COMPARATIVE ONTOGENY OF THELEBOLUS,
LASIOBOLUS, AND THECOTHEUS (PEZIZALES, ASCOMYCETES)
KENNETH EDWARD CONWAY
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
The guidance, friendship and patience of Dr. James W.
Kimbrough throughout this course of study is greatly
.appreciated. I am also indebted to the entire committee
for time spent in critical reading of the dissertation and
for their helpful comments and suggestions. Also, I wish
to tank my wife, Cynthia, for her love, encouragement, and
typing cf the dissertation. This dissertation is dedicated
to our first child, Kenna Anne, born January 28, 1973.
TABLE OF CONTENTS
LIST OF FIGURES v
MATERIALS AND METHODS 14
Spore Shooting 14
Single Spore Cultures 16
THELEBOLUS STERCOREUS 24
Vegetative Features 24
Ascogenous System 26
Cytology of the Ascus 27
LASIOnJLUS CILIATUS 35
Vegetative Features 35
Ascogenous System 38
Cytology of the Ascus 38
Thecotheus cinereus 49
Vegetative Features 49
Ascogenous System 50
Thecotheus pelletieri 51
Vegetative Features 51
Imperfect Stage 52
LITERATURE CITED 91
BIOGRAPHICAL SKETCH 97
LIST OF FIGURES
Figures 1-14 Thelebolus stercoreus 72
-Figures 15-25 Thelebolus stercoreus 74
Figures 26-36 Lasiobolus ciliatus 76
Figures 37-45 Lasiobolus ciliatus 78
Figures 46-56 Lasiobolus ciliatus 80
Figures 57-63 Thecotheus cinereus 82
Figures 64-69 Thecotneus cinereus 84
Figures 70-74 Thecotheus pelletieri 84
Figures 75-64 Thecotheus pelleticri 86
Figures 85-95 Thecotheus pelletieri 88
Figure 96 Stalked ascogonium of Coprobia
granulata (Humaria granulata); from
Gwynne-Vaughan and Williamson (1930). 90
Figure 97 Stalked ascogonium of Cheilymenia
stercorea (Lachnea stercorea); from
Fraser (1907). 90
Figure 98 Diagrammatic cross-section of
ascocarp showing the stalked
ascogonium of Scutellinia scutellata
(Lachnea scutellata); from Brown (1911).90
Figure 99 Stalked ascogonium of Lasiobolus
monascus; from Kimbrough (1973, in
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
COMPARATIVE ONTOGENY OF THELEBOLUS,
LASIOBOLUS, AND THECOTHEUS (PEZIZALES, ASCOMYCETES)
Kenneth Edward Conway
Chairman: James W. Kimbrough
Major Department: Botany
A comparative ontogenetic study was initiated on three
genera of coprophilous discomycetes; Thelebolus, Lasiobolus,
and Thecotheus. These organisms were chosen because they
represented fungi that possess different numbers of asci and
ascospores per apothecium. These genera have been placed at
one time or another in the family Thelebolaceae. The
variability of characteristics for these three genera and
others previously reported seems to indicate that the famil-
ial limits of Thelebolaceae should be much more restricted.
Thelebolus stercoreus shows an ascocarp ontogeny similar
to Trichobolus zukalii. The mycelium of T. stercoreus is
uninucleate. Ascogonial initiation begins with an evagination
from a parent hypha. No croziers are formed as the single
ascus grows directly from a privileged cell of the ascogonial
coil. Nuclear divisions in the ascus have been followed and
photographed. Thelebolus stercoreus represents perhaps the
highest evolutionary form found in the coprophilous habitat.
Its unique structures must limit the family Thelebolaceae to
Thelebolus, Trichobolus and perhaps Ascozonus and Caccobius.
The latter two genera need further study before a familial
alliance can be established.
The ontogeny of Lasiobolus ciliatus begins with an
evagination from a parent hypha that develops into a multi-
cellular stalked ascogonium. Hyphae from cells below the
ascogonium and from surrounding hyphae ensheath the
ascogonium. This development is similar to that shown for
Cheilynenia stercorea, Scutellinia scutellata and Coprobia
granulata. Ascogenous hyphal growth is terminated by the
development of croziers (pleurorhynque). In the present
study ascal cytology has been described and photographed.
The mycelium of L. ciliatus is uninucleate. The development
of the diagnostic hairs of Lasiobolus is presented. The
study of ascocarp ontogeny was facilitated with plastic
embedding. It is suggested that Lasiobolus has familial
affinities with the Aleuriaceae.
Ontogenetic features of Thecotheus were investigated
using two species, Thecotheus cinereus, an eight-spored
species and Thecotheus pelletieri, a 32-spored species. The
ascogenous system of Thecotheus consists of a series of
ascogonial cells similar to that of Ascobolus citrinus. The
ascogonia give rise to ascogenous hyphae that terminate with
the formation of croziers (pleurorhynque). The mycelium of
both species is mostly multinucleate. Both species also
produce "primordial humps" that consist of bulbous cells
that resemble the microconidia of Ascobolus carbonarius.
This stage precedes ascocarp initiation, and they appear to
only function in excipular formation. A sympodial imperfect
stage of T. pelletieri is described and photographed.
Sympodial conidia have also been described for the
Aleuriaceae. It is thought until more information is avail-
able for Thecotheus and other genera, especially in the
Aleuriaceae, the best placement is in the Iodophaneae of
Many mycologists have commented on the "ubiquitous
fungi," and this ever-present characteristic is perhaps
best illustrated by those fungi that are coprophilous. A
world of fascinating and useful organisms is opened for an
observer, representing taxa from all major classes of fungi.
Most coprophilous fungi possess a life cycle that
includes ingestion by animals. The spores of these fungi
are passed through the animal and deposited in the feces.
In most cases this passage through the animal seems to be
required for spore germination. This habitat specificity
and special pre-germination treatment often makes it
difficult to germinate, culture and sporulate these organ-
isms in the laboratory.
If one considers the dung substrate as an island in a
sea of grass, some problems of dispersal become evident.
Many coprophilous organisms have developed certain ecolog-
ical adaptations which insure the survival of these peculiar
organisms. One of tnese adaptations is the development of
a projectile mechanism which hurls the spores from the dung
specimen to blades of the surrounding grass where the spores
are eaten by grazing herbivores to be "treated" and re-
Another ecological adaptation is the phototropic
response.- This response is closely correlated with spore
ejection so that maximum distance is obtained by the pro-
jectile. Spore ejection usually takes place in the early
afternoon coinciding with maximum elevation of the spore-
Perhaps the phycomycetous genus, Pilobolus, has been
studied the most (Buller, 1934; Ingold, 1965; Page, 1964).
It is known that a single multispored sporangium can be
projected a considerable distance and with surprising
accuracy. This trait is paralleled by other coprophilous
forms. Most of these fungi that propel their spores are
multispored, a characteristic which better insures the
continued survival of the organism since some of the spores
discharged from the dung are more likely to reach a suitable
Other unusual coprophilous forms which appear to have
special adaptive features for this habitat include Sporormia,
Basidiobolus and Sphaerobolus. In Soorormia, a genus of the
Sporormiaceae, rather than an increased number of spores per
ascus, the eight original spores fragment to produce the
multispored ascus. In Basidiobolus, a saprobic genus of the
Entomophthoraceae, a conidium is propelled by the action of
the conidiophore. In culture the projectile mechanism, as
well as the phototropic response of the conidiophores, can
be observed. The result is a progressive march of the
organism across the agar surface as conidia germinate to
produce new conidiophores. Sphaerobolus of the
Sphaerobolaceae displays similar tendencies in the
Basidiomycetes. The peridiole is surrounded by several
layers of cells which reduce desiccation, and each peridiole
can be forcibly discharged.
The coprophilous discomycetes show these ecological
adaptations plus several additional peculiarities. There is
a tendency for the coprophilous forms to have more than eight
spores per ascus. Very rarely will one encounter a multi-
spored member of the Pezizales outside the coprophilous forms.
Ingold (1965) states that the larger spores or spore masses
tend to be shot a greater distance and stand a better chance
of effective dispersal than smaller ones.
An increased number of spores per ascus is accompanied
by decreases in spore size and the number of asci per
apothecium. A decrease in the number of asci is also corre-
lated with the angiocarpic development of the ascocarp.
Several workers (Kimbrough, 1966b; Kimbrough and Korf, 1967;
Eckblad, 1968) have shown that the number of asci per
apothecium is variable and is related to ecological conditions,
the number of spores per ascus being greater in the uniascal
forms. This would seem to indicate that each ascocarp has a
certain potential for spore production which may be reached
through various combinations in the number of asci and
ascospores. Recently, however, cultural work with the genus
Thelebolus (Wicklow and Malloch, 1971) indicates that the
reported variability of asci per apothecium is actually the
result of several different closely related organisms of the
same genus inhabiting the same specimen and that the number
of asci per apothecium is not necessarily subject to varia-
Desiccation is another factor which affects the surviv-
al of the multispored discomycetes. Single spores in
species with eight-spored asci often possess thick pigmented
walls which help prevent harmful effects of sunlight and
desiccation. The multispored forms eject their spores in a
mucilaginous mass. These spores are usually smaller and
possess thin walls. The multispored feature may insure the
survival of many spores since the inner core of spores is
surrounded with an expendable, dead outer layer. The thinner
walls of the surviving spores may require less treatment
than the thicker walled spores and, therefore, germinate
more readily on a suitable substrate.
As these unusual forms were studied and more information
gathered by research, mycologists turned to the task of
classifying the various coprophilous fungi. These
discomycetes were originally placed in the Ascobolaceae.
Boudier (1869) saw differences in spore color and using this
criterion separated the Ascobolaceae into two groups:
Ascobolei Spurii, for the hyaline spored species, and
Ascobolei Genuini for the dark spored forms. Into the
Ascobolei Spurii he placed three genera, separating each
according to the number of spores per ascus; Ascophanus with
eight or sixteen spores, and Ryparobius and Thecotheus, both
multispored. Thecotheus possessed thicker walled spores,
more cylindrical asci, more elongate paraphyses and larger
apothecia than Ryparobius.
Saccardo (1884) recognized differences in apothecial
characteristics of Ascophanus and erected the genus Lasiobolus
for the setose species.
New insight into the coprophilous discomycetes was
offered by Chenantais (1918) when he examined specimens of
Ascophanus cinereus (Cr. & Cr.) Boud. and determined that the
affinities of this eight-spored species lay within the iLaulti-
spored genus Thecotheus. Until this time the artificial
classification, using spore number per ascus established by
Boudier, had been used to separate genera. This contribu-
tion of Chenantais was later reinforced by the work of
Kimbrough (1966b) and Kimbrough and Korf (1967).
Kimbrough and Korf (1967) erected the tribe Theleboleae
of the Pezizaceae to include those genera with hyaline, eight-
to multispored asci which are non-amyloid, operculate or
irregularly dehiscing. Their major emphasis was on the
nature of the ascus, ascospores, ascocarps and cultural
characteristics. They rejected the taxonomic importance of
the spore number for separation of genera. They maintained
that there is close correlation between these emphasized
features within each group found in the tribe Theleboleae.
The Pseudoascoboleae was abandoned because it was not based
on the name of a genus.
Van Brummelen (1967) elevated the tribe Theleboleae to
.a subfamily Theleboloideae of the Ascobolaceae. He excluded
several species included by Kimbrough and Korf in their tribe
Theleboleae. Later, Eckblad (1968) and Rifai (1968) raised
the subfamily to familial status, Thelebolaceae. Eckblad
extended Van Brummelen's characterization of the
Theleboloideae to include the inoperculate genera of
Kimbrough and Korf (1967) and Thecotheus. His reason for
familial status was that a taxon containing both operculate
and inoperculate forms should be separated from other exclu-
sively operculate taxa at the familial level.
