Asci of the operculate Discomycetes (Pezizales)

MISSING IMAGE

Material Information

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

Subjects

Subjects / Keywords:
Pezizales   ( lcsh )
Discomycetes   ( lcsh )
Asci   ( lcsh )
Fungi -- Classification   ( lcsh )
Botany thesis Ph. D
Dissertations, Academic -- Botany -- UF
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

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

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 000012427
oclc - 03983152
notis - AAB5218
System ID:
AA00004916:00001

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
















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.

















TABLE OF CONTENTS


Page
ACKNOWLEDGEMENTS . . .. ii

LIST OF TABLES . . v

LIST OF FIGURES . . vi

ABSTRACT . . . ix

GENERAL INTRODUCTION . . 1


CHAPTER I


CHAPTER II


CHAPTER III


MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUSES OF REPRESENTA-
TIVES WITH IODINE-POSITIVE ASCI .. 9

Introduction. . 9
Materials and Methods. . 15
Results . .. .. 18
Discussion. . . 27

MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUSES OF REPRESENTA-
TIVES IN THE OTIDEA-ALEURIA COMPLEX 48


Introduction . .
Materials and Methods . .
Results . . .
Discussion . .

MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUSES OF EUGYMNO-
HYMENIAL REPRESENTATIVES .


S 48
S 55
S 57
S 74



S117


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. . .


iii


. 143

















CHAPTER V


CHAPTER VI


Page

Introduction. .. . .143
Materials and Methods . .. .147
Results . ... 148
Discussion. . . 155

MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUS OF THELEBOLUS 170

Introduction. . 170
Materials and Methods .. . 176
Results ... ....... 177
Discussion. .... . .... 187

MORPHOLOGY, DEVELOPMENT AND CYTOCHEMISTRY
OF THE APICAL APPARATUS OF TRICHOBOLUS
ZUKALII . .. .214


Introduction. ... . 214
Materials and Methods .... .. .217
Results . . 217
Discussion . 220

SUMMARY . . .. 229

BIBLIOGRAPHY . ... . 245

BIOGRAPHICAL SKETCH . ... .. .. .253
















LIST OF TABLES


Page


Chapter I

Table 1


Chapter II


Table 1


Chapter III


Table 1


Classifications of genera with iodine
positive asci . .


Classifications of genera of the Otidea-
Aleuria complex . .



Classifications of eugymnohymenial genera .


Chapter IV


Table 1 Classifications of the Helvellaceae .


144


Chapter V


Table 1 Classifications of the Thelebolaceae. .


119


172
















LIST OF FIGURES


Page


Chapter I


Figures 1-10


Figures 11-19


Figures 20-28


Figures 29-35


Figures 36-50


Figure 51a-d


The apical apparatus of Peziza
succosa. . .

The apical apparatus of Ascobolus
crenulatus . .

The apical apparatus of Saccobolus
depauperatus . .

The apical apparatus of Thecotheus
pelletieri . .

The apical apparatus of Iodophanus
granulipolaris . .

Drawings of apical apparatuses found
in iodine-positive asci. .


Chapter II


Figures 1-9


Figures 10-18


Figures 19-34


Figures 35-42


Figures 43-57


Figures 58-68


The apical apparatus of Otidea
leporina . .

The apical apparatus of Jafnea
fusicarpa. . .


The apical apparatus of Humaria
hemisphaerica. . .

The apical apparatus of Sphaero-
sporella brunnea . .

The apical apparatus of Aleuria
aurantia . .

The apical apparatus of Anthracobia
melaloma . .


S. 90


92


96


98


102









Page


Chapter II

Figures 69-83


Figures 84-94


Figures 95-101


Figures 102-109


Figure 110 A-D


Figure 111 E-H




Chapter III

Figures 1-8


Figures 9-16


Figures 17-25


Figures 26-35


Chapter IV

Figures 1-10


Figures 11-18


Figures 19-24


Figures 25-33


The apical apparatus of Scutellinia
scutellata . .. 104

The apical apparatus of Ascozonus
woolhopensis . .. 108

The apical apparatus of Geopyxis
majalis. . 110

The apical apparatus of Sowerbyella
imperialis ... .112

Apical apparatuses redrawn from
Chadefaud (1942) .. 114

Illustrations made from electron
microscopic observations of apical
apparatuses of the Otidea-Aleuria
complex. ... 116



The apical apparatus of Pyronema
domesticum . 136

The apical apparatus of Ascodesmis
sphaerospora . .. 138

The apical apparatus of Coprotus
winterii . .... 140

The apical apparatus of Coprotus
lacteus ... .142



The apical apparatus of Helvella
crispa .... . 161

The apical apparatus of Morchella
esculenta. . .. .163

The apical apparatus of Rhizina
undulata . 165

The apical apparatus of Discina
ancilis. . 167


vii









Page


Chapter IV


Figures 34-51


The apical apparatus of Gyromitra
rufula . .


Chapter V


Figures 1-9


Figures 10-19


Figures 20-35


Figures 36-53


Figure 54A-D


Chapter VI

Figures 1-18


The apical apparatus of Thelebolus
microsporus. . .

The apical apparatus of Thelebolus
crustaceus . .

The apical apparatus of Thelebolus
polysporus . .

The apical apparatus of Thelebolus
stercoreus . .

Development of the apical apparatus
in Thelebolus polysporus .



The apical apparatus of Trichobolus
zukalii. . .


viii


169


197


199


201


207


213


224








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








was paid to morphology, development and cytochemistry of the

apical apparatus as a whole, to peculiar structures, and to

the time cf their appearance during ascosporogenesis.

Morphological, cytochemical and ultrastructural evi-

dence revealed that the apical apparatus fell into six

groups. (1) Members typically with iodine-positive asci,

including species of Peziza, Ascobolus, Saccobolus and

Thecotheus, possess annular indented zones of dehiscence.

Exogenous mucilaginous coats are believed to be responsible

for the blue reaction. Although lodophanus is iodine-

positive, very few properties of this taxon are shared

with those of the annular indented species or any other

member of the Pezizales. (2) Eugymnohymenial discomycetes

such as Pyronema, Ascodesmis and Coprotus have ascal walls

which taper in thickness at the tip. Wide zones of de-

hiscence are formed in the outer layer of Coprotus and

Ascodesmis. In Pyronema the outer layer of the operculum

is differentially stained from the rest of the ascal wall.

(3) Species placed in either the Otideaceae or the Aleuriaceae

lack conspicuous dehiscent zones. The presence of subapical

rings in Aleuria, Anthracobia, Ascozonus, Humaria, Jafnea,

Otidea, Scutellinia and Sphaerosporella distinguish the

ascal tips of these taxa from the rest of the operculate

Discomycetes. (4) Members with large, variously shaped

apothecia, i.e., Morchella and Helvella, possess distinct

zones of dehiscence in the inner layer. Mature lateral

walls vary little in thickness from the apical walls.









Opercular delimitation with the light microscope is observed

in Rhizina and Discina when stained with Congo red.

(5) Four species of Thelebolus exhibit thick-walled apical

domes that are subtended by distinctly stained rings. Wall

structure most closely resembles that of the bitunicate

ascus. The inner layer consists of microfibrils organized

in a banded pattern. Ascal dehiscence occurs in a modified

jack-in-the-box manner. (6) Trichobolus zukalii possesses

a large operculum which is functionally inoperative. The

ascal wall is heavily stratified and lacks distinctive

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 lodophanus and

may be considered as separate forms. The apical apparatuses

of Thelebolous demonstrate very few microchemical, develop-

mental and morphological similarities with those found in

the other species. The taxonomic placement of Thelebolus

within the Pezizales is in serious doubt. Examination of

the apical apparatus can be useful as a systematic tool in

helping resolve ambiguous taxonomic relationships and

determine phylogeny within the Pezizales.
















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 (Boudier, 1879,

1885), the first in which ascal features were exploited.

The significance of this concept has not been fully appreci-

ated until recently.

During the last 35 years, ascal structure has been

recognized as one of the fundamental diagnostic characters

within the Ascomycotina. Studies by Chadefaud (1942) and

Miller (1949) on asci within the Discomycetes and

Pyrenomycetes revived interest in this area of taxonomic

research. Miller (1949) used various features, including

wall thickness, dimensions of the ascus, and presence or

absence of a pore or cap at the ascal apex to separate

major orders and families of the Pyrenomycetes. Chadefaud

(1942, 1946) focused mainly on the tip of the ascus in both

the Pyrenomycetes and Discomycetes. He paid particular

attention to all components directly involved in ascospore








expulsion. He used the term "apical apparatus" in defining

that region of the ascus. In addition to these works,

Luttrell (1951) separated the Pyrenomycetes into two groups,

the unitunicates and the bitunicates, basing the division on

essential structural features of the ascal wall.

Within the discomycete order Pezizales, ascal charac-

ters have provided an important contribution for the dis-

tinction of families, subfamilies and genera (Eckblad, 1968;

Kimbrough, 1970; and van Brummelen, 1974). Typically, the

ascospores are liberated through a circumscissile rupture,

the operculum, at the tip of the ascus. Most studies con-

cerned with dehiscence of the ascus have been restricted to

representatives within the smaller suborder Sarcoscyphineae

(Chadefaud, 1946; LeGal, 1946a, 1946b, 1953; van Brummelen,

1975; Samuelson, 1975). LeGal and Chadefaud noted distinct

characters that appeared similar to the apical apparatus of

various members within the inoperculate Discomycetes. They

proposed a phylogenetic relationship between representatives

of the Sarcoscyphineae and certain representatives of the

Helotiales. Recent ultrastructural studies (van Brummelen,

1975; Samuelson, 1975) demonstrated that all asci were

truly operculate in six genera of the Sarcoscyphineae.

Marked differences were described in the dimensions of the

bilayered ascal wall surrounding the operculum. Samuelson

(1975) structurally defined the structure of this region as

the suboperculum and noted two basic forms, eccentric and

noneccentric. The two forms, however, were shown to be









quite variable, representing intergrades that linked the two

extreme examples, Cookiena sulcipes (Berk.) Kuntze and

Pseudoplectania nigrella (Pers. ex Fr.) Fuckel. Taxa that

had the noneccentric form displayed significant features

that were in common with the apical apparatus of an

operculate species, Ascobolus stercorarius (Bull. ex St.

Amans) Schroet (Wells, 1972). Phylogenetically, ascal

dehiscence within the Sarcoscyphineae is believed to be

affiliated most closely with that observed in the true

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)









theorized that the asci of these fungi have become modified

through the process of evolutionary regression.

In a recent systematic treatment of the ascus,

Chadefaud (1973) separated ascus types into three cate-

gories, the Archaeasces, the Nassasces and the Annelasces.

The division was based both on ascal wall structure and the

presence and absence of principal components that comprised

the apical apparatus. He retained the view that a funda-

mental unit of ascus structure exists. The prototype appara-

tus depicted in his earlier study (1942) is basically simi-

lar to the type depicted for the Annelasce. Chadefaud

(1960, 1973) renamed the region of the "coussinet apical"

and "pontuation apicale" of the operculate asci to be

"pendentif" and "chamber oculaire," respectively.

The remainder of the studies related to the structure

of operculate apical apparatuses have been made primarily

within the last decade. Of these, van Brummelen's (1974)

work with Ascozonus woolhopensis (Berk. and Br. apud Renny)

Hans. was the only one specifically concerned with the de-

hiscence apparatus. The number of investigations made on

the apical apparatus of inoperculate Discomycetes (Bellemere,

1969, 1975; Campbell, 1973; Corlett and Elliott, 1974;

Schoknecht, 1975) and Pyrenomycentes (Beckett and Crawford,

1973; Greenhalgh and Evans, 1967; Griffiths, 1973; Reeves,

1971) have been considerably greater. The apparent lack of

interest in the operculate apical apparatus may reflect an

attitude of many investigators that most operculate apical









structures appear identical. Several previous findings sug-

gest that the opposite may be true. In a study on ascus and

ascospore development of Ascobolus strecorarius, Wells

(1972) discovered that as the spores approached maturity

an indented circular band was formed at the tip of the ascus,

delimiting the operculum. Schrantz (1970) demonstrated in

his work with Peziza and Tarzetta that the ascal apex con-

sisted of a thicker outer layer, a feature not reported pre-

viously within the Pezizales. The study of Ascozonus

woolhopensis by van Brummelen (1974) showed that the inner

layer of the ascal tip initially became swollen before the

layer underwent a process of localized disintegration which

was followed by spore release. The data of these research-

ers reveal remarkable differences between dehiscence

mechanisms of the selected taxa.

