Citation
Asci of the operculate Discomycetes (Pezizales)

Material Information

Title:
Asci of the operculate Discomycetes (Pezizales)
Creator:
Samuelson, Don Arthur, 1948-
Publication Date:
Language:
English
Physical Description:
xi, 253 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Asci ( jstor )
Ascospores ( jstor )
Diameters ( jstor )
Electrons ( jstor )
Genera ( jstor )
Mycology ( jstor )
Silver ( jstor )
Species ( jstor )
Taxa ( jstor )
Trucks ( jstor )
Asci ( lcsh )
Botany thesis Ph. D
Discomycetes ( lcsh )
Dissertations, Academic -- Botany -- UF
Fungi -- Classification ( lcsh )
Pezizales ( lcsh )
City of Gainesville ( local )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 245-252.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Don Arthur Samuelson.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
020620126 ( ALEPH )
03983152 ( OCLC )
AAB5218 ( NOTIS )

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Full Text













ASCI OF THE OPERCULATE DISCOMYCETES (PEZIZALES)


By

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


1977




ASCI OF THE OPERCLATE DISCOMYCETES (PEZIZALES)
By
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


ACKNOWLEDGEMENTS
\ 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.
11


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
LIST OF TABLES V
LIST OF FIGURES vi
ABSTRACT ix
GENERAL INTRODUCTION 1
CHAPTER I 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
CHAPTER II MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUSES OF REPRESENTA
TIVES IN THE OTIDEA-ALEURIA COMPLEX .... 48
Introduction 48
Materials and Methods 55
Results ..... 57
Discussion 74
CHAPTER III MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUSES OF EUGYMNO-
HYMENIAL REPRESENTATIVES 117
Introduction 117
Materials and Methods 123
Results 124
Discussion 130
CHAPTER IV MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUSES IN MORCHELLA
ESCULENTA AND REPRESENTATIVES OF THE
HELVELLACEAE 143
iii


Page
Introduction 143
Materials and Methods 147
Results 148
Discussion 155
CHAPTER V MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUS OF THELEBOLUS . .170
Introduction 17 0
Materials and Methods 176
Results 177
Discussion 187
CHAPTER VI 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
IV


LIST OF TABLES
Page
Chapter I
Table 1
Classifications of genera with iodine
positive asci 13
Chapter II
Table 1
Classifications of genera of the Otidea-
Aleuria complex 4 9
Chapter III
Table 1
Classifications of eugymnohymenial genera 119
Chapter IV
Table 1
Classifications of the Helvellaceae .... 144
Chapter V
Table 1
Classifications of the Thelebolaceae. . 172
v


LIST OF FIGURES
Page
Chapter I
Figures 1-10 The apical apparatus of Peziza
succosa 35
Figures 11-19 The apical apparatus of Ascobolus
crenulatus 37
Figures 20-28 The apical apparatus of Saccobolus
depauperatus 39
Figures 29-35 The apical apparatus of Thecotheus
pelletieri 41
Figures 36-50 The apical apparatus of Iodophanus
granulipolaris 43
Figure 51 a-d Drawings of apical apparatuses found
in iodine-positive asci 47
Chapter II
Figures 1-9 The apical apparatus of Otidea
leporina 88
Figures 10-18 The apical apparatus of Jafnea
fusicarpa 90
Figures 19-34 The apical apparatus of Humaria
hemisphaerica 92
Figures 35-42 The apical apparatus of Sphaero-
sporella brunnea 96
Figures 43-57 The apical apparatus of Aleuria
aurantia 98
Figures 58-68 The apical apparatus of Anthracobia
melaloma ¡ ¡ I ^ ¡ 102
vi


Page
Chapter II
Figures 69-83
The apical apparatus
scutellata .....
of
Scutellinia
104
Figures 84-94
The apical apparatus
woolhopensis ....
of
Ascozonus
108
Figures 95-101
The apical apparatus
majalis
of
Geopyxis
110
Figures 102-109
The apical apparatus
imperialis
of
Sowerbyella
112
Figure 110 A-D
Apical apparatuses redrawn from
Chadefaud (1942)
114
Figure 111 E-H
Chapter III
Illustrations made from electron
microscopic observations of apical
apparatuses of the Otidea-Aleuria
complex
116
Figures 1-8
The apical apparatus
domesticum
of
Pyronema
136
Figures 9-16
The apical apparatus
sphaerospora ....
of
Ascodesmis
138
Figures 17-25
The apical apparatus
winterii
of
Coprotus
140
Figures 26-35
Chapter IV
The apical apparatus
lacteus
of
Coprotus
142
Figures 1-10
The apical apparatus
crispa
of
Helvetia
161
Figures 11-18
The apical apparatus
esculenta
of
Morchella
163
Figures 19-24
The apical apparatus
undulata
of
Rhizina
165
Figures 25-33
The apical apparatus
ancilis
of
Discina
167
vix


Page
Chapter IV
Figures 34-51
Chapter V
Figures 1-9
Figures 10-19
Figures 20-35
Figures 36-53
Figure 54 A-D
Chapter VI
Figures 1-18
The apical apparatus of Gyromitra
rufula 169
The apical apparatus of Thelebolus
microsporus. 197
The apical apparatus of Thelebolus
crustaceus 199
The apical apparatus of Thelebolus
polysporus 201
The apical apparatus of Thelebolus
stercoreus 207
Development of the apical apparatus
in Thelebolus polysporus 213
The apical apparatus of Trichobolus
zukalii 224
viii


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)
by
Don Arthur Samuelson
December, 1977
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
and genera.
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
IX


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
fcr the blue reaction. Although Iodophanus 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,
Qtidea, 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.
x


Opercular delimitation with the light microscope is observed
in Rhizina and Piscina 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
features.
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 Iodophanus 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 phytogeny within the Pezizales.
xi


GENERAL INTRODUCTION
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 (Eoudier, 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
1


2
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


3
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.
Mans) Schroet (Wells, 1972). Phylogenetically, ascal
dehiscence within the Sarcoscyphineae is believed to be
affiliated most closely with that observed in the true
operculates (Pezizineae).
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)


4
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 Pyrenoraycentes (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


5
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, Iodophanus 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


7
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, Iodophanus, 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, Humara Fuckel, Sphaerospore11a (Svr.)
Svr. & Kub., Aleuria Fuckel, Anthracobia Boud., Scutellinia


8
(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, Helvetia L., Gyromitra Fr., Piscina (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 zukali.i 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.


I
CHAPTER I
MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY OF THE APICAL
APPARATUSES OF REPRESENTATIVES WITH IODINE-POSITIVE ASCI
Introduction
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 "blueing in iodine" in place of amyloid.
9


10
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
becomes stained.
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


11
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 "pectic" 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


12
A. stercorarlus and Peziza plebeia, respectively, displayed
little in common. In fact, their findings supported
Chadefaud's belief that the two genera are not closely
related.
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 Iodophanus 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 Iodophanus granulipolaris Kimbrough, Thecotheus
pelietieri (Crouan) Boud., Ascobolus crenulatus Karst.,
Saccobolus depauperates (Berk, and Br.) E. C. Hansen and
Peziza succosa Berk. Phylogenetic and ontogenetic compari
sons are made between the species.


13
Chapter I
Table 1
Classifications of
Genera with Iodine-positive Asci
Eckblad (1968)
Pezizaceae
Ascobolaceae
Gelatinodiscus
Ascoboloideae
Sacrosphaera
Pachyella
Peziza (= Plicaria)
Ascobolus
Saccobolus
Rifai (1968)
Ascodesmidoideae
Ascobolaceae
Ascodesmis
Boudiera
Svrekia
Ascobolus
Saccobolus
Pezizaceae
Pezizaceae
Peziza
Plicaria
Marcelleina
Psilopezia
Discomycetella
Peziza
Plicaria
Boudiera
Sarcosphaera
Pachyella
Gelatinodiscus
Thelebolaceae
Thecotheus
Iodophanus
Thecotheus
Sphaerosoma
Pyronemaceae
Kimbrough (1970)
Iodophanus
Ascobolaceae
Korf (1973)
Ascoboloideae
Ascobolaceae
Ascobolus
Saccobolus
Ascoboleae
Ascodesmidoideae
Saccobolus
Ascobolus
Ascodesmis
Boudiera
lodophaneae
Svrekia
Boudiera
Sphaerosoma
Iodophanus
Thecotheus
Pezizaceae
Peziza
Plicaria
Sarcosphaera


14
Table 1 (Cont'd.)
Pezizaceae
Pachyella
Iodophanus
Thecotheus
Psilopezia


15
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 from the Culture Collection of the Rancho
Santa Ana Botanic Garden, courtesy of R. K. Benjamin.
lodophanus granulipolaris was isolated from cow dung col
lected near Gainesville by J. Milam. Apothecia of A.
crenulatus and 1^. 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 £5. 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 1^. granulipolaris were used to determine the
stage of ascal ontogeny for the majority of the apothecia in
a particular Petri plate.


16
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 ][. 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 cn a small drop of 0.01% sodium borate and dried at
60C. 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 1. granulipolaris, and


17
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 4C. 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 60C.
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 furtner enhanced by posttreating unstained sections
with silver methenamine, a preferential stain for poly
saccharides (Martino and Zamboni, 1967). Ultrathin sections


18
were then examined with an Hitachi HU-11 E electron
microscope.
Results
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
treated individually.
The Apical Apparatus of Peziza succosa
The diploid ascus reaches a length of 140-180 ym and a
diameter of 10-12 ym. 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 ym and a diameter of 12-14 ym.
When stained with Melzer's reagent, the deep blueing at the
tip is less intense and extends 10-14 ym 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 pressue on a cover slip and
sliding the cover slip back and forth. The tips of mature


19
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 ym
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 ym.
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


20
ascal wall narrows approximately 0.1 ym as it approaches the
operculum.
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 ym long and 6-10 ym wide. As the spores mature, the
ascus grows to a length of 115-130 ym and width of 12-15 ym.
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


21
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 ym.
When the spores approach complete development, the
width of the ascal tip increases from 5.9-6.3 ym (Fig. 15)
to 8.0-8.4 ym (Figs. 17, 18). At the same time, the diameter
of the operculum has widened to 5.7-5.9 ym. 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


22
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 ym long.
The Apical Apparatus of Saccobolus depauperatus
The mature ascus is broadly clavate, being 55-80 ym
long and 12-15 ym 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
the ascus.
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 rmicilaginous 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


23
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 ym. 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 ym 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 ym 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 subopercalum. 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 ym
and a width of 50-55 ym (Fig. 29). The ascal wall becomes
diffusely blue when treated with Melzer's reagent. The


24
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 ym 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 mu toward the base. The length
of the suboperculum is 6.5-7.5 ym 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.


25
The Apical Apparatus of Iodophanus granulipolaris
The diploid ascus is broadly cylindrical, being 100-
140 ym long and 15-20 ym 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 ym in length and 30-35 ym 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


26
(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-
210 nm.
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


27
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
(Fig. 50).
Discussion
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.


28
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


29
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 cytologicai 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 "pectic"
external cover and an "amyloid" inner hood or "manchn"
(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


30
comparable stage in development to that of the tip seen in
Fig. 4 for P. succosa. He apparently based his evidence on
immature asci.
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).


31
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


32
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
A. crenulatus.
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


33
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.


Chapter I
Figures 1-10. Peziza succosa
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.
Figure 9.
Figure 10.
Tip of diploid ascus showing iodine-positive
reaction. Stained with Melzer's reagent.
XI,000.
Diffuse iodine-positive reaction at mature
ascal tip. Spore (SP). XI,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).
X7,800.
Presence of annular indentation (AI) by late
ascosporogenesis. X9,000.
Ascal tip at late ascosporogenesis. Muci
laginous coat (MC). Stained with silver
methenamine. X6,000.
Mature apical apparatus. Distinct annular
indentation (AI) delimits operculum (O).
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
methenamine. X20,000.
Region of dehiscent zone which is demarcated
by annular indentation (AI). X24,000.


35


Chapter I
Figures 11-
-19. Ascobolus crenulatus
Figure 11.
Young ascal tip with developing ascospores
shows distinct apical ring (AR) at this
time. Stained with Congo red. Xl,250.
Figure 12.
Mature tip is thinner-walled at region of
the operculum (0). Stained with Congo red.
X1,000.
Figure 13.
Vesiculated apex of diploid ascus with sub
tending ring of glycogen (G). X9,800.
Figure 14.
Ascal tip during early spore (SP) develop
ment. Stained with silver methenamine.
X10,500.
Figure 15.
Early formation of apical apparatus showing
differential increase in thickness of
opercular (0) wall. Annular indentation
(AI). X11,000.
Figure 16.
Inner layer (IL) and outer layer (OL) of
developing apical apparatus. Spore (SP).
Stained with silver methenamine. X10,500.
Figure 17.
Mature apical apparatus with umbonately
shaped operculum (0). X9,500.
Figure 18.
Mature apical apparatus shows layering of
opercular and subopercular (SO) regions of
the ascus. Annular indentation (AI).
Stained with silver methenamine. X8,500.
Figure 19.
Region of annular indentation in mature
apical apparatus. Inner layer (IL). Outer
layer (0L). X48,000.


37


Chapter I
Figures 20-28. Saccobolus depauperatus
Figure 20.
Developing apical apparatus with pronounced
ring. X2,000.
Figure 21.
Apical apparatus at later developmental
stage. Stained with Congo red. X2,000.
Figure 22.
Thickening of apical wall (AW) during early
spore development. Mucilaginous coat (MC).
X3,400.
Figure 23.
Mature apical apparatus with distinct
annular indentation (AI). X8,200.
Figure 24.
Region of annular indentation with zone of
dehiscence (ZD). Mucilaginous coat (MC).
X39,000.
Figure 25.
Mature apical apparatus stained with silver
methenamine. Operculum (0). Suboperculum
(SO). X6,500.
Figure 26.
Upper portion of suboperculum showing sub-
opercular flange (SF), Inner layer (IL).
Outer layer (OL). Stained with silver
methenamine. Xll,000.
Figure 27.
Incipient spore release. Episporal sac (ES)
Spore (SP). X7,60G.
Figure 28.
Dehisced ascus with outwardly extended sub-
opercular flange (SF). Ascostome (A).
Stained with silver methenamine. X6,200.


39


Chapter I
Figures 29
Figure 29.
Figure 30.
Figure 31.
Figure 32.
Figure 33.
Figure 34.
Figure 35.
35. Thecotheus pelletieri
Mature ascus stained with Congo red. X160
Ascal tip with wide apical ring (AR)
delimiting a conical operculum. XI,250.
Ascus after spore release. Ascostome (A).
X160.
Opercular region of mature ascus. Annular
indentation (AI). X5,400.
Mature apical apparatus. Operculum (0).
Suboperculum (SO). Stained with silver
methenamine. X2,000.
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
indentation. X34,000.


41


Chapter I
Figures 36-42. Iodophanus granulipolaris
Figure
36.
Uninucleated ascus stained with Melzer's
reagent. X400.
Figure
37.
Apex of uninucleated ascus subtended by
broad amorphous cylinder. One-micron
section stained with toluidine blue.
XI,000.
Figure
38.
Apical region of uninucleated ascus.
Glycogen (G). X6,100.
Figure
39.
Small lomasomes (L) at ascal tip. Endo
plasmic reticulum (ER). X20,000.
Figure
40.
Mitochondria (M) adjacent to large mass of
glycogen (G). X15,000.
Figure
41.
Large vacuole (V) at tip during early
ascosporogenesis. X4,700.
Figure
42.
Distal portion of apical wall (AW) stained
with silver methenamine. X4,700.


43


Chapter I
Figures 43-50.
Figure 43.
Figure 44.
Figure 45.
Figure 46.
Figure 47.
Figure 43.
Figure 49.
Figure 50.
Iodophanus granulipolaris
Mature tip stained with Congo red. XI,250.
Operculum (O) partially connected to ascus
at spore liberation. Stained with Melzer's
reagent. X400.
Thickening of lateral wall (LW) during spore
maturation. Apical wall (AW). X3,100.
Numerous small vesicles (V) at ascal tip.
X6,300.
Mature apical apparatus showing distinct
zone of dehiscence (ZD), inner layer (IL)
and outer layer (OL). Operculum (O).
Stained with silver methenamine. X13,500.
Portion of mature apical wall. X12,500.
Operculum (O) attached at spore release.
Outer layer (OL). Stained with silver
methenamine. X4,700.
Dehisced ascus with thickened inner layer
(IL). Suboperculum (SO). Stained with
silver methenamine. X8,700.


45


Figure 51.
a.
b.
c.
Chapter I
Drawings of apical apparatuses found in
iodine-positive asci.
Apical apparatus of Ascobolus furfuraceus.
Operculum (O). Coussinet (C). Redrawn from
Chadefaud (1942).
Ascal tip of Peziza plebeia illustrating layers
of the wall. Exoascus (EX). Endoascus (EN).
Bourrelet (B). Redrawn from Schrantz (1970).
Illustration made from electron microscopic
observations of the wall layering in mature
ascal tips of Ascobolus crenulatus. Outer
layer (OL). Inner layer (IL).
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).
d.


47


CHAPTER II
MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY OF THE APICAL
APPARATUSES OF REPRESENTATIVES IN THE OTIDEA-ALEURIA COMPLEX
Introduction
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
48


49
Chapter II
Table 1
Classifications of the Otidea-Aleuria Complex
Dennis (1968)
Eckblad (1968)
Huraariaceae
Pyronemaceae
Lachneae
Iodophanus
Pyronema
Sepultarla
Leucoscypha
Thricharia
Humaria
Trichophaea
Pseudoomphilia
Pyronemella
Lamprospora
Pulvinula
Caloscypha
Octospora
Leucoscypha
Coprobia
Ciliareae
Cheilymenia
Scutellinia
Scutellinia
Cheilymenia
Sphaerosporella
Neottiella
Melastiza
Anthracobia
Humaria
Tricharia
Sphaerosporella
Sepultarla
Jafnea
Fimaria
Aleurieae
Pseudombrophila
Anthracobia
Melastiza
Aleuria
Pulvinula
Coprobia
Geopyxis
Fimaria
Octospora
Psilpezia
Boudiera
Caloscypha
Lamprospora
Aleuria
Otideaceae
Geopyxis
Pustulina
Sowerbyella
Otidea
Ascosparassis
Pezizaceae
Rifai (1968)
Otideae
Humariaceae
Otidea
Barlacina
Pseudotis
Sowerbyella
Pustularia
Otideae
Otideae
Marcelleina
Lachneae


50
Table 1 (Cont'd.)
Humariaceae
Jafnea
Jafneadelfus
Nothoj afnea
Sphaerosporella
Ciliarieae
Rhizoblepharia
Scutellinia
Cheilymenia
Coprobia
Aleuriaceae
Anthracobia
Melastiza
Aleuria
Leucoscypha
Geopyxis
Octospora
Inermisia
Pulvinula
Lamprospora
Kimbrough (1970)
Otidiaceae
Otidea
Pustulina
Jafnea
Nothojafnea
Jafneadelfus
Sowerbyella
Ascosparassis
Sepultarla
Tricharia
Mycolachnea
Pseudotis
Barlaeina
Pseudombrophila
Trichophaea
Aleuriaceae
Coprobia
Cheilymenia
Aleuriaceae
Scutellinia
Geopyxis
Aleuria
Melastiza
Octospora
Anthracobia
Caloscypha
Sowerbyella
Fimaria
Leucoscypha
Inermisia
Genosperma
Rhizoblepharis
Korf (1973)
Pyronemataceae
Ascodesmidoideae
Ascodesmis
Sphaerozone
Pulparia
Jafneadelfus
Pyronematoideae
Pyronemateae
Pyronema
Karstenelleaea
Karstenella
Ascophanoideae
Geopyxideae
Geopyxis
Apapaphysaria
Pseudombrophileae
Selenaspora
Tricharina
Rhizoblepharina


51
Table 1 (Cont'd.)
Pyronemataceae
Trichophaeopsis
Pseudombrophila
Fimaria
Otideoideae
Jafneae
Jafnea
Tarzetta
Otieeae
Psilopezia
Otidea
Ascosparassis
Mycolachneeae
Nothojafneae
Geopora
Humara
Trichophaea
Pyronemataceae
Aleurieae
Anthracobia
Hiemsia
Melastiza
Leucoscypha
Aleuria
Pseudocollema
Pulvinula
Lamprospora
Octospora
Byssonectria
Kotlabaea
Scutellinioideaea
Scutellinieae
Coprobia
Scutellinia
Cheilymenia
Sowerbyelleae
Sowerbyella
Caloscypha
cervus
Phaedropezia


52
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
conclusions.
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 constellatio (Berk and Br.) Boud., Scutellinia
hirta (Schwm. ex Fr.) 0. Ktze., Octospora leucoloma Hedw.
ex. S. F. Gray (as Humaria leucoloma), Humaria wrightii
(Berk, and Cooke) Boud. and Sepultara arenosa (Fuckel)


53
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 in Fig. 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


54
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
similarly misinterpreted.


55
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


56
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
observations.
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.


57
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 O. 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.
Results
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
treated individually.
The Apical Apparatus of Otidea leporina
The mature ascus is narrowly cylindric, reaching a length
of 180-200 ym and a diameter of 9-12 ym. 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.


58
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 ym while its diameter expands to 10-15 ym
(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 ym and a uniform
thickness of 170-180 nm. The suboperculum is 3.7-4.0 ym
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 ym while
its thickness decreases by 40-60 nm. The upper extremity of


59
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 ym.
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 ym x 18-22 ym. 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
v/all (Fig. 14 ) .
Electron microscopic examination of a four-nucleated ascus
reveals for the most part the presence of a thick, lateral
v/all, 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


60
o 480-520 run. The apex is wider, having expanded to a
diameter of 9.2-9.4 ym. 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 ym 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 ym long, has thickened to 950-980 nm at its lower ex
tremity where the subopercular ring has become greatly
enlarged.
The Apical Apparatus of Humaria hemisphaerica
Asci containing immature ascospores are 160-240 ym long
and 12-16 ym 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 ym) 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


61
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 ym 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


62
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 ym and a breadth of 380-420 nm.
The suboperculum is 4.9-5.2 ym long.
The Apical Apparatus of Sphaerosporella brunnea
The mature ascus is broadly cylindric (150-190 ym x
16-20 ym). The rounded to blunt tip appears thinner-walled
than the rest of the ascus (Fig. 35). The diameter of the
ascospores (13-16 ym) is greater than that of the ascal
tip (9-10 ym) 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 ym. 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


63
(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 ym 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 ym 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


64
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 urn and a diameter of 12-16 ym. No dis
tinctive features are observed in the rounded ascal tip
(Fig. 51). At ascal dehiscence, the operculum, 4-5 ym 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 ym below the tip, the wall
protrudes into the ascoplasm, forming a subopercular ring.
A piasmalemmasome 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


65
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 ym and a uniform thickness of 260-
290 nm. After spore release (Fig. 57), the suboperculum,
which is 5.8-6.2 ym 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 ym x 10-14 ym (Fig.
58). The ascal wall appears to be thicker throughout the


66
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 ym 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


67
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 ym 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


68
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 ym x 8-12 ym. 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 ym and a diameter of 14-18 ym. 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


69
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 ym 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


70
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 ym. 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 ym and diameter of 14-18 ym. They are broadly
clavate and possess a conical 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 ym in length and 4-6 ym 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


71
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 ym 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


72
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 ym and a
thickness of 550-600 nm.
The Apical Apparatus of Geopyxis majalis
Mature asci are narrow and cylindric, 260-300 ym x
10-14 ym. 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 biiayered 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 ym
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


73
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 ym 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 ym, is observed with the aid of
phase-light microscopy.
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 ym. At spore release,


74
the suboperculum, which is 4.6-5.1 um long, has increased an
additional 40-60 nm (Fig. 109).
Discussion
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 imperialis. 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


75
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
of Ascozonus.
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 Plumaria 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 O. 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 0. leporina, seen with the light microscope,


76
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, O. leporina
and S. brunnea. The outer layer decreased in thickness
toward the tip while the inner layer decreased toward the


77
base. In A. melaloma, O. 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 Iodophanus 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


78
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


79
asci in Aleuria aurantia and Scutellinia hirta (Fig. HOB, 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


80
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.


81
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 winteri (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.


82
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, £>. 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, O. 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
imperialis demonstrated general similarities with the other
taxa. Although revived material was used, wall layering of


83
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
ring.
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


84
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 me1aloma
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


85
(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 O. 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 Iodophanus 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 thse taxa than shown in


86
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
operculate groups.


