5.: / u r

V

ABSTRACT

THE DEVELOPMENT OF REPRODUCTIVE STRUCTURES IN SELECTED
SPECIES OF MUCORALES

By

James Leslie Hiser

Twelve representatives of the Mucorales were selected for analyses
of the reproductive structure. The cultures were selected so as to re-
present each of the major types of asexual reproduction present in the
class Mucorales. Those chosen to represent the columellate sporangia

type of reproduction were: Zygorhynchus moelleri Vuillemin, Circinella

 

umbellata van Tiegham et LeMonnier, flgggg ramannianus Moeller, figsjgjg_
coerulea Bainier and Absidia glauca Hagem, Thamnidium elegans Link was
selected because it possesses both a terminal columellate sporangia and
sub-terminal sporangioles. Representatives of monosporic sporangiola
selected were: Cokeromyces pgitrasii Benjamin, Mycotypha microspora
Fenner, Mycotypha africana Novak and Backus and Cunninghamella
echinulata (Thaxter) Thaxter. Syncephalastrum racemosum Cohn ex.
Schroter was chosen to represent the merosporangiferous Mucorales.
Zygosporogenesis was also studied in Zygorhynchus moelleri, Circinella
umbellata, Absidia Llauca, A. coerulea and 5. £939.21 Lendt)Lendner.
This study was the first scanning electron microscope (SEM) pre-
sentation of comparative developmental morphology of asexual reproductive

propagules in the Mucorales. Significant findings included:

James Leslie Hiser

l) The presence of rugost hyphal apices prior to the development
of sporangia in Circinella umbellata.

2) The presence of a relatively thin transparent membrane in
Circinella umbellata which allowed observations of spore development.

3) Observations of spines on the outer surface of sporangia and
sporangioles and an explanation of their possible function.

4) Observations of sporangiospore development and formation of
polyhedral patterns on sporangia prior to spore dispersal in Muggg
ramannianus.

5) Comparison of freeze-etch and SEM photomicrographs of spines
of Mucor ramannianus, Syncephalastrum racemosum and Mycotypha micro-
5295;.

6) Proposal of Cokeromyces poitrasii as an intermediate stage in
monosporic sporangioles.

7) Support by SEM studies of the current definition of monosporic
sporangiola in Mycotypha and Cunninghamella with observations of
development of sporangiola in both genera.

8) Zygosporogenesis in Zygochynchus moellerii was observed for the
first time in SEM. The development of septal and cell wall formation
was studied.

Comparative morphological studies were made of zygospores in Absidia.

glauca, A, coerulea. and A, ramaso as viewed in the SEM.

I l I ll, | E II l1 ’1']? It'll .u’llll [I i.l‘|'\ l:l\'l\[' .‘II‘II ,’ \II \II' I, .l I ll

THE DEVELOPMENT OF REPRODUCTIVE STRUCTURES IN
SELECTED SPECIES OF MUCORALES

By

James Leslie Hiser

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1976

To Bill
Whose presence brought warmth
Whose absence now brings sorrow
but also inspiration.

I will never forget.

ii

 

lllll‘l'l'fl‘llll- .".II\I| illl'rll.ll}' ’I,‘

ACKNOWLEDGMENTS

I wish to express thanks to my major professor Dr. E. S. Beneke'
for his assistance throughout this study, and to Dr. E. J. K105 and
Dr. S. E. Bromley for their helpful criticism of this work.

Special appreciation is extended to Dr. G. R. Hooper for the many
hours of patience, understanding and guidance he provided throughout
this research. Without the influence of Dr. Hooper I would of found
it difficult indeed to complete this work.

Thanks is due to Dr. P. Volz for his kindness and the many
valuable experiences he made possible during my graduate career.

I would also like to thank Dr. Stan Fleger and Dr. Kerry O'Donnell
for their technical assistance and friendship. Sincere appreciation is
extended to Karen Baker for her help in the preparation of this thesis.
The experiences, mycological and otherwise shared with these three
individuals will long be remembered.

The encouragement and material assistance of my parents Mr. and
Mrs. John Hiser helped me through some very difficult times. Their
presence has and will always be deeply appreciated.

Grateful recognition is extended to Mrs. Phyllis Robertson for
the many hours of agony she must of endured in the typing of this thesis.
Her patience and humor inspired completion of this work.