Microchemical properties of apical apparatuses within

the Pezizales have displayed a variety of differences be-

tween numerous species. Characteristically, within the

Sarcoscyphineae the inner layer of the ascus is deeply

stained in Congo red, and in certain members the opercula

have been shown to be microchemically distinct from the

surrounding wall of the ascus (LeGal, 1946a; Samuelson,

1975). Cytochemical observations of representatives within

the true operculates have been more useful for taxonomy.

The most widely applied stain has been Melzer's reagent, an

iodide solution that produces typically a blue reaction

(Korf, 1973). In the genus Peziza (Dill.) L., taxa were








distinguished by the variable staining of the ascal tips.

Nylander (1869) was one of the first to use the iodine

reaction as a method for generic and specific delimitation.

In other operculate representatives, lodophanus Korf,

Thecotheus Boud., and Psilopezia Berk., entire asci have

been reported to be diffusely stained (Eckblad, 1968;

Kimbrough, 1969 and 1970). Within the largely coprophilic

family Thelebolaceae (sensu Kimbrough, 1970), asci in a num-

ber of taxa have been observed to rupture irregularly upon

spore release. Uniascal, multispored forms of Thelebolus

and Trichobolus appeared to have more pronounced structural

features associated with their apical apparatuses. Similar-

ly, Kimbrough (1974) discovered in the genus Lasiobolus

that the uniascal species L. monascus Kimb. forms a chemi-

cally differentiated band below the apex of the ascus and

that similar though less notable structures exist in eight-

spored species. In Caccobius, he noted that the tip of the

ascus contains a distinct, apical plug which stains in

Waterman's blue black ink but remains hyaline in Congo red

(Kimbrough, 1972). Conversely, preparations using Congo red

and acid fuchsin in lactic acid have demonstrated the pres-

ence of a prominent apical ring located within the multi-

layered ascal wall of Thelebolus (Kimbrough, 1972).

Until recently, little attention has been paid to the

ultrastructure or cytochemistry of the structures associated

with dehiscence of the ascus. Consequently, these features

have played a small role in the taxonomy of operculate








Discomycetes. Increasing information on ascal walls and

ascal tips, in particular, suggests the strong possibility

that apical apparatuses within the Pezizales are signifi-

cantly diverse and can be implemented as a useful systematic

tool. This study presents a comprehensive survey of the

apical apparatus within the Pezizineae, the primary concern

being with morphological and cytochemical features. The in-

vestigation includes a conspectus of apical apparatuses on

family, subfamily and tribe levels, using one or more major

representatives from each taxon. Over 30 species, represent-

ing 26 genera, were inspected throughout the course of the

study. Instead of attempting to describe all of the species

at one time, it has been more feasible to segregate them

into morphological groups. Special attention was paid to

the development of the apical apparatus as a whole and to

peculiar structures and the time of their appearance during

ascosporogenesis. Chapter I examines the asci and the

apical apparatus of representatives that are associated with

the iodine-positive reaction. Included are species of

Peziza L. per St. Amans, lodophanus, Ascobolus Pers. per

Hooker, Saccobolus Boud., and Thecotheus Boud. Chapter II

studies the apical apparatuses of members found throughout

the largest of all operculate groups, the Pyronemataceae

(sensu Korf, 1973), a group which has been referred to here

as the Otidea-Aleuria complex. Species of Otidea (Pers.)

Bon., Jafnea Korf, Humaria Fuckel, Sphaerosporella (Svr.)

Svr. & Kub., Aleuria Fuckel, Anthracobia Boud., Scutellinia









(Cooke) Lamb., Ascozonus (Renny) Hansen, Sowerbyella

Nannft., and Geopyxis (Pers.) Sacarrdo were used. The api-

cal apparatuses of representatives of Ascodesmis van

Tieghem, Coprotus Korf & Kimb., and Pyronema Carus are

described in Chapter III. Characters not found in the

apical apparatuses of other representatives shared by

the three members have resulted in the formation of this

grouping. In Chapter IV, the apical apparatus of the

Morchella-Helvella group are examined. This group repre-

sents members which typically form the largest ascocarps

within the operculate Discomycetes. Species of Morchella

St. Amans, Helvella L., Gyromitra Fr., Discina (Fr.) Fr.,

and Rhizina Fr. per Fr. were used. Chapter V examines the

ascal walls and the associated apical apparatuses of four

species of Thelebolus Tode per Fr. Dehiscence mechanism,

wall layering, ontogeny and cytochemistry separate this

genus from all members of the operculate Discomycetes.

Similarly, Trichobolus zukalii Heimerl is treated by itself

in Chapter VI.

The objective of this study was to learn the nature of

the operculate apical apparatus and to observe what phylo-

genetic correlations can be made within the Pezizales.
















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 blueingg in iodine" in place of amyloid.









During the last decade, Rifai (1968), Kimbrough (1970)

and Korf (1973) have placed all members of the Pezizineae

that have the "amyloid" property into two families, the

Ascobolaceae and Pezizaceae. Of the genera included in

these families, only Ascobolus has been shown to have species

that do not turn blue in iodine. Even in this genus approxi-

mately 50% of the species were observed to give an iodine

positive reaction (van Brummelen, 1967). Except for Peziza,

the blueing reaction has been found to occur diffusely over

the entire wall of the ascus. In Peziza, many species are

distinctly iodine-positive at the apex. In certain species

such as P. vesiculosa Bull. ex Fr., the blueing is restricted

to a ring while the others as much as one-half of the ascus

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









Fr. revealed the presence of a modified apparatus which

lacked a distinct funnel and tract. He depicted the ascal

tip as having a remarkably differentiated opercular dome and

subtending pad (Fig. 51a). Consequently, he reasoned that

Ascobolus was a very specialized group which represented a

line of evolutionary regression.

Although Moore (1963), Reeves (1967) and Carroll (1969)

had previously studied the fine structure of operculate

asci, Schrantz (1970) was the first to examine the ultra-

structure and cytochemistry of the ascal walls of several

ascomycetous representatives, including Peziza plebeia

(LeGal) Nannf. (as Galactinia plebeia). He described the

ascal wall of P. plebeia as consisting mostly of a thick,

"chitinous-callosic" inner layer, which became thinner

towards the apex, and a thin, "pectic-amyloid" outer layer,

which conversely thickened at the apex (Fig.51b). His elec-

tron microscopic examination demonstrated the two layers,

thus reinforcing the light microscopic observations. He was

unable, however, to distinguish from each other the "amyloid"

and the pecticc" coats, which formed the outer layer.

Wells (1972) in his work with the ascus and ascospore

ontogeny in Ascobolus stercorarius (Bull. ex St. Amans)

Schroet. showed that towards the end of spore maturation an

annular indentation was formed at the tip, delimiting the

operculum. Simultaneously, the inner layer of the ascal

wall increased in thickness throughout the apical region.

Wells' and Schrantz' accounts of the ascal tips of









A. stercorarius and Peziza plebeia, respectively, displayed

little in common. In fact, their findings supported

Chadefaud's belief that the two genera are not closely

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 lodophanus to the Pyronemaceae

and Thecotheus to the Thelebolaceae (Table 1).

The usefulness of the iodine test for systematic schemes

is seriously in doubt. This study examines morphological,

developmental and cytochemical aspects of the iodine-positive

ascus and its associated mechanism of dehiscence in order to

learn whether other characteristics of the ascal wall will

support or argue against the systematic use of the iodine

test. Five members of the Pezizaceae and Ascobolaceae

(sensu Rifai, 1968; Kimbrough, 1970; Korf, 1973) are used.

They are lodophanus granulipolaris Kimbrough, Thecotheus

pelletieri (Crouan) Boud., Ascobolus crenulatus Karst.,

Saccobolus depauperatus (Berk. and Br.) E. C. Hansen and

Peziza succosa Berk. Phylogenetic and ontogenetic compari-

sons are made between the species.








Chapter I
Table 1

Classifications of Genera with Iodine-positive Asci


Eckblad (1968)

Ascobolaceae

Ascoboloideae

Ascobolus
Saccobolus

Ascodesmidoideae

Ascodesmis
Boudiera
Svrekia

Pezizaceae

Peziza
Plicaria
Marcelleina
Psilopezia
Discomycetella

Thelebolaceae

Thecotheus

Pyronemaceae

Iodophanus


Korf (1973)

Ascobolaceae

Ascoboleae

Saccobolus
Ascobolus

Iodophaneae

Boudiera
Sphaerosoma
Iodophanus
Thecotheus


Pezizaceae

Gelatinodiscus
Sacrosphaera
Pachyella
Peziza (= Plicaria)


Rifai (1968)

Ascobolaceae

Ascobolus
Saccobolus

Pezizaceae

Peziza
Plicaria
Boudiera
Sarcosphaera
Pachyella
Gelatinodiscus
Thecotheus
lodophanus
Sphaerosoma


Kimbrough (1970)

Ascobolaceae

Ascoboloideae

Ascobolus
Saccobolus

Ascodesmidoideae

Ascodesmis
Boudiera
Svrekia

Pezizaceae

Peziza
Plicaria
Sarcosphaera





14


Table 1 (Cont'd.)


Pezizaceae

Pachyella
lodophanus
Thecotheus
Psilopezia








Materials and Methods


Collection and Developmental Determination of Material

Young and mature apothecia of Peziza succosa were col-

lected from a sandy basin near the Devil's Millhopper in

Gainesville, Florida. Specimens of Thecotheus pelletieri

and Saccobolus depauperatus were found on cow dung collected

near Gainesville. A culture of Ascobolus crenulatus, no. D-

1476, was obtained fromthe Culture Collection of the Rancho

Santa Ana Botanic Garden, courtesy of R. K. Benjamin.

Iodophanus granulipolaris was isolated from cow dung col-

lected near Gainesville by J. Milam. Apothecia of A.

crenulatus and I. granulipolaris were grown on DOA (Dung-

Oatmeal Agar; Benedict and Tyler, 1962) and WSHDD (Weitzman

Silva Hutner medium with dung decoction) respectively. Por-

tions of the apothecia of P. succosa and A. crenulatus were

free-hand sectioned for light microscopic observation to

determine the stage of ascal development. The mature and

immature stages were then separated for further examination.

Apothecia of different developmental stages of T. pelletieri

and S. depauperatus were removed from the substrate and

placed in thin layers of solidifying water agar and tempo-

rarily refrigerated. Whole squash mounts of individual

apothecia of I. granulipolaris were used to determine the

stage of ascal ontogeny for the majority of the apothecia in

a particular Petri plate.








Procedures for Light Microscopic Observations

Three to five millimeter squares from the apothecia

of P. succosa and entire apothecia of A. crenulatus were

placed in a 40% mucilage mixture, frozen and sectioned with

a cryostat at a thickness of approximately ten micrometers

(ym). Sections were mounted on a drop of Melzer's reagent

(Korf, 1973) on a slide and viewed with a Zeiss microscope.

The Melzer's reagent was used as a general differential

stain. More importantly, this reagent tested for the iodine-

positive or "amyloid" reaction at the tip or throughout the

ascal wall. The Congo red stain (Samuelson, 1975) was also

used frequently for comparison. Similarly, whole ascocarps

of T. pelletieri, S. depauperatus and I. granulipolaris were

squash-mounted in either Melzer's reagent or Congo red.