Chapter II
Figures 1-9. Otidea leporina
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Subapical region of mature ascus is distinctly
heliotropic. X400.
Tip of mature ascus. Spore (SP). Stained
with Congo red. XI,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
contrast. X400.
Figure 5.
Figure 6.
Figure 7.
Figure 8.
Figure 9.
Dehisced ascus with operculum (O) laterally
attached. Stained with Congo red. XI,500.
Distal portion of suboperculum. Arrows point
to region where opercular dehiscence will
occur. X37,000.
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
(O). X14,000.
Dehisced ascus showing shrunken suboperculum
(SO). Ascostome (A). Outer layer (OL).
X15,000.


88


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PAGE 267

81,9(56,7< 2) )/25,'$


ASCI OF THE OPERCÃœLATE DISCOMYCETES (PEZIZALES)
By
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

ACKNOWLEDGEMENTS
\ 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.
11

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
LIST OF TABLES V
LIST OF FIGURES vi
ABSTRACT ix
GENERAL INTRODUCTION 1
CHAPTER I 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
CHAPTER II MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUSES OF REPRESENTA¬
TIVES IN THE OTIDEA-ALEURIA COMPLEX .... 48
Introduction 48
Materials and Methods 55
Results ..... 57
Discussion 74
CHAPTER III MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUSES OF EUGYMNO-
HYMENIAL REPRESENTATIVES 117
Introduction 117
Materials and Methods 123
Results 124
Discussion 130
CHAPTER IV MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUSES IN MORCHELLA
ESCULENTA AND REPRESENTATIVES OF THE
HELVELLACEAE 143
iii

Page
Introduction 143
Materials and Methods 147
Results 148
Discussion 155
CHAPTER V MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUS OF THELEBOLUS . . .170
Introduction 17 0
Materials and Methods 176
Results 177
Discussion 187
CHAPTER VI 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
IV

LIST OF TABLES
Page
Chapter I
Table 1
Classifications of genera with iodine
positive asci 13
Chapter II
Table 1
Classifications of genera of the Otidea-
Aleuria complex 4 9
Chapter III
Table 1
Classifications of eugymnohymenial genera . 119
Chapter IV
Table 1
Classifications of the Helvellaceae .... 144
Chapter V
Table 1
Classifications of the Thelebolaceae. . . . 172
v

LIST OF FIGURES
Page
Chapter I
Figures 1-10 The apical apparatus of Peziza
succosa 35
Figures 11-19 The apical apparatus of Ascobolus
crenulatus 37
Figures 20-28 The apical apparatus of Saccobolus
depauperatus 39
Figures 29-35 The apical apparatus of Thecotheus
pelletieri 41
Figures 36-50 The apical apparatus of Iodophanus
granulipolaris 43
Figure 51 a-d Drawings of apical apparatuses found
in iodine-positive asci 47
Chapter II
Figures 1-9 The apical apparatus of Otidea
leporina 88
Figures 10-18 The apical apparatus of Jafnea
fusicarpa 90
Figures 19-34 The apical apparatus of Humaria
hemisphaerica 92
Figures 35-42 The apical apparatus of Sphaero-
sporella brunnea 96
Figures 43-57 The apical apparatus of Aleuria
aurantia 98
Figures 58-68 The apical apparatus of Anthracobia
melaloma I ¡ I ^ ¡ . 102
vi

Page
Chapter II
Figures 69-83
The apical apparatus
scutellata .....
of
Scutellinia
104
Figures 84-94
The apical apparatus
woolhopensis ....
of
Ascozonus
108
Figures 95-101
The apical apparatus
majalis
of
Geopyxis
110
Figures 102-109
The apical apparatus
imperialis
of
Sowerbyella
112
Figure 110 A-D
Apical apparatuses redrawn from
Chadefaud (1942)
114
Figure 111 E-H
Chapter III
Illustrations made from electron
microscopic observations of apical
apparatuses of the Otidea-Aleuria
complex
116
Figures 1-8
The apical apparatus
domesticum
of
Pyronema
136
Figures 9-16
The apical apparatus
sphaerospora ....
of
Ascodesmis
138
Figures 17-25
The apical apparatus
winterii
of
Coprotus
140
Figures 26-35
Chapter IV
The apical apparatus
lacteus
of
Coprotus
142
Figures 1-10
The apical apparatus
crispa
of
Helvetia
161
Figures 11-18
The apical apparatus
esculenta
of
Morchella
163
Figures 19-24
The apical apparatus
undulata
of
Rhizina
165
Figures 25-33
The apical apparatus
ancilis
of
Discina
167
vix

Page
Chapter IV
Figures 34-51
Chapter V
Figures 1-9
Figures 10-19
Figures 20-35
Figures 36-53
Figure 54 A-D
Chapter VI
Figures 1-18
The apical apparatus of Gyromitra
rufula 169
The apical apparatus of Thelebolus
microsporus. 197
The apical apparatus of Thelebolus
crustaceus 199
The apical apparatus of Thelebolus
polysporus 201
The apical apparatus of Thelebolus
stercoreus 207
Development of the apical apparatus
in Thelebolus polysporus 213
The apical apparatus of Trichobolus
zukalii 224
viii

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)
by
Don Arthur Samuelson
December, 1977
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
and genera.
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
IX

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
fcr the blue reaction. Although Iodophanus 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,
Qtidea, 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.
x

Opercular delimitation with the light microscope is observed
in Rhizina and Piscina 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
features.
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 Iodophanus 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 phytogeny within the Pezizales.
xi

GENERAL INTRODUCTION
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 (Eoudier, 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
1

2
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

3
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.
Mans) Schroet (Wells, 1972). Phylogenetically, ascal
dehiscence within the Sarcoscyphineae is believed to be
affiliated most closely with that observed in the true
operculates (Pezizineae).
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)

4
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 Pyrenoraycentes (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

5
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, Iodophanus 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, 197 2) . 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

7
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, Iodophanus, 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, Humaría Fuckel, Sphaerospore11a (Svr.)
Svr. & Kub., Aleuria Fuckel, Anthracobia Boud., Scutellinia

8
(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, Helvetia L., Gyromitra Fr., Piscina (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 zukali.i 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.

I
CHAPTER I
MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY OF THE APICAL
APPARATUSES OF REPRESENTATIVES WITH IODINE-POSITIVE ASCI
Introduction
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 "blueing in iodine" in place of amyloid.
9

10
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
becomes stained.
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

11
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 "pectic" 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

12
A. stercorarlus and Peziza plebeia, respectively, displayed
little in common. In fact, their findings supported
Chadefaud's belief that the two genera are not closely
related.
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 Iodophanus 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 Iodophanus granulipolaris Kimbrough, Thecotheus
pelietieri (Crouan) Boud., Ascobolus crenulatus Karst.,
Saccobolus depauperates (Berk, and Br.) E. C. Hansen and
Peziza succosa Berk. Phylogenetic and ontogenetic compari¬
sons are made between the species.

13
Chapter I
Table 1
Classifications of
Genera with Iodine-positive Asci
Eckblad (1968)
Pezizaceae
Ascobolaceae
Gelatinodiscus
Ascoboloideae
Sacrosphaera
Pachyella
Peziza (= Plicaria)
Ascobolus
Saccobolus
Rifai (1968)
Ascodesmidoideae
Ascobolaceae
Ascodesmis
Boudiera
Svrekia
Ascobolus
Saccobolus
Pezizaceae
Pezizaceae
Peziza
Plicaria
Marcelleina
Psilopezia
Discomycetella
Peziza
Plicaria
Boudiera
Sarcosphaera
Pachyella
Gelatinodiscus
Thelebolaceae
Thecotheus
Iodophanus
Thecotheus
Sphaerosoma
Pyronemaceae
Kimbrough (1970)
Iodophanus
Ascobolaceae
Korf (1973)
Ascoboloideae
Ascobolaceae
Ascobolus
Saccobolus
Ascoboleae
Ascodesmidoideae
Saccobolus
Ascobolus
Ascodesmis
Boudiera
lodophaneae
Svrekia
Boudiera
Sphaerosoma
Iodophanus
Thecotheus
Pezizaceae
Peziza
Plicaria
Sarcosphaera

14
Table 1 (Cont'd.)
Pezizaceae
Pachyella
Iodophanus
Thecotheus
Psilopezia

15
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 from the Culture Collection of the Rancho
Santa Ana Botanic Garden, courtesy of R. K. Benjamin.
lodophanus granulipolaris was isolated from cow dung col¬
lected near Gainesville by J. Milam. Apothecia of A.
crenulatus and 1^. 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 £5. 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 3^. granulipolaris were used to determine the
stage of ascal ontogeny for the majority of the apothecia in
a particular Petri plate.

16
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 JE. 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
60°C. 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 1. granulipolaris, and

17
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 (= 23°C). 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 4°C. 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 60°C.
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 furtner enhanced by posttreating unstained sections
with silver methenamine, a preferential stain for poly¬
saccharides (Martino and Zamboni, 1967). Ultrathin sections

18
were then examined with an Hitachi HU-11 E electron
microscope.
Results
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
treated individually.
The Apical Apparatus of Peziza succosa
The diploid ascus reaches a length of 140-180 ym and a
diameter of 10-12 ym. 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 ym and a diameter of 12-14 ym.
When stained with Melzer's reagent, the deep blueing at the
tip is less intense and extends 10-14 ym 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 pressue on a cover slip and
sliding the cover slip back and forth. The tips of mature

19
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 ym
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 ym.
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

20
ascal wall narrows approximately 0.1 ym as it approaches the
operculum.
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 ym long and 6-10 ym wide. As the spores mature, the
ascus grows to a length of 115-130 ym and width of 12-15 ym.
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

21
become rounder in form, still consists of a single, uniform
layer, 85-55 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 ym.
When the spores approach complete development, the
width of the ascal tip increases from 5.9-6.3 ym (Fig. 15)
to 8.0-8.4 ym (Figs. 17, 18). At the same time, the diameter
of the operculum has widened to 5.7-5.9 ym. 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

22
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 ym long.
The Apical Apparatus of Saccobolus depauperatus
The mature ascus is broadly clavate, being 55-80 ym
long and 12-15 ym 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
the ascus.
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 rmicilaginous 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

23
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 ym. 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 ym 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 ym 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 subopercalum. 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 ym
and a width of 50-55 ym (Fig. 29). The ascal wall becomes
diffusely blue when treated with Melzer's reagent. The

24
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 ym 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 ym 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.

25
The Apical Apparatus of Iodophanus granulipolaris
The diploid ascus is broadly cylindrical, being 100-
140 ym long and 15-20 ym 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 ym in length and 30-35 ym 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

26
(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-
210 nm.
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

27
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
(Fig. 50).
Discussion
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.

28
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

29
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 cytologicai 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 "pectic"
external cover and an "amyloid" inner hood or "manchón"
(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

30
comparable stage in development to that of the tip seen in
Fig. 4 for P. succosa. He apparently based his evidence on
immature asci.
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) .

31
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

32
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
A. crenulatus.
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

33
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.

Chapter I
Figures 1-10. Peziza succosa
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.
Figure 9.
Figure 10.
Tip of diploid ascus showing iodine-positive
reaction. Stained with Melzer's reagent.
XI,000.
Diffuse iodine-positive reaction at mature
ascal tip. Spore (SP). XI,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).
X7,800.
Presence of annular indentation (AI) by late
ascosporogenesis. X9,000.
Ascal tip at late ascosporogenesis. Muci¬
laginous coat (MC). Stained with silver
methenamine. X6,000.
Mature apical apparatus. Distinct annular
indentation (AI) delimits operculum (O).
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
methenamine. X20,000.
Region of dehiscent zone which is demarcated
by annular indentation (AI). X24,000.

35

Chapter I
Figures 11-
-19. Ascobolus crenulatus
Figure 11.
Young ascal tip with developing ascospores
shows distinct apical ring (AR) at this
time. Stained with Congo red. Xl,250.
Figure 12.
Mature tip is thinner-walled at region of
the operculum (0). Stained with Congo red.
X1,000.
Figure 13.
Vesiculated apex of diploid ascus with sub¬
tending ring of glycogen (G). X9,800.
Figure 14.
Ascal tip during early spore (SP) develop¬
ment. Stained with silver methenamine.
X10,500.
Figure 15.
Early formation of apical apparatus showing
differential increase in thickness of
opercular (0) wall. Annular indentation
(AI). X11,000.
Figure 16.
Inner layer (IL) and outer layer (OL) of
developing apical apparatus. Spore (SP).
Stained with silver methenamine. X10,500.
Figure 17.
Mature apical apparatus with umbonately
shaped operculum (0). X9,500.
Figure 18.
Mature apical apparatus shows layering of
opercular and subopercular (SO) regions of
the ascus. Annular indentation (AI).
Stained with silver methenamine. X8,500.
Figure 19.
Region of annular indentation in mature
apical apparatus. Inner layer (IL). Outer
layer (0L). X48,000.

37

Chapter I
Figures 20-28. Saccobolus depauperatus
Figure 20.
Developing apical apparatus with pronounced
ring. X2,000.
Figure 21.
Apical apparatus at later developmental
stage. Stained with Congo red. X2,000.
Figure 22.
Thickening of apical wall (AW) during early
spore development. Mucilaginous coat (MC).
X3,400.
Figure 23.
Mature apical apparatus with distinct
annular indentation (AI). X8,200.
Figure 24.
Region of annular indentation with zone of
dehiscence (ZD). Mucilaginous coat (MC).
X39,000.
Figure 25.
Mature apical apparatus stained with silver
methenamine. Operculum (0). Suboperculum
(SO). X6,500.
Figure 26.
Upper portion of suboperculum showing sub-
opercular flange (SF), Inner layer (IL).
Outer layer (OL). Stained with silver
methenamine. Xll,000.
Figure 27.
Incipient spore release. Episporal sac (ES)
Spore (SP). X7,60G.
Figure 28.
Dehisced ascus with outwardly extended sub-
opercular flange (SF). Ascostome (A).
Stained with silver methenamine. X6,200.

39

Chapter I
Figures 29
Figure 29.
Figure 30.
Figure 31.
Figure 32.
Figure 33.
Figure 34.
Figure 35.
35. Thecotheus pelletieri
Mature ascus stained with Congo red. X160
Ascal tip with wide apical ring (AR)
delimiting a conical operculum. XI,250.
Ascus after spore release. Ascostome (A).
X160.
Opercular region of mature ascus. Annular
indentation (AI). X5,400.
Mature apical apparatus. Operculum (0).
Suboperculum (SO). Stained with silver
methenamine. X2,000.
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
indentation. X34,000.

41

Chapter I
Figures 36-42. Iodophanus granulipolaris
Figure
36.
Uninucleated ascus stained with Melzer's
reagent. X400.
Figure
37.
Apex of uninucleated ascus subtended by
broad amorphous cylinder. One-micron
section stained with toluidine blue.
XI,000.
Figure
38.
Apical region of uninucleated ascus.
Glycogen (G). X6,100.
Figure
39.
Small lomasomes (L) at ascal tip. Endo¬
plasmic reticulum (ER). X20,000.
Figure
40.
Mitochondria (M) adjacent to large mass of
glycogen (G). X15,000.
Figure
41.
Large vacuole (V) at tip during early
ascosporogenesis. X4,700.
Figure
42.
Distal portion of apical wall (AW) stained
with silver methenamine. X4,700.

43

Chapter I
Figures 43-50.
Figure 43.
Figure 44.
Figure 45.
Figure 46.
Figure 47.
Figure 43.
Figure 49.
Figure 50.
Iodophanus granulipolaris
Mature tip stained with Congo red. XI,250.
Operculum (O) partially connected to ascus
at spore liberation. Stained with Melzer's
reagent. X400.
Thickening of lateral wall (LW) during spore
maturation. Apical wall (AW). X3,100.
Numerous small vesicles (V) at ascal tip.
X6,300.
Mature apical apparatus showing distinct
zone of dehiscence (ZD), inner layer (IL)
and outer layer (OL). Operculum (O).
Stained with silver methenamine. X13,500.
Portion of mature apical wall. X12,500.
Operculum (O) attached at spore release.
Outer layer (OL). Stained with silver
methenamine. X4,700.
Dehisced ascus with thickened inner layer
(IL). Suboperculum (SO). Stained with
silver methenamine. X8,700.

45

Figure 51.
a.
b.
c.
Chapter I
Drawings of apical apparatuses found in
iodine-positive asci.
Apical apparatus of Ascobolus furfuraceus.
Operculum (O). Coussinet (C). Redrawn from
Chadefaud (1942).
Ascal tip of Peziza plebeia illustrating layers
of the wall. Exoascus (EX). Endoascus (EN).
Bourrelet (E). Redrawn from Schrantz (1970).
Illustration made from electron microscopic
observations of the wall layering in mature
ascal tips of Ascobolus crenulatus. Outer
layer (OL). Inner layer (IL).
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).
d.

47

CHAPTER II
MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY OF THE APICAL
APPARATUSES OF REPRESENTATIVES IN THE OTIDEA-ALEURIA COMPLEX
Introduction
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
48

49
Chapter II
Table 1
Classifications of the Otidea-Aleuria Complex
Dennis (1968)
Eckblad (1968)
Huraariaceae
Pyronemaceae
Lachneae
Iodophanus
Pyronema
Sepultarla
Leucoscypha
Thricharia
Humaria
Trichophaea
Pseudoomphilia
Pyronemella
Lamprospora
Pulvinula
Caloscypha
Octospora
Leucoscypha
Coprobia
Ciliareae
Cheilymenia
Scutellinia
Scutellinia
Cheilymenia
Sphaerosporella
Neottiella
Melastiza
Anthracobia
Humaria
Tricharia
Sphaerosporella
Sepultarla
Jafnea
Fimaria
Aleurieae
Pseudombrophila
Anthracobia
Melastiza
Aleuria
Pulvinula
Coprobia
Geopyxis
Fimaria
Octospora
Psilpezia
Boudiera
Caloscypha
Lamprospora
Aleuria
Otideaceae
Geopyxis
Pustulina
Sowerbyella
Otidea
Ascosparassis
Pezizaceae
Rifai (1968)
Otideae
Humariaceae
Otidea
Barlacina
Pseudotis
Sowerbyella
Pustularia
Otideae
Otideae
Marcelleina
Lachneae

50
Table 1 (Cont'd.)
Humariaceae
Jafnea
Jafneadelfus
Nothoj afnea
Sphaerosporella
Ciliarieae
Rhizoblepharia
Scutellinia
Cheilymenia
Coprobia
Aleuriaceae
Anthracobia
Melastiza
Aleuria
Leucoscypha
Geopyxis
Octospora
Inermisia
Pulvinula
Lamprospora
Kimbrough (1970)
Otidiaceae
Otidea
Pustulina
Jafnea
Nothojafnea
Jafneadelfus
Sowerbyella
Ascosparassis
Sepultarla
Tricharia
Mycolachnea
Pseudotis
Barlaeina
Pseudombrophila
Trichophaea
Aleuriaceae
Coprobia
Cheilymenia
Aleuriaceae
Scutellinia
Geopyxis
Aleuria
Melastiza
Octospora
Anthracobia
Caloscypha
Sowerbyella
Fimaria
Leucoscypha
Inermisia
Genosperma
Rhizoblepharis
Korf (1973)
Pyronemataceae
Ascodesmidoideae
Ascodesmis
Sphaerozone
Pulparia
Jafneadelfus
Pyronematoideae
Pyronemateae
Pyronema
Karstenelleaea
Karstenella
Ascophanoideae
Geopyxideae
Geopyxis
Apapaphysaria
Pseudombrophileae
Selenaspora
Tricharina
Rhizoblepharina

51
Table 1 (Cont'd.)
Pyronemataceae
Trichophaeopsis
Pseudombrophila
Fimaria
Otideoideae
Jafneae
Jafnea
Tarzetta
Otieeae
Psilopezia
Otidea
Ascosparassis
Mycolachneeae
Nothojafneae
Geopora
Humaría
Trichophaea
Pyronemataceae
Aleurieae
Anthracobia
Hiemsia
Melastiza
Leucoscypha
Aleuria
Pseudocollema
Pulvinula
Lamprospora
Octospora
Byssonectria
Kotlabaea
Scutellinioideaea
Scutellinieae
Coprobia
Scutellinia
Cheilymenia
Sowerbyelleae
Sowerbyella
Caloscypha
Ácervus
Phaedropezia

52
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
conclusions.
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 constellatio (Berk and Br.) Boud., Scutellinia
hirta (Schwm. ex Fr.) 0. Ktze., Octospora leucoloma Hedw.
ex. S. F. Gray (as Humaria leucoloma), Humaria wrightii
(Berk, and Cooke) Boud. and Sepultaría arenosa (Fuckel)

53
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 in Fig. 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

54
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
similarly misinterpreted.

55
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

56
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
observations.
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.

57
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 O. 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.
Results
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
treated individually.
The Apical Apparatus of Otidea leporina
The mature ascus is narrowly cylindric, reaching a length
of 180-200 ym and a diameter of 9-12 ym. 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.

58
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 ym while its diameter expands to 10-15 ym
(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 ym and a uniform
thickness of 170-180 nm. The suboperculum is 3.7-4.0 ym
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 ym while
its thickness decreases by 40-60 nm. The upper extremity of

59
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 ym.
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 ym x 18-22 ym. 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
v/all (Fig. 14 ) .
Electron microscopic examination of a four-nucleated ascus
reveals for the most part the presence of a thick, lateral
v/all, 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

60
oí 480-520 run. The apex is wider, having expanded to a
diameter of 9.2-9.4 ym. 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 ym 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 ym long, has thickened to 950-980 nm at its lower ex¬
tremity where the subopercular ring has become greatly
enlarged.
The Apical Apparatus of Humaria hemisphaerica
Asci containing immature ascospores are 160-240 ym long
and 12-16 ym 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 ym) 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

61
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 ym 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

62
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 ym and a breadth of 380-420 nm.
The suboperculum is 4.9-5.2 ym long.
The Apical Apparatus of Sphaerosporella brunnea
The mature ascus is broadly cylindric (150-190 ym x
16-20 ym). The rounded to blunt tip appears thinner-walled
than the rest of the ascus (Fig. 35). The diameter of the
ascospores (13-16 ym) is greater than that of the ascal
tip (9-10 ym) . 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 ym. 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

63
(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 ym 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 ym 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

64
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 urn and a diameter of 12-16 ym. No dis¬
tinctive features are observed in the rounded ascal tip
(Fig. 51). At ascal dehiscence, the operculum, 4-5 ym 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 ym below the tip, the wall
protrudes into the ascoplasm, forming a subopercular ring.
A piasmalemmasome 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

65
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 ym and a uniform thickness of 260-
290 nm. After spore release (Fig. 57), the suboperculum,
which is 5.8-6.2 ym 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 ym x 10-14 ym (Fig.
58). The ascal wall appears to be thicker throughout the

66
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 ym 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

67
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 ym 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

68
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 ym x 8-12 ym. 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 ym and a diameter of 14-18 ym. 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

69
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 ym 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

70
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 ym. 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 ym and diameter of 14-18 ym. They are broadly
clavate and possess a conical 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 ym in length and 4-6 ym 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

71
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 ym 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

72
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 ym and a
thickness of 550-600 nm.
The Apical Apparatus of Geopyxis majalis
Mature asci are narrow and cylindric, 260-300 ym x
10-14 ym. 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 biiayered 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 ym
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

73
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 ym 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 ym, is observed with the aid of
phase-light microscopy.
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 ym. At spore release,

74
the suboperculum, which is 4.6-5.1 um long, has increased an
additional 40-60 nm (Fig. 109).
Discussion
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 imperialis. 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

75
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
of Ascozonus.
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 Plumaria 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 O. 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 0. leporina, seen with the light microscope,

76
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, O. leporina
and S. brunnea. The outer layer decreased in thickness
toward the tip while the inner layer decreased toward the

77
base. In A. melaloma, O. 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 Iodophanus 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

78
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

79
asci in Aleuria aurantia and Scutellinia hirta (Fig. HOB, 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

80
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.

81
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 winteri (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.

82
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, £>. 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, O. 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
imperialis demonstrated general similarities with the other
taxa. Although revived material was used, wall layering of

83
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
ring.
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

84
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 me1aloma
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

85
(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 O. 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 Iodophanus 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 thse taxa than shown in

86
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
operculate groups.