I will always remember the conversations. celebrations and

bacchanalia of my friends Gary Mills, Joe Vargas and Jim Percich.

iii

Without their influence I may have finished years earlier. But in

/

the end "I did it my way".

iv

 

 

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

Page
LIST OF PLATES AND FIGURES ..................................... vii
INTRODUCTION ................................................... l
LITERATURE REVIEW ..... .... ..... . ............................... 2
A. Asexual Stages of Development .......................... 3
8. Sexual Stages of Development ........................... 5
C. Freeze-Etching .. ....................................... 5
MATERIALS AND METHODS .......................................... 7
. Source of Cultures ................... .. ............... 7
. Media and Culture Conditions ........................... 7
. Sporangia, sporangioles, monosporic sporangiola, and
zygospore ontogeny: Scanning electron microscopy ... 8
D. Sporangia, sporangioles, monosporic sporangioles
Surface structure: Freeze-etching .................. 8
E. Chart - Asexual structures studied ..................... lO
RESULTS ......... ....... ........................................ ll
A. Asexual Stages of Development
I. Columellate Sporangial Development
1. Circinella umbellata ......................... ll
2. Zygorhynchus moelleri ... ..... . ............... l2
3. Mucor ramannianus ...... ......... . ............ l3
~4. Absidia §p_. ..... ..... ....................... 14
II. Columellate Sporangia and Sporangioles
Thamnidium elegans . ..... . ..................... l5
III. Monosporic Sporangiola Development
1. Cokeromyces poitrasii ................ ........ l6
2. Mycotypha africana and M. microspora ......... l7
3. Cunninghamella'echinulata .................... 18
IV. Merosporangiférous MucoraTes
Syncephalastrum racemosum .... ................... 19
B. Sexual Stages of Development
1. Z orh nchus moelleri ...... ....... ........... ... 20
2. Absidia a_p, ........ ........... . ................. 21

DISCUSSION ................. ................. . ................. 82
A. Asexual Stages of Development ........................ ' 82

l. Columellate Sporangia Development .............. 83

2. Columellate Sporangia and Sporangioles ......... 89

3. Monosporic Sporangiola ..... .... ................ 9O

4. Merosporangiferous Mucorales .... ............... 93

8. Sexual Stages of Development .......................... 94
BIBLIOGRAPHY ...... ............................................ 97

vi

LIST OF PLATES AND FIGURES
Page

Figs. l-l2. Circinella umbellata. Scanning electron micrographs
offsporangial—development.

Plate 1 (Figs. l-4). Early stages of sporangial development .. 23

Plate 2 (Figs. 5-8). Unwinding of terminal sporangium and
development of ridges on sporangial wall .... ....... . ..... 25

Plate 3 (Figs. 9-12). Release of sporangiospores ............. 27

Figs. l3-20. Zygorhynchus moelleri. Scanning electron
micrographs of sporangial development

Plate 4 (Figs. l3-l6). Early sporangial development .......... 29

Plate 5 (Figs. 17-20). Sloughing of primary wall, breaking of
spines and release of spores ........... ....... ..... . ..... 3l

Figs. 2l-32. Mucor ramannianus. Scanning electron
micrographs of sporangial development

Plate 6 (Figs. 21-24). Early sporangial development . ......... 33

Plate 7 (Figs. 25-28). Breaking of sporangial wall and release
of spores. Freeze-etched sporangiospores showing
polyhedral pattern ...... ..... ........ ...... .............. 35

Plate 8 (Figs. 29-32). Sporangiospores adhering to columella
and freeze-etched spores showing spore wall and plasma
membrane .............................. ....... ............ 37

Figs. 33-42. Absidia glauca and A. coerulea. Scanning electron
micrographs of sporangia development.

Plate 9 (Figs. 133-36). Early sporangial development in
Absidia glauca ....... ........ . ..... .. ..... ... ............ 39

Plate 10 (Figs. 37-40). Release of spores in Absidia glauca
and A, coerulea ... ........ . ........ .... ..... .. ..... . ..... 41

Plate ll (Figs. 41-42). Columella of Absidia coerulea and
A, glauca .............. ....... ..... ........ .............. 43

vii

Page

Figs. 43-50. Thamnidium elegans. Scanning electron
micrographs of sporangia and sporangiolar

 

development

Figs. 51-54. Thamnidium elegans. Freeze-etch micrographs of
sporangioles

Plate 12 (Figs. 43-46). Early sporangial development ......... 45

Plate 13 (Figs. 47- 50). Sporangiolar development and spore
release ................................................. 47

Plate 14 (Figs. 5l-54). Freeze-etch micrographs of
sporangioles ..... .... .................................... 49

Figs. 55-60. Cokeromyces poitrasii. Scanning electron micro-
graphs of sporangiole development

Plate l5 (Figs. 55-58). Early stages of sporangiolar
development ........... ........... .... .................... 51

Plate 16 (Figs. 59-60). Late sporangiolar development ... ..... 53

Figs. 6l-74. Mycotypha africana and M. microspora. Scanning
electron micrographs of monosporic sporangiolar

development
Plate 17 (Figs. 61-74). Early sporangiolar development ....... 55
Plate 18 (Figs. 65-68). Development of primary and secondary
sporangioles .......... ...... .......... ................... 57
Plate 19 (Figs. 69-72). Release of sporangioles.. ............. 59