Less frequently, 1.0% lactophenol cotton blue, 2.0% aqueous

phloxine and 2.0% analine blue in 50% glycerine solution

were used to observe cytoplasmic detail and any notable

change in wall morphology.

Plastic embedded material was sectioned at 0.5 to 1.0

micrometers with glass knives. Two or three sections were

placed on a small drop of 0.01% sodium borate and dried at

600C. Single drops of either 0.25% aqueous toluidine blue

(Stevens, 1966) or 1.0% aqueous crystal violet were applied

for 30 seconds and carefully rinsed with water before being

mounted in immersion oil.

Procedures for Electron Microscopic Observations

Five-millimeter squares of apothecia of P. succosa,

entire apothecia of A. crenulatus and I. granulipolaris, and








agar blocks, containing six to eight apothecia each, of

S. depauperatus were fixed in buffered (0.2 M sodium caco-

dylate pH 7.2) 2.0% glutaraldehyde and 2.0% paraformaldehyde

solution (Karnovsky, 1965) for two hours at room tempera-

ture (= 23C). Agar blocks containing apothecia of T.

pelletieri were fixed in 1.0% permanganate solution for one

hour at room temperature. All materials were rinsed 3 times

during a 30-minute period in 50% buffer-50% distilled water

solution and postfixed in 1.0% osmium tetroxide for one

hour at room temperature. The materials were washed several

times with 0.1 M sodium cacodylate buffer and dehydrated in

an ethanol series (25% steps). Specimens were stained with

2.0% uranyl acetate in 75% ethanol overnight at 40C. They

were washed twice with acetone for one hour and subsequently

infiltrated and embedded in a low-viscosity resin (Spurr,

1969). Three changes of 100% plastic were made to ensure

removal of any residual acetone. The materials were placed

under vacuum for five minutes to remove bubbles and then

polymerized for one day at 600C.

Ultrathin sections were cut on a Sorvall MT-2 ultra-

microtome with a diamond knife and placed on single-hole,

formvar-coated grids. Sections were normally poststained in

0.5% uranyl acetate for 15 minutes and in lead citrate

(Reynolds, 1963) for 5 minutes. In addition, wall demarca-

tion was further enhanced by posttreating unstained sections

with silver methenamine, a preferential stain for poly-

saccharides (Martino and Zamboni, 1967). Ultrathin sections








were then examined with an Hitachi HU-11 E electron

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 pm and a

diameter of 10-12 pm. In Melzer's reagent a deep blue re-

action occurs and is restricted to the immediate region of

the apex (Fig. 1). The outline of the tip of the ascus ap-

pears to be rather uneven, and the cytoplasm of that region

has a distinctly granular appearance. Fully mature asci

reach a length of 200-210 pm and a diameter of 12-14 pm.

When stained with Melzer's reagent, the deep blueing at the

tip is less intense and extends 10-14 pm down the sides of

the ascal wall (Fig. 2). The outline of this region is

smooth at this time. On occasion, a blue staining coat may

be removed by gently applying pressure on a cover slip and

sliding the cover slip back and forth. The tips of mature








asci are weakly stained in Congo red. A hyaline ring is

barely visible at the apex.

Ultrastructurally, the apical wall of the diploid

ascus (Fig. 3) is distinguishable in form from the rest of

the ascal wall. Lomasomal activity occurs throughout the

upper portion of the ascus. A thin, uneven, mucilaginous

coat, 135-160 nm thick, is also present in the immediate

area of the tip. The wall at this stage in development has

a thickness of 135-170 nm.

By the early stage of ascospore delimitation, the muci-

laginous coat has expanded notably both in thickness, being

375-415 nm at the tip, and in length, extending 6.5-7.5 pm

down the lateral face of the ascus (Fig. 4). The apical

wall remains thin, being 140-170 nm thick, while the lateral

wall has increased to 380-420 nm.

By late ascosporogenesis, the apical wall has broadened

conspicuously (Fig. 5). At the same time, an annular in-

dentation is formed delimiting the operculum. When thin

sections are treated with silver methenamine, the layering

of the ascal wall becomes sharply demarcated (Fig. 6). The

increased thickening of the tip, which measures 310-360 nm,

results from the addition of an inner layer, 250-280 nm

thick. The adjacent suboperculum has a length of 6.3-7.1 pm.

The strongly stained inner layer narrows from 190-210 nm in

thickness near the annular indentation to 75-85 nm at the

base of the suboperculum. Conversely, the outer layer

expands from 90-100 nm to 280-340 nm. Overall, the lateral









ascal wall narrows approximately 0.1 pm as it approaches the

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 um long and 6-10 pm wide. As the spores mature, the

ascus grows to a length of 115-130 pm and width of 12-15 pm.

When placed in IKI, blueing of the ascal wall is not de-

tected. The tip, which appears thick-walled, has a slight

conical shape and is bordered by a barely visible ring

(Fig. 11). At maturation the tip becomes inflated and

thinner-walled (Fig. 12).

Electron microscopic observations of the apex of the

diploid ascus demonstrate the presence of localized vesi-

culation which is bounded by a ring of glycogen (Fig. 13).

During early spore development, the apical wall, which has








become rounder in form, still consists of a single, uniform

layer, 85-95 nm thick, toward the apex, and 100-110 nm thick

in the lateral region (Fig. 14). Further on in spore matura-

tion the apical wall undergoes a differential increase in

thickness resulting in the formation of an annular indenta-

tion and an operculum (Fig. 15). With the aid of silver

methenamine, the layering of the ascal wall is revealed.

Throughout the operculum the outer layer has a thickness of

85-95 nm (Fig. 16). In contrast, the inner layer is less

uniform in thickness, expanding from approximately 110-

115 nm in the distal area of the operculum to 260-280 nm at

its periphery. The operculum at this stage has a diameter

of 4.4-4.5 pm.

When the spores approach complete development, the

width of the ascal tip increases from 5.9-6.3 pm (Fig. 15)

to 8.0-8.4 pm (Figs. 17, 18). At the same time, the diameter

of the operculum has widened to 5.7-5.9 pm. The opercular

wall layers, however, are reduced in thickness, implying a

stretching action. The inner layer has decreased to 85-

95 nm distally and 130-140 nm peripherally and the outer

layer to 65-70 nm and 85-95 nm, respectively (Figs. 17, 18).

The annular indentation has a width of 545-570 nm,

being smaller than in Peziza succosa (Fig. 19). The outer

layer tapers slightly to 60-75 nm while the inner layer nar-

rows to 35-40 nm. At the distal region of the suboperculum,

(Figs. 18, 19), the breadth of the outer layer suddenly in-

creases to 110-120 nm and maintains this approximate









thickness throughout the rest of the suboperculum. The

inner layer, which is 40-50 nm thick for a length of 680-

720 nm, expands to 100-120 nm at the lower end of the sub-

operculum. The suboperculum is roughly 3.5-4.0 pm long.

The Apical Apparatus of Saccobolus depauperatus

The mature ascus is broadly clavate, being 55-80 pm

long and 12-15 pm wide. When placed in IKI, the entire

ascus stains blue. The apical apparatus is not observed

until late ascosporogenesis whereupon a thick ring is de-

tected at the tip (Fig. 20). At a slightly later stage in

development the tip appears more inflated and less con-

spicuously thick walled (Fig. 21).

Ultrathin sections of asci during early spore wall

formation show that additional development of the ascal wall

is initially restricted to the apex or to that area which

will become the operculum (Fig. 22). Subsequently, the

inner layer making up the rest of the ascus wall is laid

down. A distinct mucilaginous coat, 65-90 nm thick, covers

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 mucilaginous coat is more prominent

at this region of the ascal tip. A zone of dehiscence is

present at the basal end of the annular indentation. The








width of the indented ring is 545-600 nm, being almost

identical to that of Ascobolus crenulatus.

As before, posttreatement with silver methenamine ac-

centuates the stratified character of the ascal wall (Figs.

25, 26). Within the operculum, the outer layer remains

60-70 nm thick. The inner layer, however, ranges from

65-75 nm apically to 180-190 nm subapically. The diameter

of the operculum measures 7.1-7.4 pm. In the apical portion

of the suboperculum, the outer layer is less reactive with

the silver methenamine stain (Fig. 25). This differen-

tially stained region, 1.2-1.3 pm long, will be referred to

as the subopercular flange (Figs. 26, 28). The inner layer

has a breadth of 65-85 nm throughout the suboperculum, which

is 3.3-3.9 pm long. The outer layer is 155-165 nm thick in

the subopercular flange and decreases slightly to 140-145 nm

before it once more increases to 220-230 nm in the lower end

of the suboperculum. At incipient spore release, the

operculum begins to pull away from the suboperculum along

the annular indentation (Fig. 27). After the eight asco-

spores have been discharged as a solid unit, the flange ex-

tends outward and shrinks to 100-150 nm in length (Fig. 28).

The Apical Apparatus of Thecotheus pelletieri

Thecotheus pelletieri is the only representative of the

5 species examined that forms 32-spored asci. The mature

ascus is broadly cylindric, having a length of 300-330 pm

and a width of 50-55 pm (Fig. 29). The ascal wall becomes

diffusely blue when treated with Melzer's reagent. The








apical apparatus consists of a large, conical operculum that

is subtended by a wide ring (Fig. 30). In Congo red, the

operculum appears to be distinctly thinner-walled than the

rest of the ascus. Furthermore, the wall below the apical

ring appears to be bilayered with the outer layer being

stained by the Congo red (Fig. 30). At dehiscence of the

ascus, the lid is often removed entirely (Fig. 31).

With the aid of the electron microscope, the form of

the ascal tip is clearly shown (Fig. 32). The wide ring is

seen to be an annular indentation, similar to that in the

three previous species, but here of considerable size, meas-

uring 980-1200 nm across. The operculum has a diameter of

21-23 pm and a thickness that varies from 610-640 nm in the

apical region to 720-750 nm subapically.

When stained with silver methenamine (Figs. 33, 34), the

less reactive outer layer is seen to constitute much of the

opercular wall, being 350-440 nm thick. In the area of the

suboperculum, the outer layer increases basally from 510-

590 nm to 1,150-1,250 nm. Similarly, the subopercular inner

layer increases to 600-650 nm toward the base. The length

of the suboperculum is 6.5-7.5 pm long. A thin mucilaginous

coat, being 80-100 nm thick, covers the entire ascal wall

(Fig. 34). Both layers of the ascus are notably thinner in

the annular indentation with the inner layer being reduced

in thickness to 200-220 nm and the outer layer to 165-195 nm

(Figs. 34, 35). Furthermore, a zone of dehiscence is ob-

served at the lower end of the indented inner layer.








The Apical Apparatus of Iodophanus granulipolaris

The diploid ascus is broadly cylindrical, being 100-

140 Pm long and 15-20 pm in diameter. When placed in

Melzer's Reagent, the staining reaction of the wall is par-

tially masked by the coloration of the cytoplasm within

(Fig. 36). The area immediately below the ascal tip turns

to a light blue. Most of the ascus appears light green ex-

cept centrally where it also stains blue. One-micron sec-

tions stained with toluidine blue (Fig. 37) shows the pres-

ence of densely packed cytoplasm in the apical region of

the ascus. The apical region is bounded by a large,

amorphous cylinder of deeply stained material. This

cylinder extends to the center of the ascus where the large

diploid nucleus is observed surrounded by cytoplasm. The

remainder of the ascus is filled with another deeply stain-

ing mass of material.

The mature ascus, which retains a broadly cylindric

form, expands to 200-250 pm in length and 30-35 pm in width.

The wall stains a light blue when treated with the IKI

solution. This is most clearly observed in empty asci. The

apices of mature asci lack conspicuous features (Fig. 43).

During ascospore liberation the operculum remains partially

attached to one side of the ascus (Fig. 44).