The investigations of Kimbrough (1966b) and Kimbrough
and Korf (1967) have greatly aided the characterization of
genera in the Thelebolaceae. Thus far, the main emphasis
has been morphology of apothecia, asci and ascospores. Little
emphasis has been placed on cytological, developmental and
Recently, the work of Wicklow and Malloch (1971) has
helped to explain the many forms of Thelebolus. By culturing
various forms of Thelebolus they found that the number of spores
per ascus and the number of asci per apothecium is consistent
at the species level. Previous to this, variable numbers of
spores and asci had been reported by different authors
(Ramlow, 1906; Massee and Salmon, 1901; Kimbrough and Korf,
At the generic level, however, the phenomena of increased
'spores per ascus and reduced number of asci per apothecium
is very evident in the coprophilous Pezizales. This tend-
ency is present in seven genera of the Thelebolaceae and in
one genus of the Pezizaceae (Kimbrough, 1972a). Very little
is known of the cytological events that determine whether
asci will be eight-spored or multispored, and whether the
apothecium becomes uniascal or multiascal.
Recently there has been increasing evidence that the
Thelebolaceae may not be a natural group due to the hetero-
geneity of many characteristics (Arpin, 1968; Berthet,
1964a; Kish, 1971; Kimbrough and Korf, 1967; Kimbrough,
1970; Milam, 1971).
The mycelium of the coprophilous discomycetes possesses
a variety of nuclear conditions. Berthet (1964a) stated
that all Pezizales studied possess a coenocytic mycelium.
However, there are some organisms such as Trichobolus
(Kimbrough, 1966a) and Coprotus (Kish, 1971) that have been
shown to be uninucleate. The excipulum also possesses vary-
ing numbers of nuclei. Milam (1971) has shown that the
excipular cells and mycelium of Iodophanus granulipolaris
Kimbr. are coenocytic. Kimbrough (1966a) has shown that the
excipulum of Trichobolus zukalii Kimbr. is uninucleate.
The diversity of the coprophilous discomycetes is also
illustrated by the morphology of their ascogonia, crozier
systems and ascocarp ontogeny. Types of plasmogamy in the
coprophilous discomycetes vary greatly. In genera such as
Thelebolus and Trichobolus compatible nuclei are already in
the system. In Lasiobolus, hyphal fusion occurs, whereas,
in Coprotus lacteus fusion is the result of an ascogonium
and a trichogyne. Ascogonia vary in form from a coil of
ascogonial cells such as in Trichobolus zukalii to a well
formed Pyronema-like ascogonium complete with trichogyne as
in Coprotus lacteus (Ck. & Phill.) Kimor., Luck-Allen and
Chadefaud (1943) described several types of systems,
including acrorhynque, pleurorhynque, aporhynque, and compose.
The acrorhynque consists of a terminal uninucleate cell with
a binucleate subterminal cell. The pleurorhynque has the
greatest variability. Essentially, it is the typical crozier.
The variability depends on whether the terminal cell fuses
with the uninucleate cell beneath the subterminal cell or
whether it remains free. The aporhynque is a simplification
of the pleurorhynque type. In the aporhynque type two nuclei
undergo parallel mitoses and two different daughter nuclei
are incluaea in the terminal cell through the formation of
a cross wall. No lateral hook is formed. In the compose
type a series of binucleate cells is delimited; each cell
may be fertile and produce an ascus.
Many difficulties are encountered in the study of the
ascogenous systems and are summarized by Berthet (1964a) who
outline types of ascogenous systems known for various
discomycete taxa. From his summary and research it appears
that a majority of ascomycetes possess the pleurorhynque
system. Tne next most frequent ascogenous system is the
The Ascobolaceae, Humariaceae, and Aleuriaceae have
representative genera of both the pleurorhynque and aporhynque
types. Variability is the rule even in ascogenous systems.
Certain species, such as Galactinia ampelina, possess both
pleurorhynque and aporhynque systems within the same
There are several known examples of ascogenous systems
in the Thelebolaceae. Tricnobolus zukalii possesses no
ascogenous hypnae or croziers (Kimbrough, 1966a). However,
it could be classified as acrorhynque because the ascal mother
cell in T. zukalii is produced by septation of the parent
hypha resulting in the inclusion of two compatible nuclei
into one non-terminal cell. Kish (1971) has shown that
Coprotus lacteus possesses a compose type, and Milam (1971)
has shown that lodophanus granulipolaris possesses
a true crozier system of the pleurorhynque type. Lasiobolus
ciliatus is reported by Berthet (1964a) as possessing an
aporhynque ascogenous system.
Berthet also reports that in the Ascobolaceae,
Ascobolus carbonarius has an aporhynque system, while
Pyronema omphalodes has a fused pleurorhynque system.
Genera of the Humariaceae possessing the pleurorhynque
system include: Anthracobia melaloma, Cheilymenia aurea,
and Scutellinia sp.
Van Brummelen (1967) outlined a system of ascocarp
ontogeny according to the maturation and exposure of the
hymenium. The heterogeneity of the coprophilous forms is
again evident. The hymenium of organisms such as Thelebolus
stercoreus and Trichobolus zukalii is closed throughout its
development. Ascocarps of other genera open at various
times exposing their hymenia at different stages of
Berthet (1964b) and Eckblad (1968) summarized the
imperfect stages of the discomycetes. The majority of the
genera listed possess an Oedocephalum imperfect stage.
Oedocephalum forms blastoconidia. Operculate genera
listed as possessing an imperfect stage were found in the
families Pezizaceae, Pyronemaceae and Aleuriaceae.
Paden (1972) has reclassified many of the imperfect
stages to fit within the current taxonomy for imperfects.
The Pezizaceae now contains imperfect stages that form
blastoconiaia and alcurioconidia. The Pyronemaceae form
oiaia. The family Otideaceae possesses Botrytis-like
imperfect stages which are blastoconidia. The imperfects
of the Aleuriaceae form sympoduloconidia. Oidia occur in
the Ascobolaceae. There are no reports of imperfect stages
in tne Thelebolaceae.
Tne presence of an imperfect stage in the coprophilous
forms has been considered, until recently, of little impor-
tance. The only forms that have been linked to an imperfect
stage are Iodophanus carneus (Pers. per Pers.) Korf and two
species of Ascobolus. Korf (1958) and Gamundi and Ranalli
(1964) reported an Oedocephalum imperfect stage for I.
carneus, and Greene (1931) has shown oidia in Ascobolus.
The emphasis placed on the systematics of the
aiscomycetes in 1967 (Van Brummelen), 1968 (Eckblad and
Rifai) and 1970 (Kimbrough) was re-emphasized at a world-
wide symposium in Exeter, England.
In his summary at the symposium Korf (1972b) listed
tne current methods of study of the discomycetes. Included
in the discussion were: microanatomical studies of the
apothecia, cultural studies, studies of nuclear numbers,
ontogenetic studies of ascocarp development, ascal wall
characteristics, ana chemotaxononic and physiotaxonomic
The organisms that I have chosen to investigate show a
marked variation in spore number per ascus and In the number
of asci per apothecium. From such variations, evolutionary
tendencies perhaps can be postulated. Lasiobolus ciliatus
(Kunze and Schmidt) Boud. represents one end of a line in
which eight spores are formed per ascus and a multiascal
apothecium is characteristic. Thecotheus pelletieri (Cr. &
Cr.) Boud. represents an intermediate condition, having a 32-
spored ascus with fewer asci per apothecium. Thelebolus
stercoreus represents perhaps the most advanced coprophilous
form, for it has a multispored ascus and a uniascal
With regard to current emphasis in discomycete studies,
the following information will be included wherever possible:
vegetative features, ontogeny of initials, ascogenous system
and cytology of the ascus. Information concerning the
vegetative features includes; determination of the nuclear
condition of the mycelium, mycelial structures such as coils
and chlamydospores, rate of growth in cultures, and occur-
rence of inter-hyphal growth. Ontogeny includes types of
plasmogamy, coiling of hyphal initials (archicarp), and
significant changes occurring during ascocarp ontogeny.
Observations are included concerning the nuclear condition
of the ascogonia and the presence, absence, and type of
crozier system. Other observations concern the cytology of
the ascus, showing the nuclear divisions leading to spore
formation and the nuclear conditions of the spores.
It now appears that the family Thelebolaceae is a very
heterogenous group of organisms with their occurrence on
dung being perhaps the only common bond which unites them
as a family. Therefore, the purpose of this investigation
is to describe the significant features of three species
that have been placed, at one time, in the family. The
criteria that are developed will then be used to place these
species into more natural familial alliances.
MATERIALS AND METHODS
Collections of the dung of various animals in the
Gainesville area were made beginning in January, 1970. These
collections were placed in moist chambers to encourage the
growth of fungi. Mature apothecia appeared within 1-2 weeks
and were picked off the dung. Care was taken to avoid
contamination from bacteria and other fungi which are abun-
dant on dung.
The apothecia were fastenea on the lid of a petri plate
with petroleum jelly and inverted over dung agar. The lid
was rotated four times at 30-minute intervals, resulting in
four groups of discharged spores. Another technique for
obtaining pure cultures was to pick an apothecium off the
dung sample and put it into a petri plate. A sterile Van
Tieghem ring was placed around the apothecium and a sterile
coverslip placed on the ring. Released spores were impacted
on the coverslip. Agar blocks or coverslips with spores
were used in germination and cultural studies.
In most cases spores, upon germination, were picked
from the agar and transferred to dung oatmeal agar plates.
Sometimes, however, as in the case of Thecotheus cinereus,
the spores would not germinate and additional treatment was
required. This consisted of cutting out blocks of agar
containing the spores and placing them on sterile slides
enclosed in petri plate moist chambers (Riddell, 1950). The
blocks were treated with a drop of 2% KOH and covered with
a sterile coverslip. Spores thus treated usually germi-
nated within two days. If this treatment was unsuccessful,
the KOH treatment was modified by placing the moist chamber
in a 60 C oven for a few hours. Blocks with germinated
spores were then cut into quarters and transferred to dung
oatmeal agar plates for further growth. Slide cultures also
provided an excellent opportunity to study spore germination,
primordial development and to check for conidial stages.
In mature colonies of Thelebolus stercoreus and
Lasiobolus ciliatus, apothecia appeared in 1-2 weeks.
Thecotheus required a longer period of time for development
in deep dish agar cultures. Slant cultures were kept on
dung oatmeal agar.
Single Spore Cultures
Cultures used in determination of mating compatibility
were obtained in several ways. One method that was similar
to the germination technique was to shoot spores onto dung
agar, cut out blocks of agar and place them on a sterile
slide. The slide was placed in a moist chamber and the
spores allowed to incubate overnight at room temperature.
Germinating spores were picked off the block with a fine
needle under low power (40X) of a microscope. Macerating
a few apothecia in a drop of water and using a sterile loop
to streak the drop over the surface of a dung agar plate,
was also employed. Again, incubation was overnight at room
temperature. The germinating spores were picked from the
plate with a fine needle under low power of the microscope.
Single spore cultures were then transferred to dung oatmeal
slants and plates.
