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Ontogenetic comparisons of arbuscular mycorrhizal fungi Scutellospora

heterogama

and Scutellospora pellucida revision of taxonomic character concepts species

descriptions and phylogenetic hypotheses

M A RL ISE RA N K E N D JOSEPHMORTON

Division of Plant and Soil Sciences 401 Brooks Hall P.O. Bo x 6057 West Virginia University

Morgantown 26506-6057 U.S.A

Received February 26, 1993

FRA N K E ,

., and

MORTON,.

1994. Ontogenetic comparisons of arbuscular mycorrhizal fungi

Scutellospora heterogarna

and

Scutellospora pellucida: revision of taxonomic character concepts, species descriptions, and phylogenetic hypotheses.

Can. J . Bot . 7 2 : 122- 134.

The taxonomic significance of morphological characters in fungi of Glomales (Zygomycetes) has been based solely on

superficial resemblance. Ontogenetic comparisons among isolates of

Scutellospora pellucida

and

Scutellospora heterogama

wer e used to resolve discrete stages of differentiation in which characters w ere delimited and ordered hierarchically according

to temporal and spatial origin in development. Character concepts were revised, and both species were redescribed. A spore

wall, two inner walls, and a germination shield were designated primary characters because they appeared separately and

in linear succession. Secondary characters included distinct layers differentiated within each wall. Tertiary characters were

qualitative and quantitative properties of each layer. All characters in each developmental stage did not vary in two hosts,

in separate experiments, and am ong five isolates of each species. Stability was attributed t o causal epigenetic linkages between

stages of differentiation, wherein each new stage depended on differentiation of all characters in the preceding stage. Charac-

ters at successively lower hierarchical levels are predicted to specify progressively less inclusive taxa in cladistic analysis.

Developmental patterns will improve reinterpretations of phylogenetic relationships and provide a more empirical basis for

grouping and ranking of organisms into species and higher taxa at the morphological Level.

Key words:

evolution, morphology, mycorrhizae, taxonomy, VAM fungi.

FRA N K E ,

. , et

MORTON,

. 1 994. Ontogenetic comparisons of arbus cular mycorrhizal fungi

Scutellospora heteroga~na

nd

Scutellospora pellucida: revision of taxonomic ch aracter concepts, spe cies descriptions, and phylog enetic hypotheses.

Ca n . J . Bot. 7 2 122-134.

La valeur taxonomique des caractkres morphologiques c hez les cham pigno ns appartenant aux Gloma les (Zygomycktes) n a

t t t baste que sur la rese mb lan ce superficielle. Les auteurs ont fait appel a comparaison ontogknique d isolats du

Scutellospora

pellucida

et du

Scutellospora heteroganla

afin de m ettre en tviden ce des stades prtcis d e diff t renciat ion, dans lesquels les

caractkres sont d6l imit ts et ordonnts hi t rarchiquement selon leur origine temporel le et spat iale au cou rs du dtveloppe ment .

11s ont rev ist les concepts des caractkres et redtcr it les espkces. Ils dtsignen t com me caractkres primaires, une paroi spo rale,

deux parois internes et une armature de germination, parce qu i ls apparaissent stpa rtm ent et en succession l intaire. Les carac-

tkres secondaires incluent des couches dist inctes diff t renc i tes l int t r ieur de chaque paroi . L ensemble d es caractkres, et

ceci tous les stades du dtveloppement , ne montrent aucune variat ion chez deux hbtes, dans des exptr iences dist inctes et

entre les cinq isolats de chacune des esptce s. O n at t r ibue la stabi l i t t des l iens causals tpigtnkt iques entre les stades de diff t -

renciation, alors que chaque nouveau stade dtpen d de la diff trenciat ion de tous les caractkres du stade prtctdent . On peut

prtd ire les caractkres des niveaux hi t rarchiques inftr ieurs successifs af in de sptcif ier progressivement les taxons moins

englobant dans l analyse cladist ique. Le s patrons de dtveloppem ent permettront d am tl iorer la r t interprktat ion d es relat ions

phylogtnt t iques et d offr i r une base plus empirique pour regrouper et ordonner les organismes en espkces et en taxons supt-

rieur au niveau morphologique.

Mots elks

tvolut ion, morphologie, mycorhizes, taxonomie, champignons MVA.

[Traduit par la rtdaction]

Introduction

The distribution of highly stable morphological specializa-

tions of the fungal mycelium (arbuscules, vesicles, auxiliary

cells) provided enou gh evidence cladistically (M orton 1990) to

group endomycorrhizal fungi previously classified as members

of Endogonales (Gerdemann and Trappe 1974) into a separate

order, Glomales, and into two suborders, Glomineae and

Gigaspo rineae (Morton and Benny 1990). Despite systematic

resolution of higher taxonomic categories, groups below the

family level still were judged highly equivocal. The problem

resided in the interpretation of subcellular charac ters of spores,

which have traditionally provided the most taxonomic infor-

mation at the species level. These characters included the

number, position, type, and properties of subcellular spore wall

types (Morton 1988). Walker (1983) was the first to propose

four wall definitions based on their phenotypic appearance in

crushed spores: evanescent, laminate, membranous, and unit.

Other wall types subsequently recognized were amorph

(Morton 1986), coriaceous (Walker 1986), expanding (Be

and Koske 1986), and germinal (Spain et al. 1989). Cladi

analysis (Morto n 19 90)-reve aled that some of these charact

were correlated in their distribution among taxa, suggest

that they were neither independent nor equivalent in th

capacity to resolve different taxonomic grou ps or ranks.

Most subcellular characters of spores have previously b

thought to ha ve value in classification based on de finitions

superficial resemblance rather than their individuality and ori

in biological processes such as development (Berch 19

Morton 1993). Alberch (1985) defines ontogeny as a seque

of temproally ordered developmental stages. However, chara

evolution in spores of glomalean fungi is concentrated at

subcellular level of spore organization, so that ontogeny

more a seauence of differentiation whe,re new characters

added, replaced, or lost.

P r ~ n ~ c dn Canada lmpr m6 au C n n ~ d a

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FRANKE AND MORTON

The only experimental solution to ascertain character origin

and boundaries and the individuality of each subcellular char-

acter in spores is through comparative ontogenetic analysis of

different taxa. Few such studies have been carried out on

glomalean fungi because of difficulties in establishing single-

isolate cultures and an inability to separate the fungus from its

host plant during propagation. Giovannetti et al. 1991) and

Meier and Charvat 1992) focused on differentiation of peridium-

forming spores of Glomus species. Results provided more

detailed descriptions of diagnostic characters, but intra- or

inter-specific comparisons were not made to determine the

value of characters in defining taxonomic groups and or) their

phylogenetic relationships.