Plate 20 (Figs. 73-76). Sterigmata after spore release,
zygospores and freeze-etched micrograph of spherical
spore ......................................... ........... 61

Plate 21 (Figs. 77-78). Freeze-etched micrograph of spherical
and oval spores of M. microspora ....... . ................. 63

Figs. 79-86. Cunninghamella echinulata. Scanning electron
micrographs of monosporic sporangiolar development

Plate 22 (Figs. 79-82). Early sproangiolar development ....... 65

Plage 23 (Figs. 83-86). Abnormal vesicular formation and .
spiked sporangioles . ..... ...... ..... . ..... . .............. 67

viii

Page

Figs. 87-95. Syncephalastrum racemosum. Scanning electron
micrographs of merosporangia development

Plate 24 (Figs. 87-90). Early development of merosporangia
and development of transverse septa to delimit spores .... 69

Plate 25 (Figs. 9l-94). Delimiting of spores in merosporangia
and freeze-etch micrographs of spores ...... .... ....... ... 71

Plate 26 (Fig. 95). Freeze-etch micrograph of spore .......... 73

Figs. 96-103. Zygorhynchus moelleri. Scanning electron micro-
graphs of zygosporogenesis

Plate 27 (Figs. 96-99). Early stages of lateral hyphae
development, fusion of gametangia and formation of
zygospore ................................ ........ . ....... 75

Plate 28 (Figs. l00-l03). Disintegration of primary zygospore
wall and appearance of spiked secondary zygospore wall ... 77

Figs. l04-lO7. Absidia s2, Scanning electron micrographs of
zygosporogenesis

Plate 29 (Figs. l04-l07). Contact of opposed zygophoric hyphae
and development of zygospore in A, ramosa ................ 79

Plate 30 (Figs. l08-lll). Appendage formation and appearance'
of tertiary zygospore wall in A, glauca and A, coerulea .. 81

ix

INTRODUCTION

The development of asexual propagules in the Mucorales has inter-
ested mycologists for years. Although a number of studies have been
carried out concerning the germination of these asexual spores few
studies have emphasized the comparative development of the reproductive
structures. The aim of this study was to investigate and compare the
development of the three basic type of asexual structures. columellate
sporangia, sporangioles and merosporangia. found in the Mucorales.
Development of the sexual stages that were considered influential to

taxonomic criteria were also studied.

LITERATURE REVIEW

Asexual reproduction in the Mucorales involves three major types:
l) columellate sporangia, 2) multispored and monosporic sporangioles,
and 3) merosporangia. Detailed morphology of these asexual reproductive
structures has been of interest to mycologists for many years. Initial
studies utilized light microscopy primarily as an aid to fungal taxonomy
(Christenberry, 1940). Further light microscopy experiments examined
asexual structural relationships in relation to spore dispersal (Ingold,
l965,l963). Many transmission electron microscopy (TEM) studies in-
volving mucoralean spores have been reported (Barnicki-Garcia §t_gl,,
l968; Bracker, l968.l97l; Dykstra, 1974; Hawker gt_gl,, l970; Khan,
l975;Kawakami, l956;l<awakami g_t_gl., 1956; Young l973,l97l,l969,l968a,
l968b) but few of these studies have attempted to relatethe structure
of these asexual propagules with their functions. Little is also re-
ported concerning their ontogeny.

Spore surface structure is an important morphological character-
istic that is used to distinguish and compare related species of fungi.
Recently the scanning electron microscope (SEM) has been used to
examine these surface features as well as other diagnostic character-
istics of fungi (Bland and Couch, l973: Cole, l973a; Ellis gt_al,, 1970;
Fennell gt_§1,. l974; 0'Donnellgt_al,, 1976). These investigators
have shown that the SEM is a valuable addition to the existing

approaches utilized in morphological and taxonomic studies of fungi.

A. Asexual Stages of Development

1. Columellate Sporangia

Alterations in cell wall morphogenesis of members of this group
have been investigated (Bartnicki-Garcia and Nickerson, 1962; Bartnicki-
Garcia and Reyers, 1964). The formation of a new vegetative cell wall
during spore germination, unique to this group of fungi, was first
described by Bartnicki-Garcia gt_al, in 1968 (1968). Ontogeny of
sporangia fbrmation was first described by Dobbs (1939) and further
studied by Ingold and Zoberi (1963) who described the relationships of
asexual structures to spore dispersal. Although a survey of the sur-
face fine structure of sporangiospores has provided additional support
for taxonomic arrangements of this order (Young, 1968a), to date there
have been no studies concerned with SEN sporangiospore ontogeny.
Bracker's (1968) TEM paper described cleavage patterns associated with

sporangiospore formation in Gilbertella persicaria (Eddy) Hesseltine

 

and a monograph of sporangium ontogeny was presented by Hesseltine and
Ellis (1973).