Dung agar was used for primary isolation because it
is a better growth medium for coprophilous organisms. It
was prepared by soaking approximately 12.5 gm. of rabbit,
cow or horse dung overnight in 100 ml. of water. For full
extraction the dung was macerated into small pieces and
this mixture was filtered through several layers of cheese-
cloth. The supernatant was refiltered through a Buchner
funnel with the aid of a vacuum pump. The volume was
brought to 500 ml. with the addition of distilled water and
Difco agar was added to give a 2% medium. Kanamycin, an
antibiotic, was added at first (1:10,000) to reduce bacteri-
al contamination. However, this later was found to be
The growth medium was a dung oatmeal agar, prepared by
boiling 10 gm. of oatmeal in 500 ml. of distilled water for
five minutes. This preparation was filtered through several
layers of cheesecloth and several changes of filter paper.
A vacuum pump was again used to accelerate the process
because of the stickiness of the oatmeal which clogged the
filter paper and reduced the flow of the supernatant. The
supernatant was mixed with 500 ml. of dung media to produce
a 2% agar. Plates and slants were stored in a refrigerator
without apparent loss of nutrient value.
Several stain techniques were used with varying
success. The problems encountered with staining were ina-
bility to obtain penetration of the ascus and stain the
chromatin material, and the overstaining of the ascus
cytoplasm to partially obscure the chromatin.
A modification of the technique used by Tu, Roberts and
Kimbrough (1969) to stain Rhizoctonia worked well for stain-
ing hyphal nuclei of the organisms under investigation.
Material to be stained was fixed in Carnoy's solution for
18-24 hours. This was followed by a washing in 50% acetic
acid, hydrolysis in IN HC1 at 65 C for 20-30 minutes and
rinsing in acetic acid. The procedure used was to stain
with 2% aceto-orcein in 50% acetic acid for 6-8 hours.
Aceto-orcein and aceto-carmine were not successful in
staining the ascal nuclei. Other stains employed included:
lacto-phenol cotton blue, pentacyl blue-black, Giemsa stain
(Furtado, 1970), basic and acid fuchsin and methyl blue in
The stains giving the best results were 2% methyl blue
in Hoyer's solution and 1% pentacyl blue-black in water.
With both preparations the nucleolus stained intensely and
after several minutes the nuclear envelope also began to
stain. There was no staining of chromatiz material. Lactc-
phenol cotton blue stained the nucleolus but to a lesser
degree and did not stain the halo around the nucleolus. The
aceto-orcein and aceto-carmine stains could be enhanced by
soaking blocks of material in FeC3 solution.
Fixation overnight with Carnoy's solution was also
tried. The longer the fixation time, the better the results.
The specimen was hydrated in an alcohol series (95-70-50-
30-10-distilled water). Hydrolysis was in 1N HC1 for 70
minutes at 60 C followed by a rinse in distilled water for
10 minutes. A mordant was applied containing 4% iron alum
for 45 minutes at 60 C, followed by a brief rinse in
distilled water. An apothecium was then crushed and pinched
apart on a slide and a drop of aceto-carmine was applied.
A coverslip was immediately placed over the apothecium and
pressure was applied to further spread the material. The
intensity of the stain increased with time. Material thus
fixed produced good results. Hot lacto-phenol cotton blue
stained the apiculi of Thecotheus cinereus.
Most staining was used in conjunction with squash
mounts. Other techniques also used were sectioning with the
cryostat and plastic embedding.
Feder and O'Brien (1968) had much success with plastic
embedding using glycol methacrylate with certain higher
plant material. This plastic was obtained from Folys.ience.
The plastic kit from this supplier included a suggested
embedding schedule. Blocks of agar containing mature
apothecia were fixed overnight with 3% glutaraldehyde in
0.1M veronal buffer at 0 C.
The blocks were transferred without a water rinse to
methyl cellosolve. These were kept in a refrigerator and
changed twice in 2h hours. They were then transferred to
100% ethanol (h-24 hours), to n-propanol (h-2h hours) and
finally to n-butanol. The blocks could then be stored for
A new plastic mixture was tried similar to Moore's
(1963) plastic used in his work with Ascodesmis. He used a
plastic consisting of butyl methacrylate, ethyl methacrylate,
and 1.5% Luperco. Ethyl methacrylate was not available;
therefore, another plastic was substituted, methyl
methacrylate. This is similar to ethyl methacrylate in both
molecular weight and density. It is also very slightly
soluble in water but soluble in alcohol. The proportions
for this plastic were 3 parts butyl methacrylate, 2 parts
methyl methacrylate and 1.5% Luperco. This plastic was
clear and of a good consistency for sectioning. Initial
trials were unsuccessful for embedding due to the addition
of a cross linking monomer, ethylene glycol dimethacrylate.
The monomcr :azc added to prevent selling of ticsucs during
the staining process. When this was discontinued, the
blocks were very acceptable. Polymerization was carried out
in a 50 C oven overnight. The ratio of butyl methacrylate
to methyl methacrylate was later changed to h:2 to obtain a
Blocks with apothecia were dehydrated in a graded
alcohol series and embedded in the final plastic mixture
using vacuum infiltration. To accomplish this the blocks
were placed in disposable 50 ml. beakers and 15 ml. of
solution was used in each change. It was found that 15 ml.
of the final embedding plastic gave an acceptable block.
For the final embedding the squares of apothecia would be
oriented to any position desired and then placed in the
embedding oven. The plastic blocks were popped out of the
beaker and small sections were cut out which contained
apothecia. These were then glued to a plastic dowel using
epoxy glue. The glue was hardened in the 50 C oven. After
the glue hardened, the sections were "faced off" using
razor blades. The blocks were then ready for sectioning.
There were problems initially in using the Polyscience
plastic embedding kit. The difficulties centered around:
plastics used, inadequate instructions, and finding a
suitable means of hardening the plastic.
Early embedding problems traced their origin to eirors
in the Polyscience data sheet. Upon a re-examination of the
original work of Leduc and Bernhard (1967), we discovered
that several errors existed in the data sheet. The original
procedure included three embedding mixtures. The initial
embedding solution consisted of 80% glycol methacrylate and
20% distilled water. The next change was 97% glycol
methacrylate and 3% water. The third mixture was inaccurate,
and according to Leduc and Bernhard should have consisted of
7 parts of 97% glycol methacrylate and 3% distilled water
plus 3 parts butyl methacrylate with 2% Luperco added.
Clouding of the plastic resulted from the inability of
Luperco to go into solution. The correct procedure to insure
its solubility in the final plastic mixture is to add the
distilled water to the glycol methacrylate, and separately
add the Luperco to the butyl methacrylate. Both solutions
can then be mixed together. This procedure is necessary as
Luperco is only slightly soluble in water.
It was also found that without a U.V. light source at
0 C tne prepolymer would not harden. However, the plastic
would eventually harden if placed in a 45-50 C oven. If
the plastic was poured too thin, bubbles would distort the
block. The plastic could be sectioned at 2 a with little
damage to the razor blade.
Sectioning was accomplished on a manual rotary
microtome. The need for ultrathin sections was not antici-
pated for light microscopy. A razor blade attachment proved
unsuccessful as blades rapidly became dull. Disposable
blades for the rotary microtome were employed with better
success. Sections of 1 u thickness would be obtained and
continuous sectioning achieved due to the larger cutting
surface. Sectioning was facilitated with a water boat made
from paraffin. As the sections were cut they would float on
tne surface of the water without curling. These sections
could then be picked up with an eyelash or camel hair brush
and placed on a microscope slide. A drop of water on the
slide facilitated the removal of the section from the brush.
Sectioning with the cryostat was done at -20 C.
Several settings for section thickness were used ranging
from 1-5 p. Cryostat sectioning was not as successful as
had been expected. The mounting medium interfered with
staining and sections haa a tendency to fall apart.
Thelebolus stercoreus Tode per Fr. occurs on most
types of dung. It was first isolated from rabbit dung.
The apothecia appear white on the dung and are immersed to
semi-immersed. The spores germinated readily on dung
Pure colonies grew quite rapidly, expanding to a
diameter of 2.5 cm. in five days. Mature apothecia were
present on the plates within two weeks. Fruiting occurred
synchronously around the margin of gro;;th, producing
consecutive rings of apothecia.
Apothecia occurring in culture were more orange colored
than that observed on dung. Apothecial size was similar to
that occurring naturally, 100-250 p in diameter. Apothecia
remained closed until spore ejection at which time the top
of the ascocarp was ripped open by the expanding ascus.
Single haploid spore cultures were easily obtained by
picking single spores from a mass of spores streaked over
the surface of the agar. Spores would germinate within
24-48 hours. Fruiting occurred within two weeks. The
formation of mature apothecia indicated that T. stercoreus
is homothallic. This supports the findings of Cooke and
Sliue cultures were used to study vegetative and
reproductive features. The vegetative mycelium of
T. stercoreus is uninucleate (Fig. 1). Ramlow (1906)
reported and illustrated (Fig. 33, l.c.) uninucleate hyphae
in T. stercoreus. My own observations of the vegetative
mycelium in this species confirm his findings. The vegeta-
tive nuclei stained best with aceto-orcein. Hyphal coils
(Fig. 2) were prevalent on the surface of the agar.
Anastomoses between the hyphae of the coil were frequently
seen, allowing perhaps for the exchange of nuclei. Vegeta-
tive sphaeroid terminal swellings of the hyphae were fre-
quently observed (Figs. 3, 4) similar to those found by
Berthet (1966) in Trichophaea sp. Their function in
Tnelebolus is unknown. The swellings measured 9-10 p in
diameter, were hyaline, and contained many oil droplets.
Ascocarp initiation begins when a hypha gives rise to
a side branch. This branch is very slender at first (1-2 p)
but enlarges in diameter to produce a swollen blunt tip
5-6 p in diameter. The branch may reach a length of 30-40 p,
the tip becoming circinate (Fig. 5). This apical curving
usually ensnares another hypha (Fig. 6). The original hypha
now becomes septate and each cell becomes swollen. These
cells continue to divide to form a mass of cells surrounding
the inner hypha (Fig. 7) and eventually form the sterile
excipulum in-which the cells are of a textura angularis
The sheathing cells continue to divide to form a
rounded mass of cells. Many hyphae radiate from the devel-
oping ascocarp on the surface of the agar (Fig. 9). A
closer inspection of these hyphae (Fig. 10) shows that they
fuse to the outer layer of cells of the ascocarp. Anasto-
moses are frequently seen between these hyphae.
As the ascocarp enlarges the centrum becomes very
active as is evident by the concentration of nuclei in this
region (Fig. 11). Sections through the middle of the
ascocarp show an ascogenous system surrounded by the
exciuplar cells. One of the cells of the ascogenous system
contains compatible nuclei (Fig. 12). This cell is always
intercalary. No croziers are formed. Kimbrough (1966a)
has shown the manner of attachment of the ascus of
Trichobolus where there are two small cells at the bottom of
the ascus which would indicate that the privileged cell
would be the subterminal cell of the ascogonium. The same
system is also present in Thelebolus. The privileged cell
begins to enlarge (Fig. 13) and stains in methyl blue in
Hoyer's solution. When the ascocarp is sectioned (Fig. 14),
it appears that the layer of cells adjacent to the ascus
adheres and conforms to its expanding shape. Many cells are
crushed in the process of ascal enlargement. However, this
may be the stage of initiation of paraphyses. As the ascus
enlarges, the peripheral cells continue to add cells to the
outside. The ascus may now occupy one-third of the entire
Cytology of the Ascus
When the ascus is carefully squeezed out from the
ascocarp at this point, the 2N nucleus becomes evident
(Fig. 15). The nucleus is approximately 5 j in diameter
with a large prominent nucleolus.
During the first division of meiosis the nucleolus
disappears. In the diplotene-diakenesis stage there appear
to be six bivalents (Fig. 16). Division I takes place on an
axis perpendicular to the long axis of the ascus (Fig. 17).