Ultrastructurally, the tips of diploid asci appear to

be cytologically active, being packed with ribosomes,

endoplasmic reticula and mitochondria (Fig. 38). The large,

amorphous cylinder consists of a uniform mass of glycogen









(Figs. 38, 40). Plasmalemmasomes are associated frequently

with the apical wall and occasionally with the lateral wall

(Figs. 39, 40). The apical wall, being 155-165 nm thick,

is slightly thinner than the lateral wall, which is 200-

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








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.








The ontogenies of the apical apparatuses found in

Peziza succosa, Ascobolus crenulatus, Saccobolus depauperatus

and I. granulipolaris followed in general a three-stage se-

quence. First, wall synthesis in diploid asci occurred

throughout all regions of the cell. Synthesis appeared to

be most concentrated at or near the apex where lomasomes

(plasmalemmasomes as defined by Heath and Greenwood, 1970)

were noted most frequently. Second, during early asco-

sporogenesis, the development of the ascal walls in each

species was restricted mainly to lateral or subapical re-

gions. This was conspicuously apparent in P. succosa and

I.granulipolaris where the lateral walls became two or three

times thicker than the apical walls. Third, during ascospore

wall formation, the additional layering of the ascal wall

was differentially deposited, starting at the apex and pro-

gressing downward. None of the representatives displayed

any evidence supportive of Chadefaud's (1942) conclusion

that complete differentiation of the apical apparatus oc-

curred by the eight-nucleated stage. Instead, the ontogeny

of the apical apparatus of each species showed the most

dramatic change during late ascosporogenesis. Observations

made in the present study coincided closely with those of

van Brummelen (1967) and Wells (1972) regarding the time of

opercular development in Ascobolus species.

In the young asci of A. crenulatus and maturing asci of

I. granulipolaris, the localized accumulation of vesicles

below the tip was reminiscent of the vesicle system described









for the developing diploid ascus of Xylaria longipes

Nitschke (Beckett and Crawford, 1973; Beckett, Heath and

McLaughlin, 1974) and the growing hyphal tips of various

taxa throughout the fungal world (Grove and Bracker, 1970).

Unlike in X. longipes, an apical body (Spitzenkorper) and an

area of localized thickening were not observed during these

stages of development in the species studied by me. Wells

(1972) observed a similar state of vacuolation in Ascobolus

stercorarius prior to meiosis and noted that it was part of

an orderly sequence. He suggested that the vacuoles may

function in concentrating the ascoplasm in the regions of

growth and cytological activity.

The gross morphology of the apical apparatuses was

basically similar in all members. Except in Thecotheus

pelletieri, the inner layer was seen to be distinctly broad-

est in the opercular region of the ascus. In every species,

the outer layer increased in thickness towards the base of

the ascus. These findings compared favorably with those re-

ported for the six representatives of the Sarcoscyphineae

(Samuelson, 1975). However, they sharply contrasted with

Schrantz' (1970) description of Peziza plebeia. Schrantz

defined the outer ascal layer as consisting of a pecticc"

external cover and an "amyloid" inner hood or "manchon"

(Fig. 51b). His light and electron microscopic descriptions

of the outer layer are nearly identical in size and shape to

the mucilaginous coat of Peziza succosa (Fig.51d). His

photomicrograph of the ascal tip of P. plebeia showed a








comparable stage in development to that of the tip seen in

Fig. 4 for P. succosa. He apparently based his evidence on

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).








The discovery of an annular indentation in Peziza

succosa was not entirely unexpected. Kimbrough had noted

frequently the presence of a distinct hyaline ring in the

ascal tips of P. vesiculosa when stained with Congo red

(personal communication). My observations of P. succosa

were similar though the staining reaction appeared less pro-

nounced. We had conjectured that the ring may have been the

result of either a chemical differentiation in the wall or a

physical thinning of the wall. The latter proved to be true.

Of the representatives that had annular indentations,

the species which turned blue in Melzer's reagent, i.e.,

P. succosa, S. depauperatus and T. pelletieri, possessed

mucilaginous coats. It is believed that the iodine speci-

fically reacted with the mucilaginous coats. Asci of P.

succosa provided the most convincing evidence. The localiza-

tion of the blueing reaction in young and mature asci

coincided perfectly with the localization of their respec-

tive mucilaginous coats. Moreover, the coat could be

removed after delicate manipulation. In T. pelletieri and

and S. depauperatus, the total blueing of the ascal walls

corresponded entirely with the thin mucilaginous layer that

covered the ascus. Although asci of A. crenulatus did not

exhibit mucilaginous coats, neither did they give an

iodine-positive reaction in Melzer's reagent.

The apical apparatus of P. succosa seemed the most

isolated morphologically of all the species studied that

developed annular indentations. Major differences consisted









of the presence of a thick, localized mucilaginous coat, a

subtending cytoplasmic ring and an operculum which thickened

distally rather than thinned (Fig.51d). In addition, the

inner layer of the suboperculum was seen to taper notably

in thickness away from the tip. By comparison, the apical

apparatuses of A. crenulatus and S. depauperatus shared a

number of identical features, including the width and breadth

of their annular indentations, opercular and subopercular

flanges. The principal difference between the two members

was observed in the layering of the lower suboperculum. The

expansion of the inner layer toward the base of the ascus in

A. crenulatus in Fig. 51c (vs. S. depauperatus) was similar

to the suboperculum of T. pelletieri. The apical apparatus

of T. pelletieri was generally similar in form to that of

A. crenulatus. For the most part, the ascal wall dimensions

of T. pelletieri were approximately three times that of

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









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.


Figure 1.



Figure 2.


Figure 3.


Figure 4.



Figure 5.


Figure 6.



Figure 7.



Figure 8.



Figure 9.





Figure 10.


Peziza succosa


Tip of diploid ascus showing iodine-positive
reaction. Stained with Melzer's reagent.
X1,000.

Diffuse iodine-positive reaction at mature
ascal tip. Spore (SP). X1,000.

Thin mucilaginous coat (MC) covers immediate
region of the apex of diploid ascus. X10,000.

Ascal tip with thick mucilaginous coat (MC)
at early spore delimitation. Spore (SP).
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 (0).
Spore wall (SW). X7,600.

Opercular (0) and subopercular (SO) regions
of mature apical apparatus. Stained with
silver methenamine. X7,000.

Close-up of Fig. 8 showing cytoplasmic ring
(CR) which subtends annular indentation.
Inner layer (IL). Outer layer (OL). Muci-
laginous coat (MC). Stained with silver
methenamine. X20,000.

Region of dehiscent zone which is demarcated
by annular indentation (AI). X24,000.







0


!r


*- f
















Chapter I


Figures 11-19.


Figure 11.



Figure 12.



Figure 13.


Figure 14.



Figure 15.




Figure 16.



Figure 17.


Figure 18.




Figure 19.


Ascobolus crenulatus


Young ascal tip with developing ascospores
shows distinct apical ring (AR) at this
time. Stained with Congo red. X1,250.

Mature tip is thinner-walled at region of
the operculum (0). Stained with Congo red.
X1,000.

Vesiculated apex of diploid ascus with sub-
tending ring of glycogen (G). X9,800.

Ascal tip during early spore (SP) develop-
ment. Stained with silver methenamine.
X10,500.

Early formation of apical apparatus showing
differential increase in thickness of
opercular (0) wall. Annular indentation
(AI). X11,000.

Inner layer (IL) and outer layer (OL) of
developing apical apparatus. Spore (SP).
Stained with silver methenamine. X10,500.

Mature apical apparatus with umbonately
shaped operculum (0). X9,500.

Mature apical apparatus shows layering of
opercular and subopercular (SO) regions of
the ascus. Annular indentation (AI).
Stained with silver methenamine. X8,500.

Region of annular indentation in mature
apical apparatus. Inner layer (IL). Outer
layer (OL). X48,000.



















Chapter I


Figures 20-28.


Figure 20.


Figure 21.


Figure 22.



Figure 23.


Figure 24.



Figure 25.



Figure 26.




Figure 27.


Figure 28.


Saccobolus depauperatus


Developing apical apparatus with pronounced
ring. X2,000.

Apical apparatus at later developmental
stage. Stained with Congo red. X2,000.

Thickening of apical wall (AW) during early
spore development. Mucilaginous coat (MC).
X8,400.

Mature apical apparatus with distinct
annular indentation (AI). X8,200.

Region of annular indentation with zone of
dehiscence (ZD). Mucilaginous coat (MC).
X39,000.

Mature apical apparatus stained with silver
methenamine. Operculum (0). Suboperculum
(SO). X6,500.

Upper portion of suboperculum showing sub-
opercular flange (SF). Inner layer (IL).
Outer layer (OL). Stained with silver
methenamine. X11,000.

Incipient spore release. Episporal sac (ES).
Spore (SP). X7,600.

Dehisced ascus with outwardly extended sub-
opercular flange (SF). Ascostome (A).
Stained with silver methenamine. X6,200.









Mc


( I


dlh...,mm


,r



















Chapter I


Figures 29-35.


Figure 29.

Figure 30.


Figure 31.


Figure 32.


Figure 33.



Figure 34.





Figure 35.


Thecotheus pelletieri


Mature ascus stained with Congo red. X160.

Ascal tip with wide apical ring (AR)
delimiting a conical operculum. X1,250.

Ascus after spore release. Ascostome (A).
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.










A
I


iv
I-
r"
r"'


AR

> lei




















Chapter I


Figures 36-42.


Figure 36.


Figure 37.




Figure 38.


Figure 39.


Figure 40.


Figure 41.


Figure 42.


Iodophanus granulipolaris


Uninucleated ascus stained with Melzer's
reagent. X400.

Apex of uninucleated ascus subtended by
broad amorphous cylinder. One-micron
section stained with toluidine blue.
X1,000.

Apical region of uninucleated ascus.
Glycogen (G). X6,100.


Small lomasomes (L) at ascal tip.
plasmic reticulum (ER). X20,000.


Endo-


Mitochondria (M) adjacent to large mass of
glycogen (G). X15,000.

Large vacuole (V) at tip during early
ascosporogenesis. X4,700.

Distal portion of apical wall (AW) stained
with silver methenamine. X4,700.










-'


6'


.,

r


0



















Chapter I


Figures 43-50.


Figure 43.

Figure 44.



Figure 45.


Figure 46.


Figure 47.




Figure 48.

Figure 49.



Figure 50.


Iodophanus granulipolaris


Mature tip stained with Congo red. X1,250.

Operculum (0) 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 (0).
Stained with silver methenamine. X13,500.

Portion of mature apical wall. X12,500.

Operculum (0) attached at spore release.
Outer layer (OL). Stained with silver
methenamine. X4,700.

Dehisced ascus with thickened inner layer
(IL). Suboperculum (SO). Stained with
silver methenamine. X8,700.





45



























I .











I 1, I
.OL **
Is























Chapter I


Figure 51. Drawings of apical apparatuses found in
iodine-positive asci.

a. Apical apparatus of Ascobolus furfuraceus.
Operculum (0). Coussinet (C). Redrawn from
Chadefaud (1942).

b. Ascal tip of Peziza plebeia illustrating layers
of the wall. Exoascus (EX). Endoascus (EN).
Bourrelet (B). Redrawn from Schrantz (1970).

c. Illustration made from electron microscopic
observations of the wall layering in mature
ascal tips of Ascobolus crenulatus. Outer
layer (OL). Inner layer (IL).

d. Illustration made from electron microscopic
observations of the wall layers and mucilaginous
coat (M) in mature ascal tips of Peziza succosa.
Outer Layer (OL). Inner layer (IL).







c 0 o








O-





IL
tt
I








C II


'I/1' IV
















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






49

Chapter II
Table 1

Classifications of the Otidea-Aleuria Complex


Dennis (1968)

Humariaceae


Eckblad (1968)

Pyronemaceae


Lachneae

Sepultaria
Leucoscypha
Thricharia
Humaria
Trichophaea
Pseudoomphilia

Ciliareae

Scutellinia
Cheilymenia
Sphaerosporella
Neottiella
Melastiza
Anthracobia

Aleurieae

Aleuria
Pulvinula
Coprobia
Geopyxis
Fimaria
Octospora
Psilpezia
Boudiera
Caloscypha
Lamprospora

Pezizaceae

Otideae


Otidea
Barlacina
Pseudotis
Sowerbyella
Pustularia


lodophanus
Pyronema
Pyronemella
Lamprospora
Pulvinula
Caloscypha
Octospora
Leucoscypha
Coprobia
Cheilymenia
Scutellinia
Humaria
Tricharia
Sphaerosporella
Sepultaria
Jafnea
Fimaria
Pseudombrophila
Anthracobia
Melastiza
Aleuria

Otideaceae

Geopyxis
Pustulina
Sowerbyella
Otidea
Ascosparassis


Rifai (1968)

Humariaceae


Otideae

Otideae
Marcelleina


Lachneae









Table 1 (Cont'd.)