2. Multi- and Monsporic Sporangioles

Light microscopic studies of sporangioles in the Mucorales have
been made by many authors (Ellis and Hesseltine, 1974; Embree, 1959:
Hesseltine and Benjamin, 1957; Lythgoe, 1958, Upadhyay, 1973). These
authors presented detailed morphological descriptions of a number of
taxa to help in taxonomic placement of the several genera studied.
Transmission electron microscopy of sporangiolar development has shown
many interesting developmental features. Kawakami g§_al, (1956) used
profile photographs to illustrate sporangial spores of Choanephora.
Dykstra (1974) attempted to differentiate between conidia and

 

 

IIIVI1I’III‘IIII

4

\

sporangioles by presenting descriptions and revising definitions of
these asexual propagules based upon differences in their development.
Carbon replicas were used to study profile and find new taxonomic
criteria for the Mucorales (Young, l968a). Ingold and Zoberi (1963)
utilizing many genera of the sporangiolar type studied asexual develop-
ment in relation to spore dispersal.

The evolution of the monosporic sporangiolar (conidial) condition
from the sporangiolar and possibly the merosporangial condition is
generally accepted by mycologists. There is however still much con-
fusion as to whether the asexual stage of Mycotypha and Cunninghamella
should be called conidial or monosporous sporangiolar. Many ultra-
structural studies have attempted to resolve this question (Dykstra,
1974; Grove, 1975; Hawker, gt 31,, 1970; Ingold and Zoberi, 1963; Khan,
1975; Young, 1973,1969,1968a). A recent paper (Khan and Talbert, 1975)
dealing with the origin of the sporangiospores rather than structure of
mature germinating propagules appears to present the most conclusive
evidence Supporting a monosporous sporangiolar structure.

Scanning electron microscopy of surface structure has emphasized
ornamentation of the sporangiolar walls (Hawker gt,gl,, 1970; Jones 33_
31,, 1976). Ornamentation as viewed in the SEM has been proposed to
have taxonomic value but future studies will be needed to verify these
findings.

In this area as in previously noted circumstances few SEM studies
of sporangiolar ontogeny and structure have been carred out.

3. Merosporangiferous Mucorales

The merosporangiferous Mucorales have been thoroughly described

and classified (Benjamin, 1966,1959). Illustrations of merosporangial

5
structure and descriptions of dispersal mechanisms clearly separate the
different asexual structures (Ingold and Zoberi, 1963). Young (l968b)
utilizing transmission electron and phase contrast microscopy described
surface structure and spore liberation in Kickxellaceae, although illus-
trationsof spore liberation were not presented. Germinating spores of
merosporangia of Linderina pennispora were also studied using carbon

replicas (Young, 1971).

8. Sexual Stages of Development

Description of cytoplasmic ultrastructure as well as wall and sur-
face structure of zygospores of several mucoraceous species have been
reported (Hawker and Gooday, 1968,1967; Hawker and Beckett, 1971;
Hesseltine and Anderson, 1956; Hocking, 1967; Ling-Long, 1930; Schripper
gt_gl,, 1975; Sutter, 1976). Hawker and Beckett (1971) in one of the
most complete ultrastructural analyses illustrated surface features as
well as wall structure details. O'Donnellgt_al, showed the value of
utilizing TEM, SEM and cryofacturing in his study of zygosporogenesis in
Ehycomyces blakesleeanus (1976).

C. Freeze-Etching_

 

Replica techniques using fungal organisms have been beneficial in
some cases already described (Young, 1971,1968a,1968b,1968c). Following
the development of a reliable freeze-etch apparatus (Moor gt al,, 1961;
.Moor and Muhlethaler, 1963) ultrastructural studies of fungi were
possible. Studies by Sassen £3 91, (1967) showed that the cell wall of
Penicillium megaspgrum consisted of several layers and that the surface

of the spore is covered by 'rodlets'. Ultrastructural investigations of

6

dormant and germinating sporangiospores of Rhizopus arrhizus showed the
presence of secretory vesicles at germ tube apices (Hess and Weber, 1973).
The taxonomic value of "rodlet" patterns on the outer layer of conidia
of Penicillum sp. was discussed by Hess gt_al, (1968). The freeze-etch
technique has provided a means to investigate fine structure of fungal
spores which cannot be resolved with the SEM. The freeze-etching

technique when associated with SEM studies has become a valuable

research tool.

 

 

"" II". 'III

MATERIALS AND METHODS

A. Source of Cultures
Twelve species of Mucorales were used in this study. Cunning-
hamella echinulata (Thaxter) Thaxter strain F-402, Zygorhynchus

 

moelleri Vuillemin strain F-406, Circinella umbellata van Tiegham et

LeMonnier strain F-403, Mucor ramannianus Moeller strain F-421,

 

Thamnidium elegans Link strain F-410, Absidia coerulea Bainier strain
F-416 and Cokeromyces poitrasii Benjamin strain F-411 were obtained
from the collection of W. G. Fields, Michigan State University.