Division II is accomplished almost synchronously and
parallel to the long axis of the ascus (Figs. 18, 19).
After division II the four nuclei lie in the center of the
ascus (Fig. 20). Subsequent divisions produce the many
spores typical of Thelebolus (Figs. 21, 22). It has been
estimated that as many as 1000-2500 spores will finally fill
the ascus (Cooke and Barr, 1964; Kimbrough, 1966b).
By this time the ascus has increased in size filling
most of the cleistothecium-like apothecium (Fig. 23), the
ascus approaching the point of spore discharge. It ruptures
the cleistothecium-like apothecium (Fig. 24) and the ascal
tip becomes somewhat extruded due to the thinner wall in
that region. Kimbrough (1966b) attributes this thinner wall
to a discontinuing and abrupt thickening of the next to the
outside wall layer (Fig. 24). The layering of the ascal
walls is very prominent at higher magnifications. The spores
are then released as pressure from below the ascus forces
the spore mass to split or tear the ascal tip. When the
ascus is young and has not fully expanded, paraphyses are
present (Fig. 25). These paraphyses are very slender,
measuring approximately 2 p in diameter. They may be
branched and are usually held in close contact to the ascus.
Thelebolus stercoreus is an excellent organism to work
with. It is easily grown in culture and fruits prolifically.
Cultures for this investigation were grown on dung oatmeal
agar. However, the species has been reported to grow on
potato dextrose agar and to fruit on a modified Leonian's
agar (Cooke and Barr, 1964).
The vegetative mycelium of T. stercoreus presents
many structures that need explanation. It is known that
T. stercoreus possesses the compatible nuclei necessary to
fruit in a single spore culture (Kimbrough, 1966b). If one
looks at tne vegetative mycelium ana its structures in light
of nuclear migration and compatibility, several possibil-
ities appear. One of the most prevalent superficial struc-
tures present were hyphal coils. Anastomoses occurred
between cells of different layers of the coil and could
provide the opportunity needed for exchange of compatible
The swellings found in the vegetative phase of T.
stercoreus appear similar to structures found by Berthet
(196o) for Trichophaea confusa (Cooke) Berthet (Pezizeae).
Berthet refers to these structures as apparent conidia and
has seen them germinate. No germination of these structures
was observed in the present study.
Gordon's (1964) work on the centrum development of
Diporotheca has some interesting implications when applied
to Thelebolus. Gordon found that there was no crozier
system formed in Diporotheca, but that the penultimate cell
of an ascogonial initial divides and surrounds the terminal
cell. These cells then send out trichogynes or receptive
hyphae which fuse with other vegetative hyphae. Gordon
then contends that compatible nuclei migrate through these
receptive hyphae to form the heterokaryotic condition in
the lower part of the centrum, from which the asci ultimate-
In this study of Thelebolus one of the striking
features of the developing ascocarp was the radiating of
the hyphae from the peripheral cells. It was often apparent
that these radiating hyphae anastomosed. However, their
role as receptive hyphae is very doubtful as the compatible
nuclei are already paired in the ascogenous system before
the hyphae begin to radiate. Rather than radiating, they
appear to add new hyphal elements to the developing excipulum.
Gordon also mentioned that the developing ascus may
fuse with the inner cells of the excipulum and incorporate
their material into the ascus. The formation of the ascus
of Thelebolus would make a good electron microscope study
to determine if the young ascus crushes or envelops the
excipular cells and paraphyses.
A transfer of compatible nuclei may also occur when a
hypha branches from the vegetative mycelium and curves and
coils itself around another hypha. The curved hypha
becomes septate and ensheaths the second hypha with cells.
Compatible nuclei may pass into the ascogonium (inner hypha)
by fusion with these peripheral cells. However, this stage
may be analogous to the stage in Trichobolus (Kimbrough,
1966a) when hyphae from the parent hypha ensheath the
ascogonium without fusion occurring. In Thelebolus, as in
Trichobolus, the compatible nuclei appear to be present in
the parent hypha and to flow into the ascogonium prior to
septation of the latter. No ascogenous hyphae or croziers
are formed. However, the ascogenous system could be clas-
sified as acrorhynque in Chadefaud's (1943) system.
The development of ascogonia in T. stercoreus is
similar to that shown by Schweizer (1923) for Ascobolus
citrinus (=Ascobolus michaudii Boud., Van Brummelen, 1967),
The ascogonium is a privileged cell that has developed from
an evagination of the parent hypha.
The exact phylogenetic position of Thelebolus has long
been a controversy. Thelebolus was first erected as a genus
by Tode (1790) and was originally placed in the
Gasteromycetes (Order Angiogastres). Fuckel (1870) was the
first to consider Thelebolus as an Ascomycete, placing it
in the Perisporiacei of the Pyrenomycetes. Saccardo (1888)
transferred it to the Nidulariaceae. Earlier Zukal (1886),
having studied the development of T. stercoreus, placed it
close to Podosphaera in the Erysiphaceae.
Boudier (1869) recognized multispored taxa in the
Ascobolii spurii, but did not consider Thelebolus. Heimerl
(1889) expanded Boudier's concept, however, to include
Thelebolus. He was the first mycologist to consider
Thelebolus a discomycete. Brefeld (1891) studied the entire
life cycle and regarded Thelebolus as a single genus in the
Thelebolaceae of the Carpohemiasci. Saccardo (1892) later
established two infrageneric categories, Eu-Thelebolus and
Tricnobolus, wnich were reaffirmed by Kimbrough (1966b).
Schroeter (1893) considered Thelebolus a member of the
Ascoboleae, family Pezizaceae.
Cooke and Barr (1964) in their comparative study of
Thelebolus used Saccobolus depauperatus (Berk. & Br.) Rehm
and the mature structures of Podosphaera and Sphaerotheca
of the Erysiphaceae. They concluded that Thelebolus
possessed a greater number of characteristics similar to the
Erysiphaceae and recommended that Thelebolus be placed back
into the Erysiphaceae.
In a more recent work (Dennis, 1968) Thelebolus is
treated in the Pseudoascoboleae. Kimibrocgh and Korf (1967)
in their work concluded that the name Pseudoascoboleae was
invalid and proposed a name change to Theleboleae based on
the genus Thelebolus.
The question of the final disposition of Thelebolus seems
to revolve around several important points. Included among
these are the nature of the ascocarp and the morphology of
certain parts of the ascocarp. According to Cooke and
Barr, T. stercoreus possesses a completely enclosed
perithecium and lacks paraphyses. Kimbrough (1966b) rejects
these points by concluding that T. stercoreus is actually
a reduced discomycete and does contain paraphyses. His
discussion (1966a) of the disposition of Trichobolus would
also apply to Thelebolus. Kimbrough points out that if
Thelebolus is considered in light of other multispored
genera, its characteristics are more truly aligned with
Pseudoascoboleae and indicate that it is a reduced
It has been shown in the present study that the develop-
ment of Thelebolus is similar to the development of
Trichobolus and corresponds to Ramlow's (1906) earlier work
with a few exceptions. The ascogenous system is similar to
that of the Erysiphaceae in which a privileged cell becomes
the ascal mother cell. No ascogenous hyphae and croziers
are formed. Therefore, according to Cooke and Barr one
might deduce the same conclusion. However, if as Kimbrough
(1966b) suggests, one compares this development to species
of Ryparobius Boud. and other multispored forms of the
Pseudoascoboleae, one must conclude that Thelebolus is in the
discomycetes. He also points out the difference in ascal
structure between Thelebolus and the Erysiphaceae. Also,
if one considers the nature of growth (parasitic vs.
saprobic), spore morphology, spore release, and possession
of paraphyses, it can be concluded that Thelebolus should
indeed remain in the discomycetes. That the unique ascal
feature of Thelebolus stercoreus is found in several taxa
with variable number of asci per ascocarp and spores per
ascus further reinforces this argument. If Ingold's (1965)
hypothesis is correct that there is a selection toward
larger projectiles in spore release for coprophilous fungi,
then T. stercoreus would represent the end-point of evolu-
tion in this genus.
Kimbrough and Korf (1967) validated the tribe
Tneleboleae based on the criteria that the genera possessed
iodine negative asci, elliptical spores and showed a tendency
to be both eight-spored and multispored. Thelebolus is the
type genus for the family. Most work has concerned the
uniascal, multispored species T. stercoreus. However, other
species do exist that show multiascal tendencies with lesser
spore numbers. If T. stercoreus is the end of the evolu-
tionary line on the dung substrate, its family alliance
should be based on characteristics of the eight-spored
species. The ascal structure for all the species of
Thelebolus is remarkably stable. However, other features
like pigment and shape of paraphyses may differ in the other
forms that are evolutionarily closer to their terrestrial or
lignicolous counterpart. Based on the material presented
here and the work of Kimbrough (1966a) for Trichobolus,
it appears that the family Thelebolaceae must be much more
Lasiobolus ciliatus (Kunze and Schmidt per Pers.) Boud.
is found on a variety of dung in the Gainesville area,
including that of cow, horse and rabbit. Cultures used in
this investigation were isolated from rabbit dung occurring
in the northwest area of Gainesville.
Excellent growth of Lasiobolus was achieved on dung
oatmeal agar. Fruiting occurred within two weeks from the
time of initial isolation. Colony growth was rapid, covering
a diameter of 6 cm. in five days. Fruiting was abundant,
with approximately 9,600 apothecia produced per plate.
Lasiobolus is an excellent organism to work with due to its
rapid growth and fruiting ability.
Most of the vegetative features occurred below the
surface of the agar and as the isolate became older, sub-
sequent transfers resulted in the development of the
apothecia below the surface. Lasiobolus ciliatus is an
eight-spored species having an operculum at the top of the
ascus. The spores are inconsistently uniseriate in the
ascus. Single haploid spore cultures developed apothecia,
which proved the species is homothallic.
The spores of Lasiobolus in culture are similar in
size to those occurring in nature: 15-25 x 10-12 P. The
spore germinates at or near one end (Fig. 26). The germ
tube branches almost immediately following its exit from
the spore. Further branching of the tubes occurs after a
period of growth. A cross wall delimits the germ tube from
The mycelium is multicellular and each cell is
uninucleate (Fig. 27). Another aspect of the vegetative
mycelium is the occurrence of interhyphal growth (Fig. 28).
Ascogonial initiation is peculiar to Lasiobolus.
Activity begins when cells in a hyphal tip swell. Protuber-
ances evaginate from each cell (Fig. 29). These elongate
into a hook so that the protuberance from the terminal cell
grows toward the protuberance from the subterminal cell
(Fig. 30). Nuclei can be seen near the tips of the protu-
berances. Fusion of these protuberances was observed (Fig.
29). As fusion continues the ascogonia take on a highly
coiled appearance (Fig. 31). This process seems to attract
other hyphae which grow toward the coil and entwine them-
selves around it (Fig. 32). Other ensheathing hyphae may
grow from the same parent hyphae (Fig. 33). The ensheathing
hyphae then divide and produce a mass of cells around the
After a period of Growth an expanding ascogonium
appears within the mass of cells (Fig. 34). Additional
ascogonia are produced by other cells of the parent hyphae
developing protuberances and fusing with adjacent cells.
Usually three ascogonial cells can be observed within the
expanding mass of cells (Fig. 35).
As the archicarp develops, other hyphae from the
parent hyphae grow out and fuse with the mass of cells
(Fig. 36). The apothecium has long non-septate setae
(Fig. 37) which arise from the developing apothecium when
the mass of cells is only 140 in diameter (Fig. 38).
They arise from the base of the mass of cells (Fig. 39).