Humariaceae

Jafnea
Jafneadelfus
Nothojafnea
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
Sepultaria
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








Table 1


Pyronemataceae


Trichophaeopsis
Pseudombrophila
Fimaria

Otideoideae

Jafneae

Jafnea
Tarzetta

Otieeae

Psilopezia
Otidea
Ascosparassis

Mycolachneeae

Nothojafneae
Geopora
Humaria
Trichophaea

Scutellinioideaea

Scutellinieae

Coprobia
Scutellinia
Cheilymenia

Sowerbyelleae

Sowerbyella
Caloscypha
Acervus
Phaedropezia


(Cont'd.)


Pyronemataceae

Aleurieae

Anthracobia
Hiemsia
Melastiza
Leucoscypha
Aleuria
Pseudocollema
Pulvinula
Lamprospora
Octospora
Byssonectria
Kotlabaea









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 constellation (Berk and Br.) Boud., Scutellinia

hirta (Schwm. ex Fr.) O. Ktze., Octospora leucoloma Hedw.

ex. S. F. Gray (as Humaria leucoloma), Humaria wrightii

(Berk. and Cooke) Boud. and Sepultaria arenosa (Fuckel)









Boud. Each of the eight species was observed to have a full

complement of apical structures associated with the oper-

culate apical apparatus as shown inFig. 110A. Four morphologi-

cal variations of the apical apparatuses were demonstrated

among the representatives. They ranged from species with

exaggerated or exceptionally well-developed apical appa-

ratuses as in C. granulata to those that had rudimentary

forms as in 0. leucoloma (Fig. HOD). Chadefaud proposed that

the differences in their morphology were the result of

regressive evolution and that in the case of the distantly

related genus Ascobolus, all species had attained a similarly

reduced state by this means.-

Eckblad (1968) confirmed the presence of a funnel in

a number of unstated species and agreed with Chadefaud's

description of the operculate apical apparatus on all main

points. He stated, however, that the funnel was cytoplasmic

in origin and did not attach to the operculum. He also was

not able to detect an apical pad or cushion on the underside

of the operculum. Recent investigations of operculate

apical apparatuses (Wells, 1972; Samuelson, 1975; van

Brummelen, 1975; Chapter I) have demonstrated the presence

of a thickened, opercular inner layer which corresponds with

the apical pad. The apical globule, punctuation, funnel and

tract were not described in any of the "iodine-positive" and

suboperculate apical apparatuses.

LeGal (1953) described the ascal apices of Phaedro-

pezia epispartia (Berk and Br.) LeGal and Trichophaea









erinaceus (Schwein.) LeGal, both placed inside the Otidea-

Aleuria complex, to have apical apparatuses typical but less

apparent than that of the suboperculates. Eckblad (1968)

suggested that the occurrence of an incomplete ring in the

suboperculate apical apparatus of T. erinaceus and P.

epispartia as well as in those of the Sarcoscyphaceae were

artifacts of fixation. He believed that the apical chamber,

which contained the ring, represented only a swelling of the

inner layer of the operculum. Samuelson's (1975) and van

Brummelen's (1975) examinations of the suboperculate apical

apparatuses corroborated Eckblad's observations. Neverthe-

less, LeGal's report of suboperculate apical apparatuses in

T. erinaceus and P. epispartia indicated the possibility of

pronounced wall layering of the ascal tips, a character that

may prove to be taxonomically useful.

The only ultrastructural examination of the ascal wall

found in the Otidea-Aleuria complex was made by Schrantz

(1970) on Tarzetta cupularis (L. ex Fr.) Lamb. He noted

that the exoascus or outer layer consisted of a thin,

loosely woven sheath which thickened near the ascal tip.

By comparison, the endoascus or inner layer was thick and

finely granular and narrowed towards the tip. He likened

the form of the ascal wall to that of Peziza plebeia (LeGal)

Nannf. It was pointed out in Chapter I that the exoascus

described for P. plebeia was apparently a thick mucilaginous

coat. The ascal wall layering in T. cupularis may have been

similarly misinterpreted.









This study has incorporated morphological, develop-

mental and cytochemical examinations of the Otidea-Aleuria

apical apparatus in order to (1) ascertain the validity of

the features depicted by Chadefaud, (2) compare the mor-

phology and developmental sequences of apical apparatuses

between selected representatives, (3) determine whether

these features would be useful in the taxonomic positioning

of members within this largest of all operculate groups.

Ten members including Otidea leporina (Fr.) Fuckel,

Sphaerosporella brunnea (Alb. and Schw. ex Fr.) Svrcek and

Kub., Jafnea fusicarpa (Gerard) Korf, Humaria hemisphaerica,

Aleuria aurantia, Anthracobia melaloma (Alb. and Schw. ex

Fr.) Boud., Scutellinia scutellata (L. ex Fr.) Lamb.,

Ascozonus woolhopensis (Berk. and Br. apud Renny) E. C.

Hansen, Sowerbyella imperialis (Peck) Korf, and Geopyxis

majalis (Fr.) Sacc. were used.


Materials and Methods


Collection and Age Determination of Material

Young and mature apothecia of Anthracobia melaloma were

collected from charred wood near Gainesville, Florida. Dif-

ferent aged material of Humaria hemispherica was found grow-

ing in moss at the University of Florida's Horticultural

Farm outside Gainesville. Young and mature apothecia of

Aleuria aurantia and Jafnea fusicarpa and mature apothecia

of Otidea leporina were found on the forest floor at the

Devil's Millhopper in Gainesville. Young and mature









apothecia of Scutellinia scutellata were gathered from a

greenhouse bench of the Department of Plant Pathology at the

University of Florida. Fully developed apothecia of

Sphaerosporella brunnea were found on carbonized humus at

the Collier-Seminole State Park in Florida. Minute apothecia

of Ascozonus woolhopensis were found on rodent dung col-

lected in Macon County, North Carolina. Fresh material was

brought to the laboratory where free-hand sections were made

for light microscopic inspection to determine the stage of

ascal development. Young and mature apothecia were studied

separately when possible.

Dried specimens of Geopyxis majalis and Sowerbyella

imperialis were obtained from the Mycological Herbarium at

the University of Florida and Dr. R. P. Korf of Cornell

University, respectively. Portions of apothecia were re-

vived at room temperature in distilled water for 6 to 12

hours in a moist chamber for light and electron microscopic

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.









Procedures for Electron Microscopic Examinations

Entire apothecia of A. melaloma, A. woolhopensis and

S. scuttellata and five millimeter squares of apothecia of

A. aurantia, G. majalis, H. hemisphaerica, J. fusicarpa,

S. imperialis and S. Brunnea were fixed in buffered (0.2 M

sodium cacodylate pH 7.2) 2.0% glutaraldehyde and 2.0%

paraformaldehyde solution for two hours at room tempera-

ture. Five millimeter squares of 0. leporina and S. brunnea

were fixed in 1.0% permanganate solution for one hour at

room temperature. All materials were postfixed in osmium

tetroxide, dehydrated, embedded, section and poststained as

described in Chapter I.


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 Pm and a diameter of 9-12 pm. The subapical

region is notably heliotropic (Fig. 1). When stained in

Congo red, the ascal wall appears thick for most of the

length of the ascus. Near the tip of the ascus the wall

becomes thinner below the region of the operculum (Fig. 2).

By comparison, the opercular wall appears to be inflated.









The diameter of the ascus becomes narrower toward the tip.

After the spores have been discharged, the ascus shrinks to

a length of 150-170 pm while its diameter expands to 10-15 pm

(Fig. 4). The operculum, which usually remains attached to

one side (Fig. 5), is less conspicuous than when observed

in undischarged asci.

Ultrastructurally, the apical wall protrudes above the

subtending lateral wall, delimiting the operculum (Fig. 3).

The operculum has a diameter of 2.2-2.4 pm and a uniform

thickness of 170-180 nm. The suboperculum is 3.7-4.0 pm

long and decreases in thickness from 510-530 nm at its lower

extremity to 165-180 nm next to the operculum. The outer

layer of the operculum and distal region of the suboperculum

are loosely fibrillar and electron-dense, having a breadth

of 110-120 nm (Figs. 3, 6). Toward the base, the outer

layer thickens to 390-410 nm, having an additional internal

portion or stratum. The inner layer and the internal

stratum of the outer layer are both electron-transparent and

granular, being separated by a faint, opaque band (Figs. 3,

9). After treating the thin sections with silver methenamine,

the inner layer is stained most strongly (Fig. 7). The

internal stratum of the outer layer is stained but not as

intensely, and the electron-dense, fibrillar portion of the

outer layer remains unstained. As the time of spore release

approaches, the apical wall becomes markedly stretched

(Fig. 8). The opercular diameter increases 0.3-0.5 pm while

its thickness decreases by 40-60 nm. The upper extremity of









the suboperculum is similarly stretched. At ascal dehis-

cence, the upper extremity of the suboperculum becomes out-

wardly extended (Fig. 9). The length-of the suboperculum

shrinks to 3.0-3.3 pm.

The Apical Apparatus of Jafnea fusicarpa

During early spore formation, the ascal wall is thinner

at the apex and thicker laterally (Fig. 10). Further in

development, the apical and subapical walls are conspicu-

ously thick (Fig. 11). At maturity, the ascus is long and

narrow, 290-310 pm x 18-22 pm. The ascal tip, which is

slightly heliotropic, has formed two layers (Fig. 13). The

first spore often becomes lodged against the opercular

region of the ascus. During spore release, the operculum

is thrown to one side and the ascospores are ejected one

after another in rapid succession. The operculum typically

remains attached to the ascus and frequently returns to its

original prone position. As the spores are released they

become strongly compressed between the sides of the ascal

wall (Fig. 14).

Electron microscopic examination of a four-nucleated ascus

reveals for the most part the presence of a thick, lateral

wall, 820-840 nm (Fig. 12), which decreases in thickness to

500-540 nm at the tip. Numerous small vesicles are ob-

served in the apical region of the ascus. During early

spore wall formation, the ascal tip becomes faintly helio-

tropic (Fig. 15). The thickness of the lateral wall has

increased to 1000-1050 nm while the tip retains a thickness









of 480-520 nm. The apex is wider, having expanded to a

diameter of 9.2-9.4 pm. Several large vacuoles are present

near the tip and extend to the base of the ascus. At the

end of spore development the apical apparatus continues to

be developed (Fig. 16). The ascal tip has increased by

1.3-1.8 pm in diameter. An inner layer was deposited mainly

in the vicinity of what will become the operculum. Lateral

walls appear to be stretched, having a thickness of 780-

820 nm. Treatment with silver methenamine strongly accen-

tuates the bilayered nature of the mature ascal wall (Fig.

17). The thickness of the inner layer narrows from 400-

410 nm at the operculum to 50-65 nm at the lower extremity

of the suboperculum where a small annular bulge or ring has

formed (Fig. 17, arrows). An opercular boundary is not ob-

served at any time prior to spore release. At dehiscence,

the operculum remains partially fastened to the suboperculum

(Fig. 18). At this stage, the suboperculum, which is 5.5-

6.3 pm long, has thickened to 950-980 nm at its lower ex-

tremity where the subopercular ring has become greatly

enlarged.