It is only through rigorous tests to hypothesize homology

that morphological characters are trustworthy enough to define

phylogenetic relationships and subsequently relate pattern to pro-

cess Lauder 1981). Simplicity in design and composition of

subcellular structures in glomalean spores easily confounds dis-

tinctions between homology of common ancestry) and analogy

of independent ancestry). Two sequential, but independent,

operations are required Rieppel 1988). The first one is non-

evolutionary and consists of tests of similarity involving onto-

genetic comparisons, such as correspondence in

i )

connection

with adjacent or associated characters, ii) origin, iii) position

in an ontogenetic sequence, and iv) transformational states

Patterson 1982; Rieppel 1988; Wagner 1989). In this paper,

taxa from Scutellospora were selected for these tests because

subcellular diversity was great enough to discover ordered pat-

terns of character origin and transformational states during

spore differentiation. The first goal was to determine if differen-

tiation was discrete enough to recognize individual characters

and then to ascertain if these patterns could be grouped into dis-

crete and stable stages.

The second test of phylogeny involves cladistic analysis. It

was not carried out in this paper because too few taxa are

involved in the study. However, the taxonomic, developmental,

and phylogenetic implications of the ontogenetic results are

explored. Discrepancies in the most recent descriptions of

Scutellospora pellucida Koske and Walker 1986) and

Scutello-

spora heterogama Koske and Walker 1985) are also corrected.

Some aspects of this study were reported in Morton 1993) and

Morton and Bentivenga 1993).

aterials and methods

Experimental isolates

Species-level comparisons were carried out using cultures of

S. heterogama WV858-1 (collected by J. M orton near Manow n, W.Va.)

and S. pellucida W V872-1 (collected by J . Morton near Pum pkintown ,

W.Va.) for several reasons. First, members of both species were

hypothesized to be divergent, but related, based on accepted character

concepts (Morton 1990). Second, subcellular diversity in spores of

both fungi was among the most complex of known species. T hird, the

innermost wall in spores of both fungi produced a dextrinoid to dark

red-purple reaction in Melzer's reagent that marked the termination

of subcellular differentiation. Last, four other geographic isolates of

each species were available in the International Culture Collection of

Arbuscular and Vesicular-arbuscular Mycorrhizal Fungi (INVAM)

for morphological comparisons: BR154C-1 (Ming Lin; Campinas,

Brazil), FL31 2A-1 (D. Sylvia; Gainesville, Fla.), N Y320-2 (D. Miller;

Geneva, N.Y.), and SI722-2 (I. Louis; National University, Singapore)

of S. heterog ama; BR208

S.

Stunner, Florianopolis, Brazil), FL966-3,

NC118 (P. Schultz, Durham, N.C.), and WV205B-1 (J. Kotcon,

Kearneysville, W.Va.) of S. pellucida.

Inocula production

All organisms were propagated on Sudan grass (Sorghum sudanerlse

(Piper) Staph.) in 15-cm diameter plastic pots. Inoculum of each

organism consisted of culture medium, mycorrhizal roots, hyphae,

and spores dried in situ, m ixed, diced to lengths less than 1 cm , mixed

thoroughly in sealed 1-gallon (1 gallon 4.5 5 L) Zip-Loc bags (Dow

Corning Co.), and then diluted 1 10 (vlv) with sterile growth medium.

The grow th medium was a sandy loam soil (Lily series) mixed 1 2 (vlv)

with No. 3 quartzite sand steamed at 1 00°C for two 1-h periods sepa-

rated by a 24 h cooling period. At planting, soil pH was adjusted to 5. 9

with calcium carbonate. The growth medium contained 0.9 organic

matter, with 8.1 mg kg- ' bicarbonate-extractable phosphoru s. Plants

were maintainied in a green house with Grow-L ux high-intensity fluores-

cent lighting placed 30 cm above plants, with a photoperiod of 12 h

and a photon flux density of 428 pmol .

m-'

.

s-I. Air temperatures

ranged from 19 to 31°C. At harvest, pot contents were air dried

in situ for 2- 3 weeks and then stored at 4°C until use.

Experimental design

Inocula of S. heterogama WV858-1 and S. pellucida WV872-1

were mixed 1:5 (vlv) with a growth medium identical in composition

to that described abov e and placed in 150-cm 3 cone-tainers (S tuewe

Sons, Inc., Corvallis, Oreg.) Red clover (Trifolium praten se L .)

and Sudan grass seeds wer e surface sterilized in 15 household

bleach fo r 3 l 5 min, rinsed five times in sterile distilled water, and

air dried. Eight seeds were placed in each cone-tainer. After emer-

gence, seedlings were thinned to five per cone-tainer. Red clover

seedlings were inoculated with Rhizobium trifolii by washing 0 .1 g of

peat-based inoculum (Nitragin C o., Milwaukee, W is.) into the growth

medium at emergence. Cone-tainers were arranged in a completely

randomized block design in racks on a greenhouse bench.

Tw o cone-tainers of each host-fungus combination were collected

at 7-day intervals, beginning 6 weeks after plant emergence. Contents

of each cone-tainer were soaked in water to separate roots, and all

remaining contents were passed through two nested sieves with 250-

and 45-pm openings usini a forced water spray. T he fraction collected

on the 45-pm sieve was added to a gradient of 20 and 60 sucrose

and centrifuged at 900 x g for 2 min. Spores were collected in a

small sieve with 45-pm openings, washed in tap water, placed in a

Petri dish, and counted unde r a Bausch Lom b stereomicroscope.

All roots were blotted dry and w eighed fresh. A 100-mg sample

was separated and stained in 0.1 trypan blue using the procedure

of Koske and Gemma (1989). All remaining roots were dried to a

constant weight at 67OC. Total root length, percent mycorrhizal

colonization, and mycorrhizal root length were estimated from a

0.1-g fresh weight subsample using the grid-line intersect method

(Giovanetti and Mosse 1980).

All other fungal isolates were propagated at different times of the

year using the same culture setup, but with Sudan grass as the sole

host. Each fungus was cultured in five cone-tainers and harvested at

75 days after planting to confirm the stages of spore differentiation.

Preliminary experiments had revealed that spore production was asyn-

chronous and that all stages in the differentiation sequence were present

in

60-

to 75-day-old cultures. Extraction and mounting procedu res were

identical to those described above.

Identi5 cation of sta ges in spor e differentiation

The full complement of subcellular characters in spores of S. pellu-

c i d ~WV872-1 and S. heterogattza WV858-1 was assessed from

comparative morphological studies of mature spores in all isolates.

Spores were extracted from 4-month-old pot cultures (see Inoculum

production), mounted in polyvinyl alc oh d lactic acid glycerin

(PVLG) or PVLG m ixed 1: 1 (vlv) with Melzer's reagent and broken

with pressure applied to the cover slip.

The direction of the differentiation sequence was determined by

separating whole spores of both species into three discontinuous classes

(1-111) according to co lor and opacity of contents. Reflected co lor of

spores was compared against that from a printed chart (INVAM, West

Virginia University) exposed to the same fiber optic illumination

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CAN. J.

BOT.

