662 NS
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
42
Table S11 NI species of live cover (PI) at the upland meadow 1999-2010 Other notes as in caption to table S3
Treatment 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control - - - - - 80 134 130 84 54 51
2 Limed (in 2005) control 223 160 160 80 79 70 64 88 50 48 42
3 FYM at 6 t ha-1
annual 226 207 251 134 123 113 152 167 162 156 170
4 +FYM at 12 t ha-1
annual 278 246 306 225 154 197 188 242 226 238 221
5 +FYM at 24 t ha-1
annual 261 268 343 367 302 375 303 344 428 379 337
6 FYM at 6 t ha-1
triennially 315 210 231 140 122 130 101 140 126 95 94
7 +FYM at 12 t ha-1
triennially 257 201 257 170 122 145 172 160 147 108 97
8 +FYM at 24 t ha-1
triennially 277 240 317 238 159 178 204 177 172 172 135
9 +Inorg equivalent to Tr 4 223 199 231 127 122 145 178 187 143 108 117
10 +Inorg equivalent to Tr 5 257 222 296 212 221 207 193 245 248 272 265
11 +Inorg equivalent to Tr 7 236 166 210 108 85 139 146 120 100 76 68
12 +Inorg equivalent to Tr 8 278 203 239 175 126 171 140 142 155 100 85
13 Lime in years 1 and 7 228 147 156 71 71 64 69 90 52 39 49
14 Lime in year 1 + FYM as Tr 4 233 209 281 261 192 252 225 213 286 234 215
15 Lime in year 1 + FYM as Tr 7 272 191 267 188 150 160 183 161 130 117 103
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
43
Table S12 Repeated measures ANOVA statistics for total species-richness (species m-2
)
at the lowland meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 287 0009
Time 6 175 904 lt0001
Treat x Time 82 175 151 0012
Form x Rate Form (Fo) 1 14 1352 0003
x Frequency (FRF) Rate (R) 1 14 403 0064 NS
Frequency (Fr) 1 14 609 0027
Fo x R 1 14 003 0865 NS
Fo x Fr 1 14 006 0810 NS
R x Fr 1 14 293 0109 NS
Fo x R x Fr 1 14 268 0124 NS
Time (T) 5 83 770 lt0001
T xFo 5 83 221 0061 NS
T xR 5 83 106 0389 NS
T xFr 5 83 639 lt0001
T x Fo x R 5 83 099 0429 NS
T x Fo x Fr 5 83 237 0046
T x R x Fr 5 83 127 0285 NS
T x Fo x R x Fr 5 83 193 0098 NS
Lime99 x Lime99 (L) 1 10 001 0922 NS
FYM frequency (LFF) FYMfreq (F) 2 10 255 0127 NS
L x F 2 10 166 0239 NS
Time (T) 5 63 388 0004
T x L 5 63 076 0582 NS
T x F 10 63 112 0362 NS
T x L x F 10 63 123 0290 NS
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
44
Table S13 Totala species-richness (species m
-2) at the lowland meadow 1999-2010 Treatments 13-15 were limed in 1999 only and
Treatments 2-12 received lime in 2005 only Treatment 1 received no lime or fertilizer in any year
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 213 212 220 228 220 201 224 223 229 229 220 196
2 Limed (in 2005) control 221 210 216 212 230 178 216 221 226 230 213 210
3 FYM at 6 t ha-1
annual 223 226 221 221 241 191 226 229 201 221 192 197
4 +FYM at 12 t ha-1
annual 208 230 218 217 224 210 204 223 199 217 176 164
5 +FYM at 24 t ha-1
annual 229 229 214 202 207 182 186 174 164 154 133 140
6 FYM at 6 t ha-1
triennially 231 219 214 242 223 200 226 222 217 217 211 223
7 +FYM at 12 t ha-1
triennially 207 210 194 198 204 184 210 218 199 220 196 217
8 +FYM at 24 t ha-1
triennially 207 212 203 204 228 214 200 221 213 230 221 182