Chapter II
Figures 1-9. Otidea leporina
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Subapical region of mature ascus is distinctly
heliotropic. X400.
Tip of mature ascus. Spore (SP). Stained
with Congo red. XI,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
contrast. X400.
Figure 5.
Figure 6.
Figure 7.
Figure 8.
Figure 9.
Dehisced ascus with operculum (O) laterally
attached. Stained with Congo red. XI,500.
Distal portion of suboperculum. Arrows point
to region where opercular dehiscence will
occur. X37,000.
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
(O). X14,000.
Dehisced ascus showing shrunken suboperculum
(SO). Ascostome (A). Outer layer (OL) .
X15,000.

88

Chapter II
Figures 10-
-18. Jafnea fusicarpa
Figure 10.
Ascal tip during early spore formation.
Stained with Congo red. XI,000.
Figure 11.
Apical and subapical walls at later time in
development. Stained with Congo red.
XI,000.
Figure 12.
Apical region of four-nucleated ascus with
scattered small vesicles (V). Nucleus (N).
X4,000.
Figure 13.
Mature ascal tip showing slight heliotro-
pism. Spore (SP). XI,000.
Figure 14.
Ascospore is compressed within ascostome
(A) during its ejection. X500.
Figure 15.
Ascal tip during early spore wall develop¬
ment shows increased thickness at lateral
walls. Apical wall (AW). Vesicle (V).
X6,700.
Figure 16.
Broadened ascal tip at end of spore develop¬
ment. Operculum (0). X6,200.
Figure 17.
Mature apical apparatus with small sub-
apical, swollen ring (arrows). Inner layer
(IL). Outer ring (OL). Stained with silver
methenamine. X5,000.
Figure 18.
Dehisced ascus with attached operculum (0).
Suboperculum (SO). Subopercular ring (SR).
Stained with silver methenamine. X5,400.

90

Chapter II
Figures 19-
-26. Humaria hemisphaerica
Figure 19.
Young ascal tip during spore development.
Stained with Congo red. X1,000.
Figure 20.
Ascal tip with developing spores (SP) .
One-micron section stained with crystal
violet. XI,000.
Figure 21.
Thin apical wall (AW) of ascus at early
spore wall formation. X7,800.
Figure 22.
Ascal tip at later stage in spore (SP)
development. Arrows point to annular
protuberance at lateral wall (LW). X6,000
Figure 23.
Apical region of ascus remains filled at
end of spore development. Subapical ring
(SR). X7,500.
Figure 24.
Later developmental stage of apical appa¬
ratus. Arrows point to subapical ring.
Mucilaginous matrix (MM). Stained with
silver methenamine. X5,000.
Figure 25.
Portion of the lateral wall showing small
subapical ring (SR). X17,000.
Figure 26.
Region of subapical ring (SR) stained with
silver methenamine. Inner layer (IL) .
X21,000.

92

Chapter II
Figures 27-34.
Humaria hemisphaerica
Figure
27.
Mature apical apparatus at incipient spore
release. Operculum (0). Stained with
Congo red. XI,000.
Figure
28.
One-micron section of mature ascal tip shows
faint subapical ring (arrows). Stained with
toluidine blue. XI,000.
Figure
29.
Opercular region of mature ascal tip. Inner
layer (IL). Outer layer (OL). Spore (SP).
X32,000.
Figure
30.
Mature ascal tip stained with silver
methenamine. Inner layer (IL). Outer
layer (OL). X9,000.
Figure
31.
Ascospore pressed against tip of mature
ascus. Inner layer (IL). Outer layer
(OL). X8,200.
Figure
32.
Occasional protruding subapical ring (SR)
in mature ascus. Stained with silver
methenamine. X7,700.
Figure
33.
Dehisced ascus with dislodged operculum (0).
Suboperculum (SO). X8,200.
Figure
34.
Border of suboperculum and operculum
showing expanded inner layer (IL). Outer
layer (OL). X30,000.

94

Chapter II
Figures 35-
-42. Sphaerosporella brunnea
Figure 35.
Mature ascus with thin apical wall. Stained
with Congo red. XI,000.
Figure 36.
Opercula (0) are partially attached during
spore release. Stained with Congo red.
Ascostome (A). XI,000.
Figure 37.
Tip of mature ascus that has been fixed in
permanganate. Arrows point to subapical
band at level of first spore. Inner layer
(IL). Outer layer (OL). X9,000.
Figure 38.
Tip of mature ascus fixed in glutaraldehyde-
paraformaldehyde solution. Arrows point to
subopercular ring. X6,900.
Figure 39.
Region of subopercular ring (SR) showing an
outer (OS) and inner stratum (IS) of the
outer layer (OL) and an inner layer (IL).
Mucilaginous coat (MC). X26,000.
Figure 40.
Operculum (0) is demarcated by intense
staining of the outer layer (OL). Inner
layer (IL). Stained with silver methena-
mine. X10,000.
Figure 41.
Ascal tip showing suboperculum (SO),
operculum and suboperculum ring (arrows).
Stained with silver methenamine. X5,0Q0.
Figure 42.
Dehisced ascus with stretched subopercular
wall. Stained with silver methenamine.
Ascostome (A). Operculum (0). X8,300.

96

Chapter II
Figures 43
Figure 43.
Figure 44.
Figure 45.
Figure 46.
Figure 47.
Figure 48.
Figure 49.
Figure 50.
50. Aleuria aurantia
Ascus during early spore formation with
thin-walled apex. Stained with Congo red.
X1,000.
At later stage in spore development, ascus
becomes slightly heliotropic near the tip.
Stained with Congo red. XI,000.
Ascal tip during spore wall development
shows early formation of subopercular ring
(SR). Apical wall (AW). X10,500.
Plasmalemmasome is associated with develop¬
ment of the ring. Lateral wall (LW).
Endoplasmic reticulum (ER). X18,500.
Presence of thin inner layer (IL) during
spore wall development. Outer layer (OL).
Stained with silver methenamine. X8,200.
Asymmetrical formation of subopercular
ring (SR). X7,500.
Ascal tip filled with long, laminated
endoplasmic reticulum. Apical wall (AW).
Subopercular ring (SR). X13,000.
Prominent subopercular ring shown in
Fig. 49. Lateral wall (LW). X25,000

86

Chapter II
Figures 51-
-57. Aleuria aurantia
Figure 51.
Rounded tip of mature ascus lacks distinc¬
tive features. Stained with Congo red.
XI,000.
Figure 52.
Attached operculum (0) at spore release.
Phase contrast. X400.
Figure 53.
Mature tip with thickened inner layer (IL).
Outer layer (OL). Stained with silver
methenamine. X7,600.
Figure 54.
Wall layering in tip of mature ascus is
poorly distinguished when stained with lead
citrate and uranyl acetate. X10,000.
Figure 55.
Zone of dehiscence (ZD) and delimitation of
the operculum (0) is seen at onset of spore
release. Stained with silver methenamine.
X12,500.
Figure 56.
Dehisced ascus with partially attached
operculum (0). Suboperculum (SO). Zone of
dehiscence (ZD). Stained with silver
methenamine. X15,000.
Figure 57.
Spore is held tightly by subopercular wall
surrounding the ascostome during spore
discharge. Stained with silver methenamine
X8,000.

100

Chapter II
Figures 58-
-68. Anthracobia melaloma
Figure 58.
Mature ascus stained with aniline blue.
X400.
Figure 59.
Protuberance (arrow) of lateral wall below
ascal tip. Stained with aniline blue.
XI,000.
Figure 60.
Subapical ring (arrows) in mature ascus.
One-micron section stained with toluidine
blue. XI,000.
Figure 61.
Early formation of localized band (arrows)
at lateral wall of ascus during spore
development. Apical wall (AW). Spore
(SP). X9,600.
Figure 62.
Subapical band (arrows) is stained intensely
by silver methenamine. Lateral wall (LW).
X9,000.
Figure 63.
Thickened lateral wall during progressive
development of spores. Apical wall (AW).
Arrows point to subapical band. X8,600.
Figure 64.
Toward end of spore development subopercular
band becomes swollen ring (arrows).
X8,600.
Figure 65.
Mature apical apparatus shows delimitation
of suboperculum (SO). Arrows point to
subopercular ring. X8,600.
Figure 66.
Region of subopercular ring. Outer layer
(OL). External stratusm (ES). Internal
Stratum (IS). X22,000.
Figure 67.
Mature apical apparatus stained with silver
methenamine. Operculum (0). Subopercular
ring (SR). X8,600.
Figure 68.
Area of operculum showing outer layer (OL)
and inner layer (IL). X15,500.

ZOl
® ®

Chapter II
Figures 69-
-76. Scutellinia scutellata
Figure 69.
Young ascus during early spore wall
formation. XI,000.
Figure 70.
Mature ascus stained with aniline blue.
X300.
Figure 71.
Ascal tip at early spore wall formation
shows numerous, small vesicles (V) and
subapical ring (SR). X8,000.
Figure 72.
Area of subapical ring. Lateral wall
(LW). X50,000.
Figure 73.
Four-nucleated ascus with strongly stained
apical wall (AW). Stained with silver
methenamine. X3,600.
Figure 74.
Apex of eight-nucleated ascus shows
thickened mucilaginous coat (MC). Arrows
point to lomasomal-like band. Stained
with silver methenamine. X8,500
Figure 75.
Apex of four-nucleated ascus shown in
Fig. 73. Arrows point lomasomes. Muci¬
laginous coat (MC). Vesicle (V). Stained
with silver methenamine. Xll,500.
Figure 76.
Apical wall of ascus during spore wall
formation. Arrows point to stained regions
of the wall. Stained with silver methena¬
mine. X10,000.

frO T

Chapter II
Figures 77-83. Scutellinia scutellata
Figure 77.
Figure 78.
Figure 79.
Figure 80.
Figure 81.
Figure 82.
Figure 83.
Thin-walled tip of mature ascus. Stained
with Congo red. Spore (SP) . Xl.,000.
Ascus at spore release with partially
fastened operculum (O). X500.
Subopercular region of mature ascus shows
different layers and subopercular ring (SR).
Inner layer (IL). Middle layer (ML).
Outer layer (OL). Stained with silver
methenamine. X14,000.
Apical apparatus of mature ascus. Arrows
point to subopercular ring. Suboperculum
(SO). Stained with silver methenamine.
X7,100.
Ascal tip at incipient spore release.
Operculum (0). Subopercular ring (SR).
Stained silver methenamine. X7,300.
Tip of mature ascus showing thick muci¬
laginous coat (MC). X8,900.
Close-up of zone of dehiscence (ZD) in
Fig. 83. Outer layer (OL). Inner layer
(IL). Stained with silver methenamine.
X21,000.

106

Chapter II
Figures 84-
-94. Ascozonus woolhopensis
Figure 84.
Tip of ascus before development of asco-
spores. Stained with aniline blue. XI,000
Figure 85.
Mature tip stained with Congo red shows
distinct subapical ring (R). Inner layer
(IL). Outer layer (OL). XI,000.
Figure 86.
Forming apex of young ascus during early
ascosporogenesis. Ring (R). Inner layer
(IL). Outer layer (OL). X3,400.
Figure 87.
Ascal tip at later stage in development
stained with silver methenamine. Inner
layer (IL). Ring (R). X3,500.
Figure 88.
Small pore (arrow) at apex. Inner layer
(IL). Outer layer (OL). Stained with
silver methenamine. Xll,000.
Figure 89.
Peripheral section of mature tip shows
inner layer (IL), outer layer (OL) and
middle stratum (MS). X3,500.
Figure 90.
Peripheral section of ring shows outer
layer (OL), middle stratum (MS) and inner
layer (IL). X6,000.
Figure 91.
Area of ring stained with silver methena¬
mine. Inner layer (IL). Middle stratum
(MS). Outer layer (OL). X15,000.
Figure 92.
Tip of dehisced ascus with apical disc
(AD). Stained with silver methenamine.
X11,000.
Figure 93.
Portion of ring seen in Fig. 90 shows
different layers and mucilaginous coat (MC)
Inner layer (IL). Outer layer (OL).
Middle stratum (MS). X14,500.
Figure 94.
Ascus after spore release. Stained with
Congo red. X400.

108

Chapter II
Figures 95-101. Geopyxis majalis
Figure 95.
Figure 96.
Figure 97.
Figure 98.
Figure 99.
Figure 100.
Figure 101.
Tip of mature ascus. Stained with Congo
red. XI,000.
Younger ascus with thin apical wall, and
ascus at spore (SP) release. Stained with
Congo red. XI,000.
Apical wall of mature ascus shows inner
layer (IL) and outer layer (OL).
Operculum (O). X16,500.
Ascal tip with slightly less developed
ascospores. Apical wall (AW). Muci¬
laginous coat (MC). Stained with silver
methenamine. X9,600.
Tip of mature ascus stained with silver
methenamine shows inner layer (IL) and
outer layer (OL). Arrows point to apical
ring. X15,500.
Upper region of suboperculum (SO) pressing
against spore (SP). Stained with silver
methenamine. X8,500,
Ascal tip at incipient spore release.
Operculum (0). Inner layer (IL). Stained
with silver methenamine. X8,500.

on

Chapter II
Figures 102
Figure 102.
Figure 103.
Figure 104.
Figure 105.
Figure 106.
Figure 107.
Figure 10 8.
Figure 109.
109. Sowerbyella imperialis
Apical portion of mature ascus. Stained
with Congo red. XI,000.
Phase-contrast of mature tip. Operculum
(0). X1,000.
Thin-walled tip of mature ascus. Inner
layer (IL). Outer layer (OL). X15,000.
Portion of subapical wall showing inner
layer (IL) and outer layer (OL).
X31,000
Mature ascus stained with silver methena-
mine. Inner layer (IL). X15,000.
Subopercular region of mature ascus shows
thickened inner layer (IL) and tapered
outer layer (OL). Mucilaginous coat
(MC). Stained with silver methenamine.
X24,000.
Ascal tip stained with silver methenamine.
Arrows point to narrowed area of inner
layer at apex. X17,000.
Ascus after spore ejection. Suboperculum
(SO). Stained with silver methenamine.
X10,500.

112

Chapter II
Figure 110.
Apical apparatuses redrawn from Chadefaud
(1942).
A.
Operculate apical apparatus with complement
of components. Apical punctuation (ap).
Funnel (f). Pad (p). Operculum (o).
Tract (t).
B.
Apical apparatus of Aleuria aurantia at spore
release. Operculum (o). Pad (p).
C.
Apical apparatus of Scutellinia hirta at
spore release. Operculum (o). Pad (p).
D.
Rudimentary apical apparatus of Octospora
leucoloma. Globule (g). Apical spherules
(as) .

114

Chapter II
Figure
111. Illustrations made from electron microscopic
observations of apical apparatuses of the
Otidea-Aleuria complex.
E.
Wall layering of mature ascal tip in Jafnea
fusicarpa. Inner layer (il). Outer layer
(ol) .
F.
Wall layering of mature ascal tip in
Sphaerosporella brunnea. Inner layer (il).
Outer layer (ol).
G.
H.
Wall layering of mature ascal tip in
Anthacobia melaloma. Internal stratum (is)
of inner layer. External stratum (ES) of
inner layer. Outer layer (ol).
Wall layering of mature ascal tip in
Ascozonus woolhopensis. Inner layer (il) .
Middle stratum (ms). Outer layer (ol) .
H.

116

CHAPTER III
MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY OF THE APICAL
APPARATUSES OF EUGYMNOHYMENIAL REPRESENTATIVES
Introduction
Within the operculate Discomycetes workers have tradi
tionally distinguished two extreme types of apothecial
ontogeny. Enclosed development of the hymenium has been
referred to as "angiocarpic" (Corner, 1929; Snell and Dick
1957). Development with the hymenium exposed has been
referred to as "gymnocarpic" (Corner, 1930; Snell and Dick
1957). For apothecia which first begin with an enclosed
hymenium that later becomes exposed certain workers have
used the term "hemiangiocarpic" (Singer, 1951; Jackson,
1949). Other workers (Corner, 1929; Reijnders, 1948) have
included this latter type of development within the
"angiocarpic" category. Van Brummelen (1967) established
a set of terms clearly defining both stages and types of
apothecial development. Ascoma that had an enclosed
hymenium at least during its early formation was called
cleistohymenial. Van Brummelen pointed out that most rep¬
resentatives within the operculate Discomycetes have this
type of development. Exposure of the hymenium from the
onset of ascocarp development until ascal maturation was
called gymnohymenial. Few operculate members have been
117

118
shown to have this type of development. He subdivided this
type of development into paragymnohymenial and eugymno-
hymenial according to the extent of investment of the
ascogonium by overarching hyphae. Ascomata of the former
had overarched ascogonia while those of the latter did not.
Eugymnohymenial ascomata have only been found in Ascodesmis
Tiegh., Pyronema Carus and Coprotus Korf and Kimbr. outside
of several species of Ascobolus Pers. ex Fr. and a number of
species of Saccobolus Bond, (van Brummelen, 1967; Corner,
1929; Claussen, 1905, 1912; Kish, 1974). Developmental
studies of the apothecia of Ascobolus by Dodge (1912) re¬
vealed that cleistohymenial and gymnohymenial forms occurred
within a single genus. Consequently, the type of develop¬
ment only had importance at the species level. Van
Brummelen (1967) pointed out, however, that in Ascobolus a
type of development frequently was particular for different
sections of the genus, indicating greater relateness among
the species within a section. Similarly, taxonomic af¬
finities may occur among the three nonascobolaceous
eugymnohymenial taxa.
Ascodesmis, Pyronema and Coprotus have generally been
placed in separate families (Table 1). The taxonomic posi¬
tioning of the three genera has remained unclear. LeGal
(1949) proposed that Ascodesmis was highly evolved. She
based her proposal on spore sculpturing and the coprophilous
nature of the fungus. Obrist (1961) concurred with LeGal
and suggested that the genus Ascodesmis may be placed

119
Chapter III
Table 1
Classifications of the Eugymnohymenial Genera
Ascodesmis, Coprotus and Pyronema
Eckblad (1968)
Thelebolaceae
Coprotus
Ascobolaceae
Ascodesmidoideae
Ascodesmis
Pyronemaceae
Pyronema
Kimbrough (1970)
Thelebolaceae
Coprotus
Ascobolaceae
Ascodesmidoideae
Ascodesmis
Pyronemataceae
Pyronema
Korf (1973)
Pyronemataceae
Ascodesmidoideae
Ascodesmis
Pyronematoideae
Pyronemateae
Pyronema
Ascophanoideae
Theleboleae
Coprotus

120
tentatively in the family Humariaceae tribe Humarieae sensu
LeGal (1947) due to similarities in spore structure with
Boudiera Cooke and Lamprospora De Not. The idea that
Ascodesmis may have been highly evolved was not entirely
new. Corner (1930) postulated that the smallest and
simplest species including members of Pyronema, Ascodesmis
and Coprotus (as Ascophanus) were the most degenerate through
an evolutionary process of juvenescence. Eckblad (1968) did
not agree with Corner's views but instead followed
Nannfeldt's (1937) theory that pileate genera were advanced.
Like Obrist, he emphasized taxonomic affinities between
Ascodesmis and Boudiera. However, he disregarded the iodine¬
negative, nonprotruding nature of the asci of Ascodesmis
and placed both genera in the Ascobolaceae.
Taxonomic positioning of Pyronema has been unclear even
though morphological, ontogenetic and cytological data have
been abundant (Moore and Korf, 1963). Rifai (1968) pointed
out that comparable information was lacking from those
genera that previously had been considered to be related to
Pyronema. He stated that the true relationship of this
genus could only be made after the anatomy and the ontogeny
of brightly pigmented species of the Humariaceae and
Thelebolaceae were understood. Eckblad (1968) noted that
the presence of a reduced excipular margin, which had been
neglected or overlooked by earlier workers, made it impos¬
sible to place Pyronema in a separate family.

121
Taxonomic affiliations of Coprotus have been similarly
vague. Species which comprise this genus were treated at
one time or other as belonging to Ascophanus and (or)
Ryparobius (Kimbrough, Luck-Alien, and Cain, 1972). Re¬
duction of the number of asci, increase in the number of
ascospores, and uninucleate nature of the cells in para-
physes and excipulum were the main characters used to
justify placing Coprotus in the Thelebolaceae (Kimbrough and
Korf, 1967). Kimbrougn, Luck-Alien, and Cain (1969) noted
that eight-spored forms resembled members of Iodophanus,
Coprobia, Peziza, Psilopezia and Pyronema. Developmental
studies of Coprotus and Pyronema by Dangeard (1907) pointed
to important similarities in these genera including the
production of clustered ascogonia, eugymnohymenial ontogeny,
and the same type of ascogenous systems. In a cytological
investigation of Coprotus lacteus (Ck. and Phill.) Kimbr.,
Luck-Alien, and Cain, Kish (1974) stated that Coprotus
demonstrated much closer morphological, developmental and
cytological affinities with the Pyronemaceae sensu Eckblad
(1968) than with the Thelebolaceae and suggested that it be
transferred to the former family.
Studies of ascal structure have provided significant
contributions to fungal systematics within the Euascomycetes
(see General Introduction). Recently, a number of studies
have paid close attention to the ascal wall and its associ¬
ated mechanism of dehiscence (Corlett and Elliott, 1974;
Beckett and Heath, 1974; van Brummelen, 1974, 1975;

122
v
Bellemere, 1975; Samuelson, 1975). Dehiscence mechanisms
(described in Chapters I and II) offered useful information
that has helped determine more accurately the phylogeny of
certain taxa within the operculate Discomycetes.
Apical apparatuses of the eugymnohymenial members,
Ascodesmis, Pyronema and Coprotus have not been critically
examined. Although asci of Ascodesmis and Pyronema have
been examined ultrastructurally (Moore, 1963; Reeves, 1967),
little information was reported on the ascal wall. Moore
(1963) disclosed in Ascodesmis that the wall, which was
unitunicate, developed near maturity a "dehiscence septum."
The "dehiscence septum" consisted of a broad annular thick¬
ening that apparently developed transversely in the distal
region of the ascal wall. He proposed that the operculum
separated along this septum.
The present study examines morphological, developmental
and cytochemical features of the apical apparatus of
eugymnohymenial species of Ascodesmis, Pyronema and Coprotus
in order to understand more clearly their taxonomic related¬
ness within the operculate Discomycetes. Fresh specimens
of Ascodesmis sphaerospora Obrist, Pyronema domesticum (Sow.
ex Fr.) Sacc., Coprotus winteri (Marchal) Kimbr. and C.
lacteus were used.

123
Materials and Methods
Collection of Materials
Young and mature apothecia of Coprotus lacteus and
C. winteri were found on cow dung collected near Gainesville.
A culture of Pyronema domesticum was graciously given by
E. M. Landecker. A culture of Ascodesmis sphaerospora,
no. 1037, was obtained from the Culture Collection of the
Rancho Santa Ana Botanic Garden, courtesy of R. K. Benjamim.
Apothecia of P. domesticum and A. sphaerospora were grown
on PDA (Potato-Dextrose Agar) and DOA (Dung-Oatmeal Agar;
Benedict and Tyler, 1962), respectively. Different develop¬
mental stages of the apothecia of C. lacteus and C. winteri
were removed from the substrate and placed in thin layers
of solidifying water agar. Whole squash-mounts of individual
apothecia of A. sphaerospora and P. domesticum were used to
determine the stage of ascal ontogeny for the majority of
apothecia in a particular Petri plate.
Procedures for Light Microscopic Observations
Whole ascocarps of C. lacteus, C. winteria, A.
sphaerospora and P. domesticum were squash mounted mostly
in Congo red to stain the ascal walls (Samuelson, 1975) .
Aniline blue and lactophenol cotton blue were also used
(see Chapter I).
Procedures for Electron Microscopic Observations
Agar blocks, each containing 6 to 12 apothecia, of
C. lacteus, C. winteri and A. sphaerospora and entire

124
apothecia of P. domesticum were fixed in a buffered (0.2M
sodium cacodylate pH 7.2) 2.0% glutaraldehyde and 2.0%
paraformaldehyde solution for two hours at room temperature.
All materials were rinsed, postfixed in osmium tetroxide,
dehydrated, embedded, sectioned and poststained as described
in Chapter I.
Results
The Apical Apparatus of Pyronema domesticum
The mature ascus is cylindric to subcylindric ranging
in length from 135 to 190 ym and in diameter from 8 to 11 ym.
The ascal wall appears uniformly thick laterally and sub-
apically (Fig. 1). At the periphery of the tip, the wall
becomes slightly thinner. The apical wall seems to stay
thin and in most mounting agents is frequently concave in
outline (Fig. 1). At ascal dehiscence, the operculum re¬
mains attached to the ascus (Fig. 2). The subapical wall
is more constricted shortly beneath the ascostome.
At the ultrastructural level, the inner layer of the
ascal wall is seen during the final stages of spore develop¬
ment (Figs. 3, 4, 6). Most of the deposition of the inner
layer occurs in the opercular (Figs. 3, 4) region where the
outer layer has a thickness of 90-100 nm. Subapically, the
outer layer dramatically increases in thickness to 250-
270 nm (Fig. 6). A thin band of electron-dense cytoplasm
lies against the internal face of the inner layer in the
apical and subapical regions of the ascus (Figs. 4, 6).