VOL. 72,

1994

r

Phase

I

I

Phase

ll

Phase

Ill

i S pellucida I

I

Stage

Stage 2 Stage 3 Stage Stage 5 Stage 6

I

S heterogama

I

i Stage Stage X Stage 2 Stage 3

Stage Stage 5

I

FIG 1. Murographic representation of stages of subcellular differentiation within spores of S. pellucida WV872-1 and S. heterog

WV858-1 separated into three phases of development. Phase I, spore expansion and differentiation of the spore wall (sw); phase 11, seque

differentiation of a first (iw l) and then a second (iw2) inner wall; ph ase 111, formation of a ge rmination shield ( gs). Patterns in dicate the foll

ing characters within each wall: open, single layer; vertical dashes, laminae; diagonal lines, flexible layer of various thickness; hemisphe

highly plastic and pleiomorphic flexible layer.

,

pink (0-20 -20-0 ) to light red-purple (20-8 0-20 -0) reaction in Melzer s reagent (M

ed-purple (40 -80 -30 0) to dark red-purple (60- 80 -50 0) reaction in MR; 0 rnamentations present. Numbers in parenth

indicate percent cyan-m agenta-yellow -black in each color estimate.

(Cole Parmer Co ., Chica go, Ill.). Colors were reported by a descriptive

name and a formula based on percent cyan -magenta- yellow -black.

Classes for S. heterogntn a were (I) white to cream (0 0 -40 O),

contents opaque ; (11) pale orange (0-20 -60 -0) to orange (0 -60

100-O), contents opaqu e; and (111) oran ge (0-6 0- 100 -0) to red-

brown (40-80- 100 -o), contents translucent. Classes for S. pellucida

were (I) white to cream (0- 10-40-O ), contents opaque; (11) white

to hyaline, contents translucent; and

(111) pale orange-brown (0-20-

80-O), contents translucent. Spores in class I were the youngest,

whereas th ose in class 111 were mature. Class I1 spores consisted of

intermediate stages in differentiation, based on kinds and position of

subcellular structures in crushed spores.

All spores in classes I and I1 were collected when they numbered

less than 150 at each harvest. Anothe r 150 spore s in class 111 were

collected to obtain an adequate sampling of later stages against a

residual background of mature spores from the original inoculum.

Spores in each class were mounted in PVLG plus M elzer s reagent

and broken with pressure applied to the cover slip. Some spores were

mounted in PVLG to examine stages of differentiation in the absence

of iodine. Slides were placed in a convection oven at 6S°C for 24-48 h

and then stored for at least 7 days at room temperature to clear spore

contents. Spores were examined under a Nikon Optiphot research

microscope using differential interference contrast optics. Selected

images depicting characters in each stage were captured through a

Sony CDD video camera mounted on the microscope, viewed on a

Sony Trinitron color monitor, and printed from a Sony Mavigraph

video image printer (B B Microscope Co., Pittsburgh, Penn.). All

slides of permanently mounted specimens were numbered and stored

at room temperature as permanent vouchers in the INVAM slide col-

lection at West Virginia University.

A repeat experiment of identical design was carried out 6 mo

later using S. pellucida WV872-1 and S. heterogama WV858-

exam ine the relationship between spore growth (exp ansion) and st

of differentiation. Spores were collected at 7-day intervals from

cone-tainers of each fungus. The size distribution of the extra

popula tion was measured by random ly sampling 1 00- 150 spore

each harvest. Then, spores were separated manually with a pas

pipette into classes of 20-pm increments. All were mounted in PV

plus Melzer s reagent, measured again, broken, and then the stag

differentiation determined in each spore. Data in each size class w

pooled from different harvests until they exceeded 65 measurem

in each class. Correlation between spore growth and any stage

differentiation was determined by the mean of the product of the

quency of spores in a stage and the size increment in which that s

was found.

Discontinu ous stages in spore differentiation was separated by

criteria. The first was de novo appearance of a character know

be present in mature spores but absent in all preceding stages.

second concerned transformational stages within a character fro

state not found in mature spores to one that was present. Som e ph

typic states in layers of different walls were continuous because

were staees in a transformational vrocess. Discontinuities in t

transformations were definable only when the juvenile state co

sponded to the terminal (mature) state in spores of at least one o

Scutellospora species.

Results

G r ow th an d d i f f er en t ia t ion of the f unga l soma could no

subdiv ided in to d i sc r e te s tages because gr ow th in r oo t s

patchy and indeterm inate . In isolates of both spec ies , auxil

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FRANKE A N D

MORTON

TABLE. A hierarchy of morphological characters in spores of S

pellucida

and

S. heterogama

ordered

by their origin in stages of differentiation from primary to secondary to tertiary characters

Primary Secondary

Spore wall Outer layer

Laminae (originating

as a single layer)

First inner wall First layer

Second layer

Second inner wall First layer

Second layer

Germination shield General shape

Length and width

Color

Margin properties

Tertiary

Color

Rigidity

Presence or absence of ornamentations

Kinds and dimensions of ornamentations when present

Thickness

Reaction in Melzer s reagent

Color

Rigidity

Thickness

Reaction

in

Melzer s reagent

Thickness

Plasticity

Reaction in Melzer s reagent

Thickness

Plasticity

Reaction in Melzer s reagent

Thickness

Plasticity

Reaction in M elzer s reagent

Thickness

Plasticity

Reaction in Melzer s reagent

cells were the first discrete structures differentiated on hy phae

entering roots and branching into so il from initial entry points.

Arbuscular differentiation within roots proceeded rapidly, but

colonization showed no informative pattern. The first early

juvenile spores were detected on stained roots 6 weeks after

seedling emergence in all host-fungus combinations. At that

time, my corrhizal root length of

S

pellucida averaged 883 cm

in red clover and 1181 cm in Sudan grass, whereas that of

S heterogama

averaged 297 and 351 cm in the two hosts,

respectively. Auxiliary cells were abundant prior to sporula-

tion and appeared to peak at the 8-week sampling. However,

two replications of each host -fungus combination w ere not

sufficient to define any conclusive trends.

Stages in spore differentiation were discrete enough to be

recognized consistently (Fig. 1). They did not vary in either

host, in repeated experiments at different times of the year, or

in different geographic isolates of either species. Patterns der-

ived from the sequence of character emergence in each stage

revealed that interpretations of characters could not be made

using conventional definitions (see Morton 1988; Morton and

Benny 1990). Characters were ordered hierarchically (Table

1)

according to their temporal and spatial origin in the process

of spore differentiation. Therefore, we distinguish characters

at each hierarchical level using terminology borrowed from

Kendrick (1965). Primary characters consisted of a spore

wall, two inner flexible walls, and a germination shield, each

of which originated in discrete temporal and spatial succes-

sion during ontogenesis. The three walls corresponded most

closely to wall groups as defined by Wa lker (19 83). Second ary

characters originated and differentiated within primary struc-

tures. In the three walls, each seco ndary character was recog-

nized consistently a s phenotypically distinct layers correspo nding

to the wall types currently defined in the literature (see M orton

1988). Secondary characters of the germination shield (Table l ),

other than color, were not studied. Tertiary characters con-

sisted of qualitative (e.g., color , flexibility, type of ornamen -

tation) and quantitative (e.g., size, thickness, dimensions of

ornamentations) variation within secondary layers of walls.