9 +Inorg equivalent to Tr 4 233 218 219 228 234 223 206 230 204 220 201 203
10 +Inorg equivalent to Tr 5 226 224 193 216 206 210 207 216 213 216 191 184
11 +Inorg equivalent to Tr 7 224 220 219 246 219 211 239 236 231 240 229 220
12 +Inorg equivalent to Tr 8 213 212 218 237 212 206 240 227 213 217 213 214
13 Lime in year 1 199 234 210 223 231 206 219 222 228 229 206 219
14 Lime in year 1 + FYM as Tr 4 196 201 178 189 194 189 172 193 191 201 194 187
15 Lime in year 1 + FYM as Tr 7 211 228 212 223 212 201 222 223 217 226 213 206 aBryophytes grouped as a single species
Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
45
Table S14 Repeated measures ANOVA statistics for PI species m-2
at the lowland
meadow 1999-2010 Other caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 409 lt0001
Time 11 92 2598 lt0001
Treat x Time 90 192 155 0006
Form x Rate Form (Fo) 1 14 1414 0002
x Frequency (FRF) Rate (R) 1 14 262 0128 NS
Frequency (Fr) 1 14 2017 lt0001
Fo x R 1 14 058 0459 NS
Fo x Fr 1 14 004 0844 NS
R x Fr 1 14 166 0219 NS
Fo x R x Fr 1 14 057 0463 NS
Time (T) 6 93 2202 lt0001
T xFo 6 93 184 0100 NS
T xR 6 93 092 0484 NS
T xFr 6 93 636 lt0001
T x Fo x R 6 93 057 0753 NS
T x Fo x Fr 6 93 061 0722 NS
T x R x Fr 6 93 087 0520 NS
T x Fo x R x Fr 6 93 188 0092 NS
Lime99 x Lime99 (L) 1 10 038 0551 NS
FYM frequency (LFF) FYMfreq (F) 2 10 440 0043
L x F 2 10 148 0274 NS
Time (T) 4 50 805 lt0001
T x L 4 50 192 0122 NS
T x F 8 50 209 0054 NS
T x L x F 8 50 082 0589 NS
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
46
Table S15 PI species m-2
at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 578 533 567 611 633 567 633 589 633 522 478 444
2 Limed (in 2005) control 544 500 533 500 589 567 589 544 567 500 467 456
3 FYM at 6 t ha-1
annual 533 489 511 567 544 489 544 522 356 422 367 411
4 +FYM at 12 t ha-1
annual 500 533 456 511 522 478 500 511 400 356 356 367
5 +FYM at 24 t ha-1
annual 533 522 478 433 489 444 400 344 278 244 278 300
6 FYM at 6 t ha-1
triennially 544 500 456 567 556 544 611 544 478 500 456 456
7 +FYM at 12 t ha-1
triennially 533 511 522 533 522 489 544 489 489 456 400 444
8 +FYM at 24 t ha-1
triennially 489 500 433 522 533 556 511 500 489 456 478 411
9 +Inorg equivalent to Tr 4 589 533 478 533 533 522 511 522 467 422 378 411
10 +Inorg equivalent to Tr 5 600 567 444 489 478 511 489 478 467 422 356 367
11 +Inorg equivalent to Tr 7 511 533 489 578 533 600 611 556 578 544 511 489
12 +Inorg equivalent to Tr 8 578 511 533 589 544 567 622 556 533 500 478 467
13 Lime in year 1 478 489 433 500 544 456 489 500 556 500 467 433
14 Lime in year 1 + FYM as Tr 4 500 467 322 467 422 433 333 422 400 322 389 356
15 Lime in year 1 + FYM as Tr 7 533 544 522 533 556 578 611 567 544 533 511 478 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
47
Table S16 Repeated measures ANOVA statistics for PI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 051 0907 NS
Time 6 193 2871 lt0001
Treat x Time 90 193 106 0365 NS
Form x Rate Form (Fo) 1 14 011 0745 NS
x Frequency (FRF) Rate (R) 1 14 002 0890 NS
Frequency (Fr) 1 14 052 0483 NS
Fo x R 1 14 002 0890 NS
Fo x Fr 1 14 000 1000 NS
R x Fr 1 14 043 0523 NS