The Apical Apparatus of Humaria hemisphaerica

Asci containing immature ascospores are 160-240 pm long

and 12-16 pm wide. They appear thick-walled except at the

apex (Figs. 19, 20). In mature asci, which are long and

cylindric (280-340 x 16-20 pm) the tips have become notably

thicker (Fig. 28). An inner layer is detected in the oper-

cular and subopercular region of the ascus after staining of









one-micron plastic sections with toluidine blue. Sub-

apically, a faint bulge is detectable at the level of the

first spore (Fig. 28). In asci that are about to rupture

(Fig. 27), a hyaline ring delimits the operculum. The ascus

appears stretched and thinner-walled at this stage.

The fine structure of ascal tips at early spore wall

formation (Fig. 21) shows the ascal wall to be relatively

thin (190-210 nm) in the immediate region of the tip. At

the level of the first spore the wall increases to 520-

550 nm in thickness. During ontogeny, the asci and para-

physes are embedded in a mucilaginous matrix (Figs. 21-26,

29-33). As the ascospores continue to mature (Fig. 22), the

wall dimensions stay approximately the same. Within 3.1-

3.5 pm of the apex an annular protuberance is faintly de-

tected on the inner surface of the ascal wall (Fig. 22).

At spore maturity (Figs. 23, 25), this subapical ring be-

comes slightly more defined. The thickness of the apical

and lateral ascal walls has not changed. A large vacuole

exists throughout most of the ascus except at the tip which

remains filled with cytoplasm (Fig. 23). Figure 24 represents

a later time in development. The apical and subapical walls

have increased in thickness to 310-350 nm and 700-750 nm,

respectively, due to the addition of an inner layer (Fig.

26). The subapical ring, which is stained by silver

methenamine, appears to be confluent with the inner layer

(Fig. 26). At a subsequent stage, the inner layer, irregu-

lar in outline, thickens to 140-180 nm in the vicinity of








the operculum (Figs. 29, 30, 31). The subapical or sub-

opercular ring is observed infrequently at this time (Fig.

26). Demarcation of the operculum is not apparent in any

developmental stage of intact asci. At ascal dehiscence,

the dislodged operculum is observed occasionally (Fig. 33),

having a diameter of 5.8-6.2 pm and a breadth of 380-420 nm.

The suboperculum is 4.9-5.2 pm long.

The Apical Apparatus of Sphaerosporella brunnea

The mature ascus is broadly cylindric (150-190 um x

16-20 pm). The rounded to blunt tip appears thinner-walled

than the rest of the ascus (Fig. 35). The diameter of the

ascospores (13-16 pm) is greater than that of the ascal

tip (9-10 pm). Consequently, the first ascospore rests

near the tip but is not closely appressed against the oper-

culum. At spore release the operculum remains attached to

one side of the ascus (Fig. 36). The ascostome appears to

have a wider diameter than that of the operculum, which at

this time is 5-6 pm. The lateral ascal wall collapses and

folds as well.

Ultrastructurally, the tip of a mature ascus consists of

a flattened, thin wall, 420-470 nm thick, which broadens to

680-740 nm towards the ascal base (Figs. 37, 38). When ex-

amining potassium permanganate-fixed material (Fig. 37), the

wall layers are sufficiently distinguished. The outer layer

is composed of a rough, electron-dense external stratum,

which broadens from 45-65 nm subapically to 130-150 nm at

the apex, and an electron-transparent internal stratum









(Figs. 37, 39). In general, the outer layer increases in

thickness from 280-320 nm throughout the opercular region

of the ascus to 500-550 nm at the base of the suboperculum

which is marked by the presence of a subopercular ring

(Figs. 37, 39, 41). The subopercular ring is 450-480 nm

long and consists of an electron-dense band, 45-55 nm thick,

which has been deposited within the inner layer against the

internal stratum of the outer layer. Like the outer layer,

the inner layer thickens from 130-150 nm at the tip to 190-

220 nm at the level of the subopercular ring (Fig. 37).

Asci fixed in a glutaraldehyde-paraformaldehyde solution and

poststained in lead citrate and uranyl acetate do not demon-

strate wall layering (Fig. 38). Slight bulges (arrows) in-

dicate the locality of the subopercular ring. Treating the

thin sections with silver methenamine sharply defined the

two wall layers (Figs. 40, 41). More importantly, the in-

tense staining of the internal stratum of the outer layer

in the operculum distinguishes this region from the sub-

operculum (Fig. 40). The suboperculum is 9.7-10.4 pm long

and tapers in thickness from 680-740 nm at the level of the

subopercular ring to 390-420 nm near the operculum. The

operculum is 4.8-5.2 pm in diameter, which compares closely

with the diameter of opercula seen in dehisced asci under

the light microscope. In dehisced asci (Fig. 42), the lower

extremity of the suboperculum is 380-400 nm thick. The wall

immediately below the subopercular ring, however, has a

thickness of 550-580 nm. It would appear that prior to








spore release the suboperculum becomes greatly stretched and

remains so after spore release.

The Apical Apparatus of Aleuria aurantia

During early spore formation, the ascal wall appears thin

at the tip and progressively becomes thicker toward the

base (Fig. 43). At a later stage in development, the ascus,

which is more inflated at the apex, is seen to be slightly

heliotropic (Fig. 44). By maturity the ascus reaches a

length of 200-240 Pm and a diameter of 12-16 vm. No dis-

tinctive features are observed in the rounded ascal tip

(Fig. 51). At ascal dehiscence, the operculum, 4-5 pm in

diameter, remains partially affixed to the lateral wall

(Fig. 52). The diameter of the ascostome is roughly similar

in size to that of the operculum.

Ultrastructural observations of asci approaching the end

of ascospore development reveal thick lateral walls, 400-

420 nm, which taper at the apices to a thickness of 140-

170 nm (Fig. 45). At 4.0-4.4 Pm below the tip, the wall

protrudes into the ascoplasm, forming a subopercular ring.

A plasmalemmasome of considerable size is associated with

the early development of this ring (Figs. 45, 46). When

stained with silver methenamine at this stage of development,

the ascal wall consists primarily of a thick outer layer

(Fig. 47) and a thin (25-35 nm thick), strongly stained

inner layer. The small plasmalemmasome in Fig. 47 is also

stained intensely. The formation of the subopercular ring

appears to be initiated asymmetrically (Fig. 48). At times









the subopercular ring becomes prominent, being 800-840 nm

thick and 320-420 nm long (Figs. 49, 50). The ascal tip is

filled with long, laminated endoplasmic reticulum (Figs. 45,

46, 49) which is associated with mitochondria, lipid bodies,

ribosomes and small vesicles.

The apical wall of the mature ascus broadens to 280-

300 nm (Fig. 54). Staining with silver methenamine demon-

strates that the increase in thickness is due to the thick-

ening of the inner layer, which is 140-160 nm throughout the

opercular and much of the subopercular regions (Fig. 53).

The outer layer is 100-130 nm thick apically and increases

to 280-300 nm toward the base of the ascus. A subopercular

ring was never detected at this stage in development

(Fig. 53). At incipient ascal dehiscence (Fig. 55), the

inner layer of the operculum is pulled away from the sub-

operculum. The opercular boundary is observed only at this

time and in dehisced asci (Fig. 56). The operculum has a

diameter of 4.0-4.2 pm and a uniform thickness of 260-

290 nm. After spore release (Fig. 57), the suboperculum,

which is 5.8-6.2 pm long, thickens from 210-240 nm at the

distal end to 360-440 nm at the level where the subopercular

ring was previously observed. The inner layer broadens from

110-120 nm to 170-190 nm for a short distance in the lower

extremity of the suboperculum.

The Apical Apparatus of Anthracobia melaloma

Mature asci are cylindric, 160-200 pm x 10-14 pm (Fig.

58). The ascal wall appears to be thicker throughout the








lateral face of the ascus and thinner at the tip (Fig. 59).

Near the ascal tip a slight protuberance of the lateral wall

is observed occasionally (Fig. 59). An apical funnel and

continuous tract, which lead to the first spore, have been

detected in asci that were placed in Congo red or aniline

blue. One-micron sections stained in toluidine blue (Fig.

60, arrows) exhibited a subapical ring or band.

Electron microscopic observations of asci at early spore

wall formation show that a localized band starts to form at

3.2-3.8 pm from the tip (Fig. 61, arrows). Lomasomal ac-

tivity is seen throughout the apical region of the ascal

wall at this stage in spore development. As the spores

continue to mature (Figs. 62, 63) the subopercular band

becomes more conspicuous, consisting of an electron-dense

area, 560-600 nm long and 50-60 nm thick. When treated with

silver methenamine (Fig. 62) only the internal face of the

subopercular band is stained. The wall at the immediate

region of the tip is 140-170 nm thick. Subapically, the

ascal wall reaches a thickness of 250-290 nm by a distance of

200-250 nm from the tip and stays at that thickness for the

remainder of the ascus (Fig. 63). Near the end of asco-

sporogenesis, the inner layer is formed throughout the ascal

wall (Fig. 64). Lomasomes are again observed, being most

abundant in the vicinity of the apex. The subopercular band

develops into a distinct, swollen ring. At spore maturity,

formation of the inner layer is completed (Figs. 65, 66, 67,

68). The area of the operculum is delimited by its greater









thickness, 240-270 nm, from the distal end of the suboper-

culum. Most of the thickness of the operculum is due to the

outer layer being 165-190 nm (Figs. 66, 68). The subopercu-

lar outer layer measures 65-100 nm at its upper extremity

but increases to 210-220 nm by the level of the subopercular

ring. In its entirety, the suboperculum, which is 6.8-

7.2 pm long, broadens from 190-210 nm next to the operculum

to 350-380 nm just below the subopercular ring (Figs. 65,

67). The inner layer of the suboperculum is divided into an

electron-transparent internal stratum and an electron-opaque

external stratum (Fig. 67). The demarcation between the two

strata is sharper than between the outer layer and the inner

layer's external stratum. This study treats the external

stratum as a part of the inner layer rather than refers to

it as a separate layer for the following reasons: (1) The

separation of the internal and external strata of the inner

layer cannot be made developmentally. The formation of

both strata is a continuous action. On the other hand, there

is a period when wall development ceases to occur between

outer layer and the external stratum of the inner layer.

(2) The external and internal strata of the inner layer are

combined below the operculum. Consequently, stratification

of the inner layer is not observed in the operculum. When

staining with silver methenamine the inner layer reacts

positively within the operculum. The internal stratum of

the inner layer also is richly stained throughout the sub-

operculum. The outer boundary of the inner layer's external








stratum is stained, delimiting the inner layer in this

region of the ascal wall.

The Apical Apparatus of Scutellinia scutellata

Young asci at the eight-nucleated to early spore stages

are cylindric to subcylindric, 100-150 pm x 8-12 pm. The

ascal wall, which stains strongly in Congo red, is distinctly

thin at the tip (Fig. 69). Mature asci (Fig. 70) reach a

length of 200-260 pm and a diameter of 14-18 pm. The ascal

wall appears to be uniformly thick in the apical and sub-

apical regions (Fig. 77). No distinctive features are ob-

served in the mature tip. At ascospore discharge, the

operculum is partially attached to the ascus (Fig. 78). The

lateral walls appear flaccid and typically collapse.

Ultrastructurally, the apex of a four-nucleated ascus is

thin-walled, 130-160 nm, and stains strongly in silver

methenamine (Figs. 73, 75). Numerous, small to large vesi-

cles are scattered throughout the upper half of the ascus

(Fig. 73). Large lomasomes are frequently observed in both

the apical and lateral regions of the ascal wall (Fig. 75).

The lateral wall is comparably thick, 240-255 nm, and stains

weakly in silver methenamine in its outer stratum. A thin

mucilaginous coat, 65-75 nm, covers only the tip of the

ascus (Fig. 75). In the eight-nucleated ascus (Fig. 74),

the mucilaginous coat increases to a thickness of 210-240 nm

at the tip and extends to the base of the ascus. An ex-

aggerated lomasomal-like band forms below the tip at this








stage in development. The innermost portion of the wall in

this zone reacts positively in silver methenamine (Fig. 74).