No ch aracters at this subordinate level were delimited in ger-

mination shields.

Phases of spore expansion and diflerentiation

Using these revised ch aracter interpretations, sp ore develop-

ment in

S pellucida

and

S. heterogama

was subdivided into

three distinct phases (Fig. 1). Phase I involved differentiation

of the spore wall (stages 1 and 2) concurrently with spore

growth o r expansion (Figs. 2B and 2D ). Secondary and ter-

tiary characters of the spore wall were expressed during this

period. Spores smaller than the size distribution of a mature

spore population possessed a spo re wall (stages 1 and 2 of both

species in Fig. 1) but no inner walls (Figs. 2A and 2C ). Con-

versel y, all spores in various stages of inner wall differentiation

were within the size range of mature spores and could not be

distinguished under a stereomicroscope. Phase

I

began with

synthesis of the first of two successive inner walls and ended

with complete differentiation of the second inner wall (F ig. 1).

Phase I11was recog nized by synthesis of a germination shield.

Germ-tube synthesis was not considered part of sp ore differen-

tiation but rather the initiation of a new fungal thallus.

Within ea ch phas e, subcellular differentiation could be divided

into six discrete stages for

S

pellucida and five stages for

S heterogama

(Fig. 1). Stages were ordered sequentially because

de novo appearance of primary and secondary characters

occurred in a temporally linear pattern. Each stage in spore

differentiation of each fung us is discussed separately to show

unique as well as parallel trends.

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J. BOT. VOL. 72. 1994

Stage I tage

3 D l

Stage

Stage 2 tage 4 Stage e

S. pellucida WV872 1

80- 101- 121- 141- 161- 181- 201- 221- 241- 261-

6

2 3

4 5 6

A

loo 120 140 160 180 200 220 240 260 280

Stages in Spore Differentiation

Spore Diameter pm)

100

80

fn

E

60

Z

0

U

9

Stage I Stage

2

Stage 4

Stage I X Stage

3

[mJ

Stage

S heterogamaWV858 1

20

E l o o

0 1

100

E

80 1

fn

P

g

60

U

.c

40

20

E loo

n

U

I

>

80- 101- 121- 141- 161- 181- 201- 221- 241-

D

1X 2 3 4

c lo o 120 140 460 180 200 220 240 260 Stages in Spore Differentiation

Spore Diameter pm)

U

FIG . 2. Re lationship between spore grow th and stages of differentiation by

S.

pellucida WV872-1 and S heterogama WV858-1.

A

nd

Percentage of spores at each stage of subcellular differentiation (see Fig. I ) within 20-pm increment size classes. Solid circles connected

lines indicate size distribution of mature spores extracted from 12-week-old cultures of the same inoculum source.

(B

and D Relations

between change in spore size (growth) and stage of differentiation. Mean values were calculated from the product of the frequency of spo

at each stage (Fig. 2A ) and the size of spores in which they were found in a random sam ple of the extracted population harvested at 6- 8 wee

Vertical bars denote standard error.

Stages of differentiation within spores of

Scutellospora pellucida

WV872-1

Spores in stage 1 possessed a wall with two equally thick

layers (Figs. 3 and 4) , with a composite thickness ranging from

1 to 4 pm (mean 2.4 pm). The inner layer produced a light

pink reaction in Melzer s reagent in all spores. In the absence

of Melzer s reagent, the two layers in the spore wall were

difficult to resolve.

Stage 2 involved morphological transform ations in both layers

of the spore wall. Phenotypic changes were gradual and thus

could not be subdivided further. The inner layer of the spore

wall differentiated into rigid hya line laminae that stained red-

purple (40 0 -40 -0) in Me lzer s reagent as differentiation

proceeded (Figs. 5 and 6). The outer layer showed the least

change, thickening only slightly. It never stained in Melzer s

reagent (Fig. 6) and therefore was easy to distinguish from

laminae. At the end of stage 2, the spore wall consisted of a

rigid outer layer with discernible boundaries and a variable

number of laminae of the sam e phenotype (Fig. 6). Composite

thickness of the spore wall ranged from 3 to 8 pm in this iso-

late, with a mean thickness of 6.1 pm.

Under the stereomicroscope, spores in stages 3-6 wer e

indistinguishable from mature spores (those with germination

shields) because of similar size distribution, color, and surface

appeara nce. With spores from the original inoculum present in

extracted populations, the pr oportion of juvenile t o mature

spores could not be quantified in any sample. The onset

stage 3 is marked by the d e novo s ynth esis of the first in

wall (Fig. 7). Initially, this flexib le wall differentiated into t

layers of

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FRANKE AN D MORTON 127

FIGS. 3-15. Ontogenetic stages in differentiation of spore subcellular structure in

S

pellucida WV872-1. Spores in Figs. 3- 12 were

mounted in PVLG plus Melzer s reagent (1:1, vlv); those in Figs. 13- 15 were mounted in PVLG alone. All photographs were taken using

differential interference contrast optics. Slide vouchers from which photos were taken are referenced in parentheses. Fig. 3. Stage I, the spore

wall consisting of two thin adherent layers (M64). Scale bar 10 pm. Fig. 4. Detail of the two layers in the spore wall (sw) at stage 1, the

inner layer staining pink (M64). Scale bar 5 pm. Fig. 5. Stage 2, with the spore wall fully differentiated (S1437). Scale bar 5 pm. Fig. 6.

Detail of the outer layer and laminae of the spore wall (sw) at stage 2, the inner laminae staining red-purple (S1437). Scale bar 5 pm. Fig. 7.

Stage 3, with the first inner wall differentiated (S1438). Scale bar 10 pm. Fig. 8. The two layers of the first inner wall (iwl) at stage 3

(S1438). Scale bar 5 pm. Fig. 9. Stage 4, with the second inner wall partially differentiated (M112). Scale bar 10 pm. Fig. 10. Details

of layers in both inner walls (iwl and iw2) of stage 4, the inner layer of the iw2 staining pink to light purple

M

12). Scale bar 5 pm. Fig. 11.

Stage 5, with the second inner wall (iw2) fully differentiated. The inner layer of iw2 is plastic and stains red-purple (S1438). Scale bar

10 pm. Fig. 12. Stage 6 a germination shield

gs)

formed between the two inner walls (iwl and iw2), with germ-tube formation (S1438).

Scale bar 10 pm. Fig. 13. Broken mature spore at stage 6 (M130). Scale bar 10 pm. Fig. 14. Detail of layers in the two inner walls

(iwl and iw2) at stage

6

with the plastic structure of the inner layer in iw2 evident (M130). Scale bar 5 pm. Fig. 15. Detail of the two

layers in the spore wall (sw) at stage 6 (M130). Scale bar 5 pm. See Fig. 1 for murographic illustration of stages.

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darkened to a red-purple (60 0 0 0) color . Th e separa-

tion of stages 4 and 5 was made only because mature spores

of other Scutellospora species possess a second inner wall that

corresponded to each stage in

S.

pellucida. Examples of fungi

with an equivalent second inner wall to that in stage 4 are

Scutellospora erythropa (Koske Walker) Walker Sanders

and Scutellospora weresubiae Koske Walker (J. Morton,

unpublished data). A fungus with an equivalent second inner wall

to that in stage 5 in

Scutellospora dipurpurascens

Morton

Koske. C orrespondence between juvenile and mature stages in

different species suggested discrete end points in the transfo rma-

tion series, despite what appeared to b e a continuum of change.

Stage 6 was recognized by de no vo synthesis of a pale yellow

(0-0 -20-0) to brown (0-20 -70-0) germination shield

positioned between the two inner flexible walls (Fig. 12).

Tertiary characters of the shield (Table 1) were not discrete

enough to separate any stages in its differentiation. Germ-tube

formation completed the developmental history of spores. In

PVL G alone (Figs. 13 5), all walls and the layers differen-

tiated within them were distinguishable and the plasticity of

the inner layer of the second inner wall was most noticeable.

Stages in differentiation within spores of

Scutellospora hetero-

gama WV8.58-I

Stage 1 spores wer e indistinguishable from those of S. pellu-

cida, thus indicating a 1: 1 corresponding in p rimary , secondary,

and tertiary characters (Figs. and 16). Th e rigid spor e wall

consisted of two thin adh erent layers of near-equal thickness

(1 .5 pm each). A light pink reaction of the inner layer in

Melzer's reagent distinguished the two layers (Fig. 17), although

it was less consistent than that observed for S. pellucida. Vari-

ation in Melzer's reaction could not be associated with spore

size, thickness of layers, or any other observable mo rphological

criterion.

Th e next stage in differentiation w as given a unique numb er

(IX) for two reasons: i ) the inner layer of the spore wall

underwent a transformation that was transitory and

i i )

the

transition had no morphological or developmental effect on all

subsequen ce stages of differentiation (as evidenced by corre-

spondence in secondary organization of the s pore walls of both

S. heterogama and S. pellucida in stage 2). Whole spores in

stage IX turned purple-black when immersed in Melzer's

reagent, so they were easily recognized under a stereomicro-

scope. At the subcellular level, the outer layer of the spore

wall remained unchanged except for some added thickne

(1 -2.5 pm). T he inner layer underwent a rapid transformat

(by the very low n umber of intermediate phenotypes in extrac

populations) from a thin layer that produced in pinkish r

(0-6 0-3 0- 10) reaction in Melzer's reagent (Fig. 18) to

highly plastic (amorphous) layer of variable thickness t

stained a dark red-purple (20 0 -20 -0 to 60- 80 0

in Melzer's reagent (Fig. 19). The pleiotrophic properties

this layer were detectable only in PVLG alone (Fig. 2 0), whe

it appeared to be an extension of mo re rigid material (Fig. 2

Structure (Fig s. 1 3 and 14) and histochemical properties

Melzer's reagent of this layer were identical to those of

second inner wall of mature S. pellucida spores (Fig. 12)

Stage 2 was recognized by further transformation of

amorphous layer into rigid laminae that then acquired oran

0

0 0 ) to red-brown (0 0 00-0) pigmentat

(Fig. 22). The transition from stage IX to 2 was gradual,

evidenced by presence of intermediate forms consisting of so

laminae together with an amorphous layer of variable thic

ness. As the amorph ous layer acquired rigidity, the reaction

Melzer's reagent changed to a dark red-brown color (40 0

80 -0). At this time, the outer layer had differentiated round

warts ranging from 1 to 5 pm in height (Fig. 23). At the e

of stage 2, the spore wall consisted of an ornamented ou

layer 2-4 pm thick and a variable number of laminae 4-9

(mean of 6.2 pm).

Stage 3 began with d e novo synthesis of the first inner fl

ible wall (Fig. 24). It appea red as very thin flexible struct

(< 0 .5 pm), which then went on to differentiate into two t

adherent layers. T he outer layer rarely exceeded

pm in thi

ness, and th e inner layer ranged from 1 to 2 pm thick at ma

rity (Fig. 25). Individual layers of this wall were discerni

only in approxim ately 20 of spores completing stage

Separation of the two layers usually occurred near the brok

edge of a spo re after it was crushed. At maturity, the two lay

separated more frequently in stage 3 spores of some isola

(e.g., BR154C -1, SI722-1) than in others. Neither layer reac

in Melzer's reagent.

Stage 4 was recognized by termination of differentiation

the first inner wall and de novo synthesis of a second inn

wall (Fig. 25). Th e pattern of differentiation of layers in t

wall mirrored those in the first inner wall (stage 3). How ev

the process occurred so rapidly that only a very low proporti

of spores in this stage were retrieved am ong sam ples (Fig.

FIGS.16-32. Ontogenetic stages in differentiation of spo re subc ellular structure in S heterogama WV858-1 . Spores in Figs. 16- 19 a

22-29 were mounted in PVLG plus Melzer's reagent; those in Figs. 20 , 21 , and 30-32 were mounted in PVLG alone. All photographs w

taken using differential interference contrast optics. Slide vouchers in which photos were taken are referenced in paren theses. Fig. 16. Stage

the spore wall consisting of two adherent layers M 1 7 ) . Scale bar 10 pm. Fig. 17. Detail of the two layers in the spore wall s w ) at stage

the inner layer s taining light pink M 1 7 ) . Scale bar 5 pm. Fig. 18. Beginning of s tage IX, with the inner layer of the spore wall s w ) stain

dark pink M 1 8 ) . Scale bar 10 pm. Fig. 19. End of stage IX, with inner layer staining dark red-pu rple M 1 8 ) .Scale bar 10 pm. Fig. 2

Unstained spo re wall s w ) at stage IX, showing plasticity of the inner layer M 1 8 ) . Scale bar 10 pm. Fig. 21. Detail of str uctu re in stage

where the two layers of the spore wall are adherent M 1 8 ) . Scale bar 5 pm. Fig. 22. Stage 2 , final transformation of the spore wall i

a rigid structure with a hyaline outer warty layer and red-brown laminae M 3 ) . Scale bar 10 pm. Fig. 23 . Detail of the spore wall

at stage 2 showing warts on the outer layer M 3 ) . Scale bar 5 pm. Fig. 24. Stage 3 , with the first inner wall differentiated M 3 ) . Sc

bar 10 pm. Fig. 25. Detail of the two layers in the first inner wall i w l ) at stage 3 M 3 ) . Scale bar 5 pm. Fig. 26. End of stage 4 , w

the second inner wall iw2) differentiating two layers, the innermost staining light purple M 3 ) . Scale bar 5 pm. Fig. 27. Another sp

at end of stage 4 , but with both inner walls i w l and iw2) adherent M 1 5 2 ) . Scale bar 5 pm. Fig. 28. Stage 5 , a germination shield

formed between the two inner walls i w l and iw 2 ) M42) . Scale bar 5 pm. Fig. 29 . Another spore in stage 5 , showing boundaries of

shield g s ) sandwiched between the two inner walls M 4 1 ) . Scale bar 5 pm. Fig. 30. Broken mature spore at stage 5 M 4 1 ) . Scale bar

10

pm. Fig.

31.

Detail of the fully differentiated layers in the two inner walls

iw l

and

iw2)

at stage 5

M 4 1 ) .

Scale bar

5

pm. Fig.

Detail of the spore wall sw ) at stage 5 , showing warts on the outer layer M 4 1 ) . Scale bar 5 pm. See Fig. for murographic illustrat

of stages.

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S

pellucida

S

heterogama

S

pellucida

120 140 160 180 200 220 240 260 280 ~~~~~

Spore diameter (pm) isolates

S heterogama

100 120 140 160 180 200 220 240 260 280

Fungal

Spore diameter (pm) isolates

FIG . 33. Distribution in sp ore diameter of five geographic isolates

of S ellucida and S. heterogarna. All spores were extracted from

12-week-old cultures in 15-cm diameter pots, using the sam e host

(Sudan grass) and growth medium.

During differentiation, the ou ter layer remained thin < 1 pm)

and the inner layer thickened slightly (1 -2 pm) and developed

a pink (0-2 0-20 -0) to light purple (20- 60-3 0-0 to

20 0 -20 -0) reaction in Melzer s reagent (Figs. 26 and 27).

Both layers could be delimited by the differential reaction of

the inner layer in Melzer s reagent, d espite adherence in many

spores (Fig. 27). Ev en though the secondary characters of the

second inner wall were different from those in spores of

S.

pellucida in stage 4 , the terminal reaction in Melzer s reagent

of the inner layer was similar. This correspondence provided

additional evidence of discrete gradients in composition of this

layer during the transformation process.

Stage 5 was delimited by de novo synthesis of a germination

shield between the two fully differentiated inner walls (Figs . 28

and 29). Although young germination shields could be distin-

guished by their smaller size and smooth margins, formative

events could not be subdivided into d iscrete stages.

In

PVLG

only and lower magnifications, layers of the two inner walls

of fully differentiated spores (stages 4 and 5) were not easily

seen (Fig. 30). U nder oil, these layers became more discernible

(Fig. 31). The spore wall of mature spores (Fig. 32) was

indistinguishable from that in spores of stage 2 (Fig. 22).

FIG. 34. Comp arison of murograph s depicting subcellular charac

in S. heterogama and

S.

pellucida. Those with an unshaded ba

ground were proposed by Koske and Walker (1985, 1986) for resp

tive species. Those with a shaded background are reinterpretations

reflect subcellular organization of spores. Discrete layers (second

characters) are depicted within primary structures of separate ori

(sw, spore wall; iwl, first inner wall; iw2, second inner wall).

characters are represented as open rectangles. Walls were placed

three groups (A-C) based on their degree of separation in crush

spores. Properties of each layer are described in the text (see R esul

Redescription of

Scutellospora pellucida

Nicol. Schen

Walker Sanders

Most features of S.

pellucida

spores redescribed by Ko

and Walker (1985) were consistent with our observations

five different geographic isolates. Spores of all isolates deriv

from pot cultures in this study exhibited a wider range in sp

size (Fig. 33) than those in previous descriptions (Koske a

Walker 1985; Nicolson and Schenck 1979). These differen

may be due to origin of the spores (pot culture versus fi

soil) as well as sample size (who le inoculum versus preserv

vouchers).

Koske and Walker (1986) described six separate and phe

typically distinct spore walls (Fig. 34, unshaded murogra

based on the terminology of Walker (1983) and M orton (198

These walls were placed in three groups A -C) based on th

degree of separation in crushed spores (Morton 1988). Fr

developmental evidence collected in this study, these six wa

are reduced to three primary characters: a spore wall and t

inner walls (Table 1; Fig. 34, shaded murograph). The sp

wall consists of two secondary characters:

i)

an outer ri

layer that has discrete boundaries, ranging in thickness fr

2 to 5

pm , and no reactivity in Melzer s reagent, and

ii

variable number of lam inae originating as a single layer, ran

ing in thickness from 3 to 9 pm at maturity, and usually sta

ing dark red-purple (0 -60 0 -40) in Melzer s reagent wh

spores are hyaline to white. Th e first inner wall consists of t

layers that often are adherent: one thin (

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3

stances. First, charac ter and wall group definitions were based

solely on phenotype rather than any dynamic process (e.g.,

ontogeny). Interpretations were subject to artefacts created by

mounting procedures and properties of the solutions used to

mount or preserve spo res (Morton 1988). Second, interpreta-

tion of wall types was influenced by definitions of those know n

at that time. This explains why the thicker flexible layers in

the two inner walls we re interpreted as unit walls sensu W alker

(1983). Last, the effects of mountants and biotic and abiotic

factors in field soils on subcellular structure were not well

understood or appreciated. Most of the specimens examined

by Koske and Walker (1986) were preserved in formalin or

lactophenol, which tend to cause both layers of an inner wall

as well as the inner walls themselves to adhere tightly. The

presence of a germination shield in spores would have distin-

guished the two inner walls had it been known that this struc-

ture always form s on the wall in closest proximity to the spo re

cytoplasm (see Fig. 1).

Redescription of Scutellospora heterogama Nicol. Gerd.)

Walker Sanders

Most features of

S. heterogama

spores redescribed by Koske

and Walker (1985) were in accordance with our observations

of six different geographic isolates, with the exception of

marked changes in microscopic features during development.

Spores were white to cream (0- 10-40-0) in youth, chang-

ing to a mix of orange-brown (0-60- 100-0) to red-brown

(40 0 00 -0) spores at maturity. Mature spores often were

covered with a white bloom, as reported by Nicolson and

Gerdemann (1968), and found also on spores of

Scutellospora

dipapillosa

(Koske and Walker 1985). This white coating can

be seen whether spores are air dried or immersed in water, but

it is not detectable on spores mounted in PV LG . Spo res of iso-

lates in this study had a much broader size range than those

reported in previous descriptions, a trend similar to that observed

for

S. pellucida.

Spores do not contain four individual walls in two wall

groups, as reported by Koske and Walker (1985) (Fig. 34,

unshaded murograph). Developmentally delimited characters

indicate three primary structures: a spore wall and two inner

walls (Fig. 34 , shaded murograph). The spor e wall consists of

two characters, an outer layer and inner laminae. The outer

layer is light brown (0 -20-50 -

lo) , 2- 4 pm thick, and with

rounded warts of various lengths (1 -5 pm). T hese ornamenta-

tions are most visible in PVLG -based media within 1-2 months

of mounting, after which they become difficult to discern

clearly. Th e laminae are derived from an amorphous transitory

structure present only in juvenile whlte to cream spores. At

maturity, laminae together are 4-9 pm thick, orange-brown

(0 -60 100 0) unde r transmitted light, and changing to

a darker red-brown color (20- 80- 80-0) when placed in

Melzer's reagent. The first inner wall consists of two adherent

layers with the outer layer consistently less than 0.5 pm and

the inner layer 1-2 pm thick. In at least 50 of the spores

in any population, both lay ers may be so tightly adheren t that

they appear as one structure. In field-collected spores or in

preserved specim ens, the outer layer either is not distinguishable

or gives the appearance of having a rugose surface . Th e second

inner wall consists of two thin adherent layers also, with the

inner layer (1 -2 pm) often double the thickness of the outer

layer < 1 pm). The two layers are most easily distinguished

in Melzer's reagent, where the innermost layer produces a

pinkish red (0 -60 -30-0) to light purple (20 -60 -20-0)

reaction. T he germination shield was pale yellow to pale brown

and appeared as a terminal event in spore differentiation. It

always was positioned between the two inner walls.

Spores of all isolates, whe n preserved in 0. 5 formalin for

longer than several months, appeared to h ave only one or two

flexible inner structures. This differed markedly from freshly

extracted sp ores, confirming that preservation in form alin, and

possibly other solutions, cause inner walls and layers in each

wall to become so adherent that they were unresolvable at the

light-microscope level. These changes explain the discrepancies

between this and the description by Koske and Walker (1985).

All but one of the specimens of

S. heterogama

they examined

were preserved in lactophenol or formalin (R.E. Koske, per-

sonal communication).

An omission in the original description (Nicolson and

Gerdemann 1968) and redescription (Koske and Walker 1985)

was recog nition that the walls of auxiliary cells and their attached

hyphae are pale brown (0-30- 100-0 ) to brown (20-40-

80-0). This chara cter was stable in all isolates exam ined.

Discussion

Maximum historical information on any group of organisms

is retrieved from the study of entire life cycles. Ind eterminant

growth of intraradical and extraradical hyphae, as well as

meristic and asynchronous synthesis of specialized offshoots

(intraradical arbuscules, e xtraradical aux iliary cells), precludes a

precise or consistent definition of discrete stages during somatic

ontogeny. A temporal sequ ence may exist in which hyphal types,

arbuscules, or auxiliary cells peak in abundance, but it could

not be accurately assessed in our experimental design. Sporu-

lation also is meristic and asynchronou s, ex cept that its induction

appears to require a m inimum threshold of mycorrhizal biomass

(Gazey et al. 1992). W e interpret the mycorrhizal root length

at first appearance of spore s in

S. pellucida

and

S. heterogama

to indicate such a threshold for sporulation. An a pproximately

threefold difference in these levels among isolates WV872-1

and WV 858-1 may be a stable species-level prope rty, but mycor-

rhizae were not measured in the other isolates to test this

hypothesis.

Spor es we re the only derivative parts of the fungal organism

with enough discrete internal differentiation of morphological

diversity to order ontogenetic processes. This result was not

surprising, since these chara cters have circumscribed 150 species

to date (Morton 1993). Thus, the remainder of the discussion

will focus on these ontogenetic patterns and their implications

for the understanding of developmental processes, taxonomic

patterns, and phylogenetic relationships.

Developmental considerations

The developmental history of spores in

Scutellospora

could

be divided into three phases that encompassed different pro-

cesses of spore growth (expansion) and differentiation of discrete

subcellular structures. Phase I encompasses spore expansion

and differentiation of layers

in the spore wall. Phase I1

includes all stages of inner wall differentiation after spore

expansion has terminated. Phase

I involves morphological

and physiological events in spore germination after all inner

walls are completely differentiated.

Th e ability to distinguish between phases and separate spores

in those phases (detailed in Materials and methods) has im por-

tant methodological benefits for research designed to examine

spore-related processes and to discover functional properties.

In ultrastructural studies designed to study finer detail in spo re

wall differentiation, selection of only phase I spores reduces

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132 CAN.

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V

sampling to only relevant specimens. In biochemical studies,

comparisons between phase I and phase I1 spor es may provide

new insights into kinds and magnitude of sh ifts in synthesis of

primary and secondary metabolites. In germination studies,

bias caused by different age groups in an extracted population

can be lowered or eliminated by discarding phase I spores.

W e attribute the discontinuities in boun dary conditions of all

subcellular characters to internal epigenetic interactions chan-

nelling new variation and constraining structure already in

existence (Alberch 1980; LGvtrup 1976). The high degree of

stability in these interactions ap pears to be the result of caus al

linkages between each stage in th spore differentiation sequence

for two reasons. First, each wall and the germination shield

were synthesized in a linear sequence, as was the differentia-

tion of layers within each wall. second, synthesis of each new

primary characte r did not begin until all seconda ry and tertiary

characters of the antecedent character we re completely differen-

tiated. Th e distinction between a causa l sequence and on e that

is strictly temporal is important, because only the former is

phylogenetically informative (Alberch 1985).

Th e epigenetic constraints on a character w ere most evident

in developmental transformations of the spore wall of S hetero

gama.

Th e rigid inner layer of the spore wall (stage 1) becomes

highly plastic (amorphous) and stains a dark red-purple in

Melze r's reagent at stage IX. How ever, the phenotype of this

layer is transitory a nd the amorpho us quality is transforme d to

rigid laminae of similar structure to those in the spore wall of

S

pellucida. This reversion in spore wall structure during

ontogenesis has important consequences, in that it does not

disrupt all subsequent stages of inner wall differentiation. It

also indicates that variation in the spor e wall occurs independ-

ently of inner wall differentiation, as long as primary and

secondary spore wall structure are not comprom ised.

One of the important modes by which morphological trans-

formations affect the organismal phenotype is heterochrony

(Gould 1977). Heterochrony is expressed as heritable (or evolu-

tionary) changes in timing of initiation or termination of on to-

genetic events or a change in developmental rates (McKinney

and McNamara 1991). In spores, heterochronic change is

expressed in phase I expansion as the final spore size (or

volume). A ny genetic determinants to this process are uncoupled

from ontogeny of inner walls, because expansion is linked only

to differentiation of layers and tertiary properties of the spore

wall. Differentiation of all inner walls ii uncoupled from sp ore

growth processes. However, heterochronic changes also can

be m anifested in phase I1 by rate of differentiation of each new

inner wall. For example, stage 4 in spores of S heterogama

differentiated so rapidly that few specime ns were found at any

harvest or pooled samples (Fig. 2). Th e converse was true of

stage 4 in spores of S pellucida.

Taxonomic considerations

The hierarchical ordering of diversity so evident in the logical

structure of classifications was also observed consistently in ori-

gins of characters in spores of

S

pellucida and S. heterogama.

Ontogenetic patterns were so stable that we now c onsider them

the basis for beginning a comprehensive revision of character

terminology. These changes involve a relatively simple reorien-

tation of existing concepts within a hierarchical framework

reflecting the process o f development. W e abandon the wall-

group definitions proposed by Walke r (1983). The y are arbitrary

constructs subject to considerable variation caused by differ-

ences in condition of spores, mounting procedures, and degree

of experience by investigators (Mo rton 1 993). Wall groups m

closely approximate the primary characters delimited o

genetically in this study. The important difference is that

spore wall, each inner wall, and the germination shield

consistently identifiable by their position in s pore ontogen e

Once the positional relationships among these characters

better understood in comparisons with other taxa, then ope

tional criteria for their circumscription can be derived f

mature spore m orphologies rather than ontogenetic patte

W e also discard many of the wall definitions cu rrently in ta

nomic use because they apply neither to discrete walls no

individual structures of separate origin. Instead, they ar

combination of secondary and tertiary characters that mus

distinguished by constraints on variation. For example, sec

dary cha racters (layers) a re defined only by their posi

within a primary character. Tertiary characters encompass

of the variation within each laye r, such as colo r, thickness, o

mentation, or degree of flexibility (see Table l) , and constra

appear to be most relaxed at this level. W e adopt the view

these characters must be defined narratively to avoid categ

zation of phenotypes into narrow definitions.