Fo x R x Fr 1 14 003 0865 NS
Time (T) 6 90 1541 lt0001
T xFo 6 90 071 0642 NS
T xR 6 90 085 0535 NS
T xFr 6 90 195 0081 NS
T x Fo x R 6 90 114 0346 NS
T x Fo x Fr 6 90 078 0588 NS
T x R x Fr 6 90 030 0935 NS
T x Fo x R x Fr 6 90 131 0261 NS
Lime99 x Lime99 (L) 1 10 132 0277 NS
FYM frequency (LFF) FYMfreq (F) 2 10 107 0379 NS
L x F 2 10 021 0814 NS
Time (T) 4 52 1535 lt0001
T x L 4 52 204 0102 NS
T x F 9 52 128 0270 NS
T x L x F 9 52 076 0653 NS
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
48
Table S17 PI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 397 320 257 255 252 315 256 296 270 184 288 320
2 Limed (in 2005) control 295 280 288 262 258 273 225 314 297 259 246 339
3 FYM at 6 t ha-1
annual 386 315 254 251 284 270 172 234 180 228 239 263
4 +FYM at 12 t ha-1
annual 374 288 265 221 260 233 164 161 211 179 307 317
5 +FYM at 24 t ha-1
annual 347 283 302 295 283 289 187 215 259 172 246 222
6 FYM at 6 t ha-1
triennially 413 343 334 281 246 233 256 260 214 266 299 338
7 +FYM at 12 t ha-1
triennially 305 310 258 220 259 228 202 230 269 253 259 397
8 +FYM at 24 t ha-1
triennially 379 268 277 285 208 241 173 191 196 218 292 347
9 +Inorg equivalent to Tr 4 321 304 302 241 238 227 202 235 228 201 249 293
10 +Inorg equivalent to Tr 5 459 340 338 243 242 158 171 183 217 194 277 297
11 +Inorg equivalent to Tr 7 307 273 312 227 267 259 240 273 245 213 265 366
12 +Inorg equivalent to Tr 8 287 323 262 236 255 210 201 234 280 210 266 359
13 Lime in year 1 342 324 305 266 281 225 151 185 178 181 241 336
14 Lime in year 1 + FYM as Tr 4 374 332 315 216 237 167 77 157 189 183 275 346
15 Lime in year 1 + FYM as Tr 7 443 357 205 228 246 199 151 227 214 227 309 325 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
49
Table S18 Repeated measures ANOVA statistics for NI species of live vegetation
(PI) arcsine-square root transformed data at the lowland meadow 1999-2010 Other
caption notes as Table S2
Treatment series Factor
Degrees
of
freedom F-ratio Probability Significance
All treatments Treatment 14 28 2143 lt0001
Time 7 215 3359 lt0001
Treat x Time 100 215 209 lt0001
Form x Rate Form (Fo) 1 14 965 0008
x Frequency (FRF) Rate (R) 1 14 3913 lt0001
Frequency (Fr) 1 14 11389 lt0001
Fo x R 1 14 494 0043
Fo x Fr 1 14 292 0110 NS
R x Fr 1 14 260 0129 NS
Fo x R x Fr 1 14 253 0134 NS
Time (T) 6 97 2290 lt0001
T xFo 6 97 102 0417 NS
T xR 6 97 159 0158 NS
T xFr 6 97 581 lt0001
T x Fo x R 6 97 087 0520 NS
T x Fo x Fr 6 97 067 0674 NS
T x R x Fr 6 97 107 0386 NS
T x Fo x R x Fr 6 97 070 0650 NS
Lime99 x Lime99 (L) 1 10 541 0042
FYM frequency (LFF) FYMfreq (F) 2 10 4076 lt0001
L x F 2 10 017 0846 NS
Time (T) 4 54 1529 lt0001
T x L 4 54 284 0033
T x F 9 54 353 0002
T x L x F 9 54 071 0697 NS
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
50
Table S19 NI species of live vegetation (PI) at the lowland meadow 1999-2010 Notes as in caption to Table S11
Treatment 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
1 Untreated control 51 16 59 28 07 24 27 47 81 39 17 09
2 Limed (in 2005) control 27 25 35 77 21 18 28 43 82 55 31 22
3 FYM at 6 t ha-1