Mycotypha microspora Fenner strain NRRL 684, Mycotypha africana Novak

 

and Backus strain NRRL 2978, Absidia glauca Hagem strain NRRL 1324,
Absidia ramosa (Lendt) Lendner strain NRRL 3180 and Syncephalastrum

 

racemosum Cohn ex Schroeter strain NRRL 9424 were received from the

Northern Regional Research Laboratory, Peoria, Illinois.

8. Media and Culture Conditions

All cultures except Mycotypha africana were grown on B agar medium
composed of 179 corn meal, 2 g dextrose, 39 sucrose and 19 yeast extract
per liter of distilled water. All cultures were innoculated with either
spores or hymeniael and were grown incontinuous fl uo‘rescent light at 25
2 1 C. Mycotypha africana was grown on yeast extract agar and incubated

at 15 C undercontinuous light.

3

C. Sporangia, sporangioles,_monosporic sporangiola, and zygosoore

 

ontogeny: Scanning Electron Microscopy,

Agar squares (3.5 mm) bearing various developmental stages of
sporangia, sporangioles, monosporic sporangiola and zygospores were
fixed in 0.2 M phosphate-buffered 3% glutaraldehyde at pH 7.2 for 2 hrs,
washed in phosphate buffer 15 min, postfixed in 2% phosphate buffered,
0504 2-4 hrs, and buffer washed 15 min. All previous steps were
carried out at 25 i 1 C. The buffer-washed material was dehydrated with
ethanol at 0-4 C using the series: 10,30,50,70,90% ethanol, 10 min each;
100% ethanol twice, 15 min each.

The ethanol dehydrated material was either stored overnight in
ethanol at 0-4 C or transferred directly to the critical-point dryer.
The specimens were dryed in a Bomar-900 critical-point dryer (Bomar Co.,
Tacoma, Wa.) using C02 as the carrier gas. Critical-point dried speci-
mens-were mounted on stubs with double-stick Scotch Brand Tape. The
stub surrounding the specimen was then painted with Television Tube Koat
(GC Electronics) to prevent charging. Specimens were sputter coated
with 20-30 nm of gold using an EMS-41 mini-coater (Film Va. Inc.,
Eaglewood, N.J.) and viewed in an 151 Supermini scanning electron
microscope. Photographs were obtained using Polaroid Type 105 positive-
negative film.

D. Sporangia, sporangioles, monosporic sporangioles surface structure:
Freeze-Etching,
Petri plates bearing spores on agar surfaces were washed with
distilled water and agitated to release spores. Spores were removed

from hyphae by glass-wool filtration and the spores were centrifuged to

9

ferm.a pellet. Water was drained leaving a dense spore mat. A small
portion of these spores were then mounted on a freeze-etch stub and
quickly frozen in partially solidified Freon 22 (cooled with liquid
nitrogen). No glycerol or other cryoprotective agent was used.

Freeze-etching was carried out on a Balzers 360 M apparatus. The
specimens were shadowed with Carbon/Platinum for 7 seconds at 45? and
with carbon for 10 seconds at 180°. Samples were immediately removed
and the replicas floated off onto distilled water. Replicas were clean-
ed 1 hr in 70% HZSO4 followed by 3 distilled water rinses (5 min each),
placed in Chlorox bleach 2-3 hr followed by 3 additional distilled
water rinses (10 min each) and finally picked up on 400 mesh, formvar-
coated grids. Specimens were examined and photographed with a Philips
300 transmission electron microscope.

Chart I lists the different species studied and what type of

asexual reproductive structure they possessed.

10

E. CHART I. Asexual structures studied.

 

 

 

 

 

 

 

Organism Family . Asexual Structure
zygorhynchus moelleri Mucoraceae columellate sporangia
Circinella umbellata Mucoraceae columellate sporangia
Mugor ramannianus Mucoraceae columellate sporangia
Absidia goerglga Mucoraceae columellate sporangia
Absidia glauca Mucoraceae columellate sporangia
Thamnidium elegans Thamnidiaceae columellate sporangia

and sporangioles
Cokeromyces poitrasii Thamnidiaceae sporangioles
Mycotypha africana Cunninghamellaceae monosporic sporangioles
Mycotypha microspora Cunninghamellaceae monosporic sporangioles
Cunninghamella echinulata Cunninghamellaceae monosporic sporangioles
Syngeohalastrgm racemosum Syncephalastraceae merosporangia

 

RESULTS

A. Asexual Stages of Development
I. Columellate Sporangial Development
Reproduction involving columellate sporangia was studied in five

species, Circinella umbellata, Zygorhynchus moelleri, Mucor ramannianus,

 

Absidia glauca, and Absidia coerulea.
1. Circinella umbellata van Tiegham et Le Monnier, Ann. Sci. Nat.
5-17:298, 1973.

