ASCI OF THE OPERCULATE DISCOMYCETES (PEZIZALES)
Don Arthur Samuelson
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
I wish to express gratitude to Dr. James W. Kimbrough,
Chairman of the Supervisory Committee, for his guidance,
friendship and patience throughout the course of my study.
I am also indebted to Dr. Henry C. Aldrich and the staff of
the Ultrastructure Laboratory for the use of the equipment
and facilities and for their appreciative assistance.
I wish to sincerely thank D. G. Griffin, III, for his
considerate advice and encouragement over the last five
years. I also wish to extend my thanks to Drs. D. A.
Roberts and N. C. Schenck, as members of my Supervisory
Committee along with Drs. Kimbrough, Aldrich and Griffin,
for their helpful comments and suggestions.
Sincere appreciations are accorded to Dr. G. L. Benny
and other researchers associated with the Mycological
Laboratory and for their encouragement and assistance.
TABLE OF CONTENTS
ACKNOWLEDGEMENTS . . .. ii
LIST OF TABLES . . v
LIST OF FIGURES . . vi
ABSTRACT . . . ix
GENERAL INTRODUCTION . . 1
MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUSES OF REPRESENTA-
TIVES WITH IODINE-POSITIVE ASCI .. 9
Introduction. . 9
Materials and Methods. . 15
Results . .. .. 18
Discussion. . . 27
MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUSES OF REPRESENTA-
TIVES IN THE OTIDEA-ALEURIA COMPLEX 48
Introduction . .
Materials and Methods . .
Results . . .
Discussion . .
MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUSES OF EUGYMNO-
HYMENIAL REPRESENTATIVES .
Introduction. . ... 117
Materials and Methods. .. 123
Results . .. 124
Discussion . 130
MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUSES IN MORCHELLA
ESCULENTA AND REPRESENTATIVES OF THE
HELVELLACEAE. . .
Introduction. .. . .143
Materials and Methods . .. .147
Results . ... 148
Discussion. . . 155
MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUS OF THELEBOLUS 170
Introduction. . 170
Materials and Methods .. . 176
Results ... ....... 177
Discussion. .... . .... 187
MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUS OF TRICHOBOLUS
ZUKALII . .. .214
Introduction. ... . 214
Materials and Methods .... .. .217
Results . . 217
Discussion . 220
SUMMARY . . .. 229
BIBLIOGRAPHY . ... . 245
BIOGRAPHICAL SKETCH . ... .. .. .253
LIST OF TABLES
Classifications of genera with iodine
positive asci . .
Classifications of genera of the Otidea-
Aleuria complex . .
Classifications of eugymnohymenial genera .
Table 1 Classifications of the Helvellaceae .
Table 1 Classifications of the Thelebolaceae. .
LIST OF FIGURES
The apical apparatus of Peziza
succosa. . .
The apical apparatus of Ascobolus
crenulatus . .
The apical apparatus of Saccobolus
depauperatus . .
The apical apparatus of Thecotheus
pelletieri . .
The apical apparatus of Iodophanus
granulipolaris . .
Drawings of apical apparatuses found
in iodine-positive asci. .
The apical apparatus of Otidea
leporina . .
The apical apparatus of Jafnea
fusicarpa. . .
The apical apparatus of Humaria
hemisphaerica. . .
The apical apparatus of Sphaero-
sporella brunnea . .
The apical apparatus of Aleuria
aurantia . .
The apical apparatus of Anthracobia
melaloma . .
Figure 110 A-D
Figure 111 E-H
The apical apparatus of Scutellinia
scutellata . .. 104
The apical apparatus of Ascozonus
woolhopensis . .. 108
The apical apparatus of Geopyxis
majalis. . 110
The apical apparatus of Sowerbyella
imperialis ... .112
Apical apparatuses redrawn from
Chadefaud (1942) .. 114
Illustrations made from electron
microscopic observations of apical
apparatuses of the Otidea-Aleuria
complex. ... 116
The apical apparatus of Pyronema
domesticum . 136
The apical apparatus of Ascodesmis
sphaerospora . .. 138
The apical apparatus of Coprotus
winterii . .... 140
The apical apparatus of Coprotus
lacteus ... .142
The apical apparatus of Helvella
crispa .... . 161
The apical apparatus of Morchella
esculenta. . .. .163
The apical apparatus of Rhizina
undulata . 165
The apical apparatus of Discina
ancilis. . 167
The apical apparatus of Gyromitra
rufula . .
The apical apparatus of Thelebolus
microsporus. . .
The apical apparatus of Thelebolus
crustaceus . .
The apical apparatus of Thelebolus
polysporus . .
The apical apparatus of Thelebolus
stercoreus . .
Development of the apical apparatus
in Thelebolus polysporus .
The apical apparatus of Trichobolus
zukalii. . .
Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment
of the Requirements for the Degree of
Doctor of Philosophy
ASCI OF THE OPERCULATE DISCOMYCETES (PEZIZALES)
Don Arthur Samuelson
Chairman: James W. Kimbrough
Major Department: Botany
In the taxonomy of Ascomycetes greater emphasis is
being placed on ascal structure and the mechanism of spore
release. All components associated with ascal dehiscence
are collectively referred to as the apical apparatus.
Within the operculate Discomycetes (Pezizales) ascal charac-
ters, including the apical apparatus, have provided sig-
nificant contributions in the delimitations of families
The primary purpose of this research was to understand
more clearly the nature of the operculate apical apparatus
and to determine whether this character can be implemented
as a systematic tool. To do this, ascal tips of approxi-
mately 30 species, representing 26 genera, were examined
with the aid of the light and electron microscopes. The
stains, Congo red, acid fuchsin, aniline blue, Melzer's
reagent and toluidine blue were commonly used for light
microscopy, while lead citrate, uranyl acetate and silver
methenamine were used for electron microscopy. Attention
was paid to morphology, development and cytochemistry of the
apical apparatus as a whole, to peculiar structures, and to
the time cf their appearance during ascosporogenesis.
Morphological, cytochemical and ultrastructural evi-
dence revealed that the apical apparatus fell into six
groups. (1) Members typically with iodine-positive asci,
including species of Peziza, Ascobolus, Saccobolus and
Thecotheus, possess annular indented zones of dehiscence.
Exogenous mucilaginous coats are believed to be responsible
for the blue reaction. Although lodophanus is iodine-
positive, very few properties of this taxon are shared
with those of the annular indented species or any other
member of the Pezizales. (2) Eugymnohymenial discomycetes
such as Pyronema, Ascodesmis and Coprotus have ascal walls
which taper in thickness at the tip. Wide zones of de-
hiscence are formed in the outer layer of Coprotus and
Ascodesmis. In Pyronema the outer layer of the operculum
is differentially stained from the rest of the ascal wall.
(3) Species placed in either the Otideaceae or the Aleuriaceae
lack conspicuous dehiscent zones. The presence of subapical
rings in Aleuria, Anthracobia, Ascozonus, Humaria, Jafnea,
Otidea, Scutellinia and Sphaerosporella distinguish the
ascal tips of these taxa from the rest of the operculate
Discomycetes. (4) Members with large, variously shaped
apothecia, i.e., Morchella and Helvella, possess distinct
zones of dehiscence in the inner layer. Mature lateral
walls vary little in thickness from the apical walls.
Opercular delimitation with the light microscope is observed
in Rhizina and Discina when stained with Congo red.
(5) Four species of Thelebolus exhibit thick-walled apical
domes that are subtended by distinctly stained rings. Wall
structure most closely resembles that of the bitunicate
ascus. The inner layer consists of microfibrils organized
in a banded pattern. Ascal dehiscence occurs in a modified
jack-in-the-box manner. (6) Trichobolus zukalii possesses
a large operculum which is functionally inoperative. The
ascal wall is heavily stratified and lacks distinctive
The apical apparatus of the operculate ascus has marked
variability. At least four major forms exist. In addition
two variations are found in Trichobolus and lodophanus and
may be considered as separate forms. The apical apparatuses
of Thelebolous demonstrate very few microchemical, develop-
mental and morphological similarities with those found in
the other species. The taxonomic placement of Thelebolus
within the Pezizales is in serious doubt. Examination of
the apical apparatus can be useful as a systematic tool in
helping resolve ambiguous taxonomic relationships and
determine phylogeny within the Pezizales.
For the past century ascal characters have played an
increasingly significant role in Ascomycete taxonomy. The
early work of the Crouan brothers (1857), Nylander (1869)
and Fuckel (1869) revealed notable differences in the ascus
with respect to size, shape, microchemical properties and
mode of spore release. Shortly thereafter, a broad scheme
of Discomycete classification was proposed (Boudier, 1879,
1885), the first in which ascal features were exploited.
The significance of this concept has not been fully appreci-
ated until recently.
During the last 35 years, ascal structure has been
recognized as one of the fundamental diagnostic characters
within the Ascomycotina. Studies by Chadefaud (1942) and
Miller (1949) on asci within the Discomycetes and
Pyrenomycetes revived interest in this area of taxonomic
research. Miller (1949) used various features, including
wall thickness, dimensions of the ascus, and presence or
absence of a pore or cap at the ascal apex to separate
major orders and families of the Pyrenomycetes. Chadefaud
(1942, 1946) focused mainly on the tip of the ascus in both
the Pyrenomycetes and Discomycetes. He paid particular
attention to all components directly involved in ascospore
expulsion. He used the term "apical apparatus" in defining
that region of the ascus. In addition to these works,
Luttrell (1951) separated the Pyrenomycetes into two groups,
the unitunicates and the bitunicates, basing the division on
essential structural features of the ascal wall.
Within the discomycete order Pezizales, ascal charac-
ters have provided an important contribution for the dis-
tinction of families, subfamilies and genera (Eckblad, 1968;
Kimbrough, 1970; and van Brummelen, 1974). Typically, the
ascospores are liberated through a circumscissile rupture,
the operculum, at the tip of the ascus. Most studies con-
cerned with dehiscence of the ascus have been restricted to
representatives within the smaller suborder Sarcoscyphineae
(Chadefaud, 1946; LeGal, 1946a, 1946b, 1953; van Brummelen,
1975; Samuelson, 1975). LeGal and Chadefaud noted distinct
characters that appeared similar to the apical apparatus of
various members within the inoperculate Discomycetes. They
proposed a phylogenetic relationship between representatives
of the Sarcoscyphineae and certain representatives of the
Helotiales. Recent ultrastructural studies (van Brummelen,
1975; Samuelson, 1975) demonstrated that all asci were
truly operculate in six genera of the Sarcoscyphineae.
Marked differences were described in the dimensions of the
bilayered ascal wall surrounding the operculum. Samuelson
(1975) structurally defined the structure of this region as
the suboperculum and noted two basic forms, eccentric and
noneccentric. The two forms, however, were shown to be
quite variable, representing intergrades that linked the two
extreme examples, Cookiena sulcipes (Berk.) Kuntze and
Pseudoplectania nigrella (Pers. ex Fr.) Fuckel. Taxa that
had the noneccentric form displayed significant features
that were in common with the apical apparatus of an
operculate species, Ascobolus stercorarius (Bull. ex St.
Amans) Schroet (Wells, 1972). Phylogenetically, ascal
dehiscence within the Sarcoscyphineae is believed to be
affiliated most closely with that observed in the true
Relatively few investigations related to mechanisms
of ascal dehiscence have been made on representatives of the
Pezizineae. The first comprehensive morphological study on
true operculate apical apparatuses was carried out by
Chadefaud (1942). While describing the apical apparatuses
in detail, he pointed out many similarities between opercu-
late and nonoperculate asci. Chadefaud (1942) designated
a fundamental apical apparatus that contains components
ubiquitous among discomycete and pyrenomycete asci. Many
of these components were incorporated within a majority of
the operculate apical apparatuses. In basipetal sequence
they included (1) an operculum, (2) a subapical 'bourrelet'
or cushion, and (3) an apical punctation or depression that
led to a funnel which was attached contiguously to a tract
that led to the base of the ascus. However, other elements
that were depicted in the fundamental apical apparatus were
not reported for operculate asci. Chadefaud (1942)
theorized that the asci of these fungi have become modified
through the process of evolutionary regression.
In a recent systematic treatment of the ascus,
Chadefaud (1973) separated ascus types into three cate-
gories, the Archaeasces, the Nassasces and the Annelasces.
The division was based both on ascal wall structure and the
presence and absence of principal components that comprised
the apical apparatus. He retained the view that a funda-
mental unit of ascus structure exists. The prototype appara-
tus depicted in his earlier study (1942) is basically simi-
lar to the type depicted for the Annelasce. Chadefaud
(1960, 1973) renamed the region of the "coussinet apical"
and "pontuation apicale" of the operculate asci to be
"pendentif" and "chamber oculaire," respectively.