annual 43 80 38 67 79 62 202 148 275 184 90 110
4 +FYM at 12 t ha-1
annual 32 61 86 106 84 157 158 236 192 275 200 339
5 +FYM at 24 t ha-1
annual 68 104 216 195 204 216 428 357 320 485 524 577
6 FYM at 6 t ha-1
triennially 25 42 39 83 19 13 50 49 143 80 63 30
7 +FYM at 12 t ha-1
triennially 33 37 65 99 35 48 81 89 78 105 37 90
8 +FYM at 24 t ha-1
triennially 42 56 55 88 63 78 141 163 203 286 173 90
9 +Inorg equivalent to Tr 4 58 42 73 98 90 118 152 163 234 249 182 286
10 +Inorg equivalent to Tr 5 43 91 118 197 125 96 238 260 204 371 323 259
11 +Inorg equivalent to Tr 7 31 55 71 109 23 18 99 86 66 87 89 38
12 +Inorg equivalent to Tr 8 39 78 102 120 47 46 90 127 114 173 96 142
13 Lime in year 1 12 36 54 86 42 83 107 91 105 48 49 50
14 Lime in year 1 + FYM as Tr 4 33 95 87 150 108 161 285 319 249 251 200 177
15 Lime in year 1 + FYM as Tr 7 47 37 78 99 43 66 233 178 163 161 45 31 Treatments comprising factorial series for fertilizer Form x Rate x Frequency (FRF) or Lime in 1999 x FYM frequency (LFF) are indicated by
lsquo+rsquo and lsquorsquo respectively
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
51
Table S20 Effect of treatments on five key variables at two sites in 2010 Values for positive indicator (PI) species are for MG3 and MG5 PI species for
the upland and lowland meadows respectively Treatment 1 was not included in ANOVAS adjusted for covariance with 1999 data (PI species at the
upland meadow - italicised) Values in parentheses are means of non-transformed data these are back-transformed means where data were adjusted for
covariance with 1999 data All treatments received lime in year 2005 except untreated controls (Tr1) at both sites and Trs 13-15 at lowland meadow)
Upland meadow (UM) Lowland meadow (LM)
Species m-2
Cover (arcsine-sqrt of p) Species m-2
Cover (arcsine-sqrt of p)
Treatment Total a PI
b NI PIb NI Total
a PIb NI PI NI
1 Untreated control 282 789 456 0769 (483) 0227 (51) 203 424 211 0601 (320) 0097 (09)
2 Limed (in 1995) control 254 722 367 0908 (621) 0204 (42) 216 451 278 0621 (339) 0143 (22)
3 FYM at 6 t ha-1
annual 281 665 589 0780 (494) 0423 (170) 202 413 344 0537 (263) 0322 (110)
4 FYM at 12 t ha-1
annual 268 633 578 0733 (447) 0489 (221) 164 385 367 0597 (317) 0620 (339)
5 FYM at 24 t ha-1
annual 202 542 522 0725 (440) 0618 (337) 140 302 444 0488 (222) 0865 (577)
6 FYM at 6 t ha-1
triennially 272 703 533 0845 (559) 0311 (94) 227 451 389 0620 (338) 0169 (30)
7 FYM at 12 t ha-1
triennially 280 730 522 0846 (560) 0314 (97) 219 446 378 0681 (397) 0286 (90)
8 FYM at 24 t ha-1
triennially 279 675 544 0811 (526) 0375 (135) 186 435 333 0629 (347) 0290 (90)
9 Inorg equivalent to Tr 4 268 769 533 0778 (492) 0347 (117) 203 385 456 0572 (293) 0561 (286)
10 Inorg equivalent to Tr 5 266 616 600 0671 (387) 0541 (265) 186 335 378 0575 (297) 0523 (259)
11 Inorg equivalent to Tr 7 271 783 467 0862 (576) 0262 (68) 237 501 289 0649 (366) 0196 (38)
12 Inorg equivalent to Tr 8 274 678 544 0851 (565) 0291 (85) 221 446 400 0641 (359) 0347 (142)
13 Lime in year 1 (+ yr 7 at the UM) 269 710 433 0870 (584) 0220 (49) 223 463 411 0614 (336) 0210 (50)