This species produced aerial sporangiosphores from hyaline mycelium
after 24 hrs incubation (Fig. l). Rugosity of hyphal apices was sub-
sequently observed on several different stages (Fig. 2, 3, 4). It
was not apparent whether rugosity had occurred prior to the inward curv-
ing of the sporangiophore (Fig. 2) or whether it was the initial step
in sporangial development prior to the observed unwinding of the
sheathed sporangiophore Fig. 3). The apex was observed to enlarge
while still attached to the membrane sheath hence there was some indi-
cation that the first explanation was most correct.

As the sporangiophore elongated the membrane sheath tore and the
sporangium enlarged (Fig. 4). Remnants of the membrane sheath were
observed on sporangia and sporangiophores at several stages of develop-
ment (Fig. 5, 6). Sporangia were observed at differing stages of
maturity at the time they were released from the sporangiophore (Fig.

6, 7). The sporangia continued to enlarge to a mean diameter of 60 um.

11

12
As the sporangia enlarged characteristic ridges were observed on the
surface of the sporangial walls (Fig. 8). At maturity the sporangial
wall became very thin and essentially transparent in the SEM (Fig. 9).
The ridges previously observed were then seen to be spores pressed
tightly to the inside of the wall (Fig. 9).

Dehiscence of the sporangial wall was observed to occur along a
radial rupture leading from the apex of the sporangium to the base of
the columella (Figs. 10, 11). At the time of sporangial breakage
globose to ovoid sporangiospores (4.0-7.0 u in diam) were observed still
adhering to remnants of the sporangial wall (Fig. 11). Sporangiospores,
as observed in the SEM were free of any internal mucilage material and
had a slightly roughened appearance. After release of the sporangio-
spores a diagnostic collar remained attached to the columella (Fig. 12).
Indentations, caused by displaced sporangiospores were observed on the
collumella (Fig. 11). Mature columella measured g3, 25-35 u x 40-60 u.

2. Zygorhynchus moelleri. Vuillemin, Bull. Soc. Mycol. Fr. 19:

116, 1903.

In this species young, globose to ovoid sporangia were borne on
erect mycelium (Fig. 13). Although these sporangia had a fine
echinulated appearance when young, the columella and sporangiophore
remained spikeless (Fig. 14). As the sporangia matured the spines in-
creased in number and size. Their density varied considerably among
different sporangia (Figs. 14, 16). At maturity the spines were pointed
and angular and 93, 1.5-3.0 u in length. Near maturity the outer
sporangia wall was sloughed off (Fig. 17) and accompanied by breaking
of the spines at their widened bases (Fig. 15). These fractures resulted

in irregular abrasions in the sporangial wall. Occasionally, the wall

 

Illilllllll'

 

13

was observed to break exposing a matted interior (Fig. 18), but most
often the wall was persistent with disintegration occurring along
channels initiated by holes formed by the broken spikes (Fig. 19). After
spore dispersal, the mature sporangia (1.5-4.0 u) often contained
irregularly shaped sporangiospores adhering to an applanate columella
(Fig. 20).

3. Mucor ramannianus Moeller, Ztschr. f. Forst. u Jagda 35:

330, 1903.

Swellings of aerial sporangiophores (less than 4 u in length) were
the first indications of sporangial development in this fungus (Fig.
21). At first, the spherical prosporangium appeared smooth to finely
corrugated but ridge-like patterns soon developed on the outer sporangial
wall (Fig. 22). Sporangia did not increase appreciably in size during
this ridge formation and mature sporangia measured 3;, 20 u in diameter.
As the sporangia developed the ridges delimited hexagonal and pentagonal
structures on the sporangial wall (Fig. 23). A circular ridge was formed
at the base of the sporangium to delimit the columella (Fig. 23). At
each stage of development the ridges remained relatively uniform in
height. Dehiscence of the sporangial wall followed the definite pattern
of ridges beginning at the dorsal end of the sporangium (Fig. 24). The
spores were released in spore drops which appeared as clumps in the SEM
(Figs. 28, 29), but dissemination was not initiated until dehiscence
or breakup of the sporangial wall was complete (Fig. 25). Remnants of
the outer sporangial wall that remained attached to spores at the base
of the columellae indicated that longitudinal delimitation of spores
occurred (Fig. 25). The polyhedral ornamentation of Spores allowed them
to fit tightly together, efficiently utilizing available space (Figs.

14

28, 29). Freeze-etch micrographs of polyhedral sporangiospores
demonstrated the presence of fine granules on the spore wall (Fig. 26,
27). The outer cell wall when viewed in transverse section exhibited a
granular hexagonal configuration (Fig. 30). The ridge patterns of
spores was shown to be continuous with this outer spore wall (Fig. 31).
Plasma membrane surfaces were composed of randomly distributed invagina-
tions (Fig. 32).