The remainder of the studies related to the structure
of operculate apical apparatuses have been made primarily
within the last decade. Of these, van Brummelen's (1974)
work with Ascozonus woolhopensis (Berk. and Br. apud Renny)
Hans. was the only one specifically concerned with the de-
hiscence apparatus. The number of investigations made on
the apical apparatus of inoperculate Discomycetes (Bellemere,
1969, 1975; Campbell, 1973; Corlett and Elliott, 1974;
Schoknecht, 1975) and Pyrenomycentes (Beckett and Crawford,
1973; Greenhalgh and Evans, 1967; Griffiths, 1973; Reeves,
1971) have been considerably greater. The apparent lack of
interest in the operculate apical apparatus may reflect an
attitude of many investigators that most operculate apical
structures appear identical. Several previous findings sug-
gest that the opposite may be true. In a study on ascus and
ascospore development of Ascobolus strecorarius, Wells
(1972) discovered that as the spores approached maturity
an indented circular band was formed at the tip of the ascus,
delimiting the operculum. Schrantz (1970) demonstrated in
his work with Peziza and Tarzetta that the ascal apex con-
sisted of a thicker outer layer, a feature not reported pre-
viously within the Pezizales. The study of Ascozonus
woolhopensis by van Brummelen (1974) showed that the inner
layer of the ascal tip initially became swollen before the
layer underwent a process of localized disintegration which
was followed by spore release. The data of these research-
ers reveal remarkable differences between dehiscence
mechanisms of the selected taxa.
Microchemical properties of apical apparatuses within
the Pezizales have displayed a variety of differences be-
tween numerous species. Characteristically, within the
Sarcoscyphineae the inner layer of the ascus is deeply
stained in Congo red, and in certain members the opercula
have been shown to be microchemically distinct from the
surrounding wall of the ascus (LeGal, 1946a; Samuelson,
1975). Cytochemical observations of representatives within
the true operculates have been more useful for taxonomy.
The most widely applied stain has been Melzer's reagent, an
iodide solution that produces typically a blue reaction
(Korf, 1973). In the genus Peziza (Dill.) L., taxa were
distinguished by the variable staining of the ascal tips.
Nylander (1869) was one of the first to use the iodine
reaction as a method for generic and specific delimitation.
In other operculate representatives, lodophanus Korf,
Thecotheus Boud., and Psilopezia Berk., entire asci have
been reported to be diffusely stained (Eckblad, 1968;
Kimbrough, 1969 and 1970). Within the largely coprophilic
family Thelebolaceae (sensu Kimbrough, 1970), asci in a num-
ber of taxa have been observed to rupture irregularly upon
spore release. Uniascal, multispored forms of Thelebolus
and Trichobolus appeared to have more pronounced structural
features associated with their apical apparatuses. Similar-
ly, Kimbrough (1974) discovered in the genus Lasiobolus
that the uniascal species L. monascus Kimb. forms a chemi-
cally differentiated band below the apex of the ascus and
that similar though less notable structures exist in eight-
spored species. In Caccobius, he noted that the tip of the
ascus contains a distinct, apical plug which stains in
Waterman's blue black ink but remains hyaline in Congo red
(Kimbrough, 1972). Conversely, preparations using Congo red
and acid fuchsin in lactic acid have demonstrated the pres-
ence of a prominent apical ring located within the multi-
layered ascal wall of Thelebolus (Kimbrough, 1972).
Until recently, little attention has been paid to the
ultrastructure or cytochemistry of the structures associated
with dehiscence of the ascus. Consequently, these features
have played a small role in the taxonomy of operculate
Discomycetes. Increasing information on ascal walls and
ascal tips, in particular, suggests the strong possibility
that apical apparatuses within the Pezizales are signifi-
cantly diverse and can be implemented as a useful systematic
tool. This study presents a comprehensive survey of the
apical apparatus within the Pezizineae, the primary concern
being with morphological and cytochemical features. The in-
vestigation includes a conspectus of apical apparatuses on
family, subfamily and tribe levels, using one or more major
representatives from each taxon. Over 30 species, represent-
ing 26 genera, were inspected throughout the course of the
study. Instead of attempting to describe all of the species
at one time, it has been more feasible to segregate them
into morphological groups. Special attention was paid to
the development of the apical apparatus as a whole and to
peculiar structures and the time of their appearance during
ascosporogenesis. Chapter I examines the asci and the
apical apparatus of representatives that are associated with
the iodine-positive reaction. Included are species of
Peziza L. per St. Amans, lodophanus, Ascobolus Pers. per
Hooker, Saccobolus Boud., and Thecotheus Boud. Chapter II
studies the apical apparatuses of members found throughout
the largest of all operculate groups, the Pyronemataceae
(sensu Korf, 1973), a group which has been referred to here
as the Otidea-Aleuria complex. Species of Otidea (Pers.)
Bon., Jafnea Korf, Humaria Fuckel, Sphaerosporella (Svr.)
Svr. & Kub., Aleuria Fuckel, Anthracobia Boud., Scutellinia
(Cooke) Lamb., Ascozonus (Renny) Hansen, Sowerbyella
Nannft., and Geopyxis (Pers.) Sacarrdo were used. The api-
cal apparatuses of representatives of Ascodesmis van
Tieghem, Coprotus Korf & Kimb., and Pyronema Carus are
described in Chapter III. Characters not found in the
apical apparatuses of other representatives shared by
the three members have resulted in the formation of this
grouping. In Chapter IV, the apical apparatus of the
Morchella-Helvella group are examined. This group repre-
sents members which typically form the largest ascocarps
within the operculate Discomycetes. Species of Morchella
St. Amans, Helvella L., Gyromitra Fr., Discina (Fr.) Fr.,
and Rhizina Fr. per Fr. were used. Chapter V examines the
ascal walls and the associated apical apparatuses of four
species of Thelebolus Tode per Fr. Dehiscence mechanism,
wall layering, ontogeny and cytochemistry separate this
genus from all members of the operculate Discomycetes.
Similarly, Trichobolus zukalii Heimerl is treated by itself
in Chapter VI.
The objective of this study was to learn the nature of
the operculate apical apparatus and to observe what phylo-
genetic correlations can be made within the Pezizales.
MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY OF THE APICAL
APPARATUSES OF REPRESENTATIVES WITH IODINE-POSITIVE ASCI
A number of workers have paid close attention to ascal
features as an additional means to help classify individual
representatives and major groups within the Ascomycetes.
Of these features, the ascal wall and its associated mech-
anism of spore release have been of particular interest.
(See General Introduction.)
Within the operculate Discomycetes, early workers
(Nylander, 1869, Karsten, 1869; Rhem, 1896; and Boudier,
1905-1910) demonstrated the occurrence of iodine positive
or "amyloid" asci in a number of species, and by 1907
(Boudier), this feature had been applied systematically
along tribal lines. Recently, Kohn and Korf (1975) have
pointed out that the term "amyloid," used traditionally for
the positive blueing reaction of iodine-treated material,
should be avoided. It implies the presence of amylose or
amylose-related substances when little has been known of its
chemical specificity. The present study has taken account
of their suggestion and has used the terms "iodine positive"
and blueingg in iodine" in place of amyloid.
During the last decade, Rifai (1968), Kimbrough (1970)
and Korf (1973) have placed all members of the Pezizineae
that have the "amyloid" property into two families, the
Ascobolaceae and Pezizaceae. Of the genera included in
these families, only Ascobolus has been shown to have species
that do not turn blue in iodine. Even in this genus approxi-
mately 50% of the species were observed to give an iodine
positive reaction (van Brummelen, 1967). Except for Peziza,
the blueing reaction has been found to occur diffusely over
the entire wall of the ascus. In Peziza, many species are
distinctly iodine-positive at the apex. In certain species
such as P. vesiculosa Bull. ex Fr., the blueing is restricted
to a ring while the others as much as one-half of the ascus
Until Chadefaud's (1942) extensive examination of the
apical apparatuses found throughout the Euascomycetes, no
one had investigated carefully the mechanism of dehiscence
of the representatives in the Ascobolaceae and Pezizaceae.
Working with material that had been treated with a chromo-
osmic solution, Chadefaud described the asci of Peziza
echinospora Karst. (= Aleuria umbrina Boud.) to have a full
complement of dehiscent components as described in the Gen-
eral Introduction. Furthermore, he stated that in all of
the species which have a completely developed apical appara-
tus, including P. echinospora, the formation of the apical
apparatus occurred before ascosporogenesis. His observa-
tions of living material of Ascobolus furfuraceus Pers. ex
Fr. revealed the presence of a modified apparatus which
lacked a distinct funnel and tract. He depicted the ascal
tip as having a remarkably differentiated opercular dome and
subtending pad (Fig. 51a). Consequently, he reasoned that
Ascobolus was a very specialized group which represented a
line of evolutionary regression.
Although Moore (1963), Reeves (1967) and Carroll (1969)
had previously studied the fine structure of operculate
asci, Schrantz (1970) was the first to examine the ultra-
structure and cytochemistry of the ascal walls of several
ascomycetous representatives, including Peziza plebeia
(LeGal) Nannf. (as Galactinia plebeia). He described the
ascal wall of P. plebeia as consisting mostly of a thick,
"chitinous-callosic" inner layer, which became thinner
towards the apex, and a thin, "pectic-amyloid" outer layer,
which conversely thickened at the apex (Fig.51b). His elec-
tron microscopic examination demonstrated the two layers,
thus reinforcing the light microscopic observations. He was
unable, however, to distinguish from each other the "amyloid"
and the pecticc" coats, which formed the outer layer.
Wells (1972) in his work with the ascus and ascospore
ontogeny in Ascobolus stercorarius (Bull. ex St. Amans)
Schroet. showed that towards the end of spore maturation an
annular indentation was formed at the tip, delimiting the
operculum. Simultaneously, the inner layer of the ascal
wall increased in thickness throughout the apical region.
Wells' and Schrantz' accounts of the ascal tips of
A. stercorarius and Peziza plebeia, respectively, displayed
little in common. In fact, their findings supported
Chadefaud's belief that the two genera are not closely
Similarly, Eckblad (1968) concluded that Ascobolus and
related genera have little phylogenetic connection with the
Pezizaceae but instead are more affiliated with members of
the Thelebolaceae. He suggested that the "amyloid" reaction
was not a valid phylogenetic trait. By putting great
taxonomic value on clavate form and protruding nature of
mature asci, he felt justified in placing Ascodesmis in the
Ascobolaceae and removing lodophanus to the Pyronemaceae
and Thecotheus to the Thelebolaceae (Table 1).
The usefulness of the iodine test for systematic schemes
is seriously in doubt. This study examines morphological,
developmental and cytochemical aspects of the iodine-positive
ascus and its associated mechanism of dehiscence in order to
learn whether other characteristics of the ascal wall will
support or argue against the systematic use of the iodine
test. Five members of the Pezizaceae and Ascobolaceae
(sensu Rifai, 1968; Kimbrough, 1970; Korf, 1973) are used.
They are lodophanus granulipolaris Kimbrough, Thecotheus
pelletieri (Crouan) Boud., Ascobolus crenulatus Karst.,
Saccobolus depauperatus (Berk. and Br.) E. C. Hansen and
Peziza succosa Berk. Phylogenetic and ontogenetic compari-
sons are made between the species.
Classifications of Genera with Iodine-positive Asci
Peziza (= Plicaria)
Table 1 (Cont'd.)
Materials and Methods
Collection and Developmental Determination of Material
Young and mature apothecia of Peziza succosa were col-
lected from a sandy basin near the Devil's Millhopper in
Gainesville, Florida. Specimens of Thecotheus pelletieri
and Saccobolus depauperatus were found on cow dung collected
near Gainesville. A culture of Ascobolus crenulatus, no. D-
1476, was obtained fromthe Culture Collection of the Rancho
Santa Ana Botanic Garden, courtesy of R. K. Benjamin.
Iodophanus granulipolaris was isolated from cow dung col-
lected near Gainesville by J. Milam. Apothecia of A.
crenulatus and I. granulipolaris were grown on DOA (Dung-
Oatmeal Agar; Benedict and Tyler, 1962) and WSHDD (Weitzman
Silva Hutner medium with dung decoction) respectively. Por-
tions of the apothecia of P. succosa and A. crenulatus were
free-hand sectioned for light microscopic observation to
determine the stage of ascal development. The mature and
immature stages were then separated for further examination.
Apothecia of different developmental stages of T. pelletieri
and S. depauperatus were removed from the substrate and
placed in thin layers of solidifying water agar and tempo-
rarily refrigerated. Whole squash mounts of individual
apothecia of I. granulipolaris were used to determine the
stage of ascal ontogeny for the majority of the apothecia in
a particular Petri plate.
Procedures for Light Microscopic Observations
Three to five millimeter squares from the apothecia
of P. succosa and entire apothecia of A. crenulatus were
placed in a 40% mucilage mixture, frozen and sectioned with
a cryostat at a thickness of approximately ten micrometers
(ym). Sections were mounted on a drop of Melzer's reagent
(Korf, 1973) on a slide and viewed with a Zeiss microscope.
The Melzer's reagent was used as a general differential
stain. More importantly, this reagent tested for the iodine-
positive or "amyloid" reaction at the tip or throughout the
ascal wall. The Congo red stain (Samuelson, 1975) was also
used frequently for comparison. Similarly, whole ascocarps
of T. pelletieri, S. depauperatus and I. granulipolaris were
squash-mounted in either Melzer's reagent or Congo red.
Less frequently, 1.0% lactophenol cotton blue, 2.0% aqueous
phloxine and 2.0% analine blue in 50% glycerine solution
were used to observe cytoplasmic detail and any notable
change in wall morphology.