As the apothecium continues to increase in size, more setae
arise near the initial growth (Fig. 40). The setac double
their length rapidly (Fig. 41). At maturity the setae have
the characteristic appearance typical of Lasiobolus. The
non-septate setae root in the ectal excipulum and possess a
bulbous base (Fig. 42). Concurrently numerous hyphae radiate
from the base to form the rhizoidal hyphae that anchor the
apothecium to the substrate. The difference between
rhizoidal hyphae and setae is in the thickness of the cell
walls. At maturity the base of the apothecium possesses
dozens of "rooting" hyphae (Fig. 43).
Plastic embedding was effective in the study of
ascocarp development. Plastic sections showed that the
excipulum encloses the ascogenous system until sporogenesis
is completed (Fig. 44). Lasiobolus develops angiocarpically
or is cleistohymenial, opening in the late mesohymenial
phase (Van Brummelen, 1967). Paraphyses outgrow the
developing asci and converge to an area at the top of the
ascocarp; at this point the ascocarp will eventually sepa-
rate to expose the asci (Fig. 45). Stages of meiosis in
the asci (Fig. 46) were observed in several sections.
The ascogonia within the young apothecium continue to
develop and the multinucleate condition becomes more
evident, especially when the ascogonia are teased from the
mass of surrounding cells (Fig. 47). Ascogenous hyphae
begin to grow out from the ascogonia. The ascogenous hyphae
become highly branched as croziers are formed, terminating
growth (Fig. 48). Numerous observations of croziers were
made (Figs. 48, 49).
Cytology of the Ascus
After the croziers are formed and the nuclei of the
penultimate cell unite, the 2N nucleus migrates into the
expanding ascus. The nucleus is rather large, approximately
6 p in diameter, with a centrally prominent nucleolus (Fig.
50). The 2N nucleus undergoes meiosis. The first division
tares place parallel to the long axis of the ascus (Fig.
51). The nucLeolus disappears during each division, but
reappears again. The second division is asynchronous; one
nucleus divides and the daughter nuclei take an extreme
position in the ascus through a division parallel to the
long axis of the ascus. iuclei now occupy the top and
bottom positions in the ascus.
The second nucleus now divides in a plane more or less
perpendicular to the long axis. At this stage there is a
pause in division and the four nucleoli reappear (Fig. 52).
Division begins again asynchronously and occurs first in
tne daughter nuclei in the upper ena of the ascus (Fig. 53).
Finally all nuclei divide and the resultant haploid nuclei
move to the positions along the length of the ascus (Fig.
54). The young ascus is usually thicker above and narrower
below, so that the majority of the nuclei reside in the
upper portion of the ascus. Sometimes inconsistencies occur
in spore cleavage and two nuclei are incorporated into one
spore (Fig. 55).
Tne immature spore wall is thick, but becomes thinner
with the age of the spore. The wall prevents the entrance
of stain into the spore, ana it is, therefore, necessary to
break the exospore to stain the single nucleus (Fig. 56).
Lasiobolus ciliatus is a useful organism in several
aspects: cultural, ontogenetical and cytological. The
organism is easily cultured and fruits abundantly in
younger cultures. As the isolate grew older, subsequent
transfers to new media fruited less. It is not known
whether this phenomenon is a result of some intrinsic
factor in the fungus or perhaps reflects a nutrient defi-
ciency of dung oatmeal agar. Studies of coprophilous fungi
would benefit by the establishment of a defined growth
Plastic sections were most useful in the study of
ascocarp development. Plastic embedding retains the shape
of the ascocarp and its vegetative features much better
than free hand or cryostat sections. It was desirous that
fixation and embedding of these fungi for light microscopy
could be extended to electron microscopy. Lasiobolus
appears to be more suitable to plastic embedding than
Thelebolus, because of the better penetration of the plastic
into the ascus.
Lasiobolus ciliatus possesses a uninucleate mycelium
and has a unique ascogonial ontogeny. In the genus
Thelebolus, compatible nuclei are already in the evagina-
tion of the parent hypha. However, in Lasiobolus a mecha-
nism is necessary in order for compatible nuclei to pair.
Several cells of the parent hypha swell and protuberances
are formed which grow out from each cell and fuse with the
adjacent cell. Proliferation of the protuberances and the
fusion of additional vegetative hyphae soon ensheath the
developing ascogonia. Kimbrough (personal communication)
has also found this same type of development in a recently
discovered species of Lasiobolus: Lasiobolus monascus
Kimbr. (1972b) (Fig. 99).
The ascogonial development of L. ciliatus is similar
to the type shown by Blackman and Fraser (1906), and
Gwynne-Vaughan and Williamson (1930) for Humaria granulata
(Fig. 96) which is now classified as Coprobia granulata
(Bull. ex Fr.) Boud. The development of the ascogonia in
C. granulata is characterized by the development of a
stalked ascogonium which is multinucleate. The ascogonium
is seen being enveloped by hyphae which originate from sub-
terminal cells. A similar sheathing of the ascogonia occurs
in L. ciliatus; however, in the latter species, several
ascogonial cells develop rather than one.
Fraser (1907) has shown a stalked ascogonial ontogeny
in Lachnea stercorea Pers. (=Cheilymenia stercorea Pers.)
(Fig. 97). The terminal cell becomes the ascogonium and
a wide trichogyne is formed. Fraser mentions an antheridium
which had the appearance of a sac filled with nuclei.
However, its functional role is questioned as ascogenous
hyphae are formed before fusion of the trichogyne with the
A similar development was observed by Brown (1911) in
Lachnea scutellata (=Scutellinia scutellata) (Fig. 98).
The ascogonium was produced on a 7-9 celled stalk. However,
no trichogyne or antheridium was observed.
Fraser (1913) studied Lachnea creta (=Tricharia creta
(Cke) Eckblad) and illustrated an ontogeny similar to that
shown by Milam (1971) for Iodophanus granulipolaris. The
ascogonium is a series of bulbous cells with a long
trichogyne produced terminally. No fusion with an anther-
idium was observed. This type of development with a long
trichogyne is found in Anthracobia melalzma by Cwynne-
Vaughan (1937) (as Lachnea melaloma) and Rosinski (1956).
It is also typical of many species of Ascobolus.
Recently, Durand (1970) studied the development of a
species of Lasiobolus identified as Lasiobolus equinus
(Mull.) Karst., which may be synonymous with L. ciliatus.
The development of the ascogonia is similar to that observed
in the present investigation. However, the initial stages
as described by Durand are similar to Ascodesmis nigricans
(Gaumann and Dodge, 1928). It is most difficult to inter-
pret the beginning stages of ascogonial initiation, but
Durand's drawings can also be interpreted as showing the
same type of development as that found in the current
investigation. Durand could not determine definitely the
species of the fungus he used. Therefore, two conclusions
can be drawn from his work. If the species is the same as
L. ciliatus, the development corresponds closely. A more
important aspect, however, is that if his fungus represents
another species of Lasiobolus, then this would indicate
that the development of the ascogonia is consistent in at
least three species of the genus.
Recently, related genera have been studied developmen-
tally. Milam (1971) studied Iodophanus (Pezizaceae) and
has shown a multinucleate ascogonial coil which is
ensheathed by hyphae from the parent hypha. This development
is similar to Ascobolus carbonarius (Dodge, 1912) and has
some similarities to the development of C. granulata.
Eckblad (1968) states that Coprobia and lodophanus have close
affinities and includes both in his family Pyronemaceae.
Kish (1971) followed the development of Coprotus
lacteus (Ck. & Phill.) Kimbr., a species with trichogynous
ascogonia and described the ascogenous system as similar to
that of Pyronema. He proposed transferring Coprotus to the
Berthet (1964a) in his summary of the ascogenous
systems of the discomycetes lists Lasiobolus ciliatus as
possessing the aporhynque type of ascogenous hyphae. This
is in contradiction to the results presented in this study.
Lasiobolus ciliatus has true croziers and the ascogenous
hyphae are of the pleurorhynque type.
Lasiobolus differs slightly from Thelebolus stercoreus
in ascocarp development. Lasiobolus, a multiascal species,
opens in the late mesohymenial phase as spores mature.
Thelebolus, the uniascal form, opens in the telohymenial
phase; during spore liberation. L. ciliatus also differs
developmentally from T. stercoreus by the presence of
croziers. Lasiobolus ciliatus also has an operculum at the
tip of the ascus, and several ascogonial cells per
Species of Lasiobolus were classified as members of the
genus Ascophanus by Boudier (1869) when he split the genera
of the Ascobolaceae into the Ascobolei Genuini and Ascobolei
Spurii. Included in the latter hyaline-spored group were:
Ascophanus with eight-spored asci, Thecotheus and
Ryparobius with multispored asci.
Saccardo (188h) separated Lasiobolus from Ascophanus
by virtue of its setose apothecia. Kimbrough and Korf (1967)
further delimited Lasiobolus by emphasizing the non-septate
nature of the setae. This was done to differentiate it from
Trichobolus, a multispored, uniascal form with septate setae.
The nature of the ascus is also stressed, Lasiobolus
possessing cylindric, operculate asci, while Trichobolus
possesses an ovate irregularly dehiscing ascus. Kimbrough
(1972a) has shown that basic ascal wall structure differs
also in these taxa even though in both genera there is
tremendous variation in the size of the ascus and number
Although the taxonomy of the genus Lasiobolus has
been relatively free of controversy, the opposite is true
of the species under investigation. The controversy
centers around four species: L. papillatus (Pers. per Pers.)
Sacc., L. equinus (MUll. per Pers.) Karst., L. pilosus Fr.,
and L. ciliatus (Schmidt ex Fr.) Boud. All have been placed,
at one time or another, in synonymy under each other. No
comparative study has determined if they are the same
fungus. However, a search of the literature reveals the
The name Lasiobolus papillatus (Pers. per Pers.) Sacc.
is used by Seaver (1928) who states in his book that L.
papillatus is the type for the genus. However, the holotype
no longer exists. In the description of the fungus by
Boudier (1869) it was noted that his specimen had septate
hairs. His concept of L. papillatus must, therefore, be
rejected, in keeping with the current description of
Lasiobolus, which has non-septate hairs. L. papillatus sensu
Boudier may well represent an eight-spored Trichobolus.
The name Lasiobolus equinus (MUll. per Pers.) Karst.
is used by Kimbrough and Korf (1967) and Seaver (1928).
Seaver places L. ciliatus in synonymy under L. equinus.
The name L. equinus was not validated by Fries but by
Sowerby (1821) in Gray's Natural Arrangement of British
Plants as Peziza equina. This name would not have prior-
ity over either of the two remaining names.
The name Lasiobolus pilosus Fr. was validated by Fries
(Systema Mycologicum volume 2 on page 164). This name has
been used by Van Brummelen (1967) and Eckblad (1968).
Eckblad correctly places L. equinus in synonymy under
L. pilosus because of priority. He makes no mention of
L. ciliatus. Van Brummelen states that L. pilosus is a
highly variable species.
The name Lasiobolus ciliatus (Schmidt ex Fr.) Boud.
has been used by Dennis (1968) and Rifai (1968). Van
Brummelen (1967) discussed the validity of the names;
however, because he had not seen L. ciliatus, he did not
include it in synonymy unaer L. pilosus. Rifai in his
discussion of the species states that Persoon (1822)
revalidated the name as Ascobolus ciliatus before Fries
(1822) validated Ascobolus pilosus. The point being; what
is the correct starting point for nomenclature? Is it the
date January 1, 1821 or is it names validated by Fries?
If one assumes that the date is the starting point,
Persoon's name is valid; however, if it is Fries' publica-
tion, then L. ciliatus would be rejected.