4. Absidia glauca Hagenn, Skrifter u.a. Vidensek-Selsk. Christ.

1. Math. Nat. Kl. No. 7, p. 43, 1908.
Absidia coerulea Bainier, Bull. Soc. Botan. France 36: 184,
1889.

The developmentalstages of Absidia glauca and A. coerul ea were
similar. The pyriform sporangia observed in A, glgggg were also observ-
ed in A, coerulea (Figs. 33, 38). In both species the delimitation of
a columella from the sporangium followed the establishment of the
sporangial shape. Breaking of the primary sporangial wall was initiated
near the base of the sporangium with the wall being sloughed dorsally
Fig. 34). The development of apophyses accompanied sporangial matura-
tion (Fig. 36). Rhizoids, characteristic of Absigig_species, were
located along glaucous stolons between sporangia (Fig. 35). Spores
were observed held together by an internal mucilage material prior to
dissemination (Fig. 38). This adhering property resulted in the spores

being released in drops similar to those observed in Mucor ramaniannus.

 

A distinctive feature observed during sporangial ontogeny in Absidia
glauca and A, coerulea was the unique shape of the columella. In A,
coerulea the columella often possessed papillate apical projections

1.5-3.0 u long (Fig. 41) while the columella of A, glauca possessed_

15

relatively short projections 1.0-2.0 u long (Fig. 42). In both
species a tranverse system was produced 10-15 u below the apophyses
(Fig. 42). Although many spores are released during spore dispersal
characteristically a few remained adhering to the columella (Figs. 39,
40).
II. Columellate Sporangia and Sporangioles

Thamnidium elegans Link, Berliner Mag. d. Naturf. Freunde 3:

1809.

Two types of asexual spores are found on one sporangiophore in
Thamnidium elegans. During early stages of development the larger
terminal sporangium (gg, 80 u in diameter) exhibited an outer layer of
spines similar to other mucoraceous fungi studied (Figs. 43, 44). The
spines were £3, 1 um long x 0.5 um wide, angular and blunt ended. In
young sporangia a thick, granular, spine covered sporangial covering
was observed over immature sporangiospores.(Fig. 43). As the sporangia
matured this wall decreased in thickness allowing observation of de-
limited spores within (Fig. 44). As the wall became thinner, the spines
broke at their bases (Fig. 45). The columella was covered with spines
prior to removal of the sporangial wall (Fig. 44) and was devoid of
spines after the wall was removed (Fig. 46). Remnants of the sporangial
wall and spines masked the sporangiospores to give them a false spikey
appearance (Fig. 46).

The second type of asexual spore observed in Thamnidium elegans were
sporangioles. These sporangioles possessed dehiscent walls which upon
breaking exposed groups of spores enclosed in a thin membrane (Fig. 47).
After removal of the secondary sporangiolar wall, groups of from 4 to 15

spores were observed bound together (Figs. 48, 49, 50). These deciduous

16

spores detached together at maturity and occasionally empty sporangiolar
encasements were observed attached to the sporangiophore. Also visible
after spore dispersal were reduced sporangiolar columella (Fig. 50).

The wall of Thamnidium elegans when examined using freeze-etch
techniques can be divided into several layers. The surface of I, glgggg§_
spores were covered with many short spikes (Fig. 51). In cross-fractured
spores the plasma membrane consisted of many depressions (92, 50 nm)
(Fig. 52). The cell wall was composed of three layers (Fig. 53). The
W] layer consisted of rough granular material and was the smallest layer
present. The W2 layer was thicker than W1 but similar in structure. The
W3 layer was the thickest and separated the W2 layer from the plasma
membrane. Definite thread-like cytoplasmic structures were obserVed
connecting W3 with the plasma membrane (Fig. 53). Fractured cells
revealed typical cytoplasmic features (Fig. 54); nuclei with nuclear
pores (N), vacuoles with dense globular bodies of metachromatin (v),
mitochondria (m), golgi bodies (9) and lipid granules (L).

III. Monosporic Sporangiola Development

1. Cokeromyces poitrasii Benjamin, R. K. Aliso 4(3): 523-530,

1959.

The deciduous sporangioles in Cokeromyces poitrasii arose from
spherical (occasionally elliptical) swollen apices borne at the terminal
end of simple sporangiospores (Figs. 55, 56). As the apices reached
their maximum diameter small sterigmata proturberances were formed over
the entire apex surface (Fig. 56). The onset of sporangiolar formation
was marked by swelling of the terminal ends of the elongated sterigmata
(Fig. 57). The elongation of sterigmata continued with oval to
elliptical sporangiospores being formed 3-7 u long x 1.5-3.0 u wide

17

(Fig. 58). Recurving of these fertile branches usually occurred after
full development of sporangioles (Figs. 59, 60). This recurving seems
to be caused by the added weight of the swollen sporangiole. A great
variation was observed in the length of branches with some reaching a
length of 20 u. The sporangioles were released from the swollen apices
by breaking of the sterigmata at their apical bases (Fig. 60). After
the sterigmata and spores were released, fractured sterigmata bases
remained attached to swollen apical surfaces. These sterigmata were
outlined by interconnecting hexagonal ridges which formed on the swollen
apical wall at maturity (Fig. 59).