125
By the end of the development of the apical apparatus
the opercular wall has thickened to 250-260 nm (Figs. 5, 7).
Much of the wall is comprised of the inner layer with an
overall thickness of 110-140 nm. The operculum is strongly
demarcated after treating the thin sections with silver
methenamine (Figs. 5, 7, 8). The opercular and subopercu-
lar inner layer is moderately stained (Fig. 7). The outer
layer, however, is intensely stained for most of the ascal
wall except at the point where it suddenly narrows in thick¬
ness at the tip (Figs. 5, 7), i.e., the operculum. In the
opercular region the outer layer reacts weakly with silver
methenamine offering a sharp contrast to the opercular inner
layer and the subopercular outer layer. At spore libera¬
tion, the operculum partially ruptures along this differen¬
tially stained portion of the tip (Fig. 8). The operculum
has a diameter of 7.4-7.8 ym. The suboperculum is relatively
short, 0.8-1.0 ym. The inner layer tapers from 50-60 nm at
its upper extremity to 20-30 nm, while the outer layer
broadens from 240-270 to 280-310 nm.
The Apical Apparatus of Ascodesmis sphaerospora
Mature asci are broadly clavate to ovoid, being 70-85 ym
long and 30-35 ym wide. The ascal wall which appears thin
throughout the ascus and particularly so at the tip has no
distinct features in the apical region (Fig. 9). After
ascospore discharge the operculum remains partially attached
to the ascus often returning to its original position

126
(Fig. 10). The wall region surrounding the ascostome is
hyaline when treated with Congo red (Fig. 10).
Ultrastructurally, the mature ascus is frequently ob¬
served to be asymmetrical (Fig. 11). The apical wall may
be eccentrically positioned in a heliotropic manner. The
thickness of the ascal wall narrows from 360-390 nm at its
lateral sides to 190-220 nm at the tip (Fig. 11). The inner
layer of the wall is granular and barely more electron-
transparent than the outer layer (Fig. 12). At the periphery
of the apical wall an electron-transluscent zone is observed
in the inner layer (Fig. 12), delimiting the operculum.
This zone, which is 230-310 nm long, most likely plays an
important role in ascal dehiscence. The diameter of the
operculum measures 10.5-11.1 ym.
Staining with silver methenamine markedly enhances the
appearance of the wall layering in the ascus (Figs. 13-16).
The strongly stained inner layer comprises almost one-half
of the thickness of the opercular wall, being 90-110 nm.
The inner layer tapers to 50-60 nm at the lower extremity
of the suboperculum, which is 4.8-5.4 ym long. The less
intensely stained outer layer consists of two strata (Fig.
15). The external stratum remains 60-80 nm thick throughout
the opercular and distal subopercular regions of the wall,
increasing slightly to 85-95 nm toward the base of the ascus.
The internal stratum increases from 50-60 nm in the operculum
to 250-280 nm at the lower extremity of the suboperculum.
At the region of the transparent dehiscent zone observed in

127
Fig. 12, the outer layer is less conspicuously stained
(Figs. 15, 16). At incipient spore release (Fig. 14), the
outer layer is devoid of any stain at this region and the
inner layer has become slightly indented. The strata of the
outer layer are not discerned in the vicinity of the differ¬
entially stained dehiscent zone (Fig. 14).
The Apical Apparatus of Coprotus winteri
Mature asci are broadly cylindric reading a length of
160-200 ym and a diameter of 40-55 ym. Two hundred and
fifty-six ascospores are formed within each ascus (Fig. 17).
The wall appears quite thick for most of the ascus except
at the truncate to rounded tip where the wall is considerably
thinner (Fig. 18). When placed in Congo red the ascal wall
is seen to be bilayered (Fig. 13). The outer layer is
strongly stained in all regions but the apex.
With the aid of the electron microscope, the tips of
almost mature asci (Figs. 19, 20, 21) are relatively thin-
walled, 580-620 nm. Within 1.6-1.8 ym of the apex, the
wall thickens to 1250-1300 nm (Figs. 19, 30). The apical
region of the ascus is filled with mitochondria, ribosomes
and glycogen at this stage in development. After staining
thin sections with silver methenamine, stratification of the
ascal wall is accentuated (Figs. 20, 21). The wall is com¬
prised of a single layer having three strata at this stage
(Fig. 21). The external stratum remains 90-100 nm thick for
the length of the ascus. The middle stratum decreases in
thickness from 950-1000 nm in the region of the lateral wall

128
to 450-480 nm at the apex. The internal stratum is rela¬
tively thin at this time varying from 30 to 70 nm and having
an irregular outline (Fig. 21).
By the end of spore wall development, an inner layer
is formed in the ascal wall (Figs. 22, 24, 25). This
inner layer, which is more fibrillar and electron-opaque
than the outer layer, is thickest at the tip, 190-220 nm,
and becomes reduced to 80-100 nm toward the base of the
ascus. With silver methenamine (Figs. 23, 25) the strongly
positive inner layer is sharply contrasted with the negative
outer layer. The opercular boundary is demarcated by an
annular swelling of the outer layer's middle stratum (arrows
in Fig. 25). Furthermore, the middle and internal strata
below the ring are more strongly stained. The operculum has
a diameter of 9.2-9.7 ym and a thickness of 770-820 nm. The
suboperculum, which is 5.1-5.3 ym long, thickens from 800-
820 nm at its distal end to 1500-1550 nm at its proximal end.
The increase is due to the thickening of the internal stratum
of the outer layer and the addition of the inner layer.
The Apical Apparatus of Coprotus lacteus
During early spore development, the tips of young asci
are thin-walled and round (Fig. 26). The lateral wall be¬
comes strongly broader toward the base. At maturity, the
ascus is subcylindric to clavate, 65-80 x 14-18 ym. The tip,
which becomes more blunt, remains thin-walled (Fig. 27).
At dehiscence, the operculum stays fastened at one side of

129
the ascus (Fig. 28). The wall below the ascostome becomes
constricted.
Electron microscopic observations of asci during spore
wall development demonstrate the apical wall to be notably
thin (Figs. 29-30), 200-220 nm. The lateral wall expands to
500-530 nm within a short distance from the tip. The wall
is uniformly granular and electron-transparent at this stage
in development. When staining thin sections with silver
methenamine (Fig. 30), the internal portion of the lateral
wall and the external boundary of the entire wall react
positively.
By the end of spore development, an inner layer has
formed predominantly throughout the apical and subapical
regions of the ascal wall (Fig. 31). The apical wall has
increased in thickness to 310-340 nm. The inner layer com¬
prises approximately one-half of the wall at the apex,
being 140-160 nm thick. The lateral wall has changed little
in thickness. During the release of the ascospores, the
operculum is still attached to the ascus at one side
(Figs. 32, 34, 35). The outer layer remains intact at the
point of attachment. The dehisced operculum has a diameter
of 7.5-7.7 ym and an even thickness of 420-430 nm. The
suboperculum is 6.0-6.2 ym long and increases in thickness
from 370-400 nm at the distal end to 510-540 nm toward the
base (Fig. 32) . Within the suboperculum the inner layer
decreases from 130-150 nm at the upper extremity. The wall
layering is greatly enhanced by the silver methenamine stain

130
(Fig. 33) with the inner layer being intensely stained.
The outer layer is unstained except at its external boundary
(Fig. 35). This thin portion of the outer layer, however,
is not stained at the periphery of the operculum (arrows,
Figs. 33, 35) .
Discussion
Light microscopic observations of the apical apparatus
in eugymnohymenial operculate discomycetes revealed few
notable characteristics. Subopercular rings, annular in¬
dentations, apical domes and apical rings described in
Chapters I, II and V were not observed. Mature asci of the
four representatives, however, had one major distinction.
The apices were conspicuously thin-walled. In the tips of
Coprotus lacteus and C. winteri the decrease of the sub-
apical wall toward the periphery of the apex was the sharp¬
est. Kimbrough (1970) and Kimbrough, Luck-Alien, and Cain
(1972) had previously reported this character in a number
of species of Coprotus. In Pyronema domesticum and
Ascodesmis sphaerospora the differences in thickness be¬
tween the subapical and apical regions of the ascal wall
were less notable. Although the apical walls of A.
sphaerospora tended to be more flaccid than the lateral
wall, an operculum or opercular region was not apparent.
On the other hand, the narrowing of the apical wall within
a restricted area in P. domesticum and both species of
Coprotus approximately delimited the operculum. The

131
subcylindric shape of the asci and the slight constriction
at the tips in Pyronema and Coprotus aided in the delimiting
of the opercular region. At dehiscence, the constriction of
the lateral wall was accentuated below the ascostome. Sub-
apical constrictions have not been observed in ascal tips
of other operculate species (see Chapters I, II, IV, VI) in
the present study. Their role in P. domesticum, C. winteri
and C. lacteus most likely was involved in the discharge of
the ascospores, supplying additional force during their
ejection. The apical wall in P. domesticum, which was
typically concave at maturity, resembled the ascal tip of
Otidea leporina (Fr.) Fuckel (see Chapter II). Initial ob¬
servation of the apical wall in P. domesticum gave the
impression that the wall was unusually swollen. By gradu¬
ally changing the focus of the oil-immersion objective lens
the wall was found to be thin and sunken in this region.
In O. leporina the apical swollen region was too small to
determine by this method whether the wall was similarly thin
and recessed. Fine structure of mature asci in P. domesticum
demonstrated convex apices as in 0. leporina. Light micro¬
scopic observations of the apical wall should be made
cautiously. Asci that appeared flattened or swollen at
their tips may have been dramatically influenced by mounting
and staining procedures.
Ultrastructurally, the gross morphology of the apical
apparatuses in the eugymnohymenial representatives presently
studied were similar. In each species the lateral wall

132
decreased between 80 and 100% in thickness toward the apex.
Tapering of the wall thickness was most pronounced in
Pyronema domesticum, occurring in the vicinity of the zone
of dehiscence. The inner layer of the four species de¬
creased in thickness toward the base of the ascus.
Delimitation of the opercula was greatly enhanced in
all species after staining with silver methenamine. Dif¬
ferential staining of the outer layer in P. domesticum
strongly delimited the operculum from the rest of the ascal
wall. In Coprotus winteri and Ascodesmis sphaerospora a
ring was distinguished between the suboperculum and the
operculum. At this region the outer layer, which became
slightly thickened in both species, was weakly stained by
silver methenamine in A. sphaerospora. By comparison, the
outer layer of the ascal wall of C. winteri was mostly un¬
reactive except for the boundaries of the strata and an
area immediately below the ring. In A. sphaerospora the
ring appeared to have degenerated markedly by the onset of
spore release. Unfortunately, dehisced asci were not ob¬
served with the electron microscope in either species.
Consequently, the exact point of rupture remained unclear.
The "dehiscence septum" described by Moore (1963) for A.
sphaerospora was never observed. The uneven appearance of
the circumscissle thickening in his photographs indicated
the possibility that they may have been grazing sections of
the ascal wall. Broad transverse rings would surely have
been observed by earlier workers with the light microscope.

133
The abrupt changes of the staining intensities of the outer
layer in the region of the operculum of A. sphaerospora, C.
winteri and P. domesticum indicated localized chemical
differences in the ascal wall, which most likely played a
critical role in ascal dehiscence.
Although the ascal wall of Coprotus lacteus did not
exhibit a distinct opercular ring, the outer layer's exter¬
nal boundary appeared to be cytochemically differentiated
at the region of the dehiscent zone. The shape of the
apical apparatus was almost identical to that of C. winteri.
Wall dimensions in C. winteri were roughly three times
greater than those in C. lacteus. Stratification of the
outer layer was observed best in C. winteri. Kimbrough and
Benny (1977) described comparable layering in the multi-
spored ascus of Lasiobolus monascus Kimbr. In its en¬
tirety the ascus of C. winteri was an exaggerated form of
the ascus of C. lacteus. Wall components appeared to be
highly accentuated in the multispored (greater than eight)
species. Similar findings were described in species of
Thelebolus, having different numbers of spores per ascus
(see Chapter V).
Morphological and developmental features of the apical
apparatus of Pyronema domesticum agreed more closely with
those of C. winteri and C. lacteus than with any other
operculate representative presently studied (see Chapters I,
II, IV & VI). The abrupt change in thickness between the
operculum and suboperculum and the distinct differential

134
staining of the entire outer layer of the opercular wall
clearly distinguished the apical apparatus of P. domesticum
from the apical apparatuses of the other eugymnohymenial
spp. The ascal tip of A. sphaerospora with its dehiscent
ring and stratified outer layer resembled that of C. winteri.
Apical apparatuses found in the eugymnohymenial species of
the present study were similar in form and wall thickness
to Otidea leporina (see Chapter II). The present information
supported the conclusions of Obrist (1963) , Rifai (1968) and
Kish (1974) which suggested that Ascodesmis, Pyronema and
Coprotus, respectively, were most closely related to members
of the Otideaceae and Aleuriaceae sensu Kimbrough (1970).

Chapter III
Figures 1-
-8. Pyronema domesticum
Figure 1.
Concave tip of mature ascus. Stained with
Congo red. XI,000.
Figure 2.
Dehisced ascus with attached operculum (0).
Stained with Congo red. X400.
Figure 3.
Development of apical apparatus during final
stages of spore development. Operculum (0).
Suboperculum (SO). X12,500.
Figure 4.
Opercular region of developing tip. Outer
layer (OL). Inner layer (IL). Ascal
cytoplasm (AC). X27,000.
Figure 5.
Mature apical apparatus with differentially
stained operculum (0). Arrows point to zone
of dehiscence. Stained with silver
methenamine. X9,000.
Figure 6.
Subopercular region of ascal tip shows
developing inner layer (IL). Outer layer
(OL). Suboperculum (SO). Ascal cytoplasm
(AC). X21,000.
Figure 7.
Area of dehiscent zone in mature ascus.
Inner layer (IL). Outer layer (OL). Stained
with silver methenamine. X26,500.
Figure 8.
Ruptured ascus with operculum (0) partially
fastened to ascus. Suboperculum (SO).
Stained with silver methenamine. X9,200.

136

Chapter III
Figures 9-
-16. Ascodesmis sphaerospora
Figure 9.
Apical portion of mature ascus. Stained with
Congo red. XI,000.
Figure 10.
Broad operculum (0) is attached after spore
release. Stained with Congo red. X5,000.
Figure 11.
Mature ascus with heliotropic apical wall
(AW). Lateral wall (LW). X4,500.
Figure 12.
Electron-transluscent zone (arrows) delimits
operculum. X23,000.
Figure 13.
Apical portion of mature ascus stained with
silver methenamine. Arrows point to zone of
dehiscence. Operculum (0). Suboperculum
(SO). X4,300.
Figure 14.
Zone of dehiscence (arrows) at incipient
spore release. Stained with silver
methenamine. X21,000.
Figure 15.
Region of zone of dehiscence showing inner
layer (IL) and external stratum (ES) and
internal stratum (IS) of outer layer.
Stained with silver methenamine. X19,000.
Figure 16.
Ascal tip stained with silver methenamine
shows layering of operculum (0) and suboper¬
culum (SO). Arrows point to zone of
dehiscence. X7,400.

138

Chapter III
Figures 17-
-25. Coprotus winterii
Figure 17.
Mature ascus containing approximately 250
ascospores. Stained with Congo red. X400
Figure 18.
Apical portion of mature ascus shows
operculum (0). Stained with Congo red.
XI,500.
Figure 19.
Ascal tip near end of spore development
has thin apical wall (AW). X5,000.
Figure 20.
Ascal tip near end of spore development
stained with silver methenamine. Lateral
wall (LW). X3,8 00.
Figure 21.
Apical region of almost mature ascus shows
stratified wall. External stratum (ES).
Middle stratum (MS). Internal stratum
(IS). Stained with silver methenamine.
X8,000.
Figure 22.
Mature ascal tip. Inner layer (IL).
X5,000.
Figure 23.
Mature apical apparatus with operculum (0)
and suboperculum (SO). Arrows point to
zone of dehiscence. Stained with silver
methenamine. X4,000.
Figure 24.
Figure 25.
Region of dehiscent zone showing inner
layer (IL) and outer layer. Operculum
(0). X16,000.
Region of dehiscent zone stained with
silver methenamine. Arrows point to
annular swelling (zone of dehiscence).
Outer layer consists of external stratum
(ES), middle stratum (MS) and internal
stratum (IS). Inner layer (IL). Sub¬
operculum (SO). X16,000.
Figure 25.

140

Chapter III
Figures 26-
-35. Coprotus lacteus
Figure 26.
Ascal tip during early spore development.
Stained with Congo red. XI,000.
Figure 27.
Apex of mature ascus. Stained with Congo
red.
Figure 28.
Partially attached operculum (0) at spore
liberation. Stained with Congo red.
XI,000.
Figure 29.
Ascal tip during spore wall development.
Apical wall (AW). X6,300.
Figure 30.
Ascal tip during spore wall development
stained with silver methenamine. Lateral
wall (LW). X8,100.
Figure 31.
Presence of inner layer (IL) in apical wall
of mature ascus. X11,000.
Figure 32.
Operculum remains attached to ascus during
spore release. Ascostome (A). Suboperculum
(SO). X5,0 0 0.
Figure 33.
Dehisced ascus stained with silver methena¬
mine. Arrows point to unstained area of
the outer layer's external boundary.
Operculum (0). Inner layer (IL). Outer
layer (OL). X6,800.
Figure 34.
Region of operculum (0) and zone of de¬
hiscence shown in Fig. 32. Inner layer
(IL). Outer layer (OL). Xl5,000.
Figure 35.
Dehisced ascus with attached operculum.
Arrow points to unstained external boundary
of the outer layer at the periphery of
operculum. Stained with silver methena¬
mine. X7,100.

142

CHAPTER IV
MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY OF THE APICAL
APPARATUSES OF MORCHELLA ESCULENTA AND REPRESENTATIVES
OF THE HELVELLACEAE
Introduction
Among the wide variety of representatives found within
the operculate Discomycetes, members with large, stipitate
variously shaped apothecia have been usually placed in the
Helvellaceae and Morchellaceae sensu Rifai (1968), Kimbrough
(1970) and Korf (1973). Ascospores, ascal and excipular
characters have clearly distinguished the taxa, Morchella
Dill, ex Fr., Verpa Sw. ex Fr. and Disciotis Boud., of the
Morchellaceae from the rest of the Pezizales. Placement of
genera within the Helvellaceae, however, has currently dif¬
fered among authors on the basis of which characters are
emphasized (Table 1). LeGal (1947) classified Piscina (Fr.)
Fr. and Rhizina Fr. ex Fr. in the Pezizaceae due to their
apiculate or ornamented ascospores. Berthet (1964), however,
demonstrated that a number of characters such as the unique
tetranucleated and guttulated ascospores, the habit, habitat
and pigmentation of the apothecia were features of these
genera that were shared with Helvella L., Wynnella Boud. and
Gyromitra Fr. Consequently, he included Piscina and Rhizina
in the Helvellaceae. Eckblad (1968) removed Piscina, Rhizina,
143

144
Chapter IV
Table 1
Classifications of the Helvellaceae
LeGal (1947)
Kimbrough (1970)
Helvellaceae
Helvellaceae
Helvelleae
Underwoodia
Helvella
Helvella
Leptopodia
Cyanthiopoda
Acetabula
Macropodia
Discina
Rhizina
Neogyromitra
Gyromitra
Wynnella
Wynnelleae
Rifai (1968)
Wynne11a
Helvellaceae
Physomitreae
Discina
Physomitra
Rhizina
Neogyromitra
Aleuriaceae
Gyromitra
Helvella
Discineae
Wynnella
Rhizina
Gyromitra
Discina
Korf (1973)
Helvellaceae
Eckblad (1968)
Helvelleae
Helvellaceae
Underwoodia
Helvella
Wynnella
Wynnella
Helvella
Gyromitreae
Rhizinaceae
Discina
Rhizina
Gyromitra
Pseudorhizina
Gyromitra
Discineae
Rhizina
Discina

145
Gyromitra and Pseudorhizina Jacevskij from the Helvellaceae
and placed them in a separate family, Rhizinaceae, on the
strength of their spore markings and unilayered excipulum.
Kimbrough (1970) pointed out that the characteristic spore
markings used in part by Eckblad for the erection of the
Rhizinaceae were present on species of Helvetia and
Underwoodia Peck as well. He concluded that the characters
associated with the Rhizinaceae sensu Eckblad (1968) were
insufficient to warrant the formation of this family.
During the last three decades the structure of the as-
cus and the mechanism of spore release have received a con¬
siderable amount of attention in the Discomycetes (Bellemére,
1969, 1975; van Brummelen, 1974, 1975; Chadefaud, 1940, 1942,
1969, 1973; Corlett and Elliot, 1974; LeGal, 1946a,b, 1953;
Moore, 1963; Samuelson, 1975; Schrantz, 1970; and Wells,
1972). Chadefaud (1942) and LeGal (1946b) were the first
authors to report the phylogenetic importance of the compo¬
nents associated with ascal dehiscence, which both workers
called the apical apparatus. Chadefaud (1942) described
six variations of the operculate apical apparatuses. The
apical apparatus of Helvetia sulcata Afz. was one of the
variations, having differed from the others on two points.
First, the apical tract was very thin and taut and did not
give rise to an apical punctuation. Secondly, in a young
state the ascospores were surrounded by abundant amounts of
osmiophilic lipid globules. He suggested that the tract may
connect the ascospores to the apical apparatus and

146
participate in their nutrition during the course of their
development. Chadefaud (1949) later investigated the ascal
characters of the Morchellaceae. Apical structures in asci
of Disciotis venosa (Pers. ex Fr.) Boud. and Morchella
rotunda (Fr.) Boud. were found to be consistent with the
operculate type (see Chapter II). He noted the presence of
lipid globules around the spores throughout ascosporogenesis
although they were less abundant than in H. sulcata.
Chapters I, II and III of the present study have shown
that the apical apparatus of the operculate ascus has been
useful as a systematic tool. Although cytoplasmic compo¬
nents of the operculate apical apparatus proposed by
Chadefaud (1942) were infrequently observed with the light
microscope and not detected with the electron microscope
(see Chapter II), other characters such as the length and
thickness of the opercular and subopercular ascal walls, the
layering and staining of the wall and the presence of a
subopercular ring proved useful in distinguishing the apical
apparatuses of different taxa. In addition, the fine struc¬
ture of ascal tips revealed certain cytoplasmic features
which were peculiar to certain representatives (see Chapters
I and II). The broad cylinder of glycogen in Iodophanus
granulipolaris Kimbr. and the large, laminated endoplasmic
reticulum in Aleuria aurantia (Oed. ex Fr.) Fuckel were not
seen in apices of asci of any other representative currently
studied. Chadefaud's (1942, 1949) description of lipid
globules in members of the Morchellaceae and Helvellaceae

147
indicated similarly that this substance could be peculiar
to species within the two families and thus have taxonomic
significance.
In the present study I examined the morphology, de¬
velopment and cytochemistry of the apical apparatuses in
Helvetia crispa (Scop.) Fr., Morchella esculenta (L) Pers.,
Gyromitra infula (Schaeff. ex Fr.) Quel., Piscina ancilis
(Pers.) Sacc. and Rhizina undulata Fr. ex Fr. Comparisons
between the species were made in order to determine degrees
of similarities and differences.
Materials and Methods
Collection of Material
Fresh apothecia of Helvetia crispa were collected at
the Devil's Millhopper five miles north of Gainesville,
Florida. The material was brought to the laboratory where
free-hand sections were made for light microscopic examina¬
tion to determine the stage of ascal development.
Dried specimens of Morchella esculenta, Rhizina undulata
and Discina ancilis were obtained from the Mycological
Herbarium at the University of Florida. Dr. R. P. Korf of
Cornell University generously contributed dried specimens
of Gyromitra rufula. Portions of apothecia were revived at
room temperature in distilled water for 6 to 12 hours in a
moist chamber for light and electron microscopic observa¬
tions.