One point overlooked is that Fries also validated
Ascobolus (Lasiobolus) ciliatus in his work in 1822 (also
in volume 2, page 164). Ascobolus ciliatus appears before
A. pilosus and if both are the same fungus, then the name
Lasiobolus ciliatus (Schmidt ex Fr.) Pers. has priority.
The differences that exist between Thelebolus and
Lasiobolus make it necessary to remove Lasiobolus from the
Thelebolaceae. Although Lasiobolus shows the same evolu-
tionary trends as does Thelebolus on dung (Kimbrough, 1972a)
with uniascal and nultispored forms, its development and
ascal structure take priority. The ascogonial development
of Lasiobolus ciliatus and L. monascus may give clues to
their taxonomic disposition. Lasiobolus may represent an
evolutionary excursion onto dung from terrestrial species
similar to Scutellinia, Cheilymenia, Coprobia or some
intermediate genus. It is felt that L. ciliatus would be
best placed in the Aleuriaceae (Arpin, 1968). Arpin's
criterion for inclusion into this family was the possession
of both gamma and beta carotenoid pigments. Lasiobolus has
not been shown to possess carotenoid pigments; however, as
Nannfeldt (1972) has suggested, can we be sure that the
species is really devoid of carotenoids? Is it not possible
to assume that these are present in the shape of colorless
precursors or colorless derivatives?
Lasiobolus ciliatus could also be placed in Rifai's
family Humariaceae tribe Ciliarieae along with Scutellinia,
Cheilymenia and Coprobia according to ontogeny. Anthracobia
of the Aleurieae and Tricharia of the Lachneae have an
ontogeny similar to lodophanus.
Thecotheus cinereus (Cr. & Cr.) Chenantais
Cultures of T. cinereus were received from Dr. J. W.
Paden, who had isolated the fungus from dung in British
Columbia. The cultures were not derived from single spores
and after a month of growth in deep dish cultures, several
fruit bodies were discovered. The early stages of fruiting
were sectioned and they consisted of a mat of bulbous cells
(Fig. 57). These patches of cells were designated as
"primordial humps" as they may function as part of an
The mycelium is mostly multinucleate, although many
individual cells may be uninucleate. Interhyphal growth
occurred in culture. The first stage in fruiting is an
intertwining of the vegetative mycelium. Coiling continues
as a mass of intertwining hyphae is produced. This coiling
takes place underneath the primordial humps.
Within this mass certain cells begin to swell and form
ascogonial cells (Fig. 58). Compatible nuclei were assumed
to enter the ascus mother cells before septation. A branch-
ing ascogenous system then develops from the ascogonia.
Thecotheus may have several ascogonial cells per apothecium.
As the ascogenous system is developing, the cell
layers surrounding the centrum continue to proliferate form-
ing a well defined medullary and ectal excipulum (Fig. 59).
The ectal excipulum is composed of bulbous cells (Fig. 60).
These bulbous cells appear to be the same as those of the
primordial humps. The emerging ascogenous system pushes the
cells upward and to the side where they form the excipulum.
These bulbous cells are uninucleate (Fig. 61); however,
the hyphae that join the cells to the base of the apothecium
are multinucleate (Fig. 62).
The centrum, meanwhile, becomes densely nucleated as
the branching ascogenous system pushes upward through the
paraphyses (Fig. 63). Finally, after a period of longi-
tudinal growth, croziers are formed (Fig. 64), and the
penultimate cell enlarges forming the ascus. Compatible
nuclei fuse and a large diploid nucleus (diameter 10-11 p)
is formed (Fig. 65). The nuclear envelope is observable as
a halo surrounding the densely stained nucleolus.
Meiosis and ascosporogenesis were not observed due to
the scarcity of material. Mature ascospores were present,
and it was observed that the wall is rather thick and smooth
at first (Fig. 66), and apiculi develop at the ends of the
spores (Fig. 67). Also the spores may become ornamented
(Fig. od). Tne apiculi stain best with hot lacto-phenol
cotton blue and this improves the longer the specimen is in
the stain. The spores of T. cinereus are uninucleate and
are irregularly uniseriate in the ascus (Fig. 69).
Thecotheus pelletieri (Cr. & Cr.) Boud.
A single haploid spore culture of T. pelletieri was
obtained by methods previously described. Actively growing
cultures were maintained for a month or more in deep dish
petri plates. The culture formed primordial humps. Bulbous
cells similar to those found in T. cinereus were present
(Fig. 70); however, apothecia failed to develop.
Interhyphal growth was evident in cultures of
T. pelletieri (Fig. 72). Also present were vegetative
mycelial loops (Fig. 71). The mycelium is septate,
approximately 3-3.5 P in diameter and is mostly multi-
nucleate (Fig. 71). Nuclei divided in the hyphae in the
loop and also in the hyphae entering the loop. The hyphae
radiating from the loop produced small side branches (Fig.
73). These small branches were similar to conidiophore
initials. Once the loops were formed, the main hypha from
the loop gave rise to several radiating hyphae. These
hyphae radiated rapidly and branched. Some of the branches
appeared to fuse with other hyphae (Fig. 74). The function
of the loops is unknown.
Fresh material from dung showed that the diploid
nucleus of T. pelletieri is relatively large (10 x 11 u),
and occupies the central portion of the ascus (Fig. 75).
The nucleolus is also large (5-6 p), and stains deeply with
methyl blue in Hoyer's solution. The nucleus is evident
only as an envelope surrounding the nucleolus. The ascus
enlarges to almost its full size while containing the
Due to the lack of fruiting in culture, cytological
stages of meiosis and ascosporogenesis could not be obtained.
Asci from dung specimens were seen that contained several
nuclei (Fig. 76), but no division stages were observed.
After the last division a spore wall is formed around each
nucleus. The wall is initially very thick (Fig. 77). The
spores are uninucleate (Fig. 78). The ascus is broadly
clavate and the spores are ejected in a gelatinous mass
(Fig. 79). The spores germinate from one end after swelling
to approximately twice their normal size (Fig. 80). The
ascocarp develops angiocarpically; however, the phase in
which the hymenium is exposed could not be determined.
Both T. pelletieri and T. cinereus produce an imperfect
stage in culture, conidial production being more abundant
in T. pelletieri. A few weeks after transferring
T. pelletieri to dung oatmeal agar, conidia were observed.
A similar imperfect stage was also discovered in cultures
and slants of T. cinereus.
The conidia of T. pelletieri are hyaline and borne on
short side branches, 15-22 p long (Figs. 85-95). The
hyaline conidiophores measure pu wide at their base and
taper to a width of 2 p at their tip. The conidiophores
begin as small evaginations on a hypha (Fig. 81). The small
protuberance elongates and a side branch may bud out from
it (Fig. 82) later developing a hooked appendage (Fig. 83).
In many cases the conidiophore develops this hooked append-
age from its base. The function of the hook is unknown.
The mycelium is superficial.
The conidia are 3-4 x 6.5-7 p. They appear to be
uninucleate (Fig. 90). Division of a nucleus takes place
near the tip of the conidiophore (Fig. 84), and the upper
nucleus migrates into the conidium. The primary conidium
seems to be blown out from the tip of the conidiophore
giving the conidium an abovate appearance (Figs. 85-87). A
cross wall delimits the conidium from the conidiophore
(Fig. 88). The tip of the conidiophore appears to be
"meristematic" and continues to increase in length just to
the side of the primary conidium (Fig. 89). A secondary
conidium is then blown out (Figs. 90-93), and becomes
delimited (Fig. 94). This process continues so that finally
as many as ten conidia are produced per conidiophore (Fig.
During the period of coniaial formation, the
conidiophore increases in length. The distance between
succeeding conidia, however, is very short and gives the
mature conidiophore a swollen appearance.
Although a thorough investigation of the two species
of Thecotheus was not possible due to their slow and scanty
growth in culture, several contributions to our understand-
ing of their ontogeny can be made. Overton (1906) was
unable to germinate the spores of T. pelletieri and relied
on an abundant supply of natural material for his research.
He noted that T. pelletieri was a late fruiting organism in
regard to its succession on dung. His source of material
was old and partly dried-up cultures of horse dung. In the
present investigation the dung specimen was almost four
weeks old, and this may be one reason that this organism is
not encountered more frequently.
Thecotheus pelletieri was obtained in a single spore
culture during this study. Although several attempts were
made to induce apothecia to form, none were successful. It
is possible that T. pelletieri is heterothallic, the absence
of compatible nuclei making fruiting impossible.
Thecotheus cinereus did fruit sparsely during the
course of this study. However, this was not a single spore
culture. Dr. Paden simply poured agar over the dung and
isolated the fungus as it grew up through the agar, but
could not obtain fruiting in culture.
Tne primordial humps may represent the end of the
vegetative phase of the fungus, fruiting being delayed only
until karyogamy of compatible nuclei. Good evidence is
presented that these primordial humps form a part of the
ectal excipulum of the Thecotheus apothecium. Kimbrough
(1969) describes and illustrates the bulbous cyanophilous
cells of the excipulum in Thecotheus. In his developmental
study, all species of Thecotheus show angiocarpic develop-
ment on dung. This supports the assumption that the primor-
dial humps are the early developmental stage of the
apothecium and excipulum of Thecotheus, and that the emerg-
ing ascogenous system pushes up through these humps.
Bulbous cells that function in excipular formation may
be found in other genera. Squash mounts of apothecia of
other genera may obliterate or alter their presence. This
may have occurred in Ascobolus albidus (Van Brummelen, 1967,
Plate 5, Fig. F-l), a species that is shown to possess
paraphyses with subglobular elements.
The bulbous cells of the primordial humps are also very
similar to the microconidia found in Ascobolus carbonarius
(Dodge, 1912). In A. carbonarius a conidium fuses with the
trichogyne ana provides the ascogonium with a compatible
male nucleus. The role of the bulbous cells in Thecotheus,
however, was not determined.
The presence of primordial humps in both species of
Tnecotheus suggests a close relationship between the two.
It may also indicate a similar ascogonial ontogeny for both
species. However, this must be confirmed by further studies.
There appears to be little difference between the develop-
ment of the two species, except for the additional mitoses
in order to produce 32 spores in T. pelletieri.
The mycelial loops found on the surface of the agar may
act as a mechanism that initiates the conidial system since
branches from the loop appear similar to the conidiophore
The imperfect stages not only possess a similar
morphology but also a similar ontogeny. These similarities
confirm a close relationship between the perfect stages of
the eight-or 32-spored species. However, this may not be
as factual as it appears because other perfect stages as
those in Nectria may have several different imperfect stages.
Also, Kimbrough and Korf (1967), in listing characteristics
of importance that should be considered in distinguishing
genera of the Theleboleae, place the use of imperfect stages
on a low priority. However, as more imperfect stages are
induced or discovered this feature may be of great use in
establishing natural relationships.
The type of coniaial ontogeny found is very similar to
Hughes' Section II (1953) which he labels "terminis spore."
The formation of the initial conidium itself is a blasto-
conidial type. More recently Kendrick and Cole (1968)
have described this type of development as a sympodial
conidium. Barron (1968) in his book on soil hyphomycetes
"would classify this specimen in his section sympodulospore.
Tne imperfect stage seems to have affinities with Sporothrix
(Hekten and Perkins) Barron and Rhinotrichella Arnaud. In
Sporotnrix the conidia are borne on denticles in an acropetal
succession. They have no septation and are truncate at the
attachment to the conidiophore. The head of the conidio-
phore, however, appears broader than the imperfect stage of
Oberwinkler, Casagrande and MUller (1967) have found
an imperfect stage of Ascocorticium anomalum (Ell. & Harkn.)