2. Aycotygha africana Novak and Backus, Mycologia 55: 793, 1963.

Mycotypha microspora Fenner, Mycologia 24: 196, 1932.

The early stages of vesicular (ampullae) development was similar
for both Aycotypha species studied. Aerial hyphae arose from vegetative
mycelium after 36 hrs. in Mycotypha africana and after 48 hrs. in AL
microspora (Fig. 61). The first signs of ampullae development were a
darkening and slight swellings of the terminal hyphal apex (Fig. 62).
Persistent sterigmata, diagonally arranged 1-1 1/2 u apart, soon
appeared in the external surface on the ampullae (Fig. 63, 64). These
primary sterigmata continued to elongate and developed over the entire
ampullae surface except for a zone at the apex which was devoid of any
sterigmata growth (Fig. 65). A second sterigmata then developed between
the primary sterigmata. This resulted in an alternating pattern of
primary and secondary sterigmata surrounding the ampullae (Fig. 66).
This type of dimorphism was observed in both species. At the time of
sporangiolar development the secondary sterigmata were not completely

developed (Fig. 67).

18
The mature spores of Aycotypha africana and A, microspora resembled

 

each other. Although more variation existed in the shape of A,
microspora spores, both species exhibited oval and spherical sporangiola
(Figs. 68, 70). After spore dispersal prominent sterigmata stalks were
observed on spores of both species (Figs. 69, 71).

Although previous studies reported circular scar zones at the base
of spores in Mycotypha africana (Young, 1968) no such scars were observed
in this tudy. In many mature spores the terminal end of the ampullae
were ruptured exposing the internal structure. Aperatures of two sizes
were observed on the inner surface of the ruptured vesicle (Fig. 72).
The sporangioles of A, microspora were liberated individually, while
those of A, africana were liberated in groups of 2 or 3 (Fig. 69, 71).
Although atypical ampullae formation was occasionally observed in A,
microspora (Fig. 74), observations of sporangiola formation indicated
the development processes were the same as normal sporophores.

Freeze-etch micrographs of oval and spherical spores showed the
stalk-like projections that remained after pedicel breakage (Figs. 76,
78). Although spore surfaces appeared smooth in SEM photographs they
exhibited a definite interwoven rodlet pattern in freeze-etched micro-
graphs (Figs. 77, 78). The only species reported to produce zygospores
is A, africana. . This species possess spiked zygospores formed
between equal suspensers (Fig. 75).

2. Cunninghamella echinulata (Thaxter) Thaxter, Rhodora 5: 98,

1903.

Cymose to indefinite branching was observed in developing sporangio-

phores of Cunninghamella echinulata (Figs. 79, 81). The branched

sporangiophores terminated in spherical vesicles covered by short

19

denticels. The vesicles and sporangioles developed at different rates
on the same sporangiophore (Fig. 81). This pattern differed from that
of C, elegy; in which a terminal swollen apex developed sporangioles
slower than the subterminal whorl of vesicles (Fig. 80).

The monosporic sporangiola of Q, echinulata possessed tapering,
blunt end spines (gA_2 u in length) on the outer cell wall (Fig. 84).
These spines were embedded in hexagonal plates on the sporangiolar wall
(Fig. 85).

Upon germination the spores lost their spiny appearance becoming
smooth to slightly roughened (Fig. 86). Germination tubes were
observed at both apical and mid-lateral regions of spores. Abnormalities
found in this species included germination of young sporangioles while
still attached to the vesicle (Fig. 82) and development a double vesciles
(Fig. 83).

IV. Merosporangiferous Mucorales

Syncephalastrum racemosum Cohn ex Schroeter, Aliso L:327, 1959.

Young sporangiophores produced spherical apical enlargements with
merosporangial initials approximately 72 hrs. after germination of
spores (Fig. 87). The merosporangia radiated from the central enlarge-
ment with various arrangements evident (Figs. 88, 89).

The protoplasm of mature merosporangium was delimited by formation
of transverse septa (Fig. 90, 91). The number of spores delimited in
this manner varied from 3-10 spores per merosporangium. As the spores
were dispersed, usually in groups of 2-6 spores, remnants of the mero-
sporangial wall was observed on the spore walls (Fig. 90).

Although relatively smooth when viewed in the SEM a spiked surface

could be seen in freeze-etch preparations (Fig. 92, 93). Transverse

20

fractured cells showed the plasma membrane covered with depressions
(Fig. 94). A magnified view of the spore wall shows that it is made of
three layers. Cytoplasmic connections were observed between the

spore wall and the plasma membrane (Fig. 95).

8. Sