franke 1994
TRANSCRIPT
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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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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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