148
Procedure for Light Microscopic Examinations
Fresh and revived apothecia were divided into blocks,
sectioned and mounted on slides as described in Chapter I.
The stain, Congo red, was most frequently used to examine
the ascal walls (Samuelson, 1975). Aniline blue and
lactophenol cotton blue were used to observe cytoplasmic
detail (see Chapter I).
Procedures for Electron Microscopic Examinations
Five millimeter squares of fresh apothecia of H. crispa
and revived apothecia of R. undulata, D. ancilis and G.
rufula were fixed in buffered (0.2M sodium cacodylate,
pH 7.2) 2.0% glutaraldehyde and 2.0% paraformaldehyde solu¬
tion for two hours at room temperature. Five millimeter
squares of M. esculenta were fixed in 1.0% permanganate
solution for one hour at room temperature. All materials
were postfixed in osmium tetroxide, dehydrated, embedded,
sectioned and poststained as described in Chapter I.
Results
Descriptions of the apical apparatus for each species
are restricted to the operculum, zone of dehiscence and the
suboperculum. Each representative is treated individually.
The Apical Apparatus of Helvella crispa
Asci with young ascospores have numerous small to large
lipid droplets at the tip and around each spore (Fig. 1,
arrow). The wall appears uniformly thin throughout the
upper half of the ascus. At maturity (Fig. 2), the ascus is

149
cylindric, reaching a length of 270-290 yin and a diamter
of 14-18 ym. As the ascus approaches dehiscence, the apical
wall becomes stretched and slighly flattened (Fig. 4). A
hyaline ring is observed at the periphery of the tip at
this time, demarcating the edge of the operculum. During
spore release the operculum remains fastened to one side
of the ascus (Fig. 5).
Electron microscopic observations of asci during early
spore development show numerous moderately-sized lipid
bodies surrounding the spores (Fig. 3). A large mass of
glycogen is found in the apical region of the ascus above
the lipid bodies (Fig. 3). The ascal wall is evenly
electron-transparent and broadens to a small extent from
240-250 nm at the tip to 310-330 nm at the lateral wall.
Toward the end of spore maturation (Fig. 6), lipid bodies
are infrequently seen. The distal region of the ascus is
filled mostly with the ascal vacuole, which is encased by
a thin layer of ascal cytoplasm (Figs. 6, 7). The apical
and lateral wall remain electron-transparent and do not
change significantly in thickness (Fig. 6). At a slightly
later stage in development (Fig. 7) the apical wall has
increased to a thickness of 320-350 nm. Staining with
silver methenamine greatly enhances the appearance of the
wall layering of the mature ascus (Fig. 7). The inner layer,
which is strongly positive (Figs. 8, 10), is 120-130 nm
thick at the operculum and upper extremities of the sub¬
operculum and decreases to 40-50 nm at the lower extremity

150
of the suboperculum (Figs. 7, 9). The suboperculum is
6.0-6.5 ym long and thickens from 300-320 nm at its distal
region to 370-400 nm at its base. The operculum is 5.2-
5.4 ym in diameter and is bordered by an unstained area of
the inner layer (Fig. 8, arrows). At ascal dehiscence the
operculum stays partially attached to the suboperculum
(Fig. 9). Rupture of the operculum occurs at the basal end
of the unstained ring (Figs. 9, 10). The opercular inner
layer and the internal region of the opercular and subopercu-
lar outer layer are distinctly less stained by silver
methenamine at this time. The operculum and the upper ex¬
tremities of the suboperculum have increased in thickness
by 14-18% and decreased in length by 12-15%.
The Apical Apparatus of Morchella esculenta
Apices of asci during early spore formation are rounded
and uniformly thin-walled (Fig. 11). The mature ascus is
long and cylindric to subcylindric, 200-240 x 18-20 ym.
The apical and subapical wall is more richly stained in
aniline blue than the younger tips and appears to be
thicker-walled (Fig. 12).
Ultrastructurally, the ascal wall during early spore
development is 290-310 nm thick at the tip and increases to
540-560 nm towards the base (Fig. 13). A thin mucilaginous
coat covers the entire ascus. A small mass of glycogen is
frequently observed below the tip at this stage. Lipid
bodies are scattered throughout the upper portion of the
ascus (Fig. 13). As the spores mature an inner layer is

151
formed, being prominent at the apical region of the ascus
(Fig. 14). The inner layer consists of two strata in the
opercular area (Figs. 14, 15). The internal stratum, GO¬
TO nm thick, ends in an electron-transluscent ring at the
periphery of the tip, delimiting the operculum (Fig. 15).
The external stratum is 90-100 nm thick and more electron-
dense than the outer layer and the internal stratum of the
inner layer. Below the electron-transluscent ring the two
strata are indistinguishable (Fig. 15). The operculum has
a diameter of 6.0-6.2 ym and a thickness of 400-420 nm. The
suboperculum is 4.0-4.5 ym long and thickens from 380-400 nm
at its upper extremity to 500-520 nm at its lower extremity.
The inner layer tapers to a thickness of 30-40 nm toward the
base of the ascus. The ascal wall is poorly stained when
treated with silver methenamine except for the internal
stratum of the inner layer at the operculum (Figs. 16, 18).
At incipient spore release the operculum starts to rupture in
the outer layer of the wall (Fig. 17).
The Apical Apparatus of Rhizina undulata
Mature asci are cylindric to subcylindric, 250-280 x
14-18 ym. The lateral wall, which is intensely stained in
Congo red, is thick-walled (Fig. 19). At ascal dehiscence
the long apiculate ascospores pass through a relatively
small ascostome (Fig. 21). As each spore is released from
the ascus it is squeezed by the distended ascostome, which
exerts additional force during ejection.

152
Ultrastructurally, the mature ascus has a thick lateral
wall (Figs. 22, 24), 460-500 nm, which narrows in thickness
to 280-300 nm at the apex. The electron-transparent inner
layer is thickest at the opercular region of the ascal wall
(80-90 nm) and tapers toward the base of the ascus to 40-
50 nm. The outer layer consists of two strata (Figs. 23,
24). The internal stratum is electron-dense at the tip,
being 100-120 nm thick, and becomes electron-transparent
toward the base of the ascus (Fig. 24), having thickened to
320-340 nm. The external stratum, which is apically less
electron-opaque, decreases in thickness from 80-90 nm at
the tip to 60-70 nm toward the base of the ascus (Figs. 23,
24). Treating the sections with silver methenamine reveals
differential staining of the apical wall (Fig. 22). The
inner layer is mildly stained throughout the length of the
ascus. The internal stratum of the outer layer, however,
is intensely positive at the opercular region and becomes
l
weakly stained toward the base of the ascus. The external
stratum of the outer layer is the least stained portion of
the ascal wall.
At spore release the operculum stays partially fastened
to the ascus (Fig. 20), not breaking entirely from the outer
layer at the zone of dehiscence. The suboperculum at this
stage is 4.0-4.5 ym long and increases in thickness from
310-340 nm at the distal end to 500-540 nm toward the base.
The operculum is roughly 5.0-5.5 ym long.

153
The Apical Apparatus of Piscina ancilis
Mature asci are long and cylindric, 320-360 x 14-18 pm.
The lateral wall appears thick and is intensely stained by
Congo red (Figs. 25, 28). In contrast, the apical wall is
thinner and less richly stained (Fig. 28, arrows). At the
periphery of the tip where the thickness of the wall de¬
creases perceptibly, the wall is slightly indented, delimit¬
ing the operculum (Fig. 28). Prior to dehiscence the
apiculate end of the first ascospore lies closely appressed
against one side of the opercular edge, most likely aiding
in the rupture of the operculum (Fig. 26).
Electron microscopic observations of apices of mature
asci show the wall to be relatively thin, 300-320 nm and
bilayered (Figs. 27, 29). Subapically, the wall broadens
to 520-540 nm (Figs. 30, 32). The entire wall is covered
with a thin mucilaginous coat which thickens from 30-40 nm
at the lateral face of the ascus to 90-100 nm at the tip
(Figs. 27, 29). Both layers of the wall are electron-
transparent with the inner layer appearing more fibrillar
(Fig. 29). When sections are poststained with silver
methenamine, wall layering is clearly distinguished (Figs. 30-
33). The strongly stained inner layer thickens from 80-90
nm at the lateral region of the wall to 190-200 nm at the
tip (Fig. 30). Conversely, the outer layer, which is weakly
positive, narrows from 400-450 to 100-120 nm (Fig. 32). The
ascal wall, which is not as rigid as that observed in
Rhizina undulata, is particularly flaccid at the tip

154
(Figs. 27, 30, 33). The suboperculum is roughly 7.0-9.0 ym
long. The area of the suboperculum surrounding the ascostome
seems to be distinctly elastic staying constricted after
dehiscence (Fig. 33).
The Apical Apparatus of Gyromitra rufula
Mature asci are cylindric, having a length of 180-
210 ym and a diameter of 12-15 ym. Both the apices and the
lateral regions of the ascus appear thin-walled (Fig. 34).
During spore ejection the operculum, which is only 4-5 ym
wide, is attached to one side of the ascus (Fig. 35). The
ascostome, being slightly wider than the diameter of the
operculum, is 6-7 ym across.
Ultrastructural examination of asci during early spore
development reveals the presence of numerous, small lipid
bodies scattered throughout the upper half of the ascus
(Figs. 36, 37). The electron-transparent ascal wall tapers
from 220-240 nm at its lateral sides to 110-130 nm at the
tip. When staining with silver methenamine most of the wall
is weakly positive at this stage in development (Fig. 37).
The innermost boundary of the wall is moderately stained.
In mature asci the wall is bilayered in the apical and
subapical portion of the ascus (Fig. 38). The inner layer
is very electron-transparent and 80-90 nm thick for much of
the apical region (Fig. 40), tapering to 40-50 nm toward the
base of the ascus. The apical wall as a whole is 150-160 nm
thick, being very flaccid in form. The outer layer, which
is electron-dense, is thinnest in this region and more than

155
doubles in thickness at the lateral sides where the wall
reaches its maximum thickness, 220-240 nm. After treating
with silver methenamine (Fig. 39), appearance of the wall
layering is particularly enhanced along the lateral wall
where discernment of the inner and outer layers in sections
poststained with uranyl acetate and lead citrate was dif¬
ficult. The inner layer is strongly stained and the outer
layer is moderately stained for the entire length of the
ascal wall (Fig. 41). Changes in the staining intensity of
either layer and the appearance of a zone of dehiscence do
not occur. Consequently, opercular and subopercular
boundaries are not delimited at any time.
Discussion
In the present study light microscopic examination of
the ascal tips demonstrated differences in wall thickness
and staining intensity between the representatives. Walls
in both Helvetia crispa and Gyromitra rufula were uniform
in thickness for most of the length of the ascus, being
slightly thinner at the tip. The ascal tip of Morchella
esculenta differed little from that of H. crispa and G.
rufula except in having a generally thicker wall. By com¬
parison, the thickness of the ascal wall in Piscina ancilis
and Rhizina undulata varied considerably from the lateral
side to the apex.
Opercular delimitation was best exhibited by D. ancilis
and R. undulata due to the weak staining of the ascal tip

156
in Congo red and the narrowing of the wall's thickness in
that region. Demarcation of opercula prior to ascal de¬
hiscence was also observed microchemically in H. crispa.
The faint hyaline ring in H. crispa resembled that seen in
Peziza succosa Berk, (see Chapter I). As in P. succosa
the ring was thought to have been the result of either a
chemical differentiation in the wall or a physical thinning
of the wall. Ultrastructural evidence demonstrated that
the former was the case.
Lipid bodies were detected most clearly in asci of
H. crispa and to a lesser extent in G. rufula and M.
esculenta. Unfortunately, the presence of lipid bodies
could not be determined in D. ancilis and R. undulata since
young developmental stages of asci were not examined.
Nevertheless, the present findings concurred with Chadefaud's
(1942, 1947) observations and strengthened the possibility
that the unique occurrence of lipid bodies in developing
asci of members of the Morchellaceae and Helvellaceae may
be taxonomically significant.
Electron microscopic examination of apical apparatuses in
the present study revealed general similarities in structure
and development. In all species the inner layer of the
ascal wall thickened toward the apex while the outer layer
became narrower in the same direction. In each case the
entire thickness of the wall increased toward the base. For
R. undulata and D. ancilis the difference in thickness
between the apical and lateral wall was more pronounced

157
than for the rest of the species. Development of the apical
apparatus in H. crispa, M. esculenta and G. rufula basically
followed the same pattern related for the iodine-positive
ascus in Chapter I and the asci of the Otidea-Aleuria complex
and eugymnohymenial species in Chapters II and III, respec¬
tively, where formation of the inner layer occurred during
late ascosporogenesis.
Ultrastructurally, distinct differences among the
apical apparatuses were most often observed in the layering
and cytochemistry of the wall. Delimitation of opercula in
intact asci was demonstrated only in H. crispa and M.
esculenta where a zone or region of dehiscence was present
in the inner layer. Similar dehiscent zones were detected
cytochemically in apical apparatuses of eugymnohymenial
representatives (see Chapter III). However, they were
found to be restricted in the outer layer of the ascal wall
in those species. Rupture of the operculum from the sub¬
operculum in BR crispa along the lower extremity of the
dehiscent zone was identical to that described for the
iodine-positive and eugymnohymenial species (Chapters I and
III). The wall of the ascus was stratified in those species
of the present study which formed the thickest walls, i.e.,
M_. esculenta and R. undulata. In both species the strati¬
fication was most conspicuous at the tip. Wall layering in
G. rufula and D. ancilis appeared to be less complex than
in the rest, not having recognizable zones of dehiscence
and strata within the layers. Still, it must be kept in

158
mind that revived material was used for the examination of
the ascal tips of G. rufula and D. ancilis. Wall features
such as stratification and the presence of a zone of de¬
hiscence may have been lost in the apical apparatuses of
these species.
Characters of the apical appratuses of H. crispa and
M. esculenta, including the presence of a distinct zone of
dehiscence and the small variability in wall thickness of
the ascal tip, suggest greater taxonomic relatedness between
these species than with any other operculate group (see
Chapters I, II, III, VI) and support the belief held by most
authors that places the families Helvellaceae and Morchel-
laceae close together (Eckblad, 1968; Rifai, 1968; Korf,
1973). Except for the differences in wall thickness in
G. rufula and D. ancilis, which may be in part due to the
differences of the sizes of their respective asci, the
apical apparatuses of G. rufula and D. ancilis are very
similar, strengthening the conclusion that the two genera
are closely related (LeGal, 1947; Berthet, 1964; Eckblad,
1968).
The apical apparatus of R. undulata is the most unique
of the species presently studied. The unusual staining
properties of the apical wall at both the light and electron
microscopic level distinguished this representative from
not only the other four members described in this chapter
but from the rest of the operculate species discussed in

159
Chapters I, II, III and VI. Although Rhizina and Discina
are taxonomically closely related, comparative analysis of
the apical appratuses of R. undulata and D. ancilis does not
provide additional support for their affiliation.

Chapter IV
Figures
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8.
Figure 9
-10. Helvella crispa
Apical portion of ascus with young asco-
spores. Arrows point to lipid droplets.
Stained with aninline blue. XI,000.
Apical portion of mature ascus. Stained
with aniline blue. X640.
Ascal tip during early spore development.
Lateral wall (LW). Lipid body (LB).
X7,100.
Before dehiscence ascal tip is stretched.
Arrows point to faint, hyaline ring. Stained
with Congo red. Xl,000.
Dehisced ascus with operculum (O) partially
attached to ascus. Stained with Congo red.
X640.
Ascal tip toward end of spore (SP) develop¬
ment. Apical wall (AVi). X6,300.
Apex of mature ascus stained with silver
methenamine shows operculum (O) and sub¬
operculum (SO). X6,000.
Operculum of mature ascus with inner layer
(IL) and outer layer (OL). Arrows point to
zone of dehiscence. Ascal cytoplasm (AC).
Stained with silver methenamine. X17,000.
Dehisced ascus shows differentially stained
operculum (0) partially attached to sub¬
operculum (SO). Stained with silver
methenamine. X7,000.
Peripheral section of dehisced ascus shows
rupture occurs at lower end of unstained
zone of dehiscence. Stained with silver
methenamine. X17,000
Figure 10.

161

Chapter IV
Figures 11-18. Morchella esculenta
Figure 11.
Figure 12.
Figure 13.
Figure 14.
Figure 15.
Figure 16.
Figure 17.
Figure 18.
Apical portion of ascus during early spore
formation. Stained with aniline blue.
XI,000.
Apical portion of mature ascus. Stained
with aniline blue. XI,000.
Tip of ascus during early spore develop¬
ment. Apical wall (AW). Glycogen (G).
Lipid body (LB). Mucilaginous coat (MC).
X7,300.
Ascal tip at later stage in spore develop¬
ment. Operculum (0). Suboperculum (SO).
X6,9 00.
Opercular region with arrows pointing to
zone of dehiscence. Opercular inner layer
(IL) consists of external stratum (ES)
and internal stratum (IS). Outer layer
(OL). X13,000.
Ascal tip during late spore development
stained with silver methenamine. Operculum
(O). X6,900.
Arrows point to rupturing outer layer at
onset of spore release. X8,500.
Opercular region shows only internal
stratum (IS) of inner layer is stained
by silver methenamine. Xll,500.

®* *
163

Chapter IV
Figures 19-24. Rhizina undulata
Figure 19.
Figure 20.
Figure 21.
Figure 22.
Figure 23.
Figure 24.
Tip of mature ascus shows hyaline apical
wall and strongly stained lateral wall (LW)
Stained with Congo red. XI,000.
Ascal tip at spore liberation with
operculum (0) still fastened to sub¬
operculum (SO). Zone of dehiscence (ZD).
Xll,000.
Dehisced ascus with spore (SP) passing
through ascostome. Stained with Congo red
XI,000.
Apex of mature ascus stained with silver
methenamine. Inner layer (IL). Outer
layer (OL). Spore (SP). Xll,000.
Opercular region shows outer layer is
comprised of external stratum (ES) and
internal stratum (IS). Inner layer (IL).
X22,000.
Apical region of mature ascus. Operculum
(0). Suboperculum (SO). X9,500.

165

Chapter IV
Figures 25-33. Piscina ancilis
Figure 25.
Apical portion of mature ascus with thick
lateral wall (LW). XI,000.
Figure 26.
Figure 27.
Figure 28.
Figure 29.
Figure 30.
Figure 31.
Figure 32.
Figure 33.
Tip of ascospore is lodged against the
inner face of operculum. Stained with
Congo red. X500.
Tip of mature ascus. Lateral wall (LW).
X8,300.
Apical portion of mature ascus. Arrows
point to opercular region. Stained with
Congo red. XI,000.
Portion of opercular wall. Inner layer
(IL). Outer layer (OL). Mucilaginous
coat. (MC). X28,000.
Mature ascus stained with silver methenamine
shows thickened inner layer at apical wall
(AW). X5,000.
Ruptured ascus at incipient spore release.
Stained with silver methenamine. Operculum
(0). Suboperculum (SO). X4,100.
Subopercular portion of ascus. Inner
layer (IL). Outer layer (OL). Stained
with silver methenamine. X18,000.
Dehisced ascus with spore (SP) below
ascostome (A). Stained with silver
methenamine. X6,100.

L 91

Chapter IV
Figures
; 34-41.
Gyromitra rufula
Figure
34.
Apex of mature ascus. Stained with Congo
red. XI,000.
Figure
35.
Operculum (0) is laterally attached during
spore release. Stained with Congo red.
XI, 000.
Figure
36.
Apical tip during early spore development.
Lateral wall (LW). Glycogen (G). Lipid
body (LB). X8,000.
Figure
37.
Ascal tip during early spore development
stained with silver methenamine. Apical
wall (AW). Lipid body (LB). X7,700.
Figure
38.
Tip of mature ascus shows bilayered apical
wall (AW). X11,000.
Figure
39.
Tip of mature ascus stained with silver
methenamine. Lateral wall (LW). X8,100.
Figure
40.
Opercular region of mature tip. Inner
layer (IL). Outer layer (OL). X14,500.
Figure
41.
Opercular region of mature tip stained with
silver methenamine. X24,500.

169

CHAPTER V
MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY OF THE APICAL
APPARATUS OF THELEBOLUS
Introduction
In a study concerned with the taxonomic position of
Trichobolus zukalii Heimerl within the operculate Dis-
comycetes, Kimbrough (1966b) compared microscopical and
microchemical examinations of representatives of Ryparobius
Boud., Ascozonus (Renny) Boud., Thecotheus Boud., Lasiobolus
Sacc., Ascophanus Boud. and Thelebolus Tode. These taxa all
possessed representatives that formed asci which contained
more than eight ascospores in each and are referred to here
as being multispored. He demonstrated that characters which
had been used at that time to separate these genera, such as
the number of ascospores per ascus and the number of asci
per ascocarp, were artificial and that true relationships
were observed to cut across traditional generic lines.
Kimbrough and Korf (1967) abandoned the name Pseudo-
ascoboleae and replaced it with Theleboleae, transferring
this tribe to the Pezizaceae. They incorporated within this
tribe those taxa which had (1) eight and multispored, non¬
amyloid, operculate or irregularly dehiscing asci which
generally protruded at maturity; (2) hyaline ascospores;
170

171
(3) minute, glabrose to setose apothecia; (4) coprophilous
habitat. The manner in which the spores were released proved
to be the most distinctive feature and was implemented to
help characterize certain genera. Ascal dehiscence occurred
by either an irregular tear, bilabiate split, apical plug,
evanescence or circumscissile rupture. Kimbrough (1966b)
relied on the staining properties of the ascal wall and the
associated mechanism of dehiscence to form more natural
groupings. As a result four new genera, Coprobolus,
Iodophanus, Trichobolus and Coprotus, were erected. Two old
genera, Ryparobius and Ascophanus were invalidated and the
genera Thelebolus and Lasiobolus were extended (see Table 1).
The type of apical apparatus for each genus was believed to
be consistent for all of its species. The work performed
by Kimbrough (1966a, b; Kimbrough and Korf, 1967; Cain and
Kimbrough, 1969) on the Pseudoascobolaceae and related taxa
was a major contribution to the taxonomy of this group of
operculate Discomycetes.
The different types of apothecial development, cleisto-
hymenial in Thelebolus, Trichobolus and Lasiobolus and
eugymnohymenial in Coprotus and possibly Ascozonus, and the
wide range of apical apparatus types found in the Thele-
bolaceae (sensu Eckblad, 1963; Kimbrough, 1970) have indi¬
cated evolutionary convergence along several more lines. Of
the seven genera presently included in the Thelebolaceae
sensu Rifai (1968) only Lasiobolus and Coprotus have been
shown to be truly operculate. Recent developmental studies

172
Chapter V
Table 1
Classifications of the Thelebolaceae
Boudier (1907)
Dennis (1968)
Ascobolaceae
Ascobolaceae
Pseudoascoboleae
Pseudoascoboleae
Boudierella
Cubonia
Thecotheus
Ascophanus
Lasiobolus
Ryparobius
Ascozonus
Thelebolus
Aphanoascus
Lasiobolus
Iodophanus
Thecotheus
Ascophanus
Ascodesmis
Ascozonus
Pyronema
Ryparobius
Sphaeridiobolus
Eckblad (1968)
Thelebolaceae
Thelebolus
Thelebolaceae
Thelebolus
Caccobius
Coprobolus
Ascozonus
Coprotus
Thecotheus
Trichobolus
Lasiobolus
Leporina
Kimbrough (1970)
Thelebolaceae
Ascozonus
Caccobius
Caprobolus
Coprotus
Lasiobolus
Thelebolus
Trichobolus

173
(Conway, 1975; Kish, 1974) proposed the transfer of both
taxa to the Pyronemaceae sensu Eckblad. Due to a number of
significant properties, the apical apparatuses of Lasiobolus
and Coprotus have been discussed separately in Chapters II
and III. Two members, Ascozonus and Coprobolus, discharge
their spores through vertical slits at the ascal apices.
Van Brummelen (1974) pointed out the presence of an apical
disc in Ascozonus woolhopensis (Berk, and Br. apud Renny)
Hansen prior to and after dehiscence. This evidence, plus
information concerning the cytochemistry, wall layering,
and developmental sequence obtained in the current investi-
gaion and by van Brummelen has resulted in categorizing the
apical apparatus of A. woolhopensis with those of the Otidea-
Aleuria complex (see Chapter II). The apical apparatus in
Caccobius strongly differed from that of all other repre¬
sentatives of the Pezizales in that it possessed a plug
which tore irregularly at the time of spore liberation.
This type of apical apparatus resembled the inoperculate
form described by Bellemere (1969, 1975). Unfortunately,
specimens of Caccobius and Coprobolus were not available for
the present study. Examination of their apical apparatus
will have to await further study. The remaining two repre¬
sentatives, Trichobolus and Thelebolus, have been reported
to release their spores through an irregular tearing of the
apical wall (Kimbrough, 1966a, b). The apical apparatus in
Trichobolus has been described separately due to its
peculiar wall structure (see Chapter VI).

174
Thelebolus was emended by Kimbrough (Kimbrough and Korf
1967) to include species formerly placed in Ascophanus,
Ryparobius and Ascozonus. Properties of the ascal wall were
greatly emphasized. When stained with Congo red the pres¬
ence of a slight to prominent ring below a hyaline apex was
common for all species. Irregularly dehiscing tips, cleisto
thecial ascocarps and thick-walled, hyaline ascospores with¬
out de Bary bubbles were other ubiquitous characters.
Cooke and Barr (1964) had proposed previously the trans
fer of Thelebolus stercoreus Tode per Fr. from within its
own family Thelebolaceae to the Erysiphales. They based
their decision on the shape of the ascus, the lateral and
apical thickness of its wall, the mechanism of ascal de¬
hiscence, the comparative development of the ascocarp and
the production of a single ascus. Kimbrough (1966b) pointed
out that if they had studied other members of the Pseudo-
ascobolaceae their conclusions and taxonomic decision would
have been different. Characters including the more thinly
walled apex and single ascus per ascocarp were superficial
and coincidental.
Delimitation of species within Thelebolus was found to
be most difficult (Kimbrough and Korf, 1967). The repeated
association of forms and broad overlapping of characters
among previously described species observed by earlier
workers (Boudier, 1869; Karsten, 1871; Barker, 1903, 1904)
and by Kimbrough and Korf suggested that the ascus number,
ascospore number and ascocarp size and color may be variable

175
in Thelebolus. Wicklow and Malloch (1971) compared the
growth and development of Thelebolus isolates over a range
of temperatures partly in order to evaluate the cultural
stability of the previously mentioned morphological charac¬
teristics and to note whether descriptive features used in
characterizing the genus could be increased. They found
that the spore number was constant within any given strain
and that the low temperature optima for apothecial growth
and development were the same for all isolates, which ex¬
plained in part why specimens have been described to contain
varying numbers of spores per ascus.
The taxonomic position of Thelebolus has remained un¬
resolved. The cylindric to spherical asci, unusual ascal
tips and associated mechanism of dehiscence are characters
that distinguish this genus from the rest of the operculate
Discomycetes. The present study was designed to examine
the morphology, development and cytochemistry of the ascal
wall and its apical apparatus in Thelebolus and to understand
more clearly the taxonomic placement of this genus in the
Euascomycetes. Four representatives, each having a different
spore number per ascus, were used. They were Thelebolus
stercoreus, T. crustaceus (Fuckel) Kimbr., Thelebolus
polysporus (Karst.) Otani and Kanzawa and T. microsporus
(Berk, and Br.) Kimbr.

176
Materials and Methods
Collections of Material
Thelebolus stercoreus was isolated from cow dung col¬
lected near Gainesville, Florida, by K. Conway. Thelebolus
polysporus and T. crustaceus were isolated by J. Kimbrough
from rodent dung collected at Bodeza, Arizona, and rabbit
dung at Mohave, California, respectively. The eight-spored
species of Thelebolus, no. F34.68, was obtained from D.
Wicklow of the University of Pittsburgh. Apothecia of each
species were grown on DOA (Dung-Oatmeal Agar; Benedict and
Tyler, 1962). Whole squash mounts of individual apothecia
were used to determine the stage of ascal ontogeny within
a circular region of the Petri plate.
Procedure for Light Microscopic Observations
Whole ascosarps of T. stercoreus, T. crustaceus, T.
eight-spored species and T. polysporus were squash-mounted
in either Congo red (Samuelson, 1975) or acid fuchsin
(Kimbrough, 1966a) to stain the layers of the ascal wall.
Plastic embedded material was sectioned, mounted and stained
in the manner described in Chapter I.
Procedures for Electron Microscopic Observations
Five-millimeter blocks of agar, each containing numer¬
ous apothecia, of T. stercoreus, T. crustaceus and T. poly¬
sporus were fixed in a buffered (0.3M sodium cacodylate
pH 7.2) 2.0% glutaraldehyde and 2.0% paraformaldehyde solu¬
tion for 12 hours at room temperature. Apothecia of T.

177
microsporus were fixed in the same buffered glutaraldehyde-
paraformaldehyde solution for two hours at room temperature.
All materials were rinsed, postfixed in osmium tetroxide,
dehydrated, embedded and poststained as described in
Chapter I.
Results
The Thelebolus ascus does not possess a true operculum
and suboperculum. Ascal dehiscence occurs by an irregular
rupture of the wall at the tip. This region of the ascal
wall is called the apical dome and is subtended by a ring
which was first reported by Kimbrough (1966b) after staining
the wall with either Congo red or acid fuchsin. Descrip¬
tions of the apical apparatus for all species will be re¬
stricted mostly to these regions of the ascus. Each
representative will be treated individually.
The Apical Apparatus of Thelebolus microsporus
Young asci at the four- and eight-nucleated stage are
small, subcylindric to clavate, 30-40 x 5-7 ym. The ascal
wall appears to be uniformly thin throughout. At maturity
the ascus reaches a length of 50-65 ym and a width of 7-10 ym
(Fig. 1). The apical region is thick-walled, forming an
apical dome (Fig. 2). When asci are placed in Congo red,
an area below the tip is strongly stained delimiting the
apical ring (Fig. 3).
Electron microscopic observations of a four-nucleated
ascus show the wall of the ascus to be fairly even in

178
thickness for most of the length of the ascus (Fig. 4). The
lateral wall is 250-260 nm thick and narrows slightly to
170-180 nm at the apex. The irregular outline of the in¬
ternal region of the wall indicates some growth at this time
in ascosporogenesis (Figs. 4, 5). In the distal portion of
the ascus numerous small to moderately large vesicles are
formed centrally. Mitochondria, ribosomes and endoplasmic
reticulum surround the vesicles. Glycogen occurs in small
packets throughout the ascus. When staining with silver
methenamine at this stage in development two strata are
distinguished (Fig. 5). The external stratum is intensely
positive and 90-100 nm thick. In contrast, the internal
stratum is weakly stained and increases from 70-90 nm at the
tip to 140-160 nm toward the base. Near the end of asco-
spore development (Fig. 6), the apical and lateral walls
have increased in thickness to 260-320 nm. The sharply ir¬
regular outline of the interior of the wall suggests pro¬
nounced wall deposition at this time. Numerous small
vesicles fill much of the ascal tip and surround each spore.
At a later stage in development (Figs. 7, 9), the apical wall
has thickened to 320-380 nm. The increase is mostly due to
the formation of an inner layer which is fibrillar and to a
small extent electron-opaque (Fig. 9). The ascus is highly
vesiculated and contains small amounts of cytoplasm appressed
against the internal surface of the wall. By spore maturity
the apical wall has reached a thickness of 450-480 nm which
extends 2.4-3.0 ym down the sides of the ascus, delimiting

179
the apical dome (Fig. 8). Below this point the wall tapers
to a thickness of 320-350 nm. Treatment with silver meth-
enamine, which enhances the appearance of the wall layering,
shows the inner layer to be strongly stained, especially
within the dome (Fig. 8). Within the apical dome the inner
layer is 150-160 nm thick and narrows to 60-70 nm toward
the base of the ascus. The internal stratum of the outer
layer is 180-200 nm thick for the entire length of the ascus
and is faintly stained at a localized region on the apical
dome (arrows in Fig. 8). This area most likely represents
the Congo red-positive apical ring in Fig. 3.
The Apical Apparatus of Thelebolus crustaceus
Young, uninucleated asci are globose to ovoid, being
25-45 ym long and 15-25 ym wide. The wall appears thin and
lacks distinct features at this stage in development. Mature
asci contain 64 ascospores which are broadly clavate to
ellipsoid, 25-30 x 85-100 ym (Fig. 10). The ascal wall is
markedly thick throughout the ascus. The wall is faintly
thinner at the ascal tip and remains hyaline when placed in
Congo red except for a thin external layer (Fig. 11). Below
the hyaline apex, which is referred to as the apical dome,
an intensely stained ring, 4-5 ym long, is detected. The
apical ring appears to be a part of an internal stratum of
the outer layer which extends most of the length of the
ascus and ends beneath the apical dome. A thin external
stratum, which is less positive in Congo red, covers the
entire ascus. Most of the ascal wall's thickness is due to

180
the presence of a thick inner layer (Figs. 10, 11), which
is not stained in Congo red.
Ultrastructurally, the uninucleated ascus is evenly
thin-walled, 320-360 nm (Figs. 12, 13, 14). When stained
with lead citrate and uranyl acetate,the primary wall of the
ascus is uniformly electron-transparent (Fig. 13). However,
when staining with silver methenamine, two strata are dis¬
tinguished (Figs. 12, 14). The strongly positive external
stratum is 210-230 nm thick. The weakly stained internal
stratum is jagged in outline and frequently has small to
large lomasomes attached to it (Figs. 13, 14). Numerous,
small packets of glycogen are scattered throughout the
vesiculated cytoplasm of the ascus (Figs. 12, 13, 14).
During early ascosporogenesis (Fig. 15), the ascal wall
has thickened conspicuously to 2100-2200 nm. The additional
thickness is primarily due to the formation of more inner
layer. Sections that have been treated with silver meth¬
enamine reveal the presence of banding within the inner
layer, which is best detected in peripheral sections (Figs.
15, 16). The ascus is filled centrally with vesicles at
this stage in development (Fig. 15). The wall of the ascus
is fully developed by the time of spore wall formation
(Fig. 17). The apex and lateral walls reach 2600-2650 nm
in thickness. When poststaining with uranyl acetate and
lead citrate the wall is uniformly electron-transparent
throughout the ascus (Fig. 19). However, after staining
with silver methenamine the apical dome and subtending ring

181
are distinguished (Figs. 17, 18). The apical dome has a
diameter of 15.0-16.5 pm and is primarily composed of a
thickened inner layer which is weakly stained by silver
methenamine except for its internal region (Fig. 18). The
positively stained apical ring, an extension of the outer
layer's external stratum, is 3.70-4.20 pm long and 1.55-
1.60 pm thick. At the dome the internal stratum tapers
abruptly (Fig. 18, AR). The external stratum remains evenly
thick, 80-100 nm, throughout the entire wall including the
apical dome.
The Apical Apparatus of Thelebolus polysporus
Uninucleated asci are globose, 10-25 pm in diameter,
and thin-walled. Fine structure of the uninucleated ascus
demonstrates an electron-transluscent primary wall, 260-
300 nm thick (Fig. 20). As in T. crustaceus, vesicles are
scattered throughout the cytoplasm, being concentrated
toward the internal face of the wall. The diploid nucleus
is centrally oriented in the ascus at this time (Fig. 20).
During early mitotic divisions the ascus expands mostly
lengthwise, being 50-55 pm long and 25-30 pm wide (Fig. 22).
The apical region is filled with small vesicles which be¬
come progressively larger toward the base. Packets of
glycogen are interspersed among the vesicles (Figs. 22, 23).
The thickness of the ascal wall has markedly increased
laterally and apically (Figs. 22, 25). However, the wall
remains thin, 280-310 nm, at the base of the ascus (Fig. 23).
An ascal pore, which is found in this vicinity (Figs. 22,

182
23), apparently connects the cytoplasm of the ascus to the
adjoining stalk cell during this stage of development. When
stained with silver methenamine the appearance of the wall
layering is sharply enhanced (Fig. 21). Most of the in¬
creased thickness of the wall is due to the formation of a
uniform inner layer which reacts weakly in silver methena¬
mine. The addition of the inner layer occurs along the
internal face of the lateral wall and at the immediate region
of the tip where the expansion of the inner layer is greatest,
being 2.9-3.0 ym thick (Fig. 25). Immediately below the
swollen tip, the inner layer narrows to 50-100 nm for a
length of 3.6-4.0 ym before broadening to 2.2-2.4 ym toward
the base of the ascus (Figs. 21, 25). The outer layer of
the ascal walls consists of two strata (Fig. 21). The
intensely stained external stratum is 200-240 nm thick for
the entire length of the ascus. In contrast, the internal
stratum, which is less strongly stained in silver methena¬
mine, increases in thickness from 550-600 nm at the lateral
wall to 1.2-1.3 ym at the region of the wall that subtends
the swollen inner layer at the apex (Figs. 21, 25). The
internal stratum then abruptly tapers in thickness below the
swollen tip. In this manner the apical dome and ring are
distinguished.
At a slightly later time in development, the wall
thickens significantly at the apical and subapical regions
and less markedly at the lateral regions (Figs. 26, 34).
One-micron sections stained in toluidine blue (Fig. 26)

183
exhibit a stratified inner layer (labelled 1&2) as well as
an outer layer (labelled 3&4). After treatment with silver
methenamine the four strata are readily distinguished (Fig.
24). The increase in thickness of the wall, which is 3.6-
3.7 pm throughout the ascus, is primarily due to the addition
of the internal stratum of the inner layer. Furthermore,
the internal stratum of the inner layer is the only area of
the wall where microfibrils of the wall are distinctly
banded (Fig. 24).
The mature ascus is broadly clavate to ovoid, 30-45 x
80-120 pm and contains approximately 250 ascospores (Fig.
27). When placed in Congo red, the wall at the tip remains
hyaline except for an extremely thin external stratum
(Fig. 29). The wall is broadly stained below the clear dome
for a short distance, 4-6 pm, delimiting the apical ring
(Figs. 27, 29). The rest of the ascal wall is stained only
in the outer layer, which is moderately thin. Most of the
wall consists of a thick, hyaline inner layer.
Ultrastructurally, the wall of the mature ascus is
changed very little in form and thickness from that observed
during early ascosporogenesis (Figs. 24, 26, 28, 34). Wall
layering in the apical and lateral regions is weakly dis¬
tinguished in sections that have been stained with uranyl
acetate and lead citrate (Figs. 28, 30). Although the in¬
ternal and external strata of the outer layer are to some
extent demarcated at the apical dome (Fig. 30), stratifica¬
tion of the outer and inner layers is most clearly observed

184
at the base of the ascus where the wall narrows sharply
(Figs. 28, 32). Several nuclei are present in this vicinity
even though the development of the ascospores is nearly com¬
pleted. The apical dome is conspicuously demarcated after
staining with silver methenamine (Fig. 31). The dome has
a diameter of 16.5-17.0 pm and a thickness of 3.1-3.2 pm.
The strongly stained subtending ring is 4.0-5.5 pm long and
2.9-3.1 pm thick. The lateral wall is 3.2-3.3 ym thick and
narrows greatly to 1.0-1.1 ym at the base of the ascus.
Prior to ascal dehiscence, the apical and subapical
regions of the wall are distinctly stretched. The numerous
ascospores are tightly lodged against the distal portion
of the ascal wall. At spore release, the apical dome is
distended beyond recognition, rupturing irregularly as the
spores are ejected from the ascus (Fig. 35). The apical
ring appears to stay intact after the spores have left the
ascus. Ultrastructural examination of incipient spore re¬
lease reveals the manner of dehiscence (Fig. 33). Initially,
the outer layer splits roughly in the middle of the apical
dome while at the same time the inner layer pushes through
this opening. The inner layer becomes greatly stretched and
ruptures often in the vicinity of the apical ring. The in¬
ternal stratum of the outer layer at the ring does not
change significantly in thickness and length before, during
and after spore release, indicating that this portion of the
ascal wall is structurally rigid. In contrast, the inner
layer throughout the ascus appears to be very flexible

185
and undergoes great changes in size and form during spore
ejection.
The Apical Apparatus of Thelebolus stercoreus
The young, multinucleated ascus is globose and very
thick-walled, up to 8-12 ym. In Congo red, face-on view of
the apex of the young ascus demonstrates a ring, 24-28 ym
in diameter (Fig. 36). As the ascus continues to grow be¬
coming subglobose to ovoid in shape, the wall decreases in
thickness, coming 8-9 ym at the lateral region and 7-8 ym
apically (Fig. 37). A hyaline apical dome, 42-44 ym in
diameter, is observed after staining with Congo red (Fig.
39). The wall below the dome is intensely stained for a
length of 12-15 ym, delimiting the apical ring. At the base
of the ascus a wide pore extends approximately two-thirds
across the wall (Fig. 43). At this point, the pore
becomes very narrow as it passes through the outer extremity
of the wall.
The mature ascus reaches a length of 160-250 ym and a
width of 100-150 ym (Fig. 38). The wall is significantly
thinner at the tip, being 5.5-6.0 ym in the region of the
dome (Fig. 40). At the same time, the apical dome is ex¬
panded to a diameter of 48-52 ym. In Congo red, the apical
ring is more distinct at this stage in development (Fig. 40).
As in T. crustaceus and T. polysporus, the broad, positive
reaction in the wall appears to be restricted to an internal
stratum of the outer layer which becomes attenuated toward
the base and narrows abruptly below the hyaline dome. A

186
thin external stratum is mildly stained in the apical dome
and the remainder of the ascus. Most of the ascus is com¬
prised of a thick inner layer, which is unstained in Congo
red as shown in Fig. 42 where the outer layer has been peeled
from the lateral wall. In acid fuchsin, layering within the
inner layer is detected (Fig. 41). A broad external stratum
is found throughout the ascal wall except at the apical ring
where it is interrupted by the unstained internal stratum
of the outer layer. Inside the inner layer's external
stratum, an internal stratum, which is faintly stained by
the acid fuchsin, is found after close examination (Fig. 41,
arrow). When external pressure is applied to the mature
ascus, the wall is markedly stretched at the tip of the
ascus (Fig. 44). The apical dome increases 10-15% in
diameter and decreases more than 50% in thickness. The
rest of the wall appears to have changed little in thickness.
Ultrastructural examination of asci during early spore-
wall development demonstrates the bilayered nature of the
lateral wall, with an electron-opaque, 340-380 nm thick
outer layer and an electron-transluscent, 7.8-7.9 ym thick
inner layer (Fig. 50). Large masses of glycogen are located
at the periphery of the ascal cytoplasm. At the base of
the ascus, the inner layer is separated more clearly into
internal and external strata (Fig. 52). In this vicinity
of the wall a large pore extends through most of the inner
layer (Fig. 53). Several nuclei are seen in the area of
the pore.

187
The mature ascus has a very prominent laminated wall
for its entire length (Fig. 45). The wall at the base and
sides of the ascus is 7.5-7.8 ym thick, consisting mostly
of a broad inner layer. Banding occurs in the internal
region of the inner layer for most of the ascal wall (Fig.
51). At the junction of the apical ring and apical dome the
wall tapers considerably to a thickness of 5.5-5.6 ym
(Figs. 45, 48). The apical ring is primarily composed of
a localized electron-opaque thickening of the internal
stratum of the outer layer (Fig. 48). When stained in
silver methenamine the internal stratum at the ring and
throughout the rest of the ascus is mildly positive (Fig.
49). By comparison, the external stratum of the outer layer
is intensely positive. The apical dome consists mostly of
a thick, electron-transluscent inner layer which appears
stratified when stained with silver methenamine (Fig. 47).
The internal stratum of the inner layer, 860-900 nm thick,
is strongly stained at the dome. Within the external
stratum of the inner layer a distinct split is frequently
seen near the outer layer throughout the dome (Figs. 46, 47).
During dehiscence the schism may aid in the release of the
spores as the inner layer becomes distally distended.
Discussion
Using fresh material placed in Congo red of the four
species presently studied similar cytochemical and morpho¬
logical characters of the apical wall of the ascus were

188
demonstrated. The presence of thick-walled apical domes,
which were subtended by distinctly stained rings, was com¬
mon features in the apical apparatus of each representative.
These findings were in complete accordance with those made
by Kimbrough (1966b; Kimbrough and Korf, 1967).
Two trends were observed in the present study with re¬
gard to the number of spores per ascus, the staining prop¬
erties of the ascal wall and the thickness of the ascal wall
at the apex. Since in each species the apical wall narrowed
markedly prior to spore release, references to the thickness
of the apical walls were made at the time when the ascus was
fully matured and the spores were still developing. In the
species with the fewest spores per ascus, T. microsporus,
the wall of the ascus was found to be thickest at the apical
region. By comparison, the thickness at the apex in the
multispored species, T. crustaceus and T. polysporus, was
approximately the same as the rest of the ascal wall, while
it was thinner than the other regions in the ascus in T.
stercoreus. Consequently, as the ascus increased in spore
number the relative thickness of the lateral wall became
more prominent. At the same time the staining reaction of
the outer layer at the apical dome was more conspicuous in
the species which had fewer spores per ascus, T. microsporus
and T. crustaceus, than in the species which had more spores
per ascus, T. stercoreus and T. polysporus.
The ascal wall in Thelebolus was previously described
to consist of three layers—a thin outer layer, a second or

189
middle layer, which was discontinuous and had a strong af¬
finity for Congo red, and a third inner layer, which was
selectively stained by acid fuchsin. After close inspection
of one-micron sections of T. polysporus stained with tolui-
dine blue and fresh material of T. stercoreus stained with
acid fuchsin, a fourth stratum was found in the present
study to line the internal region of the ascal wall.
Light microscopic observations of the apical apparatus
of the Thelebolus ascus demonstrated very few microchemical
and morphological similarites with the apical apparatuses
of the other species found in the operculate Discomycetes.
Outside of Thelebolus, occurrence of Congo red-positive
apical rings has been reported only in Ascozonus (Kimbrough,
1966b, 1972; van Brummelen, 1974). In Lasiobolus monascus
a mucilaginous-like ring was shown to be derived primarily
from the inner layer (Kimbrough and Benny, 1977). Moreover,
the ring became less prominent as the ascus approached de¬
hiscence. On the other hand, the ring in Ascozonus was
comprised mostly of a middle stratum or layer which narrowed
considerably in thickness towards the apex (see Chapter II;
van Brummelen, 1974). Consequently, at the light micro¬
scopic level microchemical properties of the wall layering
in species of Thelebolus and Ascozonus were comparable.
Nevertheless, the form of the tip of the ascus and the mode
of dehiscence clearly distinguished the apical apparatuses
of these taxa.