Stuessy (1992) advocates a phenetic concept of gloma

species, in part because they reproduce asexually. How e

he could not have forseen the decisive role of developme

constraints on m aintaining an internal coherence of spo re phe

types among populations of asexual species (see Mishler

Budd 1990 for references). M oreover, a phenetic conc

implies equal weighting of all characters, and such a m

would not recognize the hierarchy of characters so eviden

spore ontogenesis. This hierarchy provides strong evide

that spore subcellular characte rs differ in their resolution

rank (Kohn 1992) in classifications. For exam ple, second

characters of the spore wall resolve groups of glomalean fu

at the suborder level. T he permanent outer layer and the lam

structure shared by all species in Gigasporinae are phenoty

cally distinct from the sloughing outer layer (or a multit

of other layers) and laminar structure common to m ember

Glomineae (Morton and Benny 1990). Tertiary propertie

each layer in the spore wall are diverse, and all available

dence indicates they delimit species as an irreducible clu

of organisms (Cracraft 1989). The inner walls, as prim

characters, ar e so unique that they have no counterpart in fun

groups outside Glomales. Secondary and tertiary character

the inner walls do not appear to be as variable as those in

spore w all (J. Morton, unpublished data). They are likely

resolve ranks abo ve the species level that have not been rec

nized because of incorrect character interpretations. T hese is

pertaining to the grouping and ranking of taxa cannot

addressed here o r elsewhere until the distribution of ch arac

that pass all of the tests of similarity is determined am

known taxa. Cladistic analyses then must be repeated to de

mine the level of constraints on introduction of new varia

during evolution.

Phylogenetic considerations

Individual characters, and even ontogenetic stages, of S pe

c i d ~ nd

S.

heterogama spores have proved to be disc

enough to satisfy tests of similarity listed in the introduc

and thus define provisional hypotheses of homology (Albe

1985; Kluge and Strauss 1985). These tests are essentia

insure that resemblance is the result of common rather t

independent ancestry. The amorphous w d l sensu Morton (19

exemplifies how homology has been incorrectly assigned

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133

characters in the absence of validating ontogenetic criteria.

Interpreted as a discrete structure, its presence in spores of

some Acaulospora and Entrophospora species (Morton and

Benny 1990) suggested affinities with Scutel lospo ra. In reality,

it is the terminal state in ontogenetic transformation of the

innermost flexible wall, beginning as a very thin layer (m em-

branous wall sensu Walker 1983) followed by an intermediate

thickening (coriaceous wall sensu Walker 1986). Since this

transformation series is linear and likely to be conserved by

causal linkages in epigenetic interactions, we predict each of

these tertiary characters will be recapitulated in phylogeny. In

other words, they will be homologized between characters in

fully differentiated spores of on e species and in juvenile stages

of other species.

The relationship between development and phylogeny has

a long history (Gould 1977), and its application to tests of

phylogeny (e.g., cladistic analysis) was form alized by Nelson

(1978) in his biogenetic law to polarize characters as being

ancestral or derived. This comp arative method, how ever, applies

only to those ontogenetic sequences where new characters are

added successively in a linear sequen ce through the process of

terminal addition (Kluge and Strauss 1985; O'G rady 1985).

This pattern was shown to dominate the emergence of all

primary characters, secondary characters of spore and inner

walls, and some tertiary characters in spores of S. pellucida

and S. hetero gam a. It will prov ide the empirical basis for revis-

ing current hypotheses of morphological evolution (Morton

1990). The hierarchical order of characters according to their

ontogenetic origins almost assures congruence with the hier-

archy of phylogeny at the primary and secondary levels.

The problems in cladistic analysis will arise with tertiary

charac ters that are not constrained by causally linked epigenetic

interactions. Th ey a re easily replaced o r lost within secondary

characters (e.g., wall layers) without any disruption in syn-

thesis of the primary walls leading to germination. Thes e sorts

of changes in ontogeny are deviations rather than additions in

phylogeny and can be incorpo rated into phylogenetic analysis

using nondevelopmental criteria such as out-groups (Watrous

and Wheeler 1981). An obvious example is color. Pigment

changes in any layer of the spore wall would have no effect

on all subsequ ent stages in differentiation and thus are easily

introduced as new variations. M any species may arise wherein

constrained characters remain unchanged, with only color or

other tertiary chang es expressed as deviations. T hese chang es

would be small enough relative to all of the other conserved

features within spores to appear as intergrading species.

W e do not attempt a cladistic revision in this paper bec ause

the same kinds of discrepancies found in published descrip-

tions of S. pellucida and S. heterogama (Koske and Walker

1985, 1986) also occur in those of most other Scutellospora

species (at least 1 6 out of the

24

described, based on specimens

from cultures or vouchers in INVA M;

(J.

Morton, unpublished

data). W e must first conduct tests of similarity from ontogenetic

data to circumscribe char acters, define them as putative hom o-

logs, and then redescribe the species.

Th e invariance of differentiation sequences in spores of the

fungi in this study strengthens the view that developmental

constraints on m orphological structure are the main causal basis

for the hierarchical o rder and d iscreteness of subcellular char-

acters and stability of phylogenetically discrete species.

Results support the genealogical definition of species as the

smallest assemblage of reproductively isolated individuals or

populations diagnosed by epigenetic morphological or organ-

izational properties of fungal spore s that specify unique genea-

logical origin based on the criterion of monophyly (Morton

et al. 1992) in which (i) smallest signifies tertiary ch aracters of

spores defining irreducible species-level variation; (ii) assem-

blage excludes gene flow as an interactive force in species

cohesiveness; (iii) epigenetic properties indicate constraints

on variation are the more probable basis for cohesiveness;

(iv) spores are the only part of the fungal organism for which

enough morphological diversity has been expressed to define

species; (v) genealogy preserves unity within a species; and

(vi) monophyly methodologically excludes delimitation of a

species from analagous characte rs. At the very least, this defi-

nition unites disjunct organisms into a group that can then be

tested experimentally to measure other kinds of diversity.

Operational criteria for this definition must com e from tests of

similarity (see Introduction) to define provisional homologies.

The knowledge gained from the ontogenetic comparisons in

this and future studies will insure these tests can be carried out

with fewer complications than exist today.

cknowledgement

This work was supported by Hatch Funds from the West

Virginia Agricultural and Forestry Experiment Station and

National Science Foundation grant DIR-9015519.

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