At early spore wall formation, the tip, which remains

thin-walled, 140-180 nm, is filled with small vesicles and

endoplasmic reticulum (Figs. 71, 76). At 5.6-5.9 pm below

the tip a subopercular ring protrudes inwardly from the

lateral wall (Fig. 71). In Fig. 72 the annular protuber-

ance has an irregular, lomasomal appearance. After treat-

ment with silver methenamine, the innermost portion of the

ascal wall is stained as well as its outermost portion

(Fig. 76, arrows). By spore maturity, the internally stained

region of the ascal wall in Fig. 76 has developed into an

intensely stained middle layer (Figs. 79, 80, 81), which de-

creases from 110-130 nm in thickness at the upper extremity

of the ascus to 45-65 nm toward the base. In the vicinity

of the previously existing subapical protuberance, the

middle layer is weakly stained for a length of 520-540 nm

(Fig. 79). The ascal tip has broadened to 370-390 nm. The

increase is basically due to the production of an inner

layer which is 130-150 nm thick throughout the opercular

region and tapers to 30-40 nm at the level of the subopercu-

lar ring (Figs. 79, 80). Sections that are stained with

uranyl acetate and lead citrate exhibit a thick mucilaginous

coat, 540-650 nm (Fig. 82). The inner and middle wall

layers are weakly differentiated. Delimitation between the

mucilaginous coat and the outer layer is extremely difficult.

At the initiation of ascal dehiscence, the middle and outer









layers are pulled apart along a zone of dehiscence between

the operculum and suboperculum (Figs. 81, 83). The operculum

at this stage has a diameter of 6.8-7.0 pm. The lower ex-

tremity of the subopercular wall has thickened to 470-490 nm.

The Apical Apparatus of Ascozonus woolhopensis

Before ascospore delimitation, asci reach a length of

80-160 pm and diameter of 14-18 pm. They are broadly

clavate and possess aconical tip (Fig. 84). Below the tip,

a swollen ring protrudes from the thinner side of the ascal

wall. By maturity, the ascus increases an additional 20-

40 pm in length and 4-6 pm in diameter. The ring and apical

wall appear more pronounced in thickness (Fig. 85). When

placed in Congo red, the subapical ring and outer layer of

the ascal wall are strongly stained (Fig. 85). The immedi-

ate region of the tip remains hyaline. Ascal dehiscence

occurs by two or three transverse fissures (Fig. 94) that

originate at the tip.

Ultrastructurally, young asci during early ascosporo-

genesis (Fig. 86) are conspicuously thick-walled. The

apical wall, being faintly conical, consists of two broad

layers and is bordered by a prominent subapical ring. The

outer layer is thinner narrowing from 550-610 nm at the apex

to 320-280 nm at the ring. The inner layer similarly de-

creases from 900-950 nm at the apex to 350-400 nm just above

the ring. At the ring the two layers are indistinct. The

ring is 1250-1300 nm thick. The lateral wall below the ring

is 1300-1350 nm thick,being mostly comprised of a thickened









inner layer, 1000-1050 nm (Fig. 86). In Fig. 87, a stage

comparable to Fig. 84, the apical region of the ascus is

distinctly conical. The outer layer and most of the sub-

apical ring are intensely stained by silver methenamine

(Figs. 87, 91). The inner layer appears to be stratified

for a distance of 2.4-2.8 pm above the ring where demarca-

tion of the sublayers is weakened toward the tip. At the

ring the inner layer remains unstained, forming a thin,

transparent band, 25-35 nm, which broadens to 750-830 nm

below the ring. The inner layer expands from a thickness of

680-720 nm above the ring to 1790-1850 nm at the apex. A

small pore is formed at the tip during this stage of develop-

ment (Fig. 88). The outer layer and a middle stratum broad-

en from 320-350 nm above the ring to 450-480 nm below the

tip. From there the outer layer tapers to 130-190 nm at the

tip. The mature apices basically exhibit no differences in

form from that shown in Fig. 87. The thicknesses of the lat-

eral and subapical walls are reduced by 360-400 nm and 250-

280 nm, respectively, while the ring stays at 1300-1350 nm.

The attenuation of the lateral and subapical regions indi-

cates that the ascal wall is becoming stretched at this time.

The middle stratum and the inner and outer layers are ob-

served more clearly in peripheral sections (Fig. 89). Visual

distinction of the wall layering at the subapical ring is

extremely difficult to make in median sections. Peripheral

sections of the ring show that the middle stratum comprises

most of that region (Figs. 90, 93), thus reinforcing the








layering distinguished by the silver methenamine stain in

Figs. 87 and 91.

In dehisced asci, an apical disc (Fig. 92), consisting

mostly of the outer layer, is observed. The electron-

transparent inner layer appears to have shrunk or dissolved

at this stage. The disc has a diameter of 2.4-2.6 pm and a

thickness of 550-600 nm.

The Apical Apparatus of Geopyxis majalis

Mature asci are narrow and cylindric, 260-300 pm x

10-14 Pm. The ascal wall appears to be uniformly thick at

the apex and lateral sides (Fig. 95). Asci with immature

spores have thinner apical walls (Fig. 96). At dehiscence

the spores are released through a wide ascostome (Fig. 96)

with the operculum being completely detached.

Electron microscopic examinations of mature asci

demonstrate a bilayered apical wall (Fig. 97). The layers

are strongly accentuated when treated with silver methenamine

(Fig. 99). The inner and outer layers of the opercular wall

are identically 170-190 nm thick. At the periphery of the

operculum the outer layer forms a bulge increasing in thick-

ness to 210-240 nm for a length of 270-300 nm while the

inner layer decreases to 85-95 nm in this region (Fig. 99,

arrows). The inner layer briefly thickens to 125-160 nm

below the bulge before tapering to 25-40 nm at 3.2-4.0 pm

below the apex. Conversely, the outer layer briefly thins

to 125-160 nm below the bulge before it broadens to 320-

340 nm toward the base of the ascus. Younger asci do not









display a distinctly stained inner layer (Fig. 98). The

distal region of the ascus is filled with cytoplasm. The

walls of immature and mature asci are covered with a muci-

laginous coat (Figs. 98, 99). At incipient ascal dehiscence,

the subopercular and opercular inner layers are pulled apart

(Fig. 101), thus delimiting the operculum. During spore

release the upper extremity of the suboperculum is tightly

pressed against the sides of the spore as the spore passes

through the ascostome (Fig. 100).

The Apical Apparatus of Sowerbyella imperialis

The mature ascus is long and cylindric, 180-200 pm x

10-13 pm. The wall appears thin throughout the ascus,

staining lightly in Congo red (Fig. 102). The operculum,

having a diameter of 5.5-6.5 pm, is observed with the aid of

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 pm. At spore release,








the suboperculum, which is 4.6-5.1 pm 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 imperialism. Van

Brummelen (1974) demonstrated an apical disc in the ascus of

A. woolhopensis with the aid of the stain Congo red and thus

substantiated earlier findings of Vuillemin (1887).

Kimbrough (1972), however, pointed out that the nippled tips

in asci of Ascozonus cunicularius (Boud.) Marchal were un-

stained in Congo red. He did not observe a disc or lid in

asci of that species. Similarly, the apical apparatus of








A. woolhopensis in the present study did not exhibit an

apical disc in fresh material that had been stained with

Congo red. Observations of A. cunicularius (Kimbrough, 1972)

and A. woolhopensis in this study were made from material

that was collected in North America. Staining properties

of the small disc may vary according to species and isolates

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 Humaria hemisphaerica, were currently studied.

Components including the apical punctuation, tract and fun-

nel were not seen in stained and unstained fresh material

of either species. Of the remaining species in the present

examination, Anthracobia melaloma has a funnel and tract in

the apical region of its ascus. These features were vague

and infrequently observed. Apical spherules were seen in

most species from time to time but without regularity.

Opercular pads reported by LeGal (1953) in species of

Phaedropezia and Trichophaea were found in the apical ap-

paratuses of Otidea leporina. Optical view of the mature

ascal tip in 0. leporina seemed to show an expanded opercu-

lar wall. Fine structure of the apex, however, demonstrated

the opercular wall to be thinner than the subtending lateral

wall and have a convex shape. Thus, the structurally weak

ascal tips of O. leporina, seen with the light microscope,









frequently became invaginated during mounting or staining

procedures. This phenomenon was misleading in the present

study and may have misled LeGal in her analysis of

Phaedropezia and Trichophaea.

The distinct light microscopic feature of the different

ascal tips in the present investigation was the thickness of

the ascal walls. Subapical walls of asci in 0. leporina,

Sphaerosporella brunnea, Jafnea fusicarpa and Humaria

hemisphaerica appeared considerably thicker than those seen

in A. melaloma, Scutellina scutellata, Geopyxis majalis and

Sowerbyella imperialis. In multispored asci (more than eight

per ascus) of A. woolhopensis the walls were extremely thick

and displayed two layers. A bilayered ascal wall was also

observed in H. hemisphaerica and J. fusicarpa.

Ultrastructural observations of the ten species present-

ly studied reinforced the light microscopic findings. Thick-

ness of the lateral ascal walls was sharply divided between

those that were thin, 350-450 nm, as in Aleuria aurantia,

Anthracobia melaloma, Geopyxis majalis, Scutellinia

scutellata and Sowerbyella imperialis and those that were

thick, 700-1100 nm, as in Ascozonus woolhopensis, Humaria

hemisphaerica, Jafnea fusicarpa and Sphaerosporella brunnea.

Otidea leporina was the only representative that did not

fall into either division. Gross morphology of the apical

apparatuses was similar for all by A. melaloma, 0. leporina

and S. brunnea. The outer layer decreased in thickness

toward the tip while the inner layer decreased toward the








base. In A. melaloma, 0. leporina and S. Brunnea the inner

layer thickened toward the base or stayed approximately at

the same thickness throughout the ascal wall.

With the exception of A. woolhopensis, the development

of the apical apparatuses essentially followed the three-

step sequence that was outlined in Chapter I. In two spe-

cies, J. fusicarpa and H. hemisphaerica, the inner layer was

formed after spore ontogeny. Their pattern of development

resembled that seen in lodophanus granulipolaris Kimbr.

Formation of the apical apparatus in A. woolhopensis dif-

fered significantly in several respects. The inner layer

of the lateral ascal wall was formed before ascospore delimi-

tation. By early ascosporogenesis the development of the

apical apparatus was complete. At this stage a pore had

formed in the inner layer at the ascal tip. Van Brummelen

(1974) noted the appearance of the pore at a later time in

development and suggested that it was formed from a process

of localized disintegration. The pore may have been, how-

ever, the result of wall stretching as the ascus expanded in

length and width throughout ascosporogenesis. A similar

phenomenon was observed in the thick-walled apex of Cookeina

sulcipes (Berk.) Kuntze (Samuelson, 1975).

Schrantz's (1970) interpretation of the development of

the ascal wall in Tarzetta cupularis differed significantly

from that shown in the present study. He stated that in

young asci the exoascus consisted of a thin band of electron-

dense, fibrillar material and by spore formation, this layer








had increased in thickness mostly at the tip. He concluded

that most of the wall consisted of a thick endoascus which

tapered in thickness toward the tip. Apparently, Schrantz

did not examine fully ripened asci as an examination of his

photographs will confirm. His descriptions of exo- and endo-

ascal walls of T. cupularis corresponded to the mucilaginous

coat and outer layer, respectively, of a number of species

including S. scutellata, S. imperialis, and A. woolhopensis.

Cytoplasmic components of the operculate apical appa-

ratus described by Chadefaud (1942) (Fig. 110A) were not de-

tected ultrastructurally in any of the representatives

currently examined. Chadefaud's discovery of the funnel and

tract were most likely artefacts of fixation and staining.