Plastic embedded material was sectioned at 0.5 to 1.0
micrometers with glass knives. Two or three sections were
placed on a small drop of 0.01% sodium borate and dried at
600C. Single drops of either 0.25% aqueous toluidine blue
(Stevens, 1966) or 1.0% aqueous crystal violet were applied
for 30 seconds and carefully rinsed with water before being
mounted in immersion oil.
Procedures for Electron Microscopic Observations
Five-millimeter squares of apothecia of P. succosa,
entire apothecia of A. crenulatus and I. granulipolaris, and
agar blocks, containing six to eight apothecia each, of
S. depauperatus were fixed in buffered (0.2 M sodium caco-
dylate pH 7.2) 2.0% glutaraldehyde and 2.0% paraformaldehyde
solution (Karnovsky, 1965) for two hours at room tempera-
ture (= 23C). Agar blocks containing apothecia of T.
pelletieri were fixed in 1.0% permanganate solution for one
hour at room temperature. All materials were rinsed 3 times
during a 30-minute period in 50% buffer-50% distilled water
solution and postfixed in 1.0% osmium tetroxide for one
hour at room temperature. The materials were washed several
times with 0.1 M sodium cacodylate buffer and dehydrated in
an ethanol series (25% steps). Specimens were stained with
2.0% uranyl acetate in 75% ethanol overnight at 40C. They
were washed twice with acetone for one hour and subsequently
infiltrated and embedded in a low-viscosity resin (Spurr,
1969). Three changes of 100% plastic were made to ensure
removal of any residual acetone. The materials were placed
under vacuum for five minutes to remove bubbles and then
polymerized for one day at 600C.
Ultrathin sections were cut on a Sorvall MT-2 ultra-
microtome with a diamond knife and placed on single-hole,
formvar-coated grids. Sections were normally poststained in
0.5% uranyl acetate for 15 minutes and in lead citrate
(Reynolds, 1963) for 5 minutes. In addition, wall demarca-
tion was further enhanced by posttreating unstained sections
with silver methenamine, a preferential stain for poly-
saccharides (Martino and Zamboni, 1967). Ultrathin sections
were then examined with an Hitachi HU-11 E electron
Within the operculate ascus, three structural compo-
nents, the operculum, the region or zone of dehiscence, and
the adjacent lateral walls referred to as the suboperculum
(Samuelson, 1975), collectively comprise the apical appara-
tus. The apical apparatus of each species differs to some
degree developmentally and in size and shape. For clarity,
the mechanism of dehiscence for each representative will be
The Apical Apparatus of Peziza succosa
The diploid ascus reaches a length of 140-180 pm and a
diameter of 10-12 pm. In Melzer's reagent a deep blue re-
action occurs and is restricted to the immediate region of
the apex (Fig. 1). The outline of the tip of the ascus ap-
pears to be rather uneven, and the cytoplasm of that region
has a distinctly granular appearance. Fully mature asci
reach a length of 200-210 pm and a diameter of 12-14 pm.
When stained with Melzer's reagent, the deep blueing at the
tip is less intense and extends 10-14 pm down the sides of
the ascal wall (Fig. 2). The outline of this region is
smooth at this time. On occasion, a blue staining coat may
be removed by gently applying pressure on a cover slip and
sliding the cover slip back and forth. The tips of mature
asci are weakly stained in Congo red. A hyaline ring is
barely visible at the apex.
Ultrastructurally, the apical wall of the diploid
ascus (Fig. 3) is distinguishable in form from the rest of
the ascal wall. Lomasomal activity occurs throughout the
upper portion of the ascus. A thin, uneven, mucilaginous
coat, 135-160 nm thick, is also present in the immediate
area of the tip. The wall at this stage in development has
a thickness of 135-170 nm.
By the early stage of ascospore delimitation, the muci-
laginous coat has expanded notably both in thickness, being
375-415 nm at the tip, and in length, extending 6.5-7.5 pm
down the lateral face of the ascus (Fig. 4). The apical
wall remains thin, being 140-170 nm thick, while the lateral
wall has increased to 380-420 nm.
By late ascosporogenesis, the apical wall has broadened
conspicuously (Fig. 5). At the same time, an annular in-
dentation is formed delimiting the operculum. When thin
sections are treated with silver methenamine, the layering
of the ascal wall becomes sharply demarcated (Fig. 6). The
increased thickening of the tip, which measures 310-360 nm,
results from the addition of an inner layer, 250-280 nm
thick. The adjacent suboperculum has a length of 6.3-7.1 pm.
The strongly stained inner layer narrows from 190-210 nm in
thickness near the annular indentation to 75-85 nm at the
base of the suboperculum. Conversely, the outer layer
expands from 90-100 nm to 280-340 nm. Overall, the lateral
ascal wall narrows approximately 0.1 pm as it approaches the
At ascospore maturity, the annular indentation is more
defined (Figs. 7, 8) due to additional thickening of the
inner layer of the operculum. This narrowed ring (Figs. 9,
10), having a length of 620-650 nm and thickness of 210-
260 nm, consists of a greatly reduced inner layer, 130-170
nm, and a thin outer layer, 85-95 nm. Closer examination of
sections stained with silver methenamine reveals the pres-
ence of a subtending, cytoplasmic ring (Fig. 9) which may
play a critical role in ascospore discharge. The inner
layer of the operculum has increased in thickness by an ad-
ditional 20-45 nm (Fig. 8). Wall dimensions of the sub-
operculum remain essentially unchanged.
The Apical Apparatus of Ascobolus crenulatus
Diploid asci are fairly small and cylindrical, being
20-45 um long and 6-10 pm wide. As the spores mature, the
ascus grows to a length of 115-130 pm and width of 12-15 pm.
When placed in IKI, blueing of the ascal wall is not de-
tected. The tip, which appears thick-walled, has a slight
conical shape and is bordered by a barely visible ring
(Fig. 11). At maturation the tip becomes inflated and
thinner-walled (Fig. 12).
Electron microscopic observations of the apex of the
diploid ascus demonstrate the presence of localized vesi-
culation which is bounded by a ring of glycogen (Fig. 13).
During early spore development, the apical wall, which has
become rounder in form, still consists of a single, uniform
layer, 85-95 nm thick, toward the apex, and 100-110 nm thick
in the lateral region (Fig. 14). Further on in spore matura-
tion the apical wall undergoes a differential increase in
thickness resulting in the formation of an annular indenta-
tion and an operculum (Fig. 15). With the aid of silver
methenamine, the layering of the ascal wall is revealed.
Throughout the operculum the outer layer has a thickness of
85-95 nm (Fig. 16). In contrast, the inner layer is less
uniform in thickness, expanding from approximately 110-
115 nm in the distal area of the operculum to 260-280 nm at
its periphery. The operculum at this stage has a diameter
of 4.4-4.5 pm.
When the spores approach complete development, the
width of the ascal tip increases from 5.9-6.3 pm (Fig. 15)
to 8.0-8.4 pm (Figs. 17, 18). At the same time, the diameter
of the operculum has widened to 5.7-5.9 pm. The opercular
wall layers, however, are reduced in thickness, implying a
stretching action. The inner layer has decreased to 85-
95 nm distally and 130-140 nm peripherally and the outer
layer to 65-70 nm and 85-95 nm, respectively (Figs. 17, 18).
The annular indentation has a width of 545-570 nm,
being smaller than in Peziza succosa (Fig. 19). The outer
layer tapers slightly to 60-75 nm while the inner layer nar-
rows to 35-40 nm. At the distal region of the suboperculum,
(Figs. 18, 19), the breadth of the outer layer suddenly in-
creases to 110-120 nm and maintains this approximate
thickness throughout the rest of the suboperculum. The
inner layer, which is 40-50 nm thick for a length of 680-
720 nm, expands to 100-120 nm at the lower end of the sub-
operculum. The suboperculum is roughly 3.5-4.0 pm long.
The Apical Apparatus of Saccobolus depauperatus
The mature ascus is broadly clavate, being 55-80 pm
long and 12-15 pm wide. When placed in IKI, the entire
ascus stains blue. The apical apparatus is not observed
until late ascosporogenesis whereupon a thick ring is de-
tected at the tip (Fig. 20). At a slightly later stage in
development the tip appears more inflated and less con-
spicuously thick walled (Fig. 21).
Ultrathin sections of asci during early spore wall
formation show that additional development of the ascal wall
is initially restricted to the apex or to that area which
will become the operculum (Fig. 22). Subsequently, the
inner layer making up the rest of the ascus wall is laid
down. A distinct mucilaginous coat, 65-90 nm thick, covers
At maturity the tip, which has become broadly truncate,
possesses a distinct annular indentation (Figs. 23, 25).
The annular indentation consists of a broader inner layer,
65-80 nm, and a thinner outer layer, 55-60 nm (Fig. 24). As
in Peziza succosa, the mucilaginous coat is more prominent
at this region of the ascal tip. A zone of dehiscence is
present at the basal end of the annular indentation. The
width of the indented ring is 545-600 nm, being almost
identical to that of Ascobolus crenulatus.
As before, posttreatement with silver methenamine ac-
centuates the stratified character of the ascal wall (Figs.
25, 26). Within the operculum, the outer layer remains
60-70 nm thick. The inner layer, however, ranges from
65-75 nm apically to 180-190 nm subapically. The diameter
of the operculum measures 7.1-7.4 pm. In the apical portion
of the suboperculum, the outer layer is less reactive with
the silver methenamine stain (Fig. 25). This differen-
tially stained region, 1.2-1.3 pm long, will be referred to
as the subopercular flange (Figs. 26, 28). The inner layer
has a breadth of 65-85 nm throughout the suboperculum, which
is 3.3-3.9 pm long. The outer layer is 155-165 nm thick in
the subopercular flange and decreases slightly to 140-145 nm
before it once more increases to 220-230 nm in the lower end
of the suboperculum. At incipient spore release, the
operculum begins to pull away from the suboperculum along
the annular indentation (Fig. 27). After the eight asco-
spores have been discharged as a solid unit, the flange ex-
tends outward and shrinks to 100-150 nm in length (Fig. 28).
The Apical Apparatus of Thecotheus pelletieri
Thecotheus pelletieri is the only representative of the
5 species examined that forms 32-spored asci. The mature
ascus is broadly cylindric, having a length of 300-330 pm
and a width of 50-55 pm (Fig. 29). The ascal wall becomes
diffusely blue when treated with Melzer's reagent. The
apical apparatus consists of a large, conical operculum that
is subtended by a wide ring (Fig. 30). In Congo red, the
operculum appears to be distinctly thinner-walled than the
rest of the ascus. Furthermore, the wall below the apical
ring appears to be bilayered with the outer layer being
stained by the Congo red (Fig. 30). At dehiscence of the
ascus, the lid is often removed entirely (Fig. 31).
With the aid of the electron microscope, the form of
the ascal tip is clearly shown (Fig. 32). The wide ring is
seen to be an annular indentation, similar to that in the
three previous species, but here of considerable size, meas-
uring 980-1200 nm across. The operculum has a diameter of
21-23 pm and a thickness that varies from 610-640 nm in the
apical region to 720-750 nm subapically.
When stained with silver methenamine (Figs. 33, 34), the
less reactive outer layer is seen to constitute much of the
opercular wall, being 350-440 nm thick. In the area of the
suboperculum, the outer layer increases basally from 510-
590 nm to 1,150-1,250 nm. Similarly, the subopercular inner
layer increases to 600-650 nm toward the base. The length
of the suboperculum is 6.5-7.5 pm long. A thin mucilaginous
coat, being 80-100 nm thick, covers the entire ascal wall
(Fig. 34). Both layers of the ascus are notably thinner in
the annular indentation with the inner layer being reduced
in thickness to 200-220 nm and the outer layer to 165-195 nm
(Figs. 34, 35). Furthermore, a zone of dehiscence is ob-
served at the lower end of the indented inner layer.
The Apical Apparatus of Iodophanus granulipolaris
The diploid ascus is broadly cylindrical, being 100-
140 Pm long and 15-20 pm in diameter. When placed in
Melzer's Reagent, the staining reaction of the wall is par-
tially masked by the coloration of the cytoplasm within
(Fig. 36). The area immediately below the ascal tip turns
to a light blue. Most of the ascus appears light green ex-
cept centrally where it also stains blue. One-micron sec-
tions stained with toluidine blue (Fig. 37) shows the pres-
ence of densely packed cytoplasm in the apical region of
the ascus. The apical region is bounded by a large,
amorphous cylinder of deeply stained material. This
cylinder extends to the center of the ascus where the large
diploid nucleus is observed surrounded by cytoplasm. The
remainder of the ascus is filled with another deeply stain-
ing mass of material.
The mature ascus, which retains a broadly cylindric
form, expands to 200-250 pm in length and 30-35 pm in width.
The wall stains a light blue when treated with the IKI
solution. This is most clearly observed in empty asci. The
apices of mature asci lack conspicuous features (Fig. 43).
During ascospore liberation the operculum remains partially
attached to one side of the ascus (Fig. 44).
Ultrastructurally, the tips of diploid asci appear to
be cytologically active, being packed with ribosomes,
endoplasmic reticula and mitochondria (Fig. 38). The large,
amorphous cylinder consists of a uniform mass of glycogen
(Figs. 38, 40). Plasmalemmasomes are associated frequently
with the apical wall and occasionally with the lateral wall
(Figs. 39, 40). The apical wall, being 155-165 nm thick,
is slightly thinner than the lateral wall, which is 200-
By early ascosporogenesis, the ascal tip becomes highly
vacuolated as the central cylinder of glycogen appears to be
degraded (Fig. 41). Silver methenamine stains only the
distal region of the tip where the wall is 160-175 nm thick
(Fig. 42). Basally, the wall thickens to 240-265 nm.
As the ascospores continue to develop, the apical wall
changes little in form and thickness (Figs. 45, 46). The
lateral walls, however, broaden to 420-460 nm. The apical
region is filled with numerous, small vesicles.
At maturity, the ascal tip reaches a thickness of 440-
480 nm (Fig. 48). An apical apparatus is not apparent until
the sections have been treated with silver methenamine
(Fig. 47). The operculum consists of a thinner, richly
stained, outer layer, 170-200 nm thick, and a broader, less
reactive, inner layer, 270-290 nm thick. The zone of de-
hiscence is demarcated by the differential staining of the
inner layer in a small, swollen ring. The inner and outer
layers immediately below the dehiscent ring are apparently
stretched, measuring 20-40 nm and 400-420 nm in thickness,
respectively. At the lower end of the suboperculum, which
is 2.5-3.0 pm long, the breadth of the inner layer has in-
creased to 70-80 nm while the outer layer retains the same
dimensions. During spore release, the operculum usually
remains attached to the suboperculum, not breaking entirely
from the outer layer (Fig. 49). The inner layer in the
distal region of the suboperculum has thickened to 70-80 nm
Examination in the present study of the apical appa-
ratus of five species of the operculate Discomycetes demon-
strated for the most part homogeneity in form and uniformity
in development. Light microscopic observations showed that
in each representative, except Iodophanus granulipolaris,
the mature ascal tips displayed certain distinctive fea-
tures. These findings are in close agreement with those of
van Brummelen (1967) who stated that in the genus Ascobolus
the opercula have characteristic shapes depending on the
taxon in question and Kimbrough (1969), who reported similar
observations in Thecotheus.
Within the operculate Discomycetes, Kimbrough (1966a),
Kimbrough and Korf (1967) and van Brummelen (1967) reported
a bilayered condition for the unitunicate ascus. With the
light microscope, this aspect was only sufficiently deter-
mined in Thecotheus pelletieri. In each case, including this
study, the representatives studied formed large, thick-walled
asci, which permitted this observation. Electron micro-
scopic examinations of the ascal wall for each of the five
species currently studied demonstrated a double layered wall.
The ontogenies of the apical apparatuses found in
Peziza succosa, Ascobolus crenulatus, Saccobolus depauperatus
and I. granulipolaris followed in general a three-stage se-
quence. First, wall synthesis in diploid asci occurred
throughout all regions of the cell. Synthesis appeared to
be most concentrated at or near the apex where lomasomes
(plasmalemmasomes as defined by Heath and Greenwood, 1970)
were noted most frequently. Second, during early asco-
sporogenesis, the development of the ascal walls in each
species was restricted mainly to lateral or subapical re-
gions. This was conspicuously apparent in P. succosa and
I.granulipolaris where the lateral walls became two or three
times thicker than the apical walls. Third, during ascospore
wall formation, the additional layering of the ascal wall
was differentially deposited, starting at the apex and pro-
gressing downward. None of the representatives displayed
any evidence supportive of Chadefaud's (1942) conclusion
that complete differentiation of the apical apparatus oc-
curred by the eight-nucleated stage. Instead, the ontogeny
of the apical apparatus of each species showed the most
dramatic change during late ascosporogenesis. Observations
made in the present study coincided closely with those of
van Brummelen (1967) and Wells (1972) regarding the time of
opercular development in Ascobolus species.
In the young asci of A. crenulatus and maturing asci of
I. granulipolaris, the localized accumulation of vesicles
below the tip was reminiscent of the vesicle system described
for the developing diploid ascus of Xylaria longipes
Nitschke (Beckett and Crawford, 1973; Beckett, Heath and
McLaughlin, 1974) and the growing hyphal tips of various
taxa throughout the fungal world (Grove and Bracker, 1970).
Unlike in X. longipes, an apical body (Spitzenkorper) and an
area of localized thickening were not observed during these
stages of development in the species studied by me. Wells
(1972) observed a similar state of vacuolation in Ascobolus
stercorarius prior to meiosis and noted that it was part of
an orderly sequence. He suggested that the vacuoles may
function in concentrating the ascoplasm in the regions of
growth and cytological activity.
The gross morphology of the apical apparatuses was
basically similar in all members. Except in Thecotheus
pelletieri, the inner layer was seen to be distinctly broad-
est in the opercular region of the ascus. In every species,
the outer layer increased in thickness towards the base of
the ascus. These findings compared favorably with those re-
ported for the six representatives of the Sarcoscyphineae
(Samuelson, 1975). However, they sharply contrasted with
Schrantz' (1970) description of Peziza plebeia. Schrantz
defined the outer ascal layer as consisting of a pecticc"
external cover and an "amyloid" inner hood or "manchon"
(Fig. 51b). His light and electron microscopic descriptions
of the outer layer are nearly identical in size and shape to
the mucilaginous coat of Peziza succosa (Fig.51d). His
photomicrograph of the ascal tip of P. plebeia showed a
comparable stage in development to that of the tip seen in
Fig. 4 for P. succosa. He apparently based his evidence on
A substructural characteristic found in the apical ap-
paratus of all species, except I. granulipolaris, was the
presence of an annular indentation. Previously, Kimbrough
(1966b, 1969) reported the occurrence of a "well-marked
indention at the operculum" in mature asci of Thecotheus
pelletrieri and of less obvious lines of dehiscence in the
eight-spored species of Thecotheus. Van Brummelen (1967)
did not specifically describe this region in any species of
Ascobolous and Saccobolus. Instead, he stated that the
annulus, an internal ring-shaped thickening of the ascal
wall, was the place where circumscissle rendering of the lid
had occurred. Chadefaud (1942) had depicted earlier (Fig. 51a)
an open area between the operculum and the subapical
"bourrelet" or pad. Although he did not name the area or
refer to it in particular, he mentioned that the operculum
was unusually well defined. In a recent description of the
development of the ascal wall in Ascobolus stercorarius,
Wells (1972) noted that the differentiation of the operculum
began "with the appearance of an annular indentation in the
inner surface of the wall at the margin of the operculum."
His accounts of the development of the lateral and apical
walls and his measurements of their breadth and width were
very similar and in some cases identical to my observations
of A. crenulatus (Fig. 51c).
The discovery of an annular indentation in Peziza
succosa was not entirely unexpected. Kimbrough had noted
frequently the presence of a distinct hyaline ring in the
ascal tips of P. vesiculosa when stained with Congo red
(personal communication). My observations of P. succosa
were similar though the staining reaction appeared less pro-
nounced. We had conjectured that the ring may have been the
result of either a chemical differentiation in the wall or a
physical thinning of the wall. The latter proved to be true.
Of the representatives that had annular indentations,
the species which turned blue in Melzer's reagent, i.e.,
P. succosa, S. depauperatus and T. pelletieri, possessed
mucilaginous coats. It is believed that the iodine speci-
fically reacted with the mucilaginous coats. Asci of P.
succosa provided the most convincing evidence. The localiza-
tion of the blueing reaction in young and mature asci
coincided perfectly with the localization of their respec-
tive mucilaginous coats. Moreover, the coat could be
removed after delicate manipulation. In T. pelletieri and
and S. depauperatus, the total blueing of the ascal walls
corresponded entirely with the thin mucilaginous layer that
covered the ascus. Although asci of A. crenulatus did not
exhibit mucilaginous coats, neither did they give an
iodine-positive reaction in Melzer's reagent.
The apical apparatus of P. succosa seemed the most
isolated morphologically of all the species studied that
developed annular indentations. Major differences consisted
of the presence of a thick, localized mucilaginous coat, a
subtending cytoplasmic ring and an operculum which thickened
distally rather than thinned (Fig.51d). In addition, the
inner layer of the suboperculum was seen to taper notably
in thickness away from the tip. By comparison, the apical
apparatuses of A. crenulatus and S. depauperatus shared a
number of identical features, including the width and breadth
of their annular indentations, opercular and subopercular
flanges. The principal difference between the two members
was observed in the layering of the lower suboperculum. The
expansion of the inner layer toward the base of the ascus in
A. crenulatus in Fig. 51c (vs. S. depauperatus) was similar
to the suboperculum of T. pelletieri. The apical apparatus
of T. pelletieri was generally similar in form to that of
A. crenulatus. For the most part, the ascal wall dimensions
of T. pelletieri were approximately three times that of
The apical apparatus of Iodophanus granulipolaris
diverged significantly in form from the other species and
also in development and cytochemistry. The presence of a
ring of dehiscence in place of an annular indentation was
the chief morphological difference. Furthermore, the dif-
ferential staining of the inner layer along the zone of
dehiscence by silver methenamine was dissimilar to the rest
of the species. The suboperculum was considerably shorter
and less conspicuous that those of the annular indented
species. During the ontogeny of the apical apparatus, the
expansion of the apical wall was later and more restricted
in locality than observed for the other species. Since the
presence of an exogenous coat was not detected at any stage
of ascal development, the iodine-positive reaction appeared
to have been the result of iodine directly staining the wall.
The iodine-positive apical apparatus has been demon-
strated to contain distinct features that can be useful as
a systematic tool. The marked similarities in form and
development in the dehiscent structures of A. crenulatus and
S. depauperatus further substantiated the close association
of the two genera. The morphology of the apical apparatus of
T. pelletieri and its resemblance to Ascobolus supported
Korf's (1973) taxonomic treatment of this genus, placing it
in the Ascobolaceae. Although the apical apparatus of P.
succosa differed significantly from the other annular in-
dented taxa, the number of common properties shared by the
four species indicated a closer degree of relatedness than
was proposed previously by Chadefaud (1942) and Eckblad
(1968). Except in gross form and ontogeny, the apical ap-
paratus of I. granulipolaris had very few properties in
common with those of the annular indented species. The
taxonomic positioning of I. granulipolaris in either the
Pezizaceae or the Ascobolaceae received little support from
the present study. Since blueing in iodine may be the re-
sult of more than one site of activity, the iodine-positive
character should be interpreted cautiously.
Tip of diploid ascus showing iodine-positive
reaction. Stained with Melzer's reagent.
Diffuse iodine-positive reaction at mature
ascal tip. Spore (SP). X1,000.
Thin mucilaginous coat (MC) covers immediate
region of the apex of diploid ascus. X10,000.
Ascal tip with thick mucilaginous coat (MC)
at early spore delimitation. Spore (SP).
Presence of annular indentation (AI) by late
Ascal tip at late ascosporogenesis. Muci-
laginous coat (MC). Stained with silver
Mature apical apparatus. Distinct annular
indentation (AI) delimits operculum (0).
Spore wall (SW). X7,600.
Opercular (0) and subopercular (SO) regions
of mature apical apparatus. Stained with
silver methenamine. X7,000.
Close-up of Fig. 8 showing cytoplasmic ring
(CR) which subtends annular indentation.
Inner layer (IL). Outer layer (OL). Muci-
laginous coat (MC). Stained with silver
Region of dehiscent zone which is demarcated
by annular indentation (AI). X24,000.
Young ascal tip with developing ascospores
shows distinct apical ring (AR) at this
time. Stained with Congo red. X1,250.
Mature tip is thinner-walled at region of
the operculum (0). Stained with Congo red.
Vesiculated apex of diploid ascus with sub-
tending ring of glycogen (G). X9,800.
Ascal tip during early spore (SP) develop-
ment. Stained with silver methenamine.
Early formation of apical apparatus showing
differential increase in thickness of
opercular (0) wall. Annular indentation
Inner layer (IL) and outer layer (OL) of
developing apical apparatus. Spore (SP).
Stained with silver methenamine. X10,500.
Mature apical apparatus with umbonately
shaped operculum (0). X9,500.
Mature apical apparatus shows layering of
opercular and subopercular (SO) regions of
the ascus. Annular indentation (AI).
Stained with silver methenamine. X8,500.
Region of annular indentation in mature
apical apparatus. Inner layer (IL). Outer
layer (OL). X48,000.
Developing apical apparatus with pronounced
Apical apparatus at later developmental
stage. Stained with Congo red. X2,000.
Thickening of apical wall (AW) during early
spore development. Mucilaginous coat (MC).
Mature apical apparatus with distinct
annular indentation (AI). X8,200.
Region of annular indentation with zone of
dehiscence (ZD). Mucilaginous coat (MC).
Mature apical apparatus stained with silver
methenamine. Operculum (0). Suboperculum
Upper portion of suboperculum showing sub-
opercular flange (SF). Inner layer (IL).
Outer layer (OL). Stained with silver
Incipient spore release. Episporal sac (ES).
Spore (SP). X7,600.
Dehisced ascus with outwardly extended sub-
opercular flange (SF). Ascostome (A).
Stained with silver methenamine. X6,200.
Mature ascus stained with Congo red. X160.
Ascal tip with wide apical ring (AR)
delimiting a conical operculum. X1,250.
Ascus after spore release. Ascostome (A).
Opercular region of mature ascus. Annular
indentation (AI). X5,400.
Mature apical apparatus. Operculum (0).
Suboperculum (SO). Stained with silver
Region of annular indentation showing dif-
ferentially stained zone of dehisence (ZD).
Operculum (0). Inner layer (IL). Outer
layer (OL). Mucilaginous coat (MC).
Stained with silver methenamine. X20,000.
Zone of dehiscence (ZD) at base of annular
Uninucleated ascus stained with Melzer's
Apex of uninucleated ascus subtended by
broad amorphous cylinder. One-micron
section stained with toluidine blue.
Apical region of uninucleated ascus.
Glycogen (G). X6,100.
Small lomasomes (L) at ascal tip.
plasmic reticulum (ER). X20,000.
Mitochondria (M) adjacent to large mass of
glycogen (G). X15,000.
Large vacuole (V) at tip during early
Distal portion of apical wall (AW) stained
with silver methenamine. X4,700.
Mature tip stained with Congo red. X1,250.
Operculum (0) partially connected to ascus
at spore liberation. Stained with Melzer's
Thickening of lateral wall (LW) during spore
maturation. Apical wall (AW). X3,100.
Numerous small vesicles (V) at ascal tip.
Mature apical apparatus showing distinct
zone of dehiscence (ZD), inner layer (IL)
and outer layer (OL). Operculum (0).
Stained with silver methenamine. X13,500.
Portion of mature apical wall. X12,500.
Operculum (0) attached at spore release.
Outer layer (OL). Stained with silver
Dehisced ascus with thickened inner layer
(IL). Suboperculum (SO). Stained with
silver methenamine. X8,700.
I 1, I
Figure 51. Drawings of apical apparatuses found in
a. Apical apparatus of Ascobolus furfuraceus.
Operculum (0). Coussinet (C). Redrawn from
b. Ascal tip of Peziza plebeia illustrating layers
of the wall. Exoascus (EX). Endoascus (EN).
Bourrelet (B). Redrawn from Schrantz (1970).
c. Illustration made from electron microscopic
observations of the wall layering in mature
ascal tips of Ascobolus crenulatus. Outer
layer (OL). Inner layer (IL).
d. Illustration made from electron microscopic
observations of the wall layers and mucilaginous
coat (M) in mature ascal tips of Peziza succosa.
Outer Layer (OL). Inner layer (IL).
c 0 o
MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY OF THE APICAL
APPARATUSES OF REPRESENTATIVES IN THE OTIDEA-ALEURIA COMPLEX
In the area of fungal systematics, searches have long
been made for valid, new characters which would aid in
classification. With the incorporation of cytological in-
formation developed by Berthet (1964) and pigmentation
studies made by Arpin (1968) there has surfaced a general
taxonomic repositioning of the genera representing the
largest group of operculate Discomycetes (Table 1). These
members have been treated successively during the last
decade in the Humariaceae and Pezizaceae, tribe Otideae
(Dennis, 1968), Humariaceae (Rifai, 1968), Pyronemaceae and
Otideaceae (Eckblad, 1968), Aleuriaceae and Otidiaceae
(Kimbrough, 1970), and Pyronemataceae (Korf, 1973). Cir-
cumscription of groupings of these taxa, which will be
referred to as the Otidea-Aleuria complex, traditionally
has been difficult due to the occurrence of continuous
patterns of variation in several characters.
Within the Ascomycetes increasing emphasis has been
placed on ascal structure and the mechanism of ascospore
release (see General Introduction). Chadefaud (1942, 1973),
the first worker to examine the morphology of ascal tips
Classifications of the Otidea-Aleuria Complex
Table 1 (Cont'd.)
throughout the Euascomycetes, concluded that ascal apices,
in general, shared a number of common features and that the
absence or presence of certain features had phylogenetic
significance. Recent ultrastructural investigations of the
operculate ascus (van Brummelen, 1974, 1975; Samuelson,
1975; Schrantz, 1970; Wells, 1972) and that reported in
Chapter I have demonstrated that the ascal tip has a broad
variability in form. Furthermore, comparative analysis of
the morphology and ontogeny of apical apparatuses of dif-
ferent taxa may have considerable phylogenetic value as
discussed in Chapter I, thus giving support to Chadefaud's
Within the suborder Pezizineae, ascal structure has
been little studied outside of the Thelebolaceae (Kimbrough,
1966a, 1966b, 1972; Kimbrough and Korf, 1967, van Brummelen,
1974) and Ascobolaceae (van Brummelen, 1967; Wells, 1972;
Chapter I). Chadefaud's (1942) work represented the major
comprehensive study of the apical apparatus for the remainder
of the operculate Discomycetes. Of the 11 selected taxa
described, 8 were members that fell into the Otidea-Aleuria
complex. They included Aleuria aurantia (Pers. ex Hook.)
Fuckel, Humaria hemisphaerica (Wigg. ex Fr.) Fuckel, (as
Lachnea hemisphaerica), Coprobia granulata (Bull.) Boud.,
Pulvinula constellation (Berk and Br.) Boud., Scutellinia
hirta (Schwm. ex Fr.) O. Ktze., Octospora leucoloma Hedw.
ex. S. F. Gray (as Humaria leucoloma), Humaria wrightii
(Berk. and Cooke) Boud. and Sepultaria arenosa (Fuckel)
Boud. Each of the eight species was observed to have a full
complement of apical structures associated with the oper-
culate apical apparatus as shown inFig. 110A. Four morphologi-
cal variations of the apical apparatuses were demonstrated
among the representatives. They ranged from species with
exaggerated or exceptionally well-developed apical appa-
ratuses as in C. granulata to those that had rudimentary
forms as in 0. leucoloma (Fig. HOD). Chadefaud proposed that
the differences in their morphology were the result of
regressive evolution and that in the case of the distantly
related genus Ascobolus, all species had attained a similarly
reduced state by this means.-
Eckblad (1968) confirmed the presence of a funnel in
a number of unstated species and agreed with Chadefaud's
description of the operculate apical apparatus on all main
points. He stated, however, that the funnel was cytoplasmic
in origin and did not attach to the operculum. He also was
not able to detect an apical pad or cushion on the underside
of the operculum. Recent investigations of operculate
apical apparatuses (Wells, 1972; Samuelson, 1975; van
Brummelen, 1975; Chapter I) have demonstrated the presence
of a thickened, opercular inner layer which corresponds with
the apical pad. The apical globule, punctuation, funnel and
tract were not described in any of the "iodine-positive" and
suboperculate apical apparatuses.
LeGal (1953) described the ascal apices of Phaedro-
pezia epispartia (Berk and Br.) LeGal and Trichophaea
erinaceus (Schwein.) LeGal, both placed inside the Otidea-
Aleuria complex, to have apical apparatuses typical but less
apparent than that of the suboperculates. Eckblad (1968)
suggested that the occurrence of an incomplete ring in the
suboperculate apical apparatus of T. erinaceus and P.
epispartia as well as in those of the Sarcoscyphaceae were
artifacts of fixation. He believed that the apical chamber,
which contained the ring, represented only a swelling of the
inner layer of the operculum. Samuelson's (1975) and van
Brummelen's (1975) examinations of the suboperculate apical
apparatuses corroborated Eckblad's observations. Neverthe-
less, LeGal's report of suboperculate apical apparatuses in
T. erinaceus and P. epispartia indicated the possibility of
pronounced wall layering of the ascal tips, a character that
may prove to be taxonomically useful.
The only ultrastructural examination of the ascal wall
found in the Otidea-Aleuria complex was made by Schrantz
(1970) on Tarzetta cupularis (L. ex Fr.) Lamb. He noted
that the exoascus or outer layer consisted of a thin,
loosely woven sheath which thickened near the ascal tip.
By comparison, the endoascus or inner layer was thick and
finely granular and narrowed towards the tip. He likened
the form of the ascal wall to that of Peziza plebeia (LeGal)
Nannf. It was pointed out in Chapter I that the exoascus
described for P. plebeia was apparently a thick mucilaginous
coat. The ascal wall layering in T. cupularis may have been
This study has incorporated morphological, develop-
mental and cytochemical examinations of the Otidea-Aleuria
apical apparatus in order to (1) ascertain the validity of
the features depicted by Chadefaud, (2) compare the mor-
phology and developmental sequences of apical apparatuses
between selected representatives, (3) determine whether
these features would be useful in the taxonomic positioning
of members within this largest of all operculate groups.
Ten members including Otidea leporina (Fr.) Fuckel,
Sphaerosporella brunnea (Alb. and Schw. ex Fr.) Svrcek and
Kub., Jafnea fusicarpa (Gerard) Korf, Humaria hemisphaerica,
Aleuria aurantia, Anthracobia melaloma (Alb. and Schw. ex
Fr.) Boud., Scutellinia scutellata (L. ex Fr.) Lamb.,
Ascozonus woolhopensis (Berk. and Br. apud Renny) E. C.
Hansen, Sowerbyella imperialis (Peck) Korf, and Geopyxis
majalis (Fr.) Sacc. were used.
Materials and Methods
Collection and Age Determination of Material
Young and mature apothecia of Anthracobia melaloma were
collected from charred wood near Gainesville, Florida. Dif-
ferent aged material of Humaria hemispherica was found grow-
ing in moss at the University of Florida's Horticultural
Farm outside Gainesville. Young and mature apothecia of
Aleuria aurantia and Jafnea fusicarpa and mature apothecia
of Otidea leporina were found on the forest floor at the
Devil's Millhopper in Gainesville. Young and mature
apothecia of Scutellinia scutellata were gathered from a
greenhouse bench of the Department of Plant Pathology at the
University of Florida. Fully developed apothecia of
Sphaerosporella brunnea were found on carbonized humus at
the Collier-Seminole State Park in Florida. Minute apothecia
of Ascozonus woolhopensis were found on rodent dung col-
lected in Macon County, North Carolina. Fresh material was
brought to the laboratory where free-hand sections were made
for light microscopic inspection to determine the stage of
ascal development. Young and mature apothecia were studied
separately when possible.
Dried specimens of Geopyxis majalis and Sowerbyella
imperialis were obtained from the Mycological Herbarium at
the University of Florida and Dr. R. P. Korf of Cornell
University, respectively. Portions of apothecia were re-
vived at room temperature in distilled water for 6 to 12
hours in a moist chamber for light and electron microscopic
Procedures for Light Microscopic Examinations
Fresh and revived apothecia were cut into blocks, sec-
tioned and mounted on slides as described in Chapter I.
Congo red was predominantly used to stain the ascal walls
(Samuelson, 1975). Aniline blue and lactophenol cotton blue
were used to observe cytoplasmic detail (see Chapter I).
Plastic embedded material was sectioned, mounted and stained
in the manner described in Chapter I.
Procedures for Electron Microscopic Examinations
Entire apothecia of A. melaloma, A. woolhopensis and
S. scuttellata and five millimeter squares of apothecia of
A. aurantia, G. majalis, H. hemisphaerica, J. fusicarpa,
S. imperialis and S. Brunnea were fixed in buffered (0.2 M
sodium cacodylate pH 7.2) 2.0% glutaraldehyde and 2.0%
paraformaldehyde solution for two hours at room tempera-
ture. Five millimeter squares of 0. leporina and S. brunnea
were fixed in 1.0% permanganate solution for one hour at
room temperature. All materials were postfixed in osmium
tetroxide, dehydrated, embedded, section and poststained as
described in Chapter I.
Descriptions of the apical apparatuses for each species
are restricted to the three regions of the mature ascal tip;
the operculum, the zone of dehiscence and the suboperculum.
As in the previous chapter each representative has been
The Apical Apparatus of Otidea leporina
The mature ascus is narrowly cylindric, reaching a length
of 180-200 Pm and a diameter of 9-12 pm. The subapical
region is notably heliotropic (Fig. 1). When stained in
Congo red, the ascal wall appears thick for most of the
length of the ascus. Near the tip of the ascus the wall
becomes thinner below the region of the operculum (Fig. 2).
By comparison, the opercular wall appears to be inflated.
The diameter of the ascus becomes narrower toward the tip.
After the spores have been discharged, the ascus shrinks to
a length of 150-170 pm while its diameter expands to 10-15 pm
(Fig. 4). The operculum, which usually remains attached to
one side (Fig. 5), is less conspicuous than when observed
in undischarged asci.
Ultrastructurally, the apical wall protrudes above the
subtending lateral wall, delimiting the operculum (Fig. 3).
The operculum has a diameter of 2.2-2.4 pm and a uniform
thickness of 170-180 nm. The suboperculum is 3.7-4.0 pm
long and decreases in thickness from 510-530 nm at its lower
extremity to 165-180 nm next to the operculum. The outer
layer of the operculum and distal region of the suboperculum
are loosely fibrillar and electron-dense, having a breadth
of 110-120 nm (Figs. 3, 6). Toward the base, the outer
layer thickens to 390-410 nm, having an additional internal
portion or stratum. The inner layer and the internal
stratum of the outer layer are both electron-transparent and
granular, being separated by a faint, opaque band (Figs. 3,
9). After treating the thin sections with silver methenamine,
the inner layer is stained most strongly (Fig. 7). The
internal stratum of the outer layer is stained but not as
intensely, and the electron-dense, fibrillar portion of the
outer layer remains unstained. As the time of spore release
approaches, the apical wall becomes markedly stretched
(Fig. 8). The opercular diameter increases 0.3-0.5 pm while
its thickness decreases by 40-60 nm. The upper extremity of
the suboperculum is similarly stretched. At ascal dehis-
cence, the upper extremity of the suboperculum becomes out-
wardly extended (Fig. 9). The length-of the suboperculum
shrinks to 3.0-3.3 pm.
The Apical Apparatus of Jafnea fusicarpa
During early spore formation, the ascal wall is thinner
at the apex and thicker laterally (Fig. 10). Further in
development, the apical and subapical walls are conspicu-
ously thick (Fig. 11). At maturity, the ascus is long and
narrow, 290-310 pm x 18-22 pm. The ascal tip, which is
slightly heliotropic, has formed two layers (Fig. 13). The
first spore often becomes lodged against the opercular
region of the ascus. During spore release, the operculum
is thrown to one side and the ascospores are ejected one
after another in rapid succession. The operculum typically
remains attached to the ascus and frequently returns to its
original prone position. As the spores are released they
become strongly compressed between the sides of the ascal
wall (Fig. 14).
Electron microscopic examination of a four-nucleated ascus
reveals for the most part the presence of a thick, lateral
wall, 820-840 nm (Fig. 12), which decreases in thickness to
500-540 nm at the tip. Numerous small vesicles are ob-
served in the apical region of the ascus. During early
spore wall formation, the ascal tip becomes faintly helio-
tropic (Fig. 15). The thickness of the lateral wall has
increased to 1000-1050 nm while the tip retains a thickness
of 480-520 nm. The apex is wider, having expanded to a
diameter of 9.2-9.4 pm. Several large vacuoles are present
near the tip and extend to the base of the ascus. At the
end of spore development the apical apparatus continues to
be developed (Fig. 16). The ascal tip has increased by
1.3-1.8 pm in diameter. An inner layer was deposited mainly
in the vicinity of what will become the operculum. Lateral
walls appear to be stretched, having a thickness of 780-
820 nm. Treatment with silver methenamine strongly accen-
tuates the bilayered nature of the mature ascal wall (Fig.
17). The thickness of the inner layer narrows from 400-
410 nm at the operculum to 50-65 nm at the lower extremity
of the suboperculum where a small annular bulge or ring has
formed (Fig. 17, arrows). An opercular boundary is not ob-
served at any time prior to spore release. At dehiscence,
the operculum remains partially fastened to the suboperculum
(Fig. 18). At this stage, the suboperculum, which is 5.5-
6.3 pm long, has thickened to 950-980 nm at its lower ex-
tremity where the subopercular ring has become greatly
The Apical Apparatus of Humaria hemisphaerica
Asci containing immature ascospores are 160-240 pm long
and 12-16 pm wide. They appear thick-walled except at the
apex (Figs. 19, 20). In mature asci, which are long and
cylindric (280-340 x 16-20 pm) the tips have become notably
thicker (Fig. 28). An inner layer is detected in the oper-
cular and subopercular region of the ascus after staining of
one-micron plastic sections with toluidine blue. Sub-
apically, a faint bulge is detectable at the level of the
first spore (Fig. 28). In asci that are about to rupture
(Fig. 27), a hyaline ring delimits the operculum. The ascus
appears stretched and thinner-walled at this stage.
The fine structure of ascal tips at early spore wall
formation (Fig. 21) shows the ascal wall to be relatively
thin (190-210 nm) in the immediate region of the tip. At
the level of the first spore the wall increases to 520-
550 nm in thickness. During ontogeny, the asci and para-
physes are embedded in a mucilaginous matrix (Figs. 21-26,
29-33). As the ascospores continue to mature (Fig. 22), the
wall dimensions stay approximately the same. Within 3.1-
3.5 pm of the apex an annular protuberance is faintly de-
tected on the inner surface of the ascal wall (Fig. 22).
At spore maturity (Figs. 23, 25), this subapical ring be-
comes slightly more defined. The thickness of the apical
and lateral ascal walls has not changed. A large vacuole
exists throughout most of the ascus except at the tip which
remains filled with cytoplasm (Fig. 23). Figure 24 represents
a later time in development. The apical and subapical walls
have increased in thickness to 310-350 nm and 700-750 nm,
respectively, due to the addition of an inner layer (Fig.
26). The subapical ring, which is stained by silver
methenamine, appears to be confluent with the inner layer
(Fig. 26). At a subsequent stage, the inner layer, irregu-
lar in outline, thickens to 140-180 nm in the vicinity of
the operculum (Figs. 29, 30, 31). The subapical or sub-
opercular ring is observed infrequently at this time (Fig.
26). Demarcation of the operculum is not apparent in any
developmental stage of intact asci. At ascal dehiscence,
the dislodged operculum is observed occasionally (Fig. 33),
having a diameter of 5.8-6.2 pm and a breadth of 380-420 nm.
The suboperculum is 4.9-5.2 pm long.
The Apical Apparatus of Sphaerosporella brunnea
The mature ascus is broadly cylindric (150-190 um x
16-20 pm). The rounded to blunt tip appears thinner-walled
than the rest of the ascus (Fig. 35). The diameter of the
ascospores (13-16 pm) is greater than that of the ascal
tip (9-10 pm). Consequently, the first ascospore rests
near the tip but is not closely appressed against the oper-
culum. At spore release the operculum remains attached to
one side of the ascus (Fig. 36). The ascostome appears to
have a wider diameter than that of the operculum, which at
this time is 5-6 pm. The lateral ascal wall collapses and
folds as well.
Ultrastructurally, the tip of a mature ascus consists of
a flattened, thin wall, 420-470 nm thick, which broadens to
680-740 nm towards the ascal base (Figs. 37, 38). When ex-
amining potassium permanganate-fixed material (Fig. 37), the
wall layers are sufficiently distinguished. The outer layer
is composed of a rough, electron-dense external stratum,
which broadens from 45-65 nm subapically to 130-150 nm at
the apex, and an electron-transparent internal stratum
(Figs. 37, 39). In general, the outer layer increases in
thickness from 280-320 nm throughout the opercular region
of the ascus to 500-550 nm at the base of the suboperculum
which is marked by the presence of a subopercular ring
(Figs. 37, 39, 41). The subopercular ring is 450-480 nm
long and consists of an electron-dense band, 45-55 nm thick,
which has been deposited within the inner layer against the
internal stratum of the outer layer. Like the outer layer,
the inner layer thickens from 130-150 nm at the tip to 190-
220 nm at the level of the subopercular ring (Fig. 37).
Asci fixed in a glutaraldehyde-paraformaldehyde solution and
poststained in lead citrate and uranyl acetate do not demon-
strate wall layering (Fig. 38). Slight bulges (arrows) in-
dicate the locality of the subopercular ring. Treating the
thin sections with silver methenamine sharply defined the
two wall layers (Figs. 40, 41). More importantly, the in-
tense staining of the internal stratum of the outer layer
in the operculum distinguishes this region from the sub-
operculum (Fig. 40). The suboperculum is 9.7-10.4 pm long
and tapers in thickness from 680-740 nm at the level of the
subopercular ring to 390-420 nm near the operculum. The
operculum is 4.8-5.2 pm in diameter, which compares closely
with the diameter of opercula seen in dehisced asci under
the light microscope. In dehisced asci (Fig. 42), the lower
extremity of the suboperculum is 380-400 nm thick. The wall
immediately below the subopercular ring, however, has a
thickness of 550-580 nm. It would appear that prior to
spore release the suboperculum becomes greatly stretched and
remains so after spore release.
The Apical Apparatus of Aleuria aurantia
During early spore formation, the ascal wall appears thin
at the tip and progressively becomes thicker toward the
base (Fig. 43). At a later stage in development, the ascus,
which is more inflated at the apex, is seen to be slightly
heliotropic (Fig. 44). By maturity the ascus reaches a
length of 200-240 Pm and a diameter of 12-16 vm. No dis-
tinctive features are observed in the rounded ascal tip
(Fig. 51). At ascal dehiscence, the operculum, 4-5 pm in
diameter, remains partially affixed to the lateral wall
(Fig. 52). The diameter of the ascostome is roughly similar
in size to that of the operculum.
Ultrastructural observations of asci approaching the end
of ascospore development reveal thick lateral walls, 400-
420 nm, which taper at the apices to a thickness of 140-
170 nm (Fig. 45). At 4.0-4.4 Pm below the tip, the wall
protrudes into the ascoplasm, forming a subopercular ring.
A plasmalemmasome of considerable size is associated with
the early development of this ring (Figs. 45, 46). When
stained with silver methenamine at this stage of development,
the ascal wall consists primarily of a thick outer layer
(Fig. 47) and a thin (25-35 nm thick), strongly stained
inner layer. The small plasmalemmasome in Fig. 47 is also
stained intensely. The formation of the subopercular ring
appears to be initiated asymmetrically (Fig. 48). At times
the subopercular ring becomes prominent, being 800-840 nm
thick and 320-420 nm long (Figs. 49, 50). The ascal tip is
filled with long, laminated endoplasmic reticulum (Figs. 45,
46, 49) which is associated with mitochondria, lipid bodies,
ribosomes and small vesicles.
The apical wall of the mature ascus broadens to 280-
300 nm (Fig. 54). Staining with silver methenamine demon-
strates that the increase in thickness is due to the thick-
ening of the inner layer, which is 140-160 nm throughout the
opercular and much of the subopercular regions (Fig. 53).
The outer layer is 100-130 nm thick apically and increases
to 280-300 nm toward the base of the ascus. A subopercular
ring was never detected at this stage in development
(Fig. 53). At incipient ascal dehiscence (Fig. 55), the
inner layer of the operculum is pulled away from the sub-
operculum. The opercular boundary is observed only at this
time and in dehisced asci (Fig. 56). The operculum has a
diameter of 4.0-4.2 pm and a uniform thickness of 260-
290 nm. After spore release (Fig. 57), the suboperculum,
which is 5.8-6.2 pm long, thickens from 210-240 nm at the
distal end to 360-440 nm at the level where the subopercular
ring was previously observed. The inner layer broadens from
110-120 nm to 170-190 nm for a short distance in the lower
extremity of the suboperculum.
The Apical Apparatus of Anthracobia melaloma
Mature asci are cylindric, 160-200 pm x 10-14 pm (Fig.
58). The ascal wall appears to be thicker throughout the
lateral face of the ascus and thinner at the tip (Fig. 59).
Near the ascal tip a slight protuberance of the lateral wall
is observed occasionally (Fig. 59). An apical funnel and
continuous tract, which lead to the first spore, have been
detected in asci that were placed in Congo red or aniline
blue. One-micron sections stained in toluidine blue (Fig.
60, arrows) exhibited a subapical ring or band.
Electron microscopic observations of asci at early spore
wall formation show that a localized band starts to form at
3.2-3.8 pm from the tip (Fig. 61, arrows). Lomasomal ac-
tivity is seen throughout the apical region of the ascal
wall at this stage in spore development. As the spores
continue to mature (Figs. 62, 63) the subopercular band
becomes more conspicuous, consisting of an electron-dense
area, 560-600 nm long and 50-60 nm thick. When treated with
silver methenamine (Fig. 62) only the internal face of the
subopercular band is stained. The wall at the immediate
region of the tip is 140-170 nm thick. Subapically, the
ascal wall reaches a thickness of 250-290 nm by a distance of
200-250 nm from the tip and stays at that thickness for the
remainder of the ascus (Fig. 63). Near the end of asco-
sporogenesis, the inner layer is formed throughout the ascal
wall (Fig. 64). Lomasomes are again observed, being most
abundant in the vicinity of the apex. The subopercular band
develops into a distinct, swollen ring. At spore maturity,
formation of the inner layer is completed (Figs. 65, 66, 67,
68). The area of the operculum is delimited by its greater
thickness, 240-270 nm, from the distal end of the suboper-
culum. Most of the thickness of the operculum is due to the
outer layer being 165-190 nm (Figs. 66, 68). The subopercu-
lar outer layer measures 65-100 nm at its upper extremity
but increases to 210-220 nm by the level of the subopercular
ring. In its entirety, the suboperculum, which is 6.8-
7.2 pm long, broadens from 190-210 nm next to the operculum
to 350-380 nm just below the subopercular ring (Figs. 65,
67). The inner layer of the suboperculum is divided into an
electron-transparent internal stratum and an electron-opaque
external stratum (Fig. 67). The demarcation between the two
strata is sharper than between the outer layer and the inner
layer's external stratum. This study treats the external
stratum as a part of the inner layer rather than refers to
it as a separate layer for the following reasons: (1) The
separation of the internal and external strata of the inner
layer cannot be made developmentally. The formation of
both strata is a continuous action. On the other hand, there
is a period when wall development ceases to occur between
outer layer and the external stratum of the inner layer.
(2) The external and internal strata of the inner layer are
combined below the operculum. Consequently, stratification
of the inner layer is not observed in the operculum. When
staining with silver methenamine the inner layer reacts
positively within the operculum. The internal stratum of
the inner layer also is richly stained throughout the sub-
operculum. The outer boundary of the inner layer's external
stratum is stained, delimiting the inner layer in this
region of the ascal wall.
The Apical Apparatus of Scutellinia scutellata
Young asci at the eight-nucleated to early spore stages
are cylindric to subcylindric, 100-150 pm x 8-12 pm. The
ascal wall, which stains strongly in Congo red, is distinctly
thin at the tip (Fig. 69). Mature asci (Fig. 70) reach a
length of 200-260 pm and a diameter of 14-18 pm. The ascal
wall appears to be uniformly thick in the apical and sub-
apical regions (Fig. 77). No distinctive features are ob-
served in the mature tip. At ascospore discharge, the
operculum is partially attached to the ascus (Fig. 78). The
lateral walls appear flaccid and typically collapse.
Ultrastructurally, the apex of a four-nucleated ascus is
thin-walled, 130-160 nm, and stains strongly in silver
methenamine (Figs. 73, 75). Numerous, small to large vesi-
cles are scattered throughout the upper half of the ascus
(Fig. 73). Large lomasomes are frequently observed in both
the apical and lateral regions of the ascal wall (Fig. 75).
The lateral wall is comparably thick, 240-255 nm, and stains
weakly in silver methenamine in its outer stratum. A thin
mucilaginous coat, 65-75 nm, covers only the tip of the
ascus (Fig. 75). In the eight-nucleated ascus (Fig. 74),
the mucilaginous coat increases to a thickness of 210-240 nm
at the tip and extends to the base of the ascus. An ex-
aggerated lomasomal-like band forms below the tip at this
stage in development. The innermost portion of the wall in
this zone reacts positively in silver methenamine (Fig. 74).
At early spore wall formation, the tip, which remains
thin-walled, 140-180 nm, is filled with small vesicles and
endoplasmic reticulum (Figs. 71, 76). At 5.6-5.9 pm below
the tip a subopercular ring protrudes inwardly from the
lateral wall (Fig. 71). In Fig. 72 the annular protuber-
ance has an irregular, lomasomal appearance. After treat-
ment with silver methenamine, the innermost portion of the
ascal wall is stained as well as its outermost portion
(Fig. 76, arrows). By spore maturity, the internally stained
region of the ascal wall in Fig. 76 has developed into an
intensely stained middle layer (Figs. 79, 80, 81), which de-
creases from 110-130 nm in thickness at the upper extremity
of the ascus to 45-65 nm toward the base. In the vicinity
of the previously existing subapical protuberance, the
middle layer is weakly stained for a length of 520-540 nm
(Fig. 79). The ascal tip has broadened to 370-390 nm. The
increase is basically due to the production of an inner
layer which is 130-150 nm thick throughout the opercular
region and tapers to 30-40 nm at the level of the subopercu-
lar ring (Figs. 79, 80). Sections that are stained with
uranyl acetate and lead citrate exhibit a thick mucilaginous
coat, 540-650 nm (Fig. 82). The inner and middle wall
layers are weakly differentiated. Delimitation between the
mucilaginous coat and the outer layer is extremely difficult.
At the initiation of ascal dehiscence, the middle and outer
layers are pulled apart along a zone of dehiscence between
the operculum and suboperculum (Figs. 81, 83). The operculum
at this stage has a diameter of 6.8-7.0 pm. The lower ex-
tremity of the subopercular wall has thickened to 470-490 nm.
The Apical Apparatus of Ascozonus woolhopensis
Before ascospore delimitation, asci reach a length of
80-160 pm and diameter of 14-18 pm. They are broadly
clavate and possess aconical tip (Fig. 84). Below the tip,
a swollen ring protrudes from the thinner side of the ascal
wall. By maturity, the ascus increases an additional 20-
40 pm in length and 4-6 pm in diameter. The ring and apical
wall appear more pronounced in thickness (Fig. 85). When
placed in Congo red, the subapical ring and outer layer of
the ascal wall are strongly stained (Fig. 85). The immedi-
ate region of the tip remains hyaline. Ascal dehiscence
occurs by two or three transverse fissures (Fig. 94) that
originate at the tip.
Ultrastructurally, young asci during early ascosporo-
genesis (Fig. 86) are conspicuously thick-walled. The
apical wall, being faintly conical, consists of two broad
layers and is bordered by a prominent subapical ring. The
outer layer is thinner narrowing from 550-610 nm at the apex
to 320-280 nm at the ring. The inner layer similarly de-
creases from 900-950 nm at the apex to 350-400 nm just above
the ring. At the ring the two layers are indistinct. The
ring is 1250-1300 nm thick. The lateral wall below the ring
is 1300-1350 nm thick,being mostly comprised of a thickened
inner layer, 1000-1050 nm (Fig. 86). In Fig. 87, a stage
comparable to Fig. 84, the apical region of the ascus is
distinctly conical. The outer layer and most of the sub-
apical ring are intensely stained by silver methenamine
(Figs. 87, 91). The inner layer appears to be stratified
for a distance of 2.4-2.8 pm above the ring where demarca-
tion of the sublayers is weakened toward the tip. At the
ring the inner layer remains unstained, forming a thin,
transparent band, 25-35 nm, which broadens to 750-830 nm
below the ring. The inner layer expands from a thickness of
680-720 nm above the ring to 1790-1850 nm at the apex. A
small pore is formed at the tip during this stage of develop-
ment (Fig. 88). The outer layer and a middle stratum broad-
en from 320-350 nm above the ring to 450-480 nm below the
tip. From there the outer layer tapers to 130-190 nm at the
tip. The mature apices basically exhibit no differences in
form from that shown in Fig. 87. The thicknesses of the lat-
eral and subapical walls are reduced by 360-400 nm and 250-
280 nm, respectively, while the ring stays at 1300-1350 nm.
The attenuation of the lateral and subapical regions indi-
cates that the ascal wall is becoming stretched at this time.
The middle stratum and the inner and outer layers are ob-
served more clearly in peripheral sections (Fig. 89). Visual
distinction of the wall layering at the subapical ring is
extremely difficult to make in median sections. Peripheral
sections of the ring show that the middle stratum comprises
most of that region (Figs. 90, 93), thus reinforcing the
layering distinguished by the silver methenamine stain in
Figs. 87 and 91.
In dehisced asci, an apical disc (Fig. 92), consisting
mostly of the outer layer, is observed. The electron-
transparent inner layer appears to have shrunk or dissolved
at this stage. The disc has a diameter of 2.4-2.6 pm and a
thickness of 550-600 nm.
The Apical Apparatus of Geopyxis majalis
Mature asci are narrow and cylindric, 260-300 pm x
10-14 Pm. The ascal wall appears to be uniformly thick at
the apex and lateral sides (Fig. 95). Asci with immature
spores have thinner apical walls (Fig. 96). At dehiscence
the spores are released through a wide ascostome (Fig. 96)
with the operculum being completely detached.
Electron microscopic examinations of mature asci
demonstrate a bilayered apical wall (Fig. 97). The layers
are strongly accentuated when treated with silver methenamine
(Fig. 99). The inner and outer layers of the opercular wall
are identically 170-190 nm thick. At the periphery of the
operculum the outer layer forms a bulge increasing in thick-
ness to 210-240 nm for a length of 270-300 nm while the
inner layer decreases to 85-95 nm in this region (Fig. 99,
arrows). The inner layer briefly thickens to 125-160 nm
below the bulge before tapering to 25-40 nm at 3.2-4.0 pm
below the apex. Conversely, the outer layer briefly thins
to 125-160 nm below the bulge before it broadens to 320-
340 nm toward the base of the ascus. Younger asci do not
display a distinctly stained inner layer (Fig. 98). The
distal region of the ascus is filled with cytoplasm. The
walls of immature and mature asci are covered with a muci-
laginous coat (Figs. 98, 99). At incipient ascal dehiscence,
the subopercular and opercular inner layers are pulled apart
(Fig. 101), thus delimiting the operculum. During spore
release the upper extremity of the suboperculum is tightly
pressed against the sides of the spore as the spore passes
through the ascostome (Fig. 100).
The Apical Apparatus of Sowerbyella imperialis
The mature ascus is long and cylindric, 180-200 pm x
10-13 pm. The wall appears thin throughout the ascus,
staining lightly in Congo red (Fig. 102). The operculum,
having a diameter of 5.5-6.5 pm, is observed with the aid of
Ultrastructurally, the mature ascus is extremely thin-
walled and flaccid (Figs. 104, 105, 108), being 220-260 nm
thick both apically and laterally. The inner layer in-
creases from 20-30 nm laterally to 90-110 nm at the vicinity
of the tip while the outer layer decreases from 170-200 to
120-140 nm. A mucilaginous coat (Figs. 104, 107) covers the
entire ascus. When stained in silver methenamine the inner
layer reacts more intensely (Figs. 106-109). An operculum
is not readily distinguished except when the inner layer
becomes thinner at an area below the tip (Fig. 108). The
operculum has a diameter of 6.2-6.7 pm. At spore release,
the suboperculum, which is 4.6-5.1 pm long, has increased an
additional 40-60 nm (Fig. 109).
Light microscopic examination of the ascal tips of
species in the Otidea-Aleuria complex revealed general uni-
formity in form and development. Except in Ascozonus
woolhopensis, the apical wall remained thin throughout the
later stages of ascosporogenesis. Apical apparatuses of all
members but Anthracobia melaloma and A. woolhopensis lacked
distinct features by which they could be characterized.
The indented ring reported in Chapter I was not detected in
any species. Asci of A. melaloma and A. woolhopensis pos-
sessed subapical, swollen rings. In A. melaloma the ring
was observed in mature ascal walls after close inspection.
By comparison, the ring in A. woolhopensis developed much
earlier and became quite pronounced. Prior to ascal de-
hiscence the operculum was seldom seen in the mature asci
of the ten representatives, having been observed only in
Humaria hemisphaerica and Sowerbyella imperialism. Van
Brummelen (1974) demonstrated an apical disc in the ascus of
A. woolhopensis with the aid of the stain Congo red and thus
substantiated earlier findings of Vuillemin (1887).
Kimbrough (1972), however, pointed out that the nippled tips
in asci of Ascozonus cunicularius (Boud.) Marchal were un-
stained in Congo red. He did not observe a disc or lid in
asci of that species. Similarly, the apical apparatus of
A. woolhopensis in the present study did not exhibit an
apical disc in fresh material that had been stained with
Congo red. Observations of A. cunicularius (Kimbrough, 1972)
and A. woolhopensis in this study were made from material
that was collected in North America. Staining properties
of the small disc may vary according to species and isolates
Chadefaud (1942) described delicate cytoplasmic compo-
nents, which varied in size and complexity, for the apical
apparatuses of eight members placed within the Otidea-Aleuria
complex (Figs. 110A, B, C, D). Two of these species, Aleuria
aurantia and Humaria hemisphaerica, were currently studied.
Components including the apical punctuation, tract and fun-
nel were not seen in stained and unstained fresh material
of either species. Of the remaining species in the present
examination, Anthracobia melaloma has a funnel and tract in
the apical region of its ascus. These features were vague
and infrequently observed. Apical spherules were seen in
most species from time to time but without regularity.
Opercular pads reported by LeGal (1953) in species of
Phaedropezia and Trichophaea were found in the apical ap-
paratuses of Otidea leporina. Optical view of the mature
ascal tip in 0. leporina seemed to show an expanded opercu-
lar wall. Fine structure of the apex, however, demonstrated
the opercular wall to be thinner than the subtending lateral
wall and have a convex shape. Thus, the structurally weak
ascal tips of O. leporina, seen with the light microscope,
frequently became invaginated during mounting or staining
procedures. This phenomenon was misleading in the present
study and may have misled LeGal in her analysis of
Phaedropezia and Trichophaea.
The distinct light microscopic feature of the different
ascal tips in the present investigation was the thickness of
the ascal walls. Subapical walls of asci in 0. leporina,
Sphaerosporella brunnea, Jafnea fusicarpa and Humaria
hemisphaerica appeared considerably thicker than those seen
in A. melaloma, Scutellina scutellata, Geopyxis majalis and
Sowerbyella imperialis. In multispored asci (more than eight
per ascus) of A. woolhopensis the walls were extremely thick
and displayed two layers. A bilayered ascal wall was also
observed in H. hemisphaerica and J. fusicarpa.
Ultrastructural observations of the ten species present-
ly studied reinforced the light microscopic findings. Thick-
ness of the lateral ascal walls was sharply divided between
those that were thin, 350-450 nm, as in Aleuria aurantia,
Anthracobia melaloma, Geopyxis majalis, Scutellinia
scutellata and Sowerbyella imperialis and those that were
thick, 700-1100 nm, as in Ascozonus woolhopensis, Humaria
hemisphaerica, Jafnea fusicarpa and Sphaerosporella brunnea.
Otidea leporina was the only representative that did not
fall into either division. Gross morphology of the apical
apparatuses was similar for all by A. melaloma, 0. leporina
and S. brunnea. The outer layer decreased in thickness
toward the tip while the inner layer decreased toward the
base. In A. melaloma, 0. leporina and S. Brunnea the inner
layer thickened toward the base or stayed approximately at
the same thickness throughout the ascal wall.
With the exception of A. woolhopensis, the development
of the apical apparatuses essentially followed the three-
step sequence that was outlined in Chapter I. In two spe-
cies, J. fusicarpa and H. hemisphaerica, the inner layer was
formed after spore ontogeny. Their pattern of development
resembled that seen in lodophanus granulipolaris Kimbr.
Formation of the apical apparatus in A. woolhopensis dif-
fered significantly in several respects. The inner layer
of the lateral ascal wall was formed before ascospore delimi-
tation. By early ascosporogenesis the development of the
apical apparatus was complete. At this stage a pore had
formed in the inner layer at the ascal tip. Van Brummelen
(1974) noted the appearance of the pore at a later time in
development and suggested that it was formed from a process
of localized disintegration. The pore may have been, how-
ever, the result of wall stretching as the ascus expanded in
length and width throughout ascosporogenesis. A similar
phenomenon was observed in the thick-walled apex of Cookeina
sulcipes (Berk.) Kuntze (Samuelson, 1975).
Schrantz's (1970) interpretation of the development of
the ascal wall in Tarzetta cupularis differed significantly
from that shown in the present study. He stated that in
young asci the exoascus consisted of a thin band of electron-
dense, fibrillar material and by spore formation, this layer
had increased in thickness mostly at the tip. He concluded
that most of the wall consisted of a thick endoascus which
tapered in thickness toward the tip. Apparently, Schrantz
did not examine fully ripened asci as an examination of his
photographs will confirm. His descriptions of exo- and endo-
ascal walls of T. cupularis corresponded to the mucilaginous
coat and outer layer, respectively, of a number of species
including S. scutellata, S. imperialis, and A. woolhopensis.
Cytoplasmic components of the operculate apical appa-
ratus described by Chadefaud (1942) (Fig. 110A) were not de-
tected ultrastructurally in any of the representatives
currently examined. Chadefaud's discovery of the funnel and
tract were most likely artefacts of fixation and staining.
He frequently applied either Melzer's reagent or a chromium
trioxide-osmium tetroxide solution when examining the apical
apparatus. These solutions may have caused the collapse of
the ascal plasmalemma and tonoplast beneath the apical region
of the ascus, thus creating a funnel and tract. Tips of
most representatives in the present study were filled with
cytoplasm throughout ascosporogenesis. The apical cytoplasm
may account for the presence of apical punctuations observed
by Chadefaud above the funnel (Fig. 110A).
An interesting wall component that was pointed out by
Chadefaud (1942) in several species was the occurrence of a
subapical "bourrelet," i.e. pad. In the general scheme of
the operculate apical apparatus (Fig. 110A) the subapical pad
adjoined the operculum. However, his diagrams of dehisced
asci in Aleuria aurantia and Scutellinia hirta (Fig. 11OB, C)
depicted subapical pads that were further removed from the
tip. Subapical pads were not currently seen with the light
microscope in A. aurantia or S. scutellata. Ultrastructur-
ally, a subopercular, asymmetrically formed, swollen ring
was discovered in A. aurantia during late spore development.
Although the ring had disappeared by maturity, the sub-
opercular wall was thickest in that vicinity. In S.
scutellata a subopercular ring was also observed with the
aid of the electron microscope. The subapical pads de-
scribed by Chadefaud may have represented subopercular
rings. He noted that the pads were often asymmetrical as in
A. aurantia and depicted them in only mature and dehisced
asci. S. hirta and A. aurantia were both studied by
Chadefaud in a chromo-osmic solution. Wall layering in
that region of the ascus may have been abnormally affected
by this solution, thus permitting observation of the pads.
Among the ten members of the Otidea-Aleuria complex
presently studied, seven species displayed subopercular
rings. First appearance of the ring occurred at different
developmental stages for different species. In J. fusicarpa
an annular protuberance was detected after the inner layer
was formed. By comparison, the first signs of a subopercu-
lar ring in A. melaloma were observed prior to the formation
of the inner layer. In general, development of the annular
swelling occurred during the last stages of ascosporogenesis
and marked the initiation of the formation of the inner
layer. However, in A. woolhopensis the ring began to develop
shortly after meiosis in the young, truncate ascus. Similar
observations of A. woolhopensis were made by van Brummelen
(1974). Still., both investigations demonstrated that the
ring was derived from the local expansion of the inner layer.
Van Brummelen's interpretation of the wall composition
in A. woolhopensis at the ring and throughout the apical
region of the ascus differed from the present study in three
respects. (1) He recognized the presence of a middle layer
in the subapical wall which disappeared at the level of the
ring. This layer is currently described as part of the
middle stratum which extends to the base of the ascus.
Median sections of younger asci stained with silver methena-
mine and peripheral sections of mature asci established this
finding. (2) Van Brummelen demonstrated an electron-dense
internal layer at the level of the ring. Since he was not
able to follow it above or below the ring, he referred to
the area as the internal ring layer. Staining with silver
methenamine revealed that the internal ring layer was a
part of the inner layer. (3) The present study also reports
the presence of a distinct middle stratum within the inner
and outer layers. The ring was shown to consist mostly of
the middle stratum (Fig. 111H) which appears to be chemically
similar to the outer wall layer. Similar findings were ob-
served with the light microscope using Congo red. Van
Brummelen did not make these distinctions.
The smallest subopercular rings occurred in the thick-
walled species, J. fusicarpa, H. hemisphaerica and S.
brunnea. In contrast, taxa that formed conspicuous rings
during the development of their apical apparatuses, i.e.,
A. aurantia and A. melaloma, were thin-walled. A. woolhopen-
sis was the sole exception, having both thick walls and an
enormous ring. Multispored asci, in general, have been shown
to be thick-walled (Kimbrough, 1966a,b, 1969; Kimbrough and
Korf, 1967). In Chapter I wall dimensions of the 32-spored
representative Thecotheus pelletieri (Crouan) Boud. were
roughly 3 times that of the 8-spored members in the iodine-
positive group. In Chapter III the apical apparatus of the
multispored species Coprotus winter (Marchal) Kimbr. was
shown to be essentially an enlarged replica of the eight-
spored species Coprotus lacteus (Ck. and Phill.) Kimbr.
Similarly, wall dimensions of A. woolhopensis were approxi-
mately two and one-half to three times that of A. melaloma
and A. aurantia. Therefore, when taking the exaggerated
condition of the multispored ascus into consideration, the
apical apparatus of A. woolhopensis is remarkably similar in
form to that of A. melaloma.
Kimbrough and Benny (1977) have recently described in
another multispored representative, Lasiobolus monascus
Kimbr., the presence of a large subopercular ring during the
development of its apical apparatus. As in Aleuria aurantia,
the ring, which became most prominent during ascosporogene-
sis, was not apparent at the end of spore development.
Stretching of the ascal wall prior to spore release may have
been responsible for its disappearance.
Within the Otidea-Aleuria complex the function of the
subopercular ring appeared to be associated primarily with
structural support of the apical region of the ascus during
spore release. At incipient ascal dehiscence, the lateral
wall became markedly stretched, narrowing its thickness by
10-20%. In the species A. melaloma, A. aurantia, S. brunnea
and S. scutellata, where the subapical walls decreased sig-
nificantly in thickness toward the tip, the ring most likely
provided additional strength and maintained the shape of
the ascal tip. During dehiscence, the structural role that
the ring played was most evident in A. woolhopensis, keeping
the lateral wall intact as the spores shot through the
fissured opening. After dehiscence, in certain members,
J. fusicarpa, 0. leporina and H. hemisphaerica, the lateral
wall appeared to be elastic, having returned to its original
thickness. In S. brunnea elasticity of the subopercular
wall was not observed. The wall above the subopercular ring
remained stretched. Although a distinct subopercular ring
was not found in mature asci of 0. leporina, the sharp in-
crease in wall thickness at the lower extremity of the sub-
operculum may have provided strength comparable to that of
the localized rings in S. brunnea and A. melaloma.
Apical apparatuses of Geopyxis majalis and Sowerbyella
imperialism demonstrated general similarities with the other
taxa. Although revived material was used, wall layering of
the ascal tip was readily distinguished in both species.
The presence of a ring at the periphery of the operculum in
G. majalis was unique among the representatives of the
Otidea-Aleuria complex examined. The thinness of the ascal
wall in S. imperialis was equally atypical. Subopercular
rings were not observed in either representative. Since
most of the subopercular rings currently studied have been
shown to be inconspicuous, developmental examinations of
fresh specimens are necessary to verify the presence or
absence of this component within the ascal wall.
The present examination has demonstrated that within
the members studied no two species shared identical apical
apparatuses. Ascal tips consisted primarily of thin-walled
and thick-walled forms. When comparing the thick-walled
ascus of J. fusicarpa (Fig. 111E) to the thin-walled ascus
of A. melaloma (Fig. 111G), the differences were distinct.
However, the species H. hemisphaerica, S. brunnea (Fig. 111F)
and A. aurantia exhibited intergrades between the two. De-
fined types of apical apparatuses within the Otidea-Aleuria
complex could not be made on the basis of wall thickness or
any other feature of the ascal tip including the subopercular
Except in Ascozonus woolhopensis, Lasiobolus monascus
and spp. of Thelebolus subapical swollen rings have not been
observed outside of the Otidea-Aleuria complex. Variations
of the size of this unusual wall component correlated most
closely with wall thickness and, to a lesser degree, with
the spore number. Presence of subopercular rings in A.
woolhopensis and L. monascus indicated possible taxonomic
affinities with members of the Otidea-Aleuria complex.
Both species have been placed in the Theleboloceae, based
primarily on their multispored condition (see Chapter V).
In a world monograph of Lasiobolus, Bezerra and Kimbrough
(1975) stated that developmental, morphological, and micro-
chemical properties of Lasiobolus were more related to
Cheilymenia, Coprobia and Scutellinia than to Thelebolus.
They suggested that Lasiobolus belonged in the tribe
Scutellinieae sensu Korf (1973). The present study was
supportive of their findings. Cytological and developmental
studies on Ascozonus are needed to understand more clearly
its taxonomic position within the operculate Discomycetes.
Of the operculate apical apparatuses that have been ultra-
structurally examined in present and previous examinations,
A. woolhopensis most clearly resembled Anthracobia melaloma
in both structure of the ascal wall and general form.
Apical appratuses of the Otidea-Aleuria ascus were
found to be morphologically diverse. The diameter and thick-
ness of the operculum, the length and thickness of the sub-
operculum, the stratification of the wall, the size and
shape of the subopercular ring and the ontogeny of the dif-
ferent components varied from taxon to taxon. Still, sev-
eral characters of the apical apparatuses were generally
common for the representatives presently studied. A distinct
dehiscent region observed in the iodine-positive ascus
(see Chapter I), the eugymnohymenial ascus (see Chapter III)
and the suboperculate ascus (Samuelson, 1975; van Brummelen,
1975) was infrequently found in the Otidea-Aleuria complex.
The protruding apical wall in 0. leporina and the thickened
apical wall in Anthracobia melaloma were the only examples of
morphologically delimited opercula. Differential staining
of the operculum with silver methenamine, which was described
in lodophanus granulipolaris Kimbr. (see Chapter I), Pyronema
domesticum (Sow. ex Fr.) Sacc. and several representatives
of the Sarcoscyphineae (Samuelson, 1975), occurred only in
S. brunnea. The differences of the staining intensities of
the opercular wall most likely signify changes in the wall
chemistry at that area. Other operculate species may have
opercula that differ chemically from the rest of the ascal
wall but do not exhibit the differences when staining with
silver methenamine or lead citrate and uranyl acetate.
Further examination of ascal walls is needed to find various
treatments that could reveal this feature.
A fibrillar zone of dehiscence was not detected in the
ascal tips of any of the species in the present study. On
the other hand, the subopercular ring was a common feature
of the wall of the Otidea-Aleuria ascus. All of the members
that were investigated developmentally demonstrated to some
extent the presence of this component. The similarity of
the apical apparatuses and their subopercular rings in S.
brunnea and A. melaloma quite possibly suggests closer
taxonomic relatedness between those taxa than shown in
Table 1. Additional studies of the apical apparatuses in
the Otidea-Aleuria complex are needed to learn of the vari-
ability and prevalence of the subopercular ring. The
present study has shown that the apical apparatus may be
useful as a systematic tool within this largest of the
Subapical region of mature ascus is distinctly
Tip of mature ascus. Spore (SP). Stained
with Congo red. X1,500.
Raised portion at tip of mature ascus, de-
limiting operculum (0). Inner layer (IL).
Spore (SP). X14,000.
Shrunken ascus after spore release. Phase
Dehisced ascus with operculum (0) laterally
attached. Stained with Congo red. X1,500.
Distal portion of suboperculum. Arrows point
to region where opercular dehiscence will
Mature apical apparatus showing wall layering.
Stained with silver methenamine. Inner layer
(IL). Outer layer (OL). X10,000.
Ascal tip at time of spore release. Operculum
Dehisced ascus showing shrunken suboperculum
(SO). Ascostome (A). Outer layer (OL).
Ascal tip during early spore formation.
Stained with Congo red. X1,000.
Apical and subapical walls at later time in
development. Stained with Congo red.
Apical region of four-nucleated ascus with
scattered small vesicles (V). Nucleus (N).
Mature ascal tip showing slight heliotro-
pism. Spore (SP). X1,000.
Ascospore is compressed within ascostome
(A) during its ejection. X500.
Ascal tip during early spore wall develop-
ment shows increased thickness at lateral
walls. Apical wall (AW). Vesicle (V).
Broadened ascal tip at end of spore develop-
ment. Operculum (0). X6,200.
Mature apical apparatus with small sub-
apical, swollen ring (arrows). Inner layer
(IL). Outer ring (OL). Stained with silver
Dehisced ascus with attached operculum (0).
Suboperculum (SO). Subopercular ring (SR).
Stained with silver methenamine. X5,400.