190
Ultrastructural examination of the Thelebolus apical
apparatus demonstrated remarkable similarity among the multi-
spored members. Development of the apical apparatuses in
T. stercoreus, T. crustaceus and T. polysporus basically
followed a three-step sequence. (1) Before the end of
meiosis the external stratum of the outer layer is completely
formed and the internal stratum is partially formed (Fig. 54A).
Small vesicles and packets of glycogen are scattered through¬
out the ascal cytoplasm being concentrated toward the peri¬
phery of the cell. The ascus mother cell has grown by this
time to over one-half the size of the mature ascus.
(2) During subsequent mitotic divisions the internal stratum
of the outer layer continues to develop in a restricted area
near the tip resulting in the formation of a ring (Fig. 54C).
Simultaneously, an inner layer is deposited both above and
below the ring. The ascus at this time is filled with
vesicles which coallesced toward the base. (3) At a slightly
later time in development an internal stratum of the inner
layer is formed along the entire length of the ascus (Fig. 54
B). As a result the wall reaches its maximum thickness prior
to spore delimitation. The ontogeny of the apical appa¬
ratuses in the multispored species of Thelebolus differed
sharply with that of the apical apparatuses in operculate
representatives (see Chapters I-IV). In contrast, major
wall development in T. microsporus occurred considerably
later during ascosporogenesis. Deposition of the internal
stratum of the outer layer was not nearly as localized in

191
T. microsporus. Consequently, a distinct ring was not
clearly detected ultrastructurally in this species with or
without the aid of silver methenamine. Pronounced thicken¬
ing of the inner layer in T. microsporus was found only
within the apical dome. Stratification of the wall was also
less conspicuous in this species than in T. stercoreus,
T. crustaceus and T. polysporus, which possessed thicker
walls and greater spore number per ascus. Similar observa¬
tions were made in the examination of the apical apparatus
of the eugymnohymenial representative Coprotus. The apical
wall of the 32-spored species Coprotus winteri (Marchal)
Kimbr. was acutely more stratified than the apical wall of
the 8-spored species C. lacteus (Ck. and Phill.) Kimbr. (see
Chapter III).
An interesting wall feature in T. stercoreus and T.
polysporus was the comparative differences of their thickness
at the base. The absence of a thick wall at the base in
T. polysporus sharply contrasted with the uniformly thick
wall in T. stercoreus. Pores which were detected at the base
of the ascus in both species during ascosporogenesis di¬
verged greatly in size and form. Still, the differences of
the pores essentially reflected the differences of the wall
thickness in that area. Within the operculate Discomycetes
ascal pores have been previously reported in Ascodesmis
sphaerospora Obrist, Saccobolus kernerni (Cr.) Boud. and L.
monascus (Carroll, 1967; Kimbrough and Benny, 1977). In
each instance a septal pore cap, which consisted of a

192
hemispheric layer of radiating tubules, was associated with
the pore at the base of the ascus. Carroll (1967) proposed
that the pore cap may act as a subcellular sieve preventing
the ingress of certain cytoplasmic inclusions into the ascus
while allowing the free flow of small molecules. Caps or
similar structures were not found by me at the ascal pore in
Thelebolus. However, two electron-dense bodies bordered the
pore in the stalk cell of T. polysporus. Their role may be
functionally similar to that of the septal pore cap observed
in the operculate species.
The ascal walls of the species of Thelebolus in the
present study differed structurally in one major aspect from
those of the true operculate species currently described.
Throughout the internal portion of the inner layer in the
multispored species of Thelebolus stacks of microfibrils
were arranged in a banded pattern. The microfibrils were
uniformly upturned at the end of each stack. To a lesser
extent, similar banding was observed at the apical dome in
T. microsporus. In each species orientation of the bands
was essentially parallel to the cytoplasm of the ascus.
Fibrillar bands have been reported previously in the apical
apparatuses of sarcoscyphaceous species (Samuelson, 1975)
and Lasiobolus monascus (Kimbrough and Benny, 1977). How¬
ever, within the inner layer of the ascal tips of subopercu-
late species and L. monascus, the bands consisted mostly of
thick, irregular bulges which were formed for only a short
distance below the operculum.

193
Wall structure found in Thelebolus most closely re¬
sembled that of the bitunicate ascus (Reynolds, 1971;
Bellemere, 1971). The banded pattern of the secondary wall
that was illustrated by Reynolds (1971) in Limacinula theae
Syd. and Butl. was identical to the pattern observed in the
different species of Thelebolus. Furthermore, the sequen¬
tial development of the bitunicate wall in L. theae fol¬
lowed that of Thelebolus in several ways. During the major
enlargement of the ascus, only the primary wall was formed.
Deposition of the secondary or inner layer in the bitunicate
and the Thelebolus ascus began before spore formation. De¬
velopment of the inner layer occurred predominantly in the
upper half of the ascus. The presence of two layers, each
having two distinct strata in the walls of T. strecoreus and
T. polysporus, was essentially the same as that described
for the different bitunicate species reported by Bellemere
(1971). His schematic structure of each stratum, including
the banded appearance of the "inner layer of the endo-
ascus," i.e., the internal stratum of the inner layer,
fitted closely the structure of the wall layering within
species of Thelebolus. Bellemere pointed out that the ascal
tips of the different bitunicate taxa were best distinguished
from one species to another by variations of the thickness
of the four "layers" or strata. As in the present study, he
found that most of the variations in wall thickness were
restricted to the inner layer.

194
Although the type of dehiscence associated with species
of Thelebolus has been taxonomically important, the actual
mechanism involved was not known. Evidence developed by the
present study has shown that asci of Thelebolus release their
spores in a jack-in-the-box manner where the inner layer
rapidly extends beyond the boundary of the outer layer (Fig. 54
D). Dehiscence of this kind has been found only in the
bitunicate ascus. During spore discharge from a bitunicate
ascus, splitting of the outer layer, or ectoascus, occurs
either at the tip or circumscissely below the tip, throwing
off a hood-shaped portion of the outer layer. At the same
time, the inner layer expands two to three times the normal
length of the ascus. Ascal dehiscence in Thelebolus appears
to be a modification of this type of spore release with most
of the expansion being restricted to the inner layer of the
apical dome. The rigid thickening of the outer layer's
internal stratum below the dome is primarily responsible for
the limited expansion. Consequently, the extension of the
inner layer is sharply reduced, and separation of the two
layers was not visible using light microscopy.
I question the taxonomic placement of Thelebolus within
the Pezizales in light of the present data. Characters such
as the bitunicate nature of the ascus, the jack-in-the-box
type of dehiscence and the cleistohymenial development of
the ascoma affiliate this genus most closely with members of
the Loculoascomycetes. Further investigations concerned

195
with dehiscent mechanisms in the Loculoascomycetes may
be useful in discovering the appropriate positioning of
this genus.

Chapter V
Figures 1-9. Thelebolus microsporus
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.
Figure 9.
Ascocarp shows asci at different levels of
development. One-micron section stained with
toluidine blue. X500.
Mature asci with thickened apical wall.
Apical dome (AD). One-micron section stained
with toluidine blue. XI,000.
Mature ascus stained with Congo red shows
apical ring (AR) . XI,000.
Tip of four-nucleated ascus has thin apical
wall (AW). Vesicles (V). Nucleus (N).
X7,900.
Tip of four-nucleated ascus stained with
silver methenamine. External stratum (ES).
Internal stratum (IS). X12,500.
Developing tip near end of ascosporogenesis.
Spore (SP). X9,700.
Apical wall of ascus shows increased thickness
at later stage in spore development. Apical
dome (AD). X9,600.
Apical apparatus of mature ascus with apical
dome (AD). External stratum (ES) and internal
stratum (IS) of outer layer. Inner layer
(IL). Arrows point to apical ring. Stained
with silver methenamine. X12,500.
Close-up of ascal tip in Fig. 7. External
stratum (ES) and internal stratum (IS) of
outer layer. Inner layer (IL). X25,000.

197

Chapter V
Figures 10-19. Thelebolus crustaceus
Figure 10.
Figure 11.
Figure 12.
Figure 13.
Figure 14.
Figure 15.
Figure 16.
Figure 17.
Figure 18.
Figure 19.
Mature ascus with 64 ascospores. Stained
with Congo red. X500.
Apex of mature ascus shows apical dome (AD)
and apical ring (AR). Stained with Congo
red. XI,000.
Uninucleated ascus with thin primary wall
(PW). Stained with silver methenamine.
X4,000.
Portion of primary wall in uninucleated
ascus. Arrows point to lomasomes. X9,000.
Primary wall of uninucleated ascus stained
with silver methenamine shows external
stratum (ES) and internal stratum (IS).
Arrows point to lomasomes. X11,000.
Peripheral section of ascus at early
ascosporogenesis. Inner layer (IL). Outer
layer (OL). Stained with silver methena¬
mine. X3,200.
Peripheral section of lateral wall shows
microfibrillar bands (B) in inner layer
(IL). External stratum (ES) and internal
stratum (IS) of outer layer. Vesicles (V).
Stained with silver methenamine. X13,000.
Mature apical apparatus with apical dome
(AD) and apical ring (AR). Stained with
silver methenamine. X3,500.
Apical dome with subtending apical ring
(AR). Stained with silver methenamine.
X5,500.
Region of apical dome and apical ring.
X5,000.

199

Chapter V
Figures 20-26. Thelebolus polysporus
Figure 20.
Figure 21.
Figure 22.
Figure 23.
Figure 24.
Figure 25.
Figure 26.
Uninucleated ascus with thin primary wall
(PW). Nucleus (N). X4,300.
Apical region of young ascus shown in
Fig. 22 with developing inner layer (IL).
External stratum (ES) and internal stratum
(IS) of outer layer. Stained with silver
methenamine. X6,600.
Expanding ascus during early mitotic
divisions. Apical dome (AD). Nucleus
(N). Vesicles (V). Ascal pore (AP).
X2,600.
Base of young ascus shown in Fig. 22.
Arrow points to ascal pore. Lateral wall
(LW). X9,700.
Lateral wall of mature ascus stained with
silver methenamine shows four strata.
Bands (B). X7,000.
One-micron median section of apical region
of young ascus during early mitotic
divisions. Stained with toluidine blue.
X1,000.
One-micron section of apical region of
young ascus at slightly later time in
development than that in Fig. 25 shows
four strata. Stained with toluidine blue.
XI,000.

201

Chapter V
Figures 27-28. Thelebolus polysporus
Figure 27. Mature ascus with approximately 250 asco-
spores. Stained with Congo red. X400.
Mature ascus with fully developed apical
apparatus. X3,000.
Figure 28.

203

Chapter V
Figures 29-
-35. Thelebolus polysporus
Figure 29.
Apical region of mature ascus shows apical
dome (AD) and apical ring (AR). Stained
with Congo red. XI,000.
Figure 30.
Apical dome of mature ascus with internal
stratum (ISO) and external stratum (ESO)
of outer layer. X6,000.
Figure 31.
Apex of mature ascus stained with silver
methenamine. Apical dome (AD). Apical
ring (AR). X4,700.
Figure 32.
Base of mature ascus showing ascal wall with
four strata. Nuclei (N). X7,300.
Figure 33.
Incipient spore release with inner layer
(IL) extending beyond outer layer (OL).
Apical ring (AR). X4,000.
Figure 34.
Apical region of young ascus during early
mitotic divisions. Apical dome (AD).
Nuclei (N). X2,500.
Figure 35.
Dehisced ascus with torn apical dome.
Stained with Congo red. X400.

205

Chapter V
Figures 36-44.
Thelebolus stercoreus
Figure
36.
Young, multinucleated ascus showing apical
ring (AR). Viewed face-on. Stained with
Congo red. X320.
Figure
37.
Ascus at later developmental stage stained
with Congo red. X260.
Figure
38.
Mature ascus showing thinner apical and
lateral walls. Stained with Congo red.
X260.
Figure
39.
Apical region of young ascus shows hyaline
apical dome (AD). Stained with Congo red.
X640.
Figure
40.
Apical region of mature ascus with distinct
apical ring (AR). Stained with Congo red.
X640.
Figure
41.
Area of apical ring (AR) stained with acid
fuchsin. Arrows point to internal stratum
of inner layer. XI,250.
Figure
42.
Portion of lateral wall where outer layer
has been peeled away. Stained with Congo
red. Inner layer (IL). X400.
Figure
43.
Base of young, multinucleated ascus with
ascal pore (AP). Stained with aniline blue
XI,000.
Figure
44.
Apical region of mature ascus when external
pressure has been applied. Stained with
Congo red. X400.

207

Chapter V
Figure 45. Mature ascus of Thelebolus stercoreus shows
thick wall throughout its entire length.
XI,000.


Chapter V
Figures 46-53. Thelebolus stercoreus
Figure 46.
Figure 47.
Figure 48.
Figure 49.
Figure 50.
Figure 51.
Figure 52.
Figure 53.
Portion of apical dome of mature ascus
shows split within inner layer. X5,300.
Portion of apical dome stained with silver
methenamine consists mostly of external
stratum (ESI) and internal stratum (ISI)
of inner layer. X5,300.
Area of apical ring (AR) of mature ascus
shows wall narrowing markedly above the
ring. X5,300.
Area of apical ring stained with silver
methenamine. External stratum (ESO)
and internal stratum (ISO) of outer layer.
X5,300.
Lateral wall of ascus during early spore
wall formation. Outer layer (OL). X3,900.
Internal portion of mature lateral wall
shows bands (B). X6,800.
Base of ascus during early spore wall
formation. Internal stratum (ISI) and
external stratum (ESI) of inner layer.
X3,100.
Large ascal pore (AP) at base of maturing
ascus. X5,400.

211

Chapter V
Figure 54. Development of the apical apparatus in
Thelebolus polysporus.
A.
B.
C.
D.
Uninucleate ascus having thin wall. External
stratum (ESO) of outer layer.
Mature apical apparatus. Internal stratum
(ISI) and external stratum (ESI) of inner
layer.
Localized deposition of inner layer (IL) at
apical and lateral regions of ascal wall.
Internal stratum (ISO) and external stratum
(ESO) of outer layer.
Extension of inner layer (IL) at apical dome
during onset of spore release.

iso

CHAPTER VI
MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY OF THE APICAL
APPARATUS OF TRICHOBQLUS ZUKALII
Introduction
A developmental study on Trichobolus zukalii Heimerl
was made by Kimbrough (1966a) in order to determine the re¬
lationship of this species to other operculate Discomycetes.
Heimerl (1889) had originally placed this representative in
the genus Thelebolus Tode on the basis of the size and shape
of the ascus, the presence of a single ascus per ascocarp,
and the formation of numerous ascospores within a single
ascus. Kimbrough (1966a, b) pointed out, however, that a
variety of spore, ascal and apothecial characters in
Thelebolus zukalii and closely related taxa differed sig¬
nificantly from those found in Thelebolus Tode per Fr.
emend. Kimbr. Consequently, Saccardo's (1892) section
Trichobolus of Thelebolus Tode was raised by Kimbrough and
Cain (Kimbrough and Korf, 1967) to the generic level. The
most salient features which separated species of this genus
from Thelebolus were the presence of apothecial setae and
the absence of an apical ring and a hyaline dome in asci of
Trichobolus.
Studies of ascocarp development in Thelebolus by
Wicklow and Malloch (1971) demonstrated additional support
214

215
for the removal of Trichobolus from Thelebolus. They dis¬
covered that all isolates of Thelebolus Tode per Fr. emend.
Kimbr. grew best at low temperatures (5-10°C) and could be
characterized as being "psychrophilic" or "thermophobic."
In contrast, Trichobolus zukalii was found to be "thermo¬
philic," having optimal growth above 25°C.
Recently, Krug (1973) placed an eight-spored species in
Trichobolus and in doing so enlarged the concept of the genus
to include members that formed many cylindric asci, which
possessed distinct apical rings and true opercula. Further¬
more, he added that the excipulum may be "dextrinoid," a
character typically found in representatives of Lasiobolus
Sacc. (Bezerra and Kimbrough, 1975).
Recent studies of ascal structure and its associated
mechanism of spore release have made significant contribu¬
tions to our understanding of the taxonomy and phylogeny
within the Euascomycetes (Beckett and Crawford, 1973;
Bellemere, 1975; van Brummelen, 1974, 1975; Kimbrough, 1972;
Chadefaud, 1973; Reynolds, 1971). A number of examinations
on ascal structure within the Pezizineae have been made from
representatives of the Thelebolaceae sensu Rifai (1968)
(Kimbrough, 1966a, b, 1972; Kimbrough and Benny, 1977;
Kimbrough and Korf, 1967; van Brummelen, 1975) and have
aided greatly in generic delimitations within that family.
The present investigation of apical structures of
operculate asci demonstrated that members of the Thele¬
bolaceae which possessed some form of an operculum, i.e.,

216
Lasiobolus Sacc., Coprotus Kimbr. and Ascozonus (Renny)
Hansen, resembled morphologically and developmentally the
apical apparatuses of certain representatives in the
Pyronemataceae sensu Korf (1973) (see Chapters II and III).
Furthermore, these findings supported, in part, previous
studies (Kish, 1974; Conway, 1975; Bezerra and Kimbrough,
1975) that recommended the removal of Coprotus and Lasiobolus
from the Thelebolaceae.
By comparison, species of Thelebolus, which released
their spores by irregular dehiscence, were shown in the
present work to possess bitunicate asci. Ascal dehiscence
consisted of a modified "jack-in-the-box" type (see
Chapter V).
The apical apparatus of Trichobolus has been poorly
understood. Spore release was reported to occur most often
through an irregular tear at the ascal tip (Kimbrough, 1966a,
b). Kimbrough (1966a) did note occasionally opercular de¬
hiscence. In the enlarged concept of Trichobolus, Krug
(1973) referred to ascal dehiscence in the different species
of Trichobolus as being operculate. He apparently emphasized
the dehiscent mechanism of the eight-spored species which he
proposed was an adaptation for more controlled spore
discharge.
This study examines the morphology and cytochemistry of
the ascal wall of Trichobolus zukalii and its associated
mechanism of dehiscence in order to understand better the
taxonomic relationship of this taxon with other members of
the operculate Discomycetes.

217
Materials and Methods
Collections of Material
Trichobolus zukalii was obtained from the Centraal
Bureau Voor Schimmelcultures, no. 720.69. Apothecia were
grown on DOA (Dung-Oatmeal Agar; Benedict and Tyler, 1962).
Procedure for Light Microscopic Observations
Whole ascocarps of T. zukalii were squash-mounted in
Congo red (Samuelson, 1975) to stain the layers of the ascal
wall. Plastic embedded material was sectioned, mounted and
stained in the manner described in Chapter I.
Procedure for Electron Microscopic Observations
Five-millimeter blocks of agar, each containing 10-20
apothecia, were fixed in a buffered (0.2M sodium cacodylate
pH 7.2) 2.0% glutaraldehyde and 2.0% paraformaldehyde solu¬
tion for 12 hours at room temperature. The blocks were
rinsed, postfixed, dehydrated, embedded and poststained as
described in Chapter I.
Results
The mature ascus of T. zukalii is ovoid to pyriform,
being 300-425 x 325-510 ym and contains 6000-7000 ascospores
(Fig. 1). The ascal wall appears to be thinnest at the apex
(Figs. 2, 3). When stained in Congo red, wall layering at
the apex is difficult to distinguish (Fig. 2). However,
when one-micron sections are stained with toluidine blue,
an intensely stained inner layer is clearly demarcated from

218
a mildly stained outer layer at the tip and throughout the
ascal wall (Figs. 3, 6). Both layers increase in thickness
toward the lateral region (Fig. 3). In Congo red, the outer
layer of the lateral wall is comprised of two strata with
the internal stratum being more strongly stained than the
external stratum (Fig. 4). The thickened inner layer re¬
mains unstained. The outer layer does not appear to be
stratified when stained with toluidine blue (Figs. 3, 6).
At the base of the ascus the thickness of both layers
diminishes considerably (Figs. 5, 6). Even with the aid of
Congo red the two strata of the outer layer are difficult
to distinguish in this region (Fig. 5).
During spore liberation the apex of the ascus occasion¬
ally undergoes a circumscissile rupture, forming a wide
operculum (Fig. 7). The operculum remains fastened to one
side of the ascus, appearing as an attached lid. Ascal
dehiscence occurs frequently at the tip by random, irregular
tearing.
Ultrastructurally, the mature ascal wall does not ap¬
pear to possess a distinct apical apparatus (Figs. 8, 10, 11,
13). Sections that are stained with uranyl acetate and lead
citrate do not demarcate clearly the wall layering through¬
out the ascus (Figs. 11, 12). Silver methenamine staining
enhances markedly the visibility of layering in the wall
(Figs. 8-10, 13-18). At the apical region of the wall
(Fig. 8), the outer layer is composed of two strata. The
external stratum is 0.55-0.60 pm thick and appears to have

219
several substrata that lie adjacent to the internal stratum
(Fig. 13). The internal stratum and the inner layer have
approximately the same thickness, 1.10-1.20 ym. The inner
layer is not as strongly stained as the outer layer at the
ascal tip (Figs. 8, 13). The apical wall is 3.00-3.25 ym
thick and increases to 4.4-5.0 ym at the lateral region of
the ascus (Figs. 10, 15, 16). Transverse sections of the
ascal tip distinguish the two layers more distinctly (Fig.
17). Furthermore, the outer layer is seen to be strongly
ribbed. In the upper extremities of the lateral wall the
external stratum of the outer layer thickens to 0.95-1.00 ym
(Fig. 15). Delimitation of the internal stratum and the
inner layer is extremely difficult in this area of the ascal
wall. However, toward the lower extremities of the lateral
wall, the inner layer and the two strata of the outer layer
are easily distinguished (Fig. 16). The thickness of the
wall at the base of the ascus diminishes to 2.60-2.95 ym
(Fig. 14). The outer layer's external stratum thickens to
1.25-1.40 ym while the internal stratum tapers to 7.5-
8.0 ym. The inner two strata of the inner layer are stained
strongly by the silver methenamine (Figs. 9, 14, 18). By
comparison, substrata are not sharply detected in the ex¬
ternal stratum of the outer layer. Scars of the attachment
cells still remain in the outer layer at the base (Fig. 18).

220
Discussion
Wall layering of mature asci in Trichobolus zukalii
which had been stained with Congo red concurred completely
with Kimbrough's (1966a) observations. The wall appeared to
be comprised of three layers in the lateral and lower re¬
gions of the ascus. One-micron sections stained with
toluidine blue and thin sections stained with silver methena-
mine indicated, however, that only two layers exist. The
middle or third layer observed in Congo red was most likely
the internal stratum of the outer layer which thickened
conspicuously toward the base. Ultrastructural examination
of the inner layer at the lower region of the ascus cur¬
rently revealed the presence of three strata and confirmed
earlier findings made by Kimbrough (1966a).
The mature ascal wall of T. zukalii differed primarily
from that of the operculate representatives discussed in
Chapters I-IV in the amount of stratification found in the
outer and inner layers. The heavily stratified wall in
T. zukalii contrasted sharply with the walls of other taxa
including species that have more than eight spores per
ascus. Furthermore, most of the stratification in T.
zukalii occurred at the base of the ascus while in the other
operculate species sublayering of the wall was most distinct
in the area of the apical apparatus.
The apical apparatus of T. zukalii shared a number of
properties with those of the operculate species studied by

221
me. The prominence of the outer layer throughout most of
the ascal wall except at the apex in T. zukalii was a common
feature in most operculate asci. T. zukalii followed the
trend observed in other operculate taxa where multispored
species (as defined in Chapter V) develop thick, stratified
ascal walls. The marked increase of the thickness of the
ascal wall during spore formation reported by Kimbrough
(1966a) strongly resembled the pattern of development found
in most operculate apical apparatus (see Chapters I-IV).
Ascal dehiscence occurred by the circumscissle rupture of
the apex occasionally in the multispored species and typi¬
cally in the eight-spored species, T. octosporus (Kimbrough,
1966a; Krug, 1973). The infrequency of operculate de¬
hiscence in species with large, numerously spored asci
indicated that this mechanism of spore release may be func¬
tionally inadequate. The irregular tearing of the apex
during the ejection of the spores was most likely a modified
form of operculate dehiscence. The undulate or ribbed outer
layer of the apical wall possibly aided in the uneven tear¬
ing of the tip. A similar modification was observed in
Ascozonus woolhopensis (Ber. and Br. apud Renny) Hansen,
which had an apical disc that was essentially inoperative
during spore release (see Chapter II).
Although Trichobolus has been associated closely with
Thelebolus (Kimbrough, 1966a, b; Kimbrough and Korf, 1967),
having been once placed within the latter genus, the ascal
wall and the apical apparatus in T. zukalii do not resemble

222
those in Thelebolus described in Chapter V. The absence of
an apical dome subtended by a Congo red-positive ring, a
bitunicate wall and a "jack-in-the-box" type of dehiscence
clearly distinguished the apical apparatus of Trichobolus
from that of Thelebolus and supported strongly the separa¬
tion of these genera.
In terms of what is known to date, the type of apical
apparatus found in T. zukalii may be regarded as unique in
both function and structure. The lack of distinctive fea¬
tures such as an annular indentation, a subapical ring, or
a differentially stained zone of dehiscence failed to
demonstrate a direct alliance with the different forms of
apical apparatuses in the present study. Since ascospore
and apothecial characters of Trichobolus were found to be
most similar to Lasiobolus, Krug (1973) proposed that T.
octosporus Krug may represent a transitional form between
these genera. Examination of the apical apparatus in eight-
spored species of Trichobolus and Lasiobolus may reveal the
degree of their relatedness. The apical apparatus of the
large uniascal species L. monascus Kimbr. (Kimbrough and
Benny, 1977) and T. zukalii exhibit no significant similari¬
ties. The present evidence does not support Krug's proposal.