He frequently applied either Melzer's reagent or a chromium

trioxide-osmium tetroxide solution when examining the apical

apparatus. These solutions may have caused the collapse of

the ascal plasmalemma and tonoplast beneath the apical region

of the ascus, thus creating a funnel and tract. Tips of

most representatives in the present study were filled with

cytoplasm throughout ascosporogenesis. The apical cytoplasm

may account for the presence of apical punctuations observed

by Chadefaud above the funnel (Fig. 110A).

An interesting wall component that was pointed out by

Chadefaud (1942) in several species was the occurrence of a

subapical "bourrelet," i.e. pad. In the general scheme of

the operculate apical apparatus (Fig. 110A) the subapical pad

adjoined the operculum. However, his diagrams of dehisced









asci in Aleuria aurantia and Scutellinia hirta (Fig. 11OB, C)

depicted subapical pads that were further removed from the

tip. Subapical pads were not currently seen with the light

microscope in A. aurantia or S. scutellata. Ultrastructur-

ally, a subopercular, asymmetrically formed, swollen ring

was discovered in A. aurantia during late spore development.

Although the ring had disappeared by maturity, the sub-

opercular wall was thickest in that vicinity. In S.

scutellata a subopercular ring was also observed with the

aid of the electron microscope. The subapical pads de-

scribed by Chadefaud may have represented subopercular

rings. He noted that the pads were often asymmetrical as in

A. aurantia and depicted them in only mature and dehisced

asci. S. hirta and A. aurantia were both studied by

Chadefaud in a chromo-osmic solution. Wall layering in

that region of the ascus may have been abnormally affected

by this solution, thus permitting observation of the pads.

Among the ten members of the Otidea-Aleuria complex

presently studied, seven species displayed subopercular

rings. First appearance of the ring occurred at different

developmental stages for different species. In J. fusicarpa

an annular protuberance was detected after the inner layer

was formed. By comparison, the first signs of a subopercu-

lar ring in A. melaloma were observed prior to the formation

of the inner layer. In general, development of the annular

swelling occurred during the last stages of ascosporogenesis

and marked the initiation of the formation of the inner








layer. However, in A. woolhopensis the ring began to develop

shortly after meiosis in the young, truncate ascus. Similar

observations of A. woolhopensis were made by van Brummelen

(1974). Still., both investigations demonstrated that the

ring was derived from the local expansion of the inner layer.

Van Brummelen's interpretation of the wall composition

in A. woolhopensis at the ring and throughout the apical

region of the ascus differed from the present study in three

respects. (1) He recognized the presence of a middle layer

in the subapical wall which disappeared at the level of the

ring. This layer is currently described as part of the

middle stratum which extends to the base of the ascus.

Median sections of younger asci stained with silver methena-

mine and peripheral sections of mature asci established this

finding. (2) Van Brummelen demonstrated an electron-dense

internal layer at the level of the ring. Since he was not

able to follow it above or below the ring, he referred to

the area as the internal ring layer. Staining with silver

methenamine revealed that the internal ring layer was a

part of the inner layer. (3) The present study also reports

the presence of a distinct middle stratum within the inner

and outer layers. The ring was shown to consist mostly of

the middle stratum (Fig. 111H) which appears to be chemically

similar to the outer wall layer. Similar findings were ob-

served with the light microscope using Congo red. Van

Brummelen did not make these distinctions.








The smallest subopercular rings occurred in the thick-

walled species, J. fusicarpa, H. hemisphaerica and S.

brunnea. In contrast, taxa that formed conspicuous rings

during the development of their apical apparatuses, i.e.,

A. aurantia and A. melaloma, were thin-walled. A. woolhopen-

sis was the sole exception, having both thick walls and an

enormous ring. Multispored asci, in general, have been shown

to be thick-walled (Kimbrough, 1966a,b, 1969; Kimbrough and

Korf, 1967). In Chapter I wall dimensions of the 32-spored

representative Thecotheus pelletieri (Crouan) Boud. were

roughly 3 times that of the 8-spored members in the iodine-

positive group. In Chapter III the apical apparatus of the

multispored species Coprotus winter (Marchal) Kimbr. was

shown to be essentially an enlarged replica of the eight-

spored species Coprotus lacteus (Ck. and Phill.) Kimbr.

Similarly, wall dimensions of A. woolhopensis were approxi-

mately two and one-half to three times that of A. melaloma

and A. aurantia. Therefore, when taking the exaggerated

condition of the multispored ascus into consideration, the

apical apparatus of A. woolhopensis is remarkably similar in

form to that of A. melaloma.

Kimbrough and Benny (1977) have recently described in

another multispored representative, Lasiobolus monascus

Kimbr., the presence of a large subopercular ring during the

development of its apical apparatus. As in Aleuria aurantia,

the ring, which became most prominent during ascosporogene-

sis, was not apparent at the end of spore development.








Stretching of the ascal wall prior to spore release may have

been responsible for its disappearance.

Within the Otidea-Aleuria complex the function of the

subopercular ring appeared to be associated primarily with

structural support of the apical region of the ascus during

spore release. At incipient ascal dehiscence, the lateral

wall became markedly stretched, narrowing its thickness by

10-20%. In the species A. melaloma, A. aurantia, S. brunnea

and S. scutellata, where the subapical walls decreased sig-

nificantly in thickness toward the tip, the ring most likely

provided additional strength and maintained the shape of

the ascal tip. During dehiscence, the structural role that

the ring played was most evident in A. woolhopensis, keeping

the lateral wall intact as the spores shot through the

fissured opening. After dehiscence, in certain members,

J. fusicarpa, 0. leporina and H. hemisphaerica, the lateral

wall appeared to be elastic, having returned to its original

thickness. In S. brunnea elasticity of the subopercular

wall was not observed. The wall above the subopercular ring

remained stretched. Although a distinct subopercular ring

was not found in mature asci of 0. leporina, the sharp in-

crease in wall thickness at the lower extremity of the sub-

operculum may have provided strength comparable to that of

the localized rings in S. brunnea and A. melaloma.

Apical apparatuses of Geopyxis majalis and Sowerbyella

imperialism demonstrated general similarities with the other

taxa. Although revived material was used, wall layering of








the ascal tip was readily distinguished in both species.

The presence of a ring at the periphery of the operculum in

G. majalis was unique among the representatives of the

Otidea-Aleuria complex examined. The thinness of the ascal

wall in S. imperialis was equally atypical. Subopercular

rings were not observed in either representative. Since

most of the subopercular rings currently studied have been

shown to be inconspicuous, developmental examinations of

fresh specimens are necessary to verify the presence or

absence of this component within the ascal wall.

The present examination has demonstrated that within

the members studied no two species shared identical apical

apparatuses. Ascal tips consisted primarily of thin-walled

and thick-walled forms. When comparing the thick-walled

ascus of J. fusicarpa (Fig. 111E) to the thin-walled ascus

of A. melaloma (Fig. 111G), the differences were distinct.

However, the species H. hemisphaerica, S. brunnea (Fig. 111F)

and A. aurantia exhibited intergrades between the two. De-

fined types of apical apparatuses within the Otidea-Aleuria

complex could not be made on the basis of wall thickness or

any other feature of the ascal tip including the subopercular

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









the spore number. Presence of subopercular rings in A.

woolhopensis and L. monascus indicated possible taxonomic

affinities with members of the Otidea-Aleuria complex.

Both species have been placed in the Theleboloceae, based

primarily on their multispored condition (see Chapter V).

In a world monograph of Lasiobolus, Bezerra and Kimbrough

(1975) stated that developmental, morphological, and micro-

chemical properties of Lasiobolus were more related to

Cheilymenia, Coprobia and Scutellinia than to Thelebolus.

They suggested that Lasiobolus belonged in the tribe

Scutellinieae sensu Korf (1973). The present study was

supportive of their findings. Cytological and developmental

studies on Ascozonus are needed to understand more clearly

its taxonomic position within the operculate Discomycetes.

Of the operculate apical apparatuses that have been ultra-

structurally examined in present and previous examinations,

A. woolhopensis most clearly resembled Anthracobia melaloma

in both structure of the ascal wall and general form.

Apical appratuses of the Otidea-Aleuria ascus were

found to be morphologically diverse. The diameter and thick-

ness of the operculum, the length and thickness of the sub-

operculum, the stratification of the wall, the size and

shape of the subopercular ring and the ontogeny of the dif-

ferent components varied from taxon to taxon. Still, sev-

eral characters of the apical apparatuses were generally

common for the representatives presently studied. A distinct

dehiscent region observed in the iodine-positive ascus








(see Chapter I), the eugymnohymenial ascus (see Chapter III)

and the suboperculate ascus (Samuelson, 1975; van Brummelen,

1975) was infrequently found in the Otidea-Aleuria complex.

The protruding apical wall in 0. leporina and the thickened

apical wall in Anthracobia melaloma were the only examples of

morphologically delimited opercula. Differential staining

of the operculum with silver methenamine, which was described

in lodophanus granulipolaris Kimbr. (see Chapter I), Pyronema

domesticum (Sow. ex Fr.) Sacc. and several representatives

of the Sarcoscyphineae (Samuelson, 1975), occurred only in

S. brunnea. The differences of the staining intensities of

the opercular wall most likely signify changes in the wall

chemistry at that area. Other operculate species may have

opercula that differ chemically from the rest of the ascal

wall but do not exhibit the differences when staining with

silver methenamine or lead citrate and uranyl acetate.

Further examination of ascal walls is needed to find various

treatments that could reveal this feature.

A fibrillar zone of dehiscence was not detected in the

ascal tips of any of the species in the present study. On

the other hand, the subopercular ring was a common feature

of the wall of the Otidea-Aleuria ascus. All of the members

that were investigated developmentally demonstrated to some

extent the presence of this component. The similarity of

the apical apparatuses and their subopercular rings in S.

brunnea and A. melaloma quite possibly suggests closer

taxonomic relatedness between those taxa than shown in





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.


Figure 1.


Figure 2.


Figure 3.



Figure 4.


Figure 5.


Figure 6.



Figure 7.



Figure 8.


Figure 9.


Otidea leporina


Subapical region of mature ascus is distinctly
heliotropic. X400.

Tip of mature ascus. Spore (SP). Stained
with Congo red. X1,500.

Raised portion at tip of mature ascus, de-
limiting operculum (0). Inner layer (IL).
Spore (SP). X14,000.

Shrunken ascus after spore release. Phase
contrast. X400.

Dehisced ascus with operculum (0) laterally
attached. Stained with Congo red. X1,500.

Distal portion of suboperculum. Arrows point
to region where opercular dehiscence will
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
(0). X14,000.

Dehisced ascus showing shrunken suboperculum
(SO). Ascostome (A). Outer layer (OL).
X15,000.



























7e
tn


Winaa


&


0


SW
Ae
as'"


Oki
















Chapter II


Figures 10-18.


Figure 10.


Figure 11.



Figure 12.



Figure 13.


Figure 14.


Figure 15.




Figure 16.


Figure 17.




Figure 18.


Jafnea fusicarpa


Ascal tip during early spore formation.
Stained with Congo red. X1,000.

Apical and subapical walls at later time in
development. Stained with Congo red.
X1,000.

Apical region of four-nucleated ascus with
scattered small vesicles (V). Nucleus (N).
X4,000.

Mature ascal tip showing slight heliotro-
pism. Spore (SP). X1,000.

Ascospore is compressed within ascostome
(A) during its ejection. X500.

Ascal tip during early spore wall develop-
ment shows increased thickness at lateral
walls. Apical wall (AW). Vesicle (V).
X6,700.

Broadened ascal tip at end of spore develop-
ment. Operculum (0). X6,200.

Mature apical apparatus with small sub-
apical, swollen ring (arrows). Inner layer
(IL). Outer ring (OL). Stained with silver
methenamine. X5,000.

Dehisced ascus with attached operculum (0).
Suboperculum (SO). Subopercular ring (SR).
Stained with silver methenamine. X5,400.