14 Lime in year 1 + FYM as Tr 4 268 614 578 0707 (422) 0480 (215) 187 373 422 0627 (346) 0433 (177)
15 Lime in year 1 + FYM as Tr 7 269 672 544 0860 (574) 0327 (103) 211 480 322 0606 (325) 0176 (31)
SEDs All Trs except Tr 4 at the UM 1725 0583 0404 05827 00436 123 0487 074 00539 00856
Tr 4 v others at the UM 1494 0504 0350 05044 00377 - - - - - -
P for treatment effect in ANOVA 0017 0016 lt0001 lt0001 lt0001 lt0001 0019 NS NS lt0001
Residual df 31 28 31 28 31 28 27 28 28 28
Values in bold are significantly different (Plt005) from the limed control treatment (Tr 2) a Values for total species per m
2 include individual bryophyte species
b Values adjusted for covariance with 1999 data except for the untreated control treatment at the upland meadow (not present in 1999)
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
52
Table S21 Soil microbial community analysis at the upland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control Not assessed Not assessed Not assessed Not assessed
2 Limed (in 1995) control 883 339 268 169 252 0346 010 0015
3 FYM at 6 t ha-1
annual 756 169 232 061 190 0258 008 0013
4 FYM at 12 t ha-1
annual 818 976 253 339 220 0367 009 0008
5 FYM at 24 t ha-1
annual 866 288 284 165 164 0085 006 0006
6 FYM at 6 t ha-1
triennially 734 1189 222 374 231 0453 011 0019
7 FYM at 12 t ha-1
triennially 877 962 267 317 209 0312 008 0003
8 FYM at 24 t ha-1
triennially 846 496 264 207 188 0110 007 0008
9 Inorg equivalent to Tr 4 862 347 278 083 175 0045 006 0000
10 Inorg equivalent to Tr 5 727 144 236 092 181 0053 008 0004
11 Inorg equivalent to Tr 7 743 108 239 044 158 0082 007 0004
12 Inorg equivalent to Tr 8 1054 816 321 198 273 0388 009 0004
13 Lime in year 1 943 565 296 157 197 0053 007 0000
14 Lime in year 1 + FYM as Tr 4 895 1129 286 367 202 0220 007 0000
15 Lime in year 1 + FYM as Tr 7 814 378 252 085 212 0082 009 0004
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006
53
Table S22 Soil microbial community analysis at the lowland meadow total bacterial and fungal phospho-lipid fatty acid (PLFA) content (μmol g-1
) in
2010 and the ratio of fungalbacterial PLFA
Total PLFA Bacterial PLFA Fungal PLFA FungalBacterial ratio
Treatment Mean se Mean se Mean se Mean se
1 Untreated control 705 500 217 214 189 0383 009 0017
2 Limed (in 1995) control 611 502 183 148 172 0214 010 0007
3 FYM at 6 t ha-1
annual 699 1068 215 259 161 0102 008 0009
4 FYM at 12 t ha-1
annual 653 379 204 092 151 0239 008 0012
5 FYM at 24 t ha-1
annual 648 616 200 141 143 0195 007 0009
6 FYM at 6 t ha-1
triennially 750 483 224 127 225 0135 010 0012
7 FYM at 12 t ha-1
triennially 747 232 229 108 230 0283 010 0018
8 FYM at 24 t ha-1
triennially 681 794 225 296 152 0221 007 0013
9 Inorg equivalent to Tr 4 605 458 186 182 128 0129 007 0009
10 Inorg equivalent to Tr 5 627 266 204 143 129 0131 006 0009
11 Inorg equivalent to Tr 7 696 270 221 027 209 0306 010 0015
12 Inorg equivalent to Tr 8 652 425 190 072 153 0172 007 0007
13 Lime in year 1 657 546 202 152 187 0352 009 0015
14 Lime in year 1 + FYM as Tr 4 709 927 238 233 148 0110 007 0013
15 Lime in year 1 + FYM as Tr 7 698 1205 249 112 161 0255 008 0006