Earle which is morphologically similar to that of Thecotheus.
The conidiophore has a hook which closely resembles the hook
in the imperfect stage of Thecotheus. They describe the
conidial ontogeny as sympodial with the conidiophore
elongating to form a zig-zag appearance. The base of the
conidiophore is brown with the conidiogenous portion hyaline.
The conidia are ellipsoid to round (1.5-2.5 u). In discus-
sing the classification of the imperfect stage, Oberwinkler
et al. say that it resembles Tubaki's (1958) Chloriaium
minutum, but that since Hughes (1958) places C. minutum
in synonymy under Bisporomyces, it cannot be placed there.
Instead, they suggest a close relationship with
Rhinotrichella Arnaud (1953). However, they would not name
the species until more work has been done on the imperfect
-stage. Likewise, until more work is accomplished with this
imperfect stage of T. pelletieri, a genus cannot be assigned.
Several other researchers have discovered imperfect
stages in allied families. Greene (1931), working with the
Ascobolaceae, observed oidia in cultures of Ascobolus. The
oidia were formed by the separation of rectangular cells of
the aerial mycelium. These oidia were abundant in cultures
that failed to produce apothecia. This was also observed in
Thecotheus as conidial production was greater in T.
pelletieri which did not fruit. The oidia of Ascobolus
germinated but produced no fertile apothecia.
Paden (1967) found a conidial stage of Peziza
brunneoatra Desm. Conidia of P. brunneoatra are similar to
the bulbous cells found in the primordial humps of Thecotheus
(Figs. 1, 2). The bulbous cells measured 7-9 x 13-16 u,
approximately the same size as the conidial stage of P.
brunneoatra. Germination of these bulbous cells was not
observed and their function seems limited to excipular
Tubaki (1950) described an Oedocephalum imperfect
stage for lodophanus testaceus (Moug. in Fr.) Korf. This
is similar to the imperfect stage of Thecotheus except for
a more inflated apical apex of the conidiophore. An
Oedocephalum conidial apparatus was reported for Pyronema
omphalodes (Bull.) Fuckel (Hughes, 1953). Oedocephalum is
placed in Hughes' Section Ib.
The occurrence of a sympodial conidial type in
Thecotheus is significant. A review of the literature deal-
ing with imperfect stages (Berthet, 1964b; Eckblad, 1968),
indicates that the occurrence of the sympodial conidial
type is rare.
Most of the imperfect stages listed occur in the form
genus Oedocephalum. This genus is included in Section Ib of
Hughes' (1953) classification, characterized by blastoconidia.
This imperfect stage is found in three operculate families:
Pezizaceae, Pyronemaceae and Aleuriaceae.
Oidia are found frequently. Hughes placed in Section
VII those fungi that produce oidia. Oidia are now desig-
nated by other workers (Barron, 1968; Tubaki, 1958) as
arthoconidia. Oidia are found in three operculate families:
Ascobolaceae, Pezizaceae and Pyronemaceae.
No imperfect stage has been found in either the
Otideaceae or the Rhizinaceae.
Sympodial conidia have been found in two families:
Morchellaceae and Sarcoscyphaceae. These conidia are found
in Section II of Hughes.
There are thirty-two species of operculate discomycetes
known to possess an imperfect stage, of these twenty-six
possess either an Oedocephalum or oidial conidia. Two
-genera have a Botrytis imperfect stage (Section Ib) and
another possesses an Ostracoderma imperfect stage (Section
Ib). Therefore, only three genera possess a sympodial
conidia imperfect stage.
In a more recent work, Paden (1972) has re-evaluated
conidial formation of the imperfect state of the Pezizales.
Four families, Sarcoscyphaceae, Sarcosomataceae, Aleuriaceae
and the Morchellaceae possess sympoduloconidia. Cookina
sulcipes, C. tricholoma, Pithya cupressina and Sarcoscypha
coccinea (Sarcoscyphaceae) have sympoduloconidia that could
not be placed into a known genus. Desmazierella acicola
(Sarcosomataceae) has a Verticicladium imperfect stage and
Urnula craterium, Plectania nannfeldtii, and Sarcosoma
latahensis have Conoplea imperfect stages. Two species of
Geopyxis (Aleuriaceae) have Nodulisporium imperfect stages
and Caloscypha fulgens has a sympoduloconidial stage that
could not be placed into a known genus. Morchella
(Morchellaceae) has a Costantinella imperfect stage.
The Pezizaceae are reported to possess imperfect stages
representing three different genera. Peziza petersii and
Iodophanus are associated with an Oedocephalum stage, four
species are associated with an Ostracoderma stage and three
species possess aleurioconidia. Botrytis-like conidia are
found in the Otideaceae.
The Ascobolaceae possess oidia and a Papulaspora stage.
No conidia are reported for the Thelebolaceae.
Other imperfect stages have been found in inoperculate
families including, Orbiliaceae, Hyaloscyphaceae,
Helotiaceae, Geoglossaceae and Sclerotiniaceae.
Overton's (1906) observation of ascogonia was confirmed.
The ascogonial cells of T. cinereus observed were like those
found in Ascobolus citrinus (Schweizer, 1923) as Overton
illustrated for T. pelletieri. Ascus formation is initiated
by crozier formation.
Thecotheus was first described in the Ascobolaceae by
Boudier (1869) as a hyaline, multispored species of the
Ascobolei Spurii. It was separated from Ryparobius, the
other multispored form, by its larger thick walled spores,
and its larger fruiting body. Chenantais (1918) later
transferred the eight-spored Ascobolus cinereus Cr. & Cr. to
Kimbrough and Korf (19o7) removed Thecotheus and
lodophanus from the Tnelebolaceae (Pseudoascoboleae) and
placed both in the tribe Pezizeae of the Pezizaceae. Both
genera were considered similar due to the diffuse amyloid
reaction of their asci and their callose-pectic marking on
the spores. Eckblad (1968) retained Thecotheus in the
Thelebolaceae because of its simpler excipular structure,
its multispored tendency, and its protruding asci. He
placed lodophanus in the more primitive family Pyronemaceae.
Eckblad based this decision on the presence of carotenoid
paraphyses and simpler excipular structure. Kimbrough (1969)
further typified the entire genus with a morphological and
cytochemical study of four species of Thecotheus.
Milam (1971) studied lodophanus carneus (Pers. ex Fr.)
Korf both developmentally and structurally. Results from
his research and from the present study point to some of
the differences between the two genera. Iodophanus has
coenocytic excipular cells and an Oedocephalum imperfect
stage. Thecotheus has a sympodial conidial type and uni-
nucleate excipular cells. Milam also showed that the
ontogeny of I. granulipolaris consisted of a multicellular
archicarp with a long trichogyne. This type of development
has also been reported by Fraser (1913) for Lachnea creta
(=Anthracobia sp). Anthracobia is now placed in the
Aleuriaceae by Arpin (1968).
Pfister (1972) has recently described several members
of the genus Thecotheus that occur on wood. A correlation
of their ontogeny, cytology and their possible imperfect
stages would be most significant. The finding of these
terrestrial species reinforces the theory that the
coprophilous species represent an evolutionary and ecological
excursion from some terrestrial species. Many features of
Thecotheus appear similar to members of the Ascobolaceae:
diffusely amyloid asci, thick-walled ascospores, bulbous
excipular cells (at least in Ascobolus albidus), and a
perisporic sheath that encrusts the mature spore. In
Ascobolus a characteristic pigment is obvious in its outer
spore wall. However, Kimbrough (personal communication) has
noted that when the spores of Ascobolus are bleached, there
are cyanophilic markings on the inner walls of the spore
similar to Thecotheus. Another feature in common with the
Ascobolaceae is the similarity of the bulbous cells in the
primordial humps of Thecotheus and the microconidia of
Korf (1972a) has placed both Iodophanus and Thecotheus
in the lodophaneae of the Ascobolaceae. For the moment this
appear to be the best placement for Thecotheus. However,
as more is learned about the ontogeny and excipular cells
of Thecotheus a relation with the Aleuriaceae may be possible
due to the possession of sympodial conidia. It is recommend-
ed that other mycologists working with genera in the
Aleuriaceae note such features as bulbous cells in the
excipulum and also the presence of imperfect stages which
may show relationships to Thecotheus. Until these studies
are performed, a conclusive family alliance is not possible.
It has long been recognized by many authorities
(Kimbrough, 1966b; Kimbrough and Korf, 1967; Eckblad, 1968;
Rifai, 1968) that the family Thelebolaceae represents a
unique and diversified group of fungi. The purpose of this
research was to compare the ontogeny of representatives of
three genera that have been at one time classified as
members of this group. These genera were chosen because
they represented fungi with varying number of asci per
apothecium and varying number of spores per ascus.
Contrary to reports by Berthet (1964a) and Eckblad
(1968) that the mycelium of the Pezizales is coenocytic,
that of Lasiobolus ciliatus and Thelebolus stercoreus is
uninucleate. Thecotheus pelletieri and T. cinereus are
highly variable. The mycelium of each is mostly coenocytic;
however, the large cyanophilous bulbous cells that comprise
the excipulum of both genera are uninucleate.
The initiation of the ascogonia differs in all three
genera. Thelebolus stercoreus has been shown to have an
ontogeny similar to Trichobolus zukalii. Ascogonia are
initiated by evaginations from the parent hypha which
contains the compatible nuclei.
Lasiobolus ciliatus possesses an ontogeny similar to
that found in Coprobia granulata (Blackman and Fraser, 1906;
Gwynne-Vaughan and Williamson, 1930); a stalked ascogonium.
Essentially, the parent hypha swells and evaginations grow
from these swollen cells. These evaginations fuse, and it
is thought that through these fusions compatible nuclei are
introduced into the same cells. Ensheathing hypha then grow
from tne parent hypha, and also from other vegetative hyphae
and enclose the ascogonia.
Lasiobolus ciliatus has an ontogeny similar to Coprobia.
Pigment analyses may show a relationship between these fungi
and other related genera such as Scutellinia. The finding
of a multispored, uniascal species of Lasiobolus has
completed the evolutionary line within this genus. However,
the major comparison with the genus Lasiobolus to terres-
trial species will have to be determined by L. ciliatus.
This, according to Ingold (1965), would represent more
closely the ancestral type, as the uniascal form would
possess the more highly evolved mechanisms associated with
a coprophilous environment.
The presence of ectal hairs and an ontogeny similar
to Coprobia granulata indicate a close relationship to the
Aleuriaceae (Arpin, 1968). Analysis of carotenoid pigments
will be necessary to determine if L. ciliatus has the same
biochemistry as Coprobia, Scutellinia and Cheilymenia.
These genera possess gamma carotene as their najor pigment.
iifai (19io) also recognizes these genera as being closely
related and includes them in the tribe Ciliareae of the
liunariaceae. A further correlation is that they, along with
L. cilictus, possess stalked ascogonia. Rifai also erected
the tribes Lachneae, Aleurieae and Otideae. It is known that
Tricharia (Lachneae) and Anthracobia (Aleurieae) possess
ascogonia similar to lodoohanus (Milan, 1971) and Ascobolus
(Dodge, 1912). Ascogonial formation in the Otideae has not
been studied. Therefore, the closest relationship appears
to exist between L. ciliatus and the genera of the Ciliareae.
Thecotheus cinereus has an ontogeny that appears similar
to Ascobolus citrinus. This species possesses a group of
inflated ascogonial cells which are soon enclosed by
enveloping hyphae. The occurrence of bulbous cells in
primordial numps raises the possibility that these cells
may play a role in the passage of compatible nuclei by
fusion witn a trichogyne or some similar structure. It has
been shown that these bulbous cells are uninucleate and
ultimately form part of the ectal excipulum.