Chapter VI
Figures 1-7. Trichobolus zukalii
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Mature ascus containing 6,000-7,000 asco-
spores. Stained with Congo red. X280.
Ascal tip shows narrowed wall without
distinct layers. Stained with Congo red.
XI,000.
Ascal tip showing inner layer (IL) and
outer layer (OL). One-micron section
stained with toluidine blue. X500.
Deeply stained outer layer (OL) with two
strata and unstained inner layer (IL) of
lateral wall. Stained with Congo red.
XI,000.
Base of ascal wall shows inner layer (IL)
and outer layer (OL). Stained with Congo
red. XI,000.
Base of ascus. One-micron section stained
with toluidine blue. X500.
Dehisced ascus with operculum (0) partially
attached. X500.


Chapter VI
Figures 8-10. Trichobolus zukalii
Figure 8.
Figure 9.
Figure 10.
Ascal apex stained with silver methenamine.
Inner layer (IL). Outer layer (OL).
X3,100.
Base of mature ascus stained with silver
methenamine. Inner layer (IL). Outer
layer (OL). X2,000.
Distal region of lateral wall. Stained with
silver methenamine. Inner layer (IL).
Outer layer (OL). X2,000.

226

Chapter VI
Figures 11-18. Trichobolus zukalii
Figure 11.
Figure 12.
Figure 13.
Figure 14.
Figure 15.
Figure 16.
Figure 17.
Figure 18.
Portion of apical wall. X12,000.
Wall at base of ascus. X12,000.
Apical wall stained with silver methena-
mine shows external stratum (ESO) and
internal stratum (ISO) of outer layer.
Inner layer (IL). X8,400.
Base of ascal wall with stratified inner
layer (IL). Internal stratum (ESO) and
external stratum (ESO) of outer layer.
Stained with silver methenamine. X,9000.
Upper portion of lateral wall. Outer
layer (OL). Inner layer (IL). Stained
with silver methenamine. X7,600.
Lower portion of lateral wall. Inner
layer (IL). Internal stratum (ISO) and
external stratum (ESO) of outer layer.
Stained with silver methenamine. X7,600.
Transverse section of ascal tip shows
ribbed outer layer (OL). Inner layer (IL).
Stained with silver methenamine. X6,100.
Base of ascus. Arrows point to scars of
attachment cell. Inner layer (IL).
Internal stratum (ISO) and external stratum
(ESO) of outer layer. Stained with silver
methenamine. X3,900.

228

SUMMARY
The importance of ascal dehiscence in the classifica¬
tion of Discomycetes and the Ascomycetes, in general, has
long been an interesting question. By 1940, dehiscence
mechanisms were considered to be reliable criteria upon
which to base large group relations in the Ascomycetes.
Ingold (1933, 1939) demonstrated that there was considerable
variation in ascal structure in the Pyrenomycetes. Conse¬
quently, Luttrell (1951) stated "if greater emphasis were
placed on ascus structure and more extensive data obtained,
these variations might prove to be criteria of fundamental
importance in the classification of the Ascomycetes."
The present study has demonstrated distinct variability
of the operculate ascus and its apical apparatus in mor¬
phology, cytochemistry and development. Several major forms
of the apical structures were observed. Members typically
with iodine-positive asci, which included representatives
of Ascobolus, Saccobolus, Thecotheus and Peziza, possess
annular indented zones of dehiscence. Furthermore, exogenous
mucilaginous coats are found in those species, which also
stain blue in Melzer's Reagent. The mucilaginous coats seem
responsible for the staining reaction. The apical apparatus
of Iodophanus granulipolaris is the only exception. The
229

230
absence of a detectable mucilaginous coat and indented ring,
along with the presence of a differentially stained opercu¬
lar wall and zone of dehiscence, sharply distinguish the
apical apparatus of this species from the rest of the
"iodine-positive" species.
Cytochemical demarcation of the operculum and the de¬
hiscent zone was also observed in species of Coprotus,
Ascodesmis and Pyronema. The apical apparatuses of these
eugymnohymenial discomycetes represent a second major form
and differed from I. granulipolaris in several ways. Asci
of Coprotus, Ascodesmis and Pyronema are not iodine-positive
in Melzer's reagent. Their ascal walls tapered in thickness
at the tip whereas the tip in Iodophanus remained as thick
as the rest of the wall. Bands or wide zones of dehiscence
were seen in Coprotus and Ascodesmis. In contrast, the
dehiscent zone of !E. granulipolaris was composed of a small
swollen ring. Differential staining of the apical wall of
I. granulipolaris occurred only within the inner layer. By
comparison, the outer layer was the area differentially
stained in the ascal tips of the eugymnohymenial species.
Additional cytochemical demarcation was found in the
apical apparatus of Rhizina undulata, Helvetia crispa,
Morchella esculenta and Sphaerosporella brunnea. In H.
crispa and M. esculenta, which possessed the third form of
apical apparatus, differential staining of the ascal tips
was restricted to dehiscent zones similar to those in A.
sphaerospora but here were being associated with the inner

231
layer. A middle stratum of the opercular wall was differen¬
tially stained in the ascal tips of R. undulata and S.
brunnea. Until the present study, differentially stained
opercula had been found in certain representatives of the
Sarcoscyphineae (Samuelson, 1975). The present findings and
those of the suboperculates suggest chemical changes of the
wall at specific areas, which may be specifically involved
in spore ejection.
Another form of apical apparatus was observed in mem¬
bers of the Otidea-Aleuria complex. The lack of conspicuous
dehiscent zones and the occurrence of subapical rings dis¬
tinguished their ascal tips from the rest of the operculate
discomycetes. Furthermore, the morphological variation of
apical structures in the Otidea-Aleuria complex correlated
with the marked diversity of taxa placed in this group.
The examination of the different operculate apical
apparatuses in the present study supported, in general,
chemotaxonomic and cytological investigations on representa¬
tives of the Pezizales made by Arpin (1968) and Berthet
(1964). Berthet transferred the genera Otidea Fuckel,
Sepultarla (Cooke) Lamb, and Pustularia Fuckel emend Boud.
out of the Aleuriaceae sensu LeGal (1947) or Pezizaceae
sensu Dennis (1968) (see Table 1, Chapter II) into the
Humariaceae sensu LeGal on the basis of uninucleate para-
physes. The pronounced differences of the apical apparatuses
in Peziza succosa, Otidea leporina and Aleuria aurantia
strengthened Berthet's findings. Similarly, the transference

232
of the tribe Discineae from the Aleuriaceae sensu LeGal to
the Helvellaceae by Berthet was supported by the similari¬
ties of the apical apparatuses in Helvetia crispa, Gyromitra
rufula and Piscina ancilis and their differences with the
apical apparatus in P. succosa.
Arpin (1968) found that members of the tribes Ciliarieae
and Humarieae of the Humariaceae sensu LeGal possessed either
both or one of two kinds of carotenes. Consequently, he
combined the two tribes and placed them in a new family, the
Aleuriaceae, which is shown in Table 1 of Chapter II under
Aleuriaceae sensu Kimbrough. The tribe Lachneae of the
Humariaceae lacked carotene and was placed in the Otideaceae
sensu Eckblad (1968) , which Arpin emended. Comparative
analysis of different apical apparatuses of the Otidea-
Aleuria complex in Chapter II supported to some extent the
conclusions made by Arpin. The apical apparatuses of 0.
leporina, Humaría hemispherica, S. brunnea and Jafnea
fusicarpa, which would be placed in the Otideaceae Eckblad
emend. Arpin, generally consisted of thick to thin opercular
walls and thick subopercular walls with reduced annular
swellings. By comparison, the apical apparatuses of Aleuria
aurantia, Anthracobia melaloma, Scutellinia scutellata,
Sowerbyella imperialis and Geopyxis majalis, which would be
placed in the Aleuriaceae Arpin, possessed mostly thin
opercular and subopercular walls, often with conspicuous
subopercular rings. The formation of two groups of apical
apparatuses within the Otidea-Aleuria complex, however, was

233
found to be impractical after closer inspection. Differ¬
ences in the structure of the apical wall were more pro¬
nounced between J. fusicarpa and S. brunnea than between
A. melaloma and S. brunnea. The apical apparatus of the
Otidea-Aleuria complex collectively represented a wide
spectrum of one major form of operculate dehiscence.
Members of the Pezizales have been characterized
chiefly by the operculate dehiscence of their asci. Species
of cup fungi that release their spores by the dissolution of
an apical plug or in a jack-in-the-box manner have been
placed in the inoperculate Discomycetes and the Loculoas-
comycetes, respectively. However, within the Pezizales, a
group of representatives that eject their spores through a
variety of dehiscent mechanisms including operculate ap¬
paratuses has remained. They are placed in a single family,
the Thelebolaceae sensu Rifai (1968), on the basis of
similar ascal, ascospore, apothecial, and habitat characters
Recent investigators (Kish, 1974; Conway, 1975; Bezerra and
Kimbrough, 1975) proposed that Lasiobolus and Coprotus, the
only two functionally operculate genera of the Thelebolaceae
should be transferred to the Pyronemaceae sensu Eckblad
(1968). The present study supported their proposals and
also pointed out that the taxa, Ascozonus and Trichobolus,
which form nonoperative opercula, show closer affinities
with species of the Otidea-Aleuria complex than with the
nonoperculate representatives of the Thelebolaceae.

234
Two genera, Mycoarctium Jain and Cain (Jain and Cain,
1973) and Lasiothelebolus Kimbr. and Luck-Alien (Kimbrough
and Luck-Alien, 1974), have been added to the Thelebolaceae
since 1970. Both genera have setose apothecia with non-
operculate, eight-spored asci. Of the five nonoperculate
genera currently placed in the Thelebolaceae, only Thelebolus
and Lasiothelebolus appeared to be closely related to each
other. Although a critical examination of Lasiothelebolus
has not been made, light microscopic observations of the
apical apparatus demonstrated microchemical and morphologi¬
cal similarities with that of Thelebolus (Kimbrough and
Luck-Alien, 1974). Species of Thelebolus were shown in
Chapter V to have bitunicate asci and jack-in-the-box type
of dehiscence. On the basis of these and other characters
including ascal and apothecial development, Thelebolus and
most likely Lasiothelebolus do not belong in the operculate
Discomycetes. Similarly, Caccobius, with its inoperculate-
like apical apparatus, and Mycoarctium, with its evanescent
asci, most likely should not be placed in the Pezizales.
The mechanism of ascal dehiscence in Coprobolus is not under¬
stood. Cain and Kimbrough (1969) described the formation of
a bilabiate split at spore liberation. Like Ascozonus, the
apical apparatus of Coprobolus may have a modified, non¬
operative operculum. Further examination of this taxon is
needed in order to determine whether it should be placed in
the Pezizales.

235
After close inspection of a large number of coprophilous
species distributed among the Discomycetes, Pyrenomycetes
and Loculoascomycetes, Kimbrough (1972) noted that a variety
of adaptations occurred. He pointed out that there was a
tendency toward an increased spore number per ascus, accom¬
panied by a reduction of asci per ascocarp. This trait was
conspicuous in members of the Thelebolaceae which possessed
multispored species in most genera. In the developmental
study of Coprotus lacteus, Kish (1974) felt that the multi¬
spored tendency could be ignored phylogenetically because
this trait was probably an ecological adaptation. Evidence
in the present study supports his conclusion. I believe
that only representatives which have functional or nonfunc¬
tional operculate apical apparatuses should be retained in
the Pezizales. The term nonfunctional is referred to here
as that which has the capacity to form an operculum but does
not function normally in a operculate manner during spore
release.
The taxonomic placement of Thelebolus and Lasiothele-
bolus is in serious doubt. As previously mentioned, current
findings strongly favor the transfer of these genera to the
Loculoascomycetes. Unfortunately, among the Loculo¬
ascomycetes unicellular, hyaline-spored representatives (the
Botryosphaeriaceae) are neither coprophilous nor multispored,
and representatives that are coprophilous (the Sporormiaceae)
are neither hyaline-spored nor multispored, although most
members do have septate ascospores. On the basis of ascal

236
and ascocarp development, Thelebolus would appear to be most
closely related to the Pleosporales. Further investigations
concerned with dehiscence mechanisms in the Pleosporales may
be useful in discovering the appropriate positioning of
these genera.
Most Ascomycetes release ascospores in a violent and
consistent manner. Forceful discharge occurs principally
through either a pore, an operculum or a jack-in-the-box
manner. In addition, modifications of each type of de¬
hiscence mechanism have been observed. The inoperculate,
operculate and bitunicate apical apparatuses share three
important features. First, they are bilayered. Frequently,
sublayers or strata are associated with one or both layers,
and distinction of the two layers may be extremely difficult
unless the wall is studied developmentally. Second, the
mature ascus becomes conspicuously stretched prior to spore
release. The wall of the apical apparatus narrows con¬
siderably in thickness during this stage. At the same time
the spores become tightly lodged against the inner face of
the apical apparatus. Third, the spores are squeezed by the
ascal wall surrounding the ascostome during dehiscence.
This supplies an added impetus to each spore as it leaves
the ascus. As a result, the operculate, inoperculate, and
bitunicate apical apparatuses functionally behave in a
similar manner.
By comparison, the three major types of apical appara¬
tuses differ basically in structure. Furthermore, these

237
differences directly reflect the manner of their dehiscence.
Ultrastructural investigations of different representatives
of the Pyrenomycetes and inoperculate Discomycetes revealed
that the pored apical apparatus consists primarily of an
apical cushion, an apical ring (=pore cylinder or annulus)
and a manubrium (=pore plug or axial body). Studies of
Ciboria acerina Whetz. and Buchw. (Corlett and Elliot, 1974),
seven pyrenomycetes (Griffiths, 1973), Xylaria longipes
Nitschke (Beckett and Crawford, 1973) , and Sordaria fimicola
(Reeves, 1971) demonstrated that the ascal apex is considera¬
bly less complex than previously interpreted from light
microscopic observations (Chadefaud, 1942). However, in
Bulgaria inquinans Fr. Bellemere (1969) described greater
morphological complexity. Having based his terminology on
that of Chadefaud (1960), he referred to a superior ring,
an apical cap, and an apical dome in addition to the apical
cushion, apical ring, and manubrium. Nevertheless, the
pored apical apparatuses of different taxa have generally
shown various elaborations of the basic construction.
The bitunicate ascus is characteristically composed of
a thick apical and lateral wall. The apical apparatus of
the bitunicate ascus is not as clearly defined as that of
the inoperculate ascus. An ocular chamber or pore is often
formed immediately below the apex and is usually encased by
longitudinal striations that are referred to as the nasse.
Bellemere (1971) found that the wall was organized into two
pairs of sublayers or strata with one pair comprising the

238
inner layer and the other pair comprising the outer layer.
The inner layer contains banded microfibrils that are hori¬
zontally oriented in the plane of the ascal wall. Reynolds
(1971) demonstrated that in the apical wall, margins of the
bands often appear as linear striae which are visualized in
light microscopy as a "nasse apicale" (Chadefaud, 1942).
In general, the organization of specific components in in-
operculate ascal tips and their manner of dehiscence, i.e.,
the dissolution of a pore plug inside of an annulus, followed
by eversion of the pore, clearly distinguish the pored
apical apparatus from the bitunicate type.
In studying the various apical structures of asci in
the Discolichens, Chadefaud (1973; et al. 1969) called at¬
tention to the broad spectrum of forms found in that group.
Certain members formed bitunicate asci which did not release
their spores in the jack-in-the-box manner. Some members
possessed rings within their apical apparatuses as well.
On the basis of these and other findings among the different
taxa of the Discolichens, Chadefaud proposed that the pored
or annelaceous ascus and the bitunicate or nassasceous ascus
evolved from a common ancestor which shared both characters
and that the operculate ascus was derived from the pored
ascus. He and LeGal (1946b) believed that members of the
Sarcoscyphineae represented an intermediate line of evolu¬
tion between inoperculate and operculate species. Samuelson
(1975) and van Brummelen (1975), however, clearly demonstrated

239
that sarcoscyphaceous taxa were truly operculate and did not
bridge the Pezizales and the Helotiales.
The operculate ascal tip is the most distinctive of the
three major types of apical apparatuses, consisting of an
operculum, a zone or line of dehiscence, and a suboperculum.
In addition, operculate tips are generally thin-walled and
conspicuously bilayered. Like the bitunicate ascus, the
inner layer of the operculate ascus is most pronounced at
the apex. The development of the inner layer, however, oc¬
curs much later in the operculate ascus than in the bituni¬
cate ascus. In contrast, layering of the pored apical
apparatus has been difficult to determine. Codron (1973)
and Bellemere (1975) proposed that the annulus, manubrium
and apical dome were derived from the inner layer. Greenhalgh
and Evans (1967) basically agreed in their findings but
hesitated to state specifically whether a particular com¬
ponent was formed by either layer. Corlett and Elliott
(1974) pointed out that although the outer layer remained
thin at the tip of the pored ascus, the inner layer was also
being thinly deposited in the lower region of the apical
dome and manubrium. They concluded that most of the apical
dome and manubrium was developed from a middle layer which
they called ground material and that the annulus appeared
to have originated from the outer layer. Further cyto-
chemical and developmental examination of inoperculate asci
is needed to resolve this question. In any event, the pro¬
nounced differences in the construction and development

240
of the three types of apical apparatuses do not support the
theoretical schemes of Chadefaud (1942, 1964, 1973) which
are concerned with the origin and evolutionary trend of
apical apparatuses found in the Ascomycetes (see General
Introduction). At present there is no evidence to suggest
that the operculate ascus was derived from either the
bitunicate or the inoperculate ascus.
The operculate asci that I examined had a single ascal
wall, which structurally consisted of two layers. Chadefaud
(1942) similarly described asci, which have pored and jack-
in-the-box types of dehiscence, as having single bilayered
walls. Luttrell (1951) , however, divided asci into two
major types, bitunicate and unitunicate, on the basis of
wall structure. The bitunicate type incorporated those asci
that are surrounded by two distinct walls, whereas the uni¬
tunicate type incorporated asci that have a single wall. In
Lecanidion atratum (Hedw.) Endl., Butler (1939), the first
worker to report the jack-in-the-box type of dehiscence in
the Discomycetes, used the terms "ectoascus" and "endoascus"
to designate, respectively, the outer wall and the extended
inner membrane, i.e., the inner wall. Luttrell used the
same terms for his description of the bitunicate ascus. In
the course of evaluating recent light and electron micro¬
scopic investigations of ascal walls and apical apparatuses,
Chadefaud (1960, 1973) maintained that the ascal wall is
composed of two tunica, which correspond to the term "layers"
in his earlier studies. He referred to the external tunica

241
or outer layer as the exoascus and the internal tunica as
the endoascus. In addition, he called the film ^mucilagi¬
nous coat), which occasionally covers the ascus, the ecto-
ascus. Greenhalgh and Evans (1967) stated that to apply the
terms exoascus and endoascus, as used by Chadefaud, to the
inoperculate ascal wall of Hypoxylon fragiforme (Pers. ex
Fr.) Kickx. would be misleading. They believed that the
ascal wall of H. fragiforme is structurally unitunicate.
Beckett and Crawford (1973) further reiterated this point
stating "future references to unitunicate or bitunicate asci
should be made only within the limits laid down by Luttrell
(1951) and that these terms are defined with regard to both
structure and function." Nevertheless, ultrastructural
evidence of operculate and inoperculate asci (the unituni-
cates) and asci with jack-in-the-box type of dehiscence
(the bitunicates) in the present and previous studies
(Bellemere, 1971; van Brummelen, 1974, 1975; Corlett and
Elliott, 1974) demonstrated that asci generally possess a
single bilayered wall. Moreover, among the operculate dis-
comycetes, the two layers appear to be very distinct. In
accordance to the interpretation of Greenhalgh and Evans,
the operculate ascal wall would be structurally bitunicate.
Consequently, in view of the findings concerning the fine
structure of ascal walls, caution should be observed when
defining and interpreting terms connected with wall structure.
My observations of operculate asci and, in the case of
Thelebolus, bitunicate asci, conceptually agree with the

242
terminology of Chadefaud (1942, 1960, 1973). All ascal
walls can be conceived as being at least structurally be-
layered, and if one conceives that the term "tunica" refers
to layer, all asci then are bitunicate. Only ascal walls
which have separable layers, however, can be conceived as
being functionally bitunicate. Furthermore, functionally
bitunicate walls can be structurally characterized due to
the presence of bands within the inner layer. In taxa that
do not release their spores in a jack-in-the-box manner but
are structurally similar to those that do, the term
"nassasceous" can be used. Ascal walls which do not have
separable layers nor have "nassasceous" tips can be referred
to as being functionally unitunicate. As a result, the
terms "bitunicate" and "unitunicate" are perfectly acceptable
in a reconceptualized sense.
The present study shows that ascal structure and its
associated apical apparatus have marked variability in the
operculate Discomycetes. Comparative analysis of the dif¬
ferent dehiscence structures can be useful in helping de¬
termine taxonomic affinities at special, generic and familial
levels. Characters such as the apical apparatus will con¬
tinue to be significant in the phylogeny and taxonomy of
this group of organisms.
As the result of the present examination of the opercu¬
late apical apparatus, the following conclusions are made:
1. With the exception of Iodophanus granulipolaris,
iodine-positive asci appear to be useful as a
taxonomic character.

243
2. The reaction site of the stain, Melzer's reagent,
in asci which have annular indentations is an
exogenous mucilaginous coat. In 1^. granulipolaris
the reaction site appears to be the ascal wall.
3. Among the ten representatives of the Otidea-
Aleuria complex presently studied, seven form
subopercular rings. For the most part, those
species which have thick, 700-1000 nm, lateral
walls possess less conspicuous subopercular rings
than those species which have thin, 400-400 nm,
lateral walls.
4. Apical apparatuses of members in the Otidea-
Aleuria complex, in general, lack distinct zones
of dehiscence.
5. Walls of multispored asci (more than eight spores
per ascus) are typically thicker and more strati¬
fied than those of their eight-spored counterparts.
6. Morphological and cytochemical similarities of the
apical apparatuses in Ascodesmis, Pyronema and
Coprotus help demonstrate greater relationship be¬
tween these eugymnohymenial taxa and support the
belief that these taxa are most closely related to
members of the Otidea-Aleuria complex.
7. Comparative analysis of the apical apparatuses in
species of Helvetia and Morchella suggest a greater
taxonomic relationship between these members than
with any other operculate group.

244
8. Except in Ascozonus woolhopensis major development
of the operculate ascus occurs late in spore
development.
9. Although ascal dehiscence is nonoperculate in
Ascozonus and Trichobolus, both taxa do form true
opercula, which are functionally inoperative.
10. No two genera share identical apical apparatuses.
11. In Thelebolus the ascal wall is functionally
bitunicate. The ascus dehisces in a modified
jack-in-the-box manner.
12. The family Thelebolaceae, which needs revision,
does not belong in the Pezizales.
13. All asci with forceful spore discharge are at
least bilayered. The term "unitunicate" should
refer to those with an inseparable wall.

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BIOGRAPHICAL SKETCH
Don Arthur Samuelson was born August 30, 1948, in
Boston, Massachusetts. He attended grammar school in
Brunswick, Maine, until 1958, whereupon he moved with his
parents and older brother to Newton, Massachusetts. In
June, 1967, he was graduated from the Rivers Country Day
School in Weston, Massachusetts. He received his Bachelor
of Arts with a major in biology from Boston University at
Boston, Massachusetts, in May, 1971. From 1971 to 1972 he
worked as a law clerk in a corporate law firm at Boston,
Massachusetts. In September, 1972, he entered the Botany
Department of the University of Florida at the graduate level
and earned the degree of Master of Science in March, 1975.
In April, 1975, he continued to pursue his graduate studies
toward the degree of Doctor of Philosophy at the University
of Florida. He was married to the former Leslie Joyce
Gilbert on February 14, 1977. He is a member of the
Mycological Society of America, the American Phytopatho-
logical Society, the Botanical Society of America, and the
Association of Southeastern Biologists.
253

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
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I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree qf Doctor of Philosophy.
Ill
ssor of Botany
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
0. QlÁákJ
Henry C. Aldrich
Professoá: of Microbiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Daniel A. Roberts
Professor of Plant Pathology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scolarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
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A
Norman C. Schenck
Professor of Plant Pathology
This dissertation was submitted to the Graduate Faculty of
the Department of Botany in the College of Arts and Sciences
and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of
Philosophy.
December, 1977
Dean, Graduate School

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