Submitted 14 June 2019Accepted 10 December 2019Published 22 January 2020

Corresponding authorsFeinan Hu hufnnwafueducnShiwei Zhao swzhaonwafueducn

Academic editorHannah Buckley

Additional Information andDeclarations can be found onpage 15

DOI 107717peerj8382

Copyright2020 Ma et al

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

Shifts in soil nutrient concentrations andCNP stoichiometry during long-termnatural vegetation restorationRentian Ma1 Feinan Hu12 Jingfang Liu1 Chunli Wang1 Zilong Wang1Gang Liu12 and Shiwei Zhao12

1 State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau College of Natural Resourcesand Environment Northwest AampF University Yangling Shaanxi China

2 Institute of Soil and Water Conservation Chinese Academy of Sciences and Ministry of Water ResourcesYangling Shaanxi China

ABSTRACTBackground Ecological stoichiometry (CNP ratios) in soil is an important indicatorof the elemental balance in ecological interactions and processes Long-term naturalvegetation plays an important role in the accumulation and distribution of soilstoichiometry However information about the effects of long-term secondary forestsuccession on soil stoichiometry along a deep soil profile is still limitedMethods We selected Ziwuling secondary succession forest developed from farmlandas the study area investigated the concentrations and stoichiometry of soil organiccarbon (SOC) total nitrogen (TN) and total phosphorus (TP) at a depth of 0ndash100 cmalong a 90-year succession chronosequence including farmland (control) grasslandshrub early forest and climax forestResults SOC and TN concentrations significantly increased with increasing restorationage whereas soil P concentration remained relatively stable across various successionalstages SOC and TN concentrations decreased with an increase in soil depth exhibitingdistinct soil nutrient lsquolsquosurface-aggregationrsquorsquo (high nutrients concentration in the top soillayer) The soil CP andNP ratios increased with an increase in restoration age whereasthe variation of theCN ratiowas small and relatively stable across vegetation successionThe nutrient limitation changed along with vegetation succession transitioning fromlimited N in the earlier successional stages to limited P in the later successional stagesConclusion Our results suggest that more nitrogen input should be applied to earliersuccession stages and more phosphorus input should be utilized in later successionstages in order to address limited availability of these elements In general naturalvegetation restoration was an ecologically beneficial practice for the recovery ofdegraded soils in this area The findings of this study strengthen our understandingof the changes of soil nutrient concentration and nutrient limitation after vegetationrestoration and provide a simple guideline for future vegetation restoration andreconstruction efforts on the Loess Plateau

Subjects Agricultural Science Ecology Soil ScienceKeywords Vegetation restoration Soil organic carbon Nitrogen Phosphorus Secondary forest

How to cite this article Ma R Hu F Liu J Wang C Wang Z Liu G Zhao S 2020 Shifts in soil nutrient concentrations and CNP stoi-chiometry during long-term natural vegetation restoration PeerJ 8e8382 httpdoiorg107717peerj8382

INTRODUCTIONNatural vegetation restoration has beenwidely used to prevent soil degradation improve theecological environment and rehabilitate degraded environments (Mcgroddy Daufresne ampHedin 2004Gruumlnzweig et al 2007Wang et al 2014) Vegetation restoration can stimulatesoil nutrient cycling and maintain soil quality by altering plant species and communitycomposition litter quality and quantity (Cao amp Chen 2017 Li et al 2016 Zhao et al2015) root architecture and exudates (Berger Neubauer amp Glatzel 2002 Clemmensen etal 2013Deng et al 2018) andmicrobial activity (Gao et al 2014Gispert et al 2013) Thestudy of the vegetation restoration process is critical for understanding the relationshipbetween vegetation succession and the changes of soil ecological function providing aguideline for ecological environment reconstruction or restoration

In the vegetation restoration process changes inevitably occur in the composition ofsoil nutrient elements particularly in the three main elements carbon (C) nitrogen (N)and phosphorus (P) (Wei et al 2009 Zhao et al 2015) C N and P are the three primarynutrients in soils that most influence ecosystem structure and function (Agren 2008 Tianet al 2010) To some extent these elementsrsquo synergistic effects control ecological processessuch as the biological elemental cycle and energy transfer (Agren 2008 Ladanai Agren ampOlsson 2010 Laik et al 2009) CNP stoichiometry mainly focuses on the interaction andbalance of chemical elements in ecological processes (Agren 2008 Mcgroddy Daufresneamp Hedin 2004 Ren et al 2016 Wardle et al 2004) and provides a useful and effectiveway to study the distribution nutrient limitation and regulatory mechanism of nutrientcomposition in the ecosystem (Wang et al 2014) In recent years many researchers havereported the CNP stoichiometry patterns in plant organs (Bai et al 2019 Yang Liu ampAn 2018) plant communities (Fu et al 2010 Jiao et al 2013 Zhao et al 2017) standages (Zhang et al 2019) and plantations and natural secondary forests (Cao amp Chen 2017Deng et al 2016) For example Yang Liu amp An (2018) found that the CNP stoichiometryin leaves roots litter and soil varied hugely and that the plant community makeup had asignificant effect on CNP stoichiometry Li et al (2019) indicated that natural vegetationrestoration increased microbial CP and NP ratios in farmland after abandonment Caoamp Chen (2017) documented that natural forest had a greater capacity for C storage thanplantation Despite numerous studies looking at the CNP stoichiometry in terrestrialecosystems (Agren 2008 Han et al 2005) CNP stoichiometry in soils is yet to be fullydescribed (Manzoni amp Porporato 2009 Zhao et al 2017) especially in the Loess Plateau ofChina

The Loess Plateau is an ecologically fragile area in China Owing to long-termunsustainable land use and large-scale cultivation of sloping croplands native vegetationwas destroyed and soil erosion was aggravated in this area (Deng et al 2016 Fu et al2010 Wang Zhang amp Haung 2009) In order to improve the degraded land the Chinesegovernment has taken a variety ofmeasures to prevent further soil andwater loss and restorethe ecosystem (Fu et al 2000) So far a large number of croplands have been convertedto natural grassland or forest in the Loess Plateau Soil nutrient concentrations generallyundergo dynamic changes over time and these changes directly influence elemental

Ma et al (2020) PeerJ DOI 107717peerj8382 220

nutrient stoichiometry (Fan et al 2015 Ma et al 2019 Wang et al 2014) Many studieshave found that vegetation restoration enhances soil nutrient status improves microbialactivity (Fan et al 2015 Ren et al 2016 Zinn Marrenjo amp Silva 2018) and improves theCNP stoichiometry (Bai et al 2019 Yang amp Luo 2011) However other studies haveshown that land use change decreases soil nutrient concentrations (Yang et al 2012) anddid not change soil CN ratio (Zinn Marrenjo amp Silva 2018) In addition studies about theeffects of vegetation restoration on soil nutrient concentrations and CNP stoichiometryare mainly focused on topsoil (0ndash20 cm) as the topsoil is the source of the majority ofplant nutrients and is most vulnerable to human disturbances (Wang Zhang amp Haung2009) Recent studies have suggested that the nutrient status of deep soil may also beaffected by long-term vegetation restoration (Li et al 2016 Vandenbygaart et al 2011)Zhao et al (2015) demonstrated that long-term afforestation could greatly affect C and Nstocks and stoichiometry in deep soil However knowledge about the nutrient dynamicsof deep soil profiles at various succession stages in arid and semi-arid areas is still lackingwhich puts constraints on our ability to understand the geochemical cycles of nutrientelements in these environments Therefore we chose the Ziwuling secondary successionforest as the study area which is developed from farmland and has an intact series ofnaturally recovering vegetation succession The concentrations and stoichiometry of SOCTN and TP at a depth of 0ndash100 cm along a 90-year succession chronosequence in this areawere investigated The aim of this study was to (1) examine the dynamics in soil nutrientconcentrations along a long-term natural vegetation succession and the relationshipbetween soil nutrient content and a 0ndash100 cm soil profile in a secondary forest region (2)illustrate the effects of vegetation succession on soil CNP stoichiometry and determinenutrient limitations

MATERIALS AND METHODSSite descriptionThe study region is located in Lianjiabian forest farm Heshui County Gansu Provinceto the north of the Ziwuling Mountains in the Loess Plateau of China (3503primendash3637primeN10810primendash10908primeE altitude 1245ndash1285 m) (Fig 1) The climate is semi-arid monsoonwith a mean annual rainfall of 587 mm and a mean annual temperature of 74 C Thesoil in the study region is typical loessal developed from native (hillside) or secondary(valley) loess (Jia et al 2005) The soil is uniformly distributed from a depth of 50 to 130m above red earth At present the Ziwuling forest area is the most intact natural secondaryforest in the Loess Plateau During the national conflict from 1842 to 1866 many localpeople were displaced and secondary forests were established on the abandoned landOwing to the implementation of lsquolsquoGrain-for-Green Projectrsquorsquo in 2000 a large number offarmlands have gradually been abandoned In the same region a series of successionstages have formed along with different restoration ages Without human interference thevegetation succession here would progress as follows grassland (Bothriochloa ischaemumCarex lanceolata Glycyrrhiza and Stipa bungeana are the main herb species) to shrubland(Sophora davidiiHippophae rhamnoides and Spiraea pubescens are themain shrub species)

Ma et al (2020) PeerJ DOI 107717peerj8382 320

Figure 1 Location of the study site in the Loess PlateauFull-size DOI 107717peerj8382fig-1

to early forest (Populus davidiana and Betula platyphylla) to climax forest (Quercusliaotungensis) (Cheng et al 2012)

Experiment design and soil samplingAfter we obtained oral permission from the administrator (Mr Jifan Wang the head ofZiwuling Forestry Bureau) we collected the soil samples in mid-June of 2018 Accordingto the vegetation restoration age structure and community composition the typicalvegetation type for each stage of the vegetation succession was selected using space-for-time substitution (Zhao et al 2015) such as Bothriochloa ischaemum (Bi) for grasslandHippophae rhamnoides (Hr) for shrubland Betula platyphylla (Bp) and Populus davidiana(Pd) for early forest andQuercus liaotungensis (Ql) as climax forest with adjacent farmland(Fa) as control The main crop of the farmlands was corn Corn cultivation followed thetraditional cropland practice farmers plowing the soil at least twice before the crop growingseason and applying chemical fertilizers (about 70 kg N haminus1 and 23 kg P haminus1) twice peryear at sowing time and in mid-July The stand ages of all vegetation types were determinedwith the help of the local forest management bureau and confirmed using the hole-boringtechnique Three sampling sites were selected as replicates for each vegetation type andthree plots (20 m times 20 m for forest community 10 m times 10 m for shrub community and1 m times 1 m for herbaceous community and farmland) were randomly established withineach sampling site As soils below 100 cm in depth were mixed with small pieces of stonesin most sampling sites we selected 0ndash100 cm as our sampling depth After removing leaflitter and humus layer from the soil surface undisturbed soils were sampled at depths of0ndash10 10ndash20 20ndash40 40ndash70 and 70ndash100 cm using a shovel in each plot Soil samples of

Ma et al (2020) PeerJ DOI 107717peerj8382 420

Table 1 Geographical and vegetation characteristics at different successional stages in the Ziwuling forest region of the Loess Plateau Bi HrBp Pd Ql NE and NW represent Bothriochloa ischemum Hippophae rhamnoides Betula platyphylla Populus davidiana Pinus tabuliformis andQuercus liaotungensis northeast and northwest respectively

Successionalstage

Vegetationtypes

Restorationages(year)

Longitude(E)

Latitude(N)

Altitude(m)

Slope()

Aspect()

Coverage()

Other majorplantspecies

Farmland Cornfield

0 10832prime599primeprime 364prime3620primeprime 1021 0 NE10 0 ndash

Grassland Bi 20 10831prime3670primeprime 365prime313primeprime 1345 13 NW74 95 Stipa grandisGlycyrrhizauralensisArtemisiagiraldii andCarex lanceolata

Shrub Hr 30 10831prime3602primeprime 365prime581primeprime 1349 10 NW30 70 Stipa grandis andCarex lanceolata

Bp 45 10832prime507primeprime 364prime857primeprime 1339 20 NE2 58 Carex lanceolataEarlyforest Pd 60 10831prime4504primeprime 362prime5457primeprime 1449 12 NW13 68 Spiraea pubescens

Cotoneaster acuti-folius Rosa hugo-nis and Carexlanceolata

Climaxforest

Ql 90 10832prime3237primeprime 363prime526primeprime 1432 17 NE51 75 Spiraea pubescensCotoneaster acuti-folius Stipa gran-dis and Carexlanceolata

the same layer were mixed into one sampling bag at each sampling site In total 90 soilsamples (six vegetation types times three sites times five soil layers) were collected All sampleswere air-dried roots and stones were removed and the sample was passed through a 025mm sieve for SOC TN and TP analyses Table 1 shows the basic information regardingvegetation types physiographical conditions and elevation

Soil sample analysisSoil organic carbon (SOC) was determined by the H2SO4-K2Cr2O7 oxidation method(Nelson amp Sommers 1982) The 05 g soil samples were broken down with 5 ml of1 M K2Cr2O7 and 5 ml of concentrated H2SO4 at 180 C for 5 min then titrated withstandardized FeSO4 Total soil N (TN) concentration was measured using the Kjeldahlmethod after extraction with sulfuric acid (Sparks et al 1996) Total soil P (TP) wasdetermined colorimetrically after the sample was broken down with H2SO4 and HClO4

(Parkinson amp Allen 1975)

Data analysisStatistical analyses were performedusing the Statistical Package for the Social Sciences (SPSSversion 200 for Windows) Before applying parametric tests we tested for the normalityand homogeneity of the variances Nested ANOVA was used to systemically test the effectof vegetation types and soil depths on soil nutrient concentrations and stoichiometry Least

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Table 2 Concentrations of SOC TN and TP in soil profileMeans with different lowercase letters were significantly different at the 005 level fordifferent soil layers in the same nutrient elements The sample size n= 3

Soildepth (cm)

Maximum(g kgminus1)

Minimum(g kgminus1)

Mean(g kgminus1)

Standarddeviation (g kgminus1)

Coefficient ofvariation

0ndash10 4020 720 2720a 1042 04110ndash20 1693 642 1112b 295 02720ndash40 914 423 625c 133 02140ndash70 641 267 408c 092 022

SOC

70ndash100 548 257 337c 067 0200ndash10 382 098 239a 095 04010ndash20 156 085 111b 018 01620ndash40 094 037 068c 019 02840ndash70 071 025 047c 015 032

TN

70ndash100 063 022 039c 013 0320ndash10 082 060 071a 006 00910ndash20 076 053 066b 007 01120ndash40 071 051 062c 007 01140ndash70 069 054 061c 006 009

TP

70ndash100 072 051 061c 007 011

Squares Difference (LSD) post hoc tests were used for multiple comparisons Correlationsbetween C N and P concentrations in soil were determined by Pearsonrsquos correlation test at95 confidence interval A P value of less than 005 was considered statistically significantThe CN CP and NP ratios of the soil from different vegetation types were computed asmass ratios

RESULTSSOC TN and TPBasic characteristics of SOC TN and TP concentrations in soil profileThe SOC TN and TP concentrations decreased with an increase in soil depth in the studysite The SOC TN and TP concentrations in surface soil (0ndash20 cm) is high exhibitingremarkable soil nutrient lsquolsquosurface-aggregationrsquorsquo (high nutrients in the surface soil layer)(Yang Liu amp An 2018) (Table 2) The SOC concentrations varied greatly from soil depthsof 0ndash10 to 20ndash40 cm with the mean concentration ranging from 2720 to 625 g kgminus1

(decreased of 7702) The TN concentrations varied remarkably from soil depths of0ndash10 to 20ndash40 cm with the average concentration decreasing from 239 to 068 g kgminus1

(decrease of 7155) The mean concentration of TP from soil depths of 0ndash10 to 20ndash40 cmdecreased from 071 to 062 g kgminus1 (decrease of 1268) The coefficient variation (CV) isthe main index used to describe the degree of spatial variability of the variables The SOCand TN concentrations had the highest variability at 0ndash10 cm with CVs of 041 and 040respectively The variation of TP concentration was smallest with CV values between 009and 011

Ma et al (2020) PeerJ DOI 107717peerj8382 620

Changes of SOC TN and TP concentrations in different vegetation typesThe SOC concentrations in different soil layers increased gradually with advancingvegetation restoration (Fig 2A) The highest SOC concentrations of all vegetation stagesappeared in the 0ndash10 cm soil layer which illustrates that SOC mainly accumulates in thetop soil layer The SOC concentrations of climax forest (Ql) were higher than those of otherrestoration stages in 0ndash40 cm soil layers The highest SOC concentrations in the 40ndash100cm soil layers were found in shrub (Hr) whereas the lowest appeared in grassland (Bi)Overall the SOC concentrations in the 0ndash40 cm soil layers in each restoration stage variedsubstantially whereas SOC concentrations in the 40ndash100 cm soil layers in each restorationstage exhibited no obvious changes

The concentration of TN increased with vegetation succession (Fig 2B) and wassignificantly different between different restoration stages The highest TN concentrationof all vegetation stages was detected in the 0ndash10 cm soil layer displaying a similar tendencyto the SOC The concentration of TN in climax forest (Ql) were significantly higher thanthose of other restoration stages at soil depth of 0ndash20 cm The highest TN in the 20ndash100 cmsoil layers occurred in farmland whereas the lowest appeared in grassland (Bi) The TNconcentration in different successional stages decreased with increasing depth in the 0ndash100cm soil profile and all of them decreased significantly in the 0ndash40 cm soil layer Below 40cm the concentration of TN showed a tendency toward stabilization

In general variation was small for soil TP concentrations ranging from 051 to 082 gkgminus1 with an average concentration of 064 g kgminus1 (Fig 2C) The TP concentration in eachrestoration stage decreased with an increase in soil depth in the 0ndash100 cm soil layer and isdistributed evenly in the soil profiles The TP concentrations of farmland and early forest(Bp) were significantly higher than those of other vegetation types in 0ndash100 cm whereasthe TP concentration had not significant interaction with vegetation restoration

Nested ANOVA indicated that both soil depth and vegetation type significantly affectedSOC TN and TP Soil depth showed a greater effect on SOC and TN than vegetation typewhereas vegetation type had a bigger impact on TP than soil depth (Table 3)

SOC TN and TP stoichiometric characteristicsStatistical analysis of SOC TN and TP stoichiometry in soil profileThe soil CN CP and NP ratios decreased with an increase in soil depth (Table 4) TheCN ratio in the 0ndash10 cm soil layer was significantly higher than that in the 10ndash100 cm soillayers (P lt 005) The CN ratio in the 10ndash100 cm soil layer did not significantly vary Thevariation of the CN ratio was smaller and relatively stable compared with the SOC and TNconcentrations In contrast the variation of the CP ratio was large The mean value of theCP ratio from soil depths of 0ndash10 to 20ndash40 cm decreased from 3918 to 1025 (decreaseof 7384) The soil CP ratio in the 40ndash100 cm soil layer had no significant change TheNP ratio in the 0ndash20 cm soil layer was significantly larger than that in the lower soil layer(P lt 005) The average value of NP ratio from soil depths of 0ndash10 to 20ndash40 cm decreasedfrom 364 to 107 (decrease of 7060) However the soil NP ratio of the 40ndash100 cm layerdisplayed no significant changes

Ma et al (2020) PeerJ DOI 107717peerj8382 720

Figure 2 Variation of nutrient concentrations in soil profile of different vegetation types (A) Soil or-ganic carbon (B) Total nitrogen (C) Total phosphorus Data are shown as the meanplusmn standard devia-tion The sample size n= 3 Different small letters in the same vegetation type mean significant differencein different soil layers and different capital letters in the same soil layer mean significant difference in dif-ferent vegetation types at 005 level Bi Hr Bp Pd and Ql represent Bothriochloa ischemum Hippophaerhamnoides Betula platyphylla Populus davidiana Pinus tabuliformis and Quercus liaotungensis respec-tively

Full-size DOI 107717peerj8382fig-2

Ma et al (2020) PeerJ DOI 107717peerj8382 820

Table 3 Nested ANOVA results for the effects of vegetation type and soil depth on SOC TN and TP inZiwuling forest area

Factor SOC TN TP

F P F P F P

Soil depth 16200 lt0001 8170 lt0001 737 lt0001Vegetation type 6624 lt0001 2160 lt0001 6252 lt0001

Table 4 Stoichiometries of SOC TN and TP in soil profileDifferent lowercase letters in the same CNCP and NP ratios indicate significant dif-ference in different soil layers at 005 confidence interval The sample size n= 3

Soildepth (cm)

Maximum(g kgminus1)

Minimum(g kgminus1)

Mean(g kgminus1)

Standarddeviation (g kgminus1)

Coefficient ofvariation

0ndash10 1364 696 1087a 157 01510ndash20 1125 653 968b 122 01220ndash40 1193 675 951b 153 01640ndash70 1300 619 937b 175 019

CN

70ndash100 1364 631 952b 189 0200ndash10 5676 835 3918a 1220 03110ndash20 2558 844 1811b 429 02420ndash40 1351 650 1025c 251 02540ndash70 1106 469 676c 179 026

CP

70ndash100 1001 435 560c 138 0250ndash10 547 120 364a 114 03110ndash20 235 129 171b 031 01820ndash40 148 061 107c 029 02740ndash70 122 045 074c 023 030

NP

70ndash100 115 043 061c 020 032

Changes of CN CP and NP ratios in soil profiles of different vegetationtypesThe soil CN ratio was relatively stable along the gradient of vegetation restoration (Fig 3A)The highest CN ratio in the different vegetation restoration stages appeared in grassland(Bi) whereas the lowest occurred in farmland The soil CN ratio of farmlandwas lower thanthose of other vegetation types which might be the result of the high TN concentrationsin farmland The variation of the CN ratio in 0ndash100 cm soil layer was small and remainstable at about 10

In general the soil CP ratio increased gradually along with the progression of thevegetation succession and significant differences were found between different restorationstages (Fig 3B) The highest CP ratios appeared in climax forest (Ql) and early forest (Pd)whereas the lowest occurred in farmland Furthermore in the 0ndash100 cm soil profile theCP ratio decreased with an increase in soil depth Soil CP ratio in soil depth of over 40cm varied substantially according to restoration stage whereas the CP ratio in soil depthbelow 40 cm exhibited small variation

The soil NP ratio showed a similar trend to the CP ratio (Fig 3C) The NP ratio inthe different soil layers increased gradually alongside vegetation restoration In the vertical

Ma et al (2020) PeerJ DOI 107717peerj8382 920

Figure 3 CN CP and NP ratios in soil profile of different vegetation type (A) The ratio of soil or-ganic carbon to total nitrogen (B) The ratio of soil organic carbon to total phosphorus (C) The ratio oftotal nitrogen to total phosphorus Data are shown as the meanplusmn standard deviation The sample size n=3 Different small letters in the same vegetation type mean significant difference in different soil layersand different capital letters in the same soil layer mean significant difference in different vegetation typesat 005 level Bi Hr Bp Pd and Ql represent Bothriochloa ischemum Hippophae rhamnoides Betula platy-phylla Populus davidiana Pinus tabuliformis and Quercus liaotungensis respectively

Full-size DOI 107717peerj8382fig-3

Ma et al (2020) PeerJ DOI 107717peerj8382 1020

Table 5 Nested ANOVA results for the effects of vegetation type and soil depth on CNP stoichiomet-ric characteristics in Ziwuling forest area

Factor CN CP NP

F P F P F P

Soil depth 244 00027 8592 lt0001 7975 lt0001Vegetation type 3405 lt0001 4571 lt0001 2789 lt0001

Table 6 Correlation analysis between soil nutrient concentrations and ecological stoichiometry Thesample size n = 90

Index N P CN CP NP

C 0966 0241 0325 0992 0984

N 1 0290 0190 0965 0991

P 1 minus0160 0402 0449

CN 1 0251 0235

CP 1 0965

Notesplt 005plt 001

direction the NP ratio in each restoration stage decreased with increase in soil depth inthe 0ndash100 cm soil layer The highest NP ratio in different vegetation restoration stagesappeared in climax forest Ql and early forest Pd whereas the lowest occurred in farmlandSoil NP ratio in soil depth of over 40 cm in each restoration stage varied substantiallywhereas the NP ratio in soil depth below 40 cm exhibited small variations

Nested ANOVA indicated that both soil depth and vegetation type significantly affectedCN CP and NP Soil depth showed a greater effect on CP and NP than vegetation typewhereas vegetation type had a bigger impact on CN than soil depth (Table 5)

Correlations of ecological stoichiometry between SOC TN and TPSoil C N and P showed a strong significant correlation (P lt 001) (Table 6) Soil C showeda significant and positive correlation to the CN CP and NP ratios (P lt 001) Soil N washighly positively correlated to CP and NP ratios (P lt 001) but not correlated with CNratio (P gt 005) Soil P was highly positively correlated to CP and NP ratios (P lt 001)However the correlation between P and CN was negative (P gt 005) In addition positivecorrelations (P lt 005) among CN CP and NP ratios were observed

DISCUSSIONResponse of soil nutrient concentrations to vegetation successionand soil depthC N and P in soil are important for maintaining sustainable and productiveecosystems (Agren 2008 Laik et al 2009 Mcgroddy Daufresne amp Hedin 2004) Theaverage concentrations of C N and P in the study area were 2720 239 and 071 gkgminus1 respectively The average concentration of soil C was close to that of the global(2571 g kgminus1) (Cleveland amp Liptzin 2007) and national (2951 g kgminus1) (Tian et al 2010)

Ma et al (2020) PeerJ DOI 107717peerj8382 1120

average The average concentration of soil N was higher than that of the global (210 gkgminus1) (Cleveland amp Liptzin 2007) and national (230 g kgminus1) values (Tian et al 2010) Theaverage concentration of soil P was also higher than that of the national average (056g kgminus1) (Tian et al 2010) Compared to other findings on the change of soil nutrientconcentrations after vegetation restoration (Jiao et al 2013 Zhang et al 2019) the overallconcentration of soil nutrient in Ziwuling forest area was also higher This result indicatesthat the concentration of C in the Ziwuling forest area was relatively high N and Pnutrient elements were abundant and vegetation restoration increases the soil nutrientconcentration and quality in the region to an extent

Long-term natural vegetation restoration could have strong effects on soil quality andthe carbon and nitrogen cycle (Gruumlnzweig et al 2007 Vandenbygaart et al 2011) In thisstudy vegetation restoration significantly increased the concentrations of SOC and TNThe results are in agreement with those in previous studies (Fu et al 2010 Yang amp Luo2011 Zhang et al 2019 Zhao et al 2017) These results were due to the ability for long-term natural vegetation restoration to improve vegetation primary productivity therebyresulting in the improvement of the quantity and quality of litter and rhizodeposition (Caoamp Chen 2017 Deng et al 2016 Li et al 2016) and ultimately releasing a large amountof organic C and N into the soil Meanwhile soil temperature and moisture may changeduring the process of vegetation restoration which in turn could lead to the enhancementof soil microbial and enzymatic activities and increased nutrient decomposition rate(Gispert et al 2013 Zhang et al 2019) thereby increasing the soil C and N concentrationsCompared with the soil C and N soil P did not significantly change with vegetationrestoration Soil P is mainly affected by parent material vegetation type land use andbiogeochemical processes in the soil (Kooijman Jongejans amp Sevink 2005 Lane Noske ampSheridan 2011) As the climate and parent material is similar across all vegetation typesthe variation in soil P is not as obvious as that of C and N The soil nutrient concentrationsof different vegetation types were significantly different The C and N concentrationswere highest in forest (Ql) and lowest in farmland Increased C and N concentrations inforests may be due to the accumulation of surface leaves and litter whereas crop harvestson farmlands decrease the return of nutrients to the soil This indicates that reasonableagricultural management practices such as retaining manure and crop residues are vitalto maintaining and improving soil fertility (Kirkby et al 2013)

The depth of sampling is an important factor for predicting spatial variation of soilnutrients (Vandenbygaart et al 2011) Our results found that the concentrations of C NandP in the study area decreasedwith an increase in soil depthwhich is consistentwithmostexisting studies (Cao amp Chen 2017 Xu et al 2019 Zhang amp Shangguan 2018) At the sametime changes in C and N concentrations in the surface soil were clearly observed across theprogression of vegetation succession which is also supported by previous research (Tian etal 2010 Xu et al 2019) Soil nutrient is influenced not only by soil parent materials butalso by litter decomposition root architecture and exudates (Berger Neubauer amp Glatzel2002 Clemmensen et al 2013 Gao et al 2014 Xu et al 2019) Microorganism-drivenlitter decomposition mainly occurs in surface soil (0ndash20 cm) (Gao et al 2014) whichincreases the nutrient concentration in surface soil With an increase of soil depth the

Ma et al (2020) PeerJ DOI 107717peerj8382 1220

input of organic matter decreases due to the decrease of microbial decomposition activityand root exudates (Berger Neubauer amp Glatzel 2002 Clemmensen et al 2013) Deng et al(2018) found that the amount of litter and roots in surface soil (0ndash20 cm) in differentvegetation succession stages was significantly higher than that of subsoil (20ndash60 cm) whichhad a significant positive effect on SOC and soil C sequestration P is mainly affected bysoil parent material which is a sedimentary mineral with poor migration in soil (Ren et al2016 Walker amp Syers 1976 Wang Zhang amp Haung 2009) and therefore the distributionof soil P in the vertical profile is more uniform

Responses of soil stoichiometry to vegetation succession and soildepthLong-term vegetation restoration from farmland to forest had very significant effectson soil nutrient composition The variation of SOC TN and TP concentrations insoil caused changes in nutrient stoichiometric relations (Agren 2008 Tian et al 2010)The soil CN ratio is a measure of C and N nutrient balance which affects the cyclingof organic C and N and is a sensitive indicator of soil quality (Tian et al 2010) Theaverage soil CN ratio in this study was 1087 which was lower than that of the averageChinese soil (1230) and the average global forest soil (1240) (Mcgroddy Daufresne ampHedin 2004 Tian et al 2010) Other studies showed that the soil CN ratio was inverselyproportional to the decomposition rate of organic matter and soil with a low CN ratiohas a faster mineralization rate (Cleveland amp Liptzin 2007 Wang amp Yu 2008) indicatingthat the soil C accumulation organic matter decomposition and mineralization rate of theZiwuling forest area would be faster than average The CN ratio in the soil also remainedremarkably stable along the process of vegetation restoration which is in line with theresults reported for secondary forests worldwide (Yang amp Luo 2011 Zinn Marrenjo ampSilva 2018) This is due to C and N as structural components have relatively fixedratios in the process of accumulation and consumption (Cleveland amp Liptzin 2007) ZinnMarrenjo amp Silva (2018) also discovered that soil CN ratios are unresponsive to land usechange Furthermore soil CN ratios were stable across the soil profile across differentvegetation types which is consistent with previous reports (Tian et al 2010 Wang et al2014) This is due to the close temporal coupling of C and N concentrations in litter androots (Fan et al 2015Mcgroddy Daufresne amp Hedin 2004 Yang amp Luo 2011)

In general the soil CP ratio is considered a marker of soil P mineralization It is alsoan index that is used to measure the microbial mineralization of soil organic matter torelease or absorb potential P from the environment (Tian et al 2010) A low CP ratio isconducive to the release of nutrients by microorganisms via the process of organic matterdecomposition and promotes the increase of effective P in the soil On the contrary a highCP ratio leads to the limitation of P resulting from the decomposition of organic matter bymicroorganisms microorganisms will compete with the plant for soil inorganic P that isnot conducive for plant growth (Wang et al 2014) The average value of the soil CP ratioin this study was 3918 which was lower than that of the average for China soil (5270) andglobal forest soil (8190) (Mcgroddy Daufresne amp Hedin 2004 Tian et al 2010) This valueshows that the availability of P in the Ziwuling forest area is relatively high In addition SOC

Ma et al (2020) PeerJ DOI 107717peerj8382 1320

and TN were significantly correlated and the soil CP and soil NP had a similar trend inresponse to vegetation restoration The soil CP ratio increased with vegetation successionCompared across different vegetation types the CP ratio was highest in climax forest (Ql)and early forest (Pd) These results suggest that P would be a limiting element for plantgrowth during vegetation succession As P mainly comes from weathering and leaching ofrocks and showed low biological availability in the Loess Plateau (Wang Zhang amp Haung2009) the uptake of P by plants gradually increasing during vegetation succession (Ren etal 2016) results in P becoming increasingly limited Previous studies also show that a Plimitation is exacerbated during forest succession (Fan et al 2015 Huang et al 2013 Renet al 2016)

Soil NP ratio can serve as an indicator of N saturation which in turn indicates theavailability of soil nutrient elements during plant growth and is used to determine thethreshold of nutrient restriction (Guumlsewell Koerselman amp Verhoeven 2003 Tessier ampRaynal 2003) The average soil NP ratio in the Ziwuling forest area was 362 whichwas lower than that of Chinarsquos terrestrial soil with an average of 39 (Tian et al 2010) buthigher than those found by other scholars on the Loess Plateau (Bai et al 2019 Cao ampChen 2017) With the restoration of vegetation the concentration of N in soil increasedsignificantly whereas that of P remained stable resulting in an increasing soil NP ratioMoreover some studies have shown that soil NP ratio was negatively correlated withplant growth rate (Fan et al 2015) The NP ratio increases at low plant growth ratesand decreases at high growth rates In this study the soil NP ratio was lower in theearly successional stage and higher in the later stages In other words plant growth maybe subject to nitrogen limitation in the early stages of vegetation succession Previousstudies have also shown that nutrient limitation changed with vegetation succession in thedevelopment of soil ecosystem transitioning from N limitation in the early successionalstage to P limitation in the late successional stage (Reed amp Cleveland 2011 Hayes et al2014) The total amount of N in the soil decreased with an increase in soil depth and therelative stability of TP concentration at the profile level led to a decrease of the NP ratiowith an increase in soil depth

In summary CNP stoichiometry plays a key role in the structure and function ofecosystems (Agren 2008 Ladanai Agren amp Olsson 2010) In this study we only exploredthe soil CNP stoichiometry across vegetation types and age sequence on the Loess PlateauHowever the above-and below-ground ecosystemprocesses are closely integrated and theirinteractions have an important effect on ecosystem processes and properties (Wardle et al2004) Therefore further research on the CNP stoichiometry relationships between plantsand soils at different scales as well as effects of vegetation succession on soil physical andbiological properties and their relationship with soil nutrient concentration is necessaryThese results can inform management practices for forest policymakers aiming to createsustainable forest ecosystems particularly for the large-scale natural secondary forest onthe Loess Plateau China

Ma et al (2020) PeerJ DOI 107717peerj8382 1420

CONCLUSIONSWe investigated the changes of soil nutrient concentrations and CNP stoichiometry in0ndash100 cm soil profile following vegetation restoration in the Loess Plateau Region Ourstudy suggested that long-term vegetation restoration can enhance the concentrations ofSOC and TN and ratios of soil CP and NP whereas the concentrations of soil TP and theratio of CN did not improve substantially with vegetation restoration The concentrationsof SOC and TN and the ratios of CP and NP decreased with an increase of soil depth(0ndash100 cm) and they were most prone to change in surface soil (0ndash20 cm) Furthermoreour study indicated that in the early stage of succession for degraded grasslands adding Nfertilization may enhance the growth of plants and increasing P application can avoid Plimitation during the later recovery period

ADDITIONAL INFORMATION AND DECLARATIONS

FundingThis work was supported by the National Key RampD Program of China (2016YFE0202900)the Fundamental Research Funds for the Central Universities (2452019078) and theNational Natural Science Foundation of China (41601236 and 41761144060) The fundershad no role in study design data collection and analysis decision to publish or preparationof the manuscript

Grant DisclosuresThe following grant information was disclosed by the authorsNational Key RampD Program of China 2016YFE0202900Fundamental Research Funds for the Central Universities 2452019078National Natural Science Foundation of China 41601236 41761144060

Competing InterestsThe authors declare there are no competing interests

Author Contributionsbull Rentian Ma conceived and designed the experiments performed the experimentsanalyzed the data prepared figures andor tables and approved the final draftbull Feinan Hu conceived and designed the experiments analyzed the data authored orreviewed drafts of the paper and approved the final draftbull Jingfang Liu Chunli Wang and Zilong Wang performed the experiments preparedfigures andor tables and approved the final draftbull Gang Liu and Shiwei Zhao conceived and designed the experiments authored orreviewed drafts of the paper and approved the final draft

Field Study PermissionsThe following information was supplied relating to field study approvals (ie approvingbody and any reference numbers)

Ma et al (2020) PeerJ DOI 107717peerj8382 1520

The research station is called Lianjiabian forest farm and ismanaged by Ziwuling ForestryAdministration of Heshui County Gansu Province We collected very few samples in theresearch area so it would not cause damage to the local environmentWhen taking sampleswe obtained oral approval from the head of Ziwuling Forestry Administration WangjiFan and took samples with the help of the staff

Data AvailabilityThe following information was supplied regarding data availability

The raw measurements are available in the Supplemental File

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj8382supplemental-information

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Li J Liu Y Hai X Shangguan Z Deng L 2019 Dynamics of soil microbial CNPstoichiometry and its driving mechanisms following natural vegetation restora-tion after farmland abandonment Science of the Total Environment 6931ndash10DOI 101016jscitotenv2019133613

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Ren C Zhao F Kang D Yang G Han X Tong X Feng Y Ren G 2016 Link-ages of CNP stoichiometry and bacterial community in soil following af-forestation of former farmland Forest Ecology and Management 37659ndash66DOI 101016jforeco201606004

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XuH Qu Q Li P Guo ZWu LWulan E Xu X 2019 Stocks and stoichiometry of soilorganic carbon total nitrogen and total phosphorus after vegetation restoration inthe Loess Hilly Region China Forests 10(1)14 DOI 103390f10010027

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Ma et al (2020) PeerJ DOI 107717peerj8382 2020