An imperfect stage has been found in association with
Thecotheus pelletieri and T. cinereus. The imperfect stage
is classified as a sympodial conidium resembling
Rhinotrichella and Sporothrix. Conidial stages have been
found in association with only two other closely related
operculate genera that occur on dung, lodophanus and
Ascobolus. The sympodial imperfect stage of Thecotheus
differs developmentally from the other genera, which are
arthroconidial or blastoconidial. Sympodial conidia have
been found only in the Morchellaceae, Aleuriaceae,
Sarcosomataceae and Sarcosyphaceae of the Pezizales, and
are, therefore, unique for this group. The presence of
imperfect stages may play an important role as more work
is done in this area.
The use of plastic sections has been useful in follow-
ing ascocarp development. Thelebolus stercoreus is
cleistohymenial because the hymenium is not exposed until
spore release. Lasiobolus ciliatus is angiocarpic with the
hymenium opening in the late mesohymenial stage, as the
spores mature. Thecotheus cinereus is also angiocarpic;
however, it could not be determined when the hymenium
Much of the recent research by Wicklow and Malloch
(1971), Durand (1970), Kish (1971), Milam (1971) and now
this study points to the great diversity of features found
in the coprophilous discomycetes. Kish has suggested that
the affinities of Coprotus may lie with the Pyronemaceae.
Iodophanus has been placed in the Pezizaceae by Kimbrough
and Korf (1967) and more recently Korf (1972a) has erected
the tribe Iodophaneae to include lodophanus, Thecotheus,
Boudiera and Sphaerosoma in the Ascobolaceae.
The phylogenetic proximity of Thecotheus and
lodophanus is now in doubt due to the vegetative features
of both. Until further studies have been undertaken on the
terrestrial species of Thecotheus, final disposition to a
family cannot be made because of the variability of characters
when compared to established families. For instance, the
ascal structure found in Thecotheus is also found in
Ascobolus immersus (Van Brummelen, 1967). Also, subglobular
elements are found in Ascobolus albidus that are similar to
the bulbous cells of Thecotheus. The terrestrial species of
Thecotheus were identified previously as Peziza. The
imperfect stage of Thecotheus is closer morphologically to
the Aleuriaceae than the Ascobolaceae. Finally, the
ascogonia of Thecotheus are similar to the asccgonia of
Ascobolus. Until more characteristics of other families are
established, such as ontogeny, imperfect stages and excipular
features, Thecotheus seems best placed in the Iodophaneae
of the Ascobolaceae.
Thelebolus stercoreus represents the highest evolution-
ary level in the genus Thelebolus. The uniqueness of its
structures may exclude most other genera from the family
Thelebolaceae. It now appears that the Thelebolaceae may
be restricted to Thelebolus, Trichobolus, and perhaps
Ascozonus and Caccobius. The latter two genera have not
been studied enough to determine family placement.
Kimbrough (personal communication) suggests that the pore
mechanism in the ascus of Caccobius may indicate an excur-
sion onto dung from an inoperculate genus. It may well be
that the coprophilous discomycetes represent excursions onto
the dung habitat by several families that have through
.parallel evolution evolved many similar features. These
features would include an increased number of spores per
ascus, a decreased number of asci per apothecium, and
various modifications in the ascal wall itself. The irony
of Thelebolus stercoreus may be that this organism that was
once considered to be primitive, and even classified in
families outside the Ascomycetes, may well be among the
most advanced fungi in its habitat and morphology.
Figs. 1 25. Tnelebolus stercoreus.
Fig. 1. Nuclear stain of vegetative hypha showing
uninucleate (arrows) condition. X 1250.
Fig. 2. Superficial mycelial loop on agar surface. X 1000.
Figs. 3 4. Bulbous cells of the vegetative mycelium.
Figs. 5 6. Early hyphal coiling. X 1000.
Fig. 7. Proliferation of cells surrounding ascogonial
initial. X 1000.
Fig. 8. Excipular cells of textura angularis. X 1000.
Fig. 9. Developing ascocarp with radiating hyphae. X 160.
Fig. 10. Fusion of hyphae to the developing ascocarp.
Note anastomosis of these hyphae (arrow). X 1000.
Fig. 11. Increased nuclear activity in the expanding
ascocarp. X 1000.
Fig. 12. Plastic section showing ascogonial cells. X 1000.
Fig. 13. Young ascus in the cleistothecium-like apothecium.
Fig. 14. Plastic section showing the layer of cells
surrounding the ascus. X 400.
Fig. 15. Nuclear stain showing a large diploid nucleus
(arrow) in the ascus. X 1000.
Fig. 16. Nuclear stain showing Prophase I (arrow). X 1000.
Fig. 17. Ascus containing two nuclei (arrows). X 400.
Fig. 18. The start of Division II. Note the appearance
of a spindle (arrow). X 400.
Fig. 19. Higher magnification of spindle. X 1250.
Fig. 20. Four nuclei (N) present in the ascus. X 1000.
Figs. 21 22. Plastic sections of young asci showing the
prominent nucleoli (NU). X 1000.
Fig. 23. A fully expanded ascus. X 400.
Fig. 24. Ripe ascus pushing through the excipulum. Note
the characteristic ascal tip. X 400.
Fig. 25. Ascus surrounded by closely adhering paraphyses.
R O I *ir *
.... ..... ,
. I i
Figs. 26 5U. Lasiobolus ciliatus.
Fig. 26. Germinating ascospores. X 400.
Fig. 27. Nuclear stain of vegetative mycelium showing
uninucleate condition (arrows). X 1000.
Fig. 28. Interhyphal growth in culture. X 1000.
Fig. 29. Developing ascogonium. Note evaginations from
tne parent cells. X 1000.
Fig. 30. Developing ascogonium showing growth of
evaginations. Note nuclei in protuberance.
Fig. 31. Highly coiled ascogonium due to proliferation
of surrounding cells. X 1250.
Fig. 32. Ascogonium surrounded by cells. X 400.
Fig. 33. Ensheathing hypha growing from the parent hypnae.
Fig. 34. Enlarged ascogonium surrounded by a layer of
cells. X 400.
Fig. 35. Three ascogonia in a developing ascocarp. X 1000.
Fig. 36. Fusion of hyphae from the parent hypha to the
ascocarp. X 1250.
*. .D^ ::0
+ l "..
Fig. 37. Ascocarp on dung showing characteristic hairs.
Fig. 38. The beginning of the development of a hair
(arrow). X 400.
Figs. 39 41. Elongation of the hair (arrow). X 400.
Fig. 42. Higher magnification of the base of a hair
showing its attachment in the excipulum. X 1250.
Fig. 43. Rhizodial hyphae at the base of the ascocarp.
Fig. 44. Plastic section showing the enclosed hymenium.
Fig. 45. Plastic section showing paraphyses growing to a
point above the asci where the ascocarp will
eventually rupture. The result of nuclear
divisions can be seen in the asci. X 600.
I ,r r
** ^s- ~* *
Fig. 46. Plastic section showing nuclei (N) present in
an ascus. X 1000.
Fig. 47. Squash mount of an ascocarp with three multi-
nucleate ascogonia. X 1000.
Fig. 48. Ascogenous system showing asci in various stages
of development. A crozier (C) is present. X 400.
Fig. 49. A crozier. X 400.
Fig. 50. An ascus containing the large diploid nucleus.
Fig. 51. Ascus after Division I. X 1250.
Fig. 52. Ascus with four nuclei (N). X 400.
Fig. 53. Ascus with four nuclei. Two (arrows) are
beginning to undergo further division. X 1250.
Fig. 54. Ascus containing eight nuclei (N) near the start
of spore cleavage. X 1000.
Fig. 55. Ascus after spore cleavage. Note that there has
been an inconsistency in cleavage with one spore
receiving two nuclei (arrow). X 1000.
Fig. 56. A uninucleate ascospore. X 1250.
-U. *~ '
Figs. 57 69. Thecotneus cinereus.
Fig. 57. A "primordial hump" showing the bulbous cells
(arrows). X 200.
Fig. 58. Plastic section showing a series of ascogonia.
Fig. 59. A frozen section using the cryostat showing the
centrum ana excipulum. X 100.
Fig. 60. Uninucleate bulbous cells near the edge of a
primordial hump. X 400.
Fig. 61. Uninucleate bulbous cells forming the excipulum.
Fig. 62. Nuclear stain showing the multinucleate hyphae
beneath the bulbous cells. X 1250.
Fig. 63. Plastic section showing ascogenous hyphae
(arrows) pushing through the paraphyses. X 1000.
Fig. 64. Section showing a crozier (C). X 1250.
Fig. 65. Nuclear stain showing an ascus containing a
diploid nucleus (arrow). X 1000.
Fig. 66. A young ascospore with thick, smooth spore valls.
Fig. 67. A young ascospore with apiculi. X 2000.
Fig. 68. Mature ascospore with apiculi and ornamented
walls. X 1250.
Fig. 69. An ascus with irregularly uniseriate ascospores.
Figs. 70 95. Thecotheus pelletieri.
Fig. 70. Bulbous cells and filaments of the "primordial
nump". X 1000.
Fig. 71. Nuclear stain of a mycelial loop to show the
multinucleate condition. X 600.
Fig. 72. Interhyphal growth in culture. X 1000.
Fig. 73. Mycelial loop with evaginations from the loop
similar in shape to conidiophore initials (CI).
Fig. 74. Mycelial loop with radiating hyphae. Note the
fusion of hyphae (arrows). X 100.
Fig. 75. Nuclear stain showing the large diploid nucleus
(arrow) in the ascus. X 1000.
Fig. 76. An ascus with four nuclei (N). X 1000.
Fig. 77. An ascus with young thick walled ascospores.
Note the ascal tip. X 1250.
Fig. 78. Uninucleate ascospore. X 1250.
Fig. 79. Ascospores ejected from an ascus showing that
they are released in one large clump. X 440.
Fig. 80. Germinating ascospores. Note large vacuoles in
the spore ana that tne ascospores have swollen
co twice their original size. X 440.
Fig. 81. A conidiophore initial. X 1000.
Fig. 82. The initial has increased in length and a hooked
appendage has begun to form. X 1000.
Fig. 83. The conidiophore and hook have continued to
increase in length. X 1000.
Fig. 84. Nuclear division in the conidiophore tip. X 1000.
Fig. 05. The primary conidium is blown out. X 1000.
Fig. 86. The conidium elongates. X 400.
Fig. 87. The conidium continues to elongate. Note hooked
appendage at the base of the conidiophore. X 1000.
Fig. 88. A septum is formed between the conidium and the
conidiophore. X 1000.
Fig. 89. Tne tip of the conidiophore becomes meristematic
and begins to elongate to one side of the
primary conidium. X 1000.
Figs. 90 93. A secondary conidium is blown out and
elongates. The conidium is uninucleate. X 1000.
Fig. 94. The secondary conidium is delimited from the
conidiopnore by a septum. X 1000.
Fig. 95. A conidiophore with a head of conidia. X 1000.
Fig. 9o. Stalked ascogonium of Coprobia granulata
(Humaria granulata); from Gwynne-Vaughan and
Fig. 97. Stalked ascogonium of Cheilymenia stercorea
(Lacnnea stercorea); from Fraser (1907).
Fig. 98. Diagrammatic cross-section of ascocarp showing
tne stalked ascogonium of Scutellinia scutellata
(Lacnnea scutellata); from Brown (1911).
Fig. 99. Stalked ascogonium of Lasiobolus monascus; from
Kimbrough (1973, in press).
:;b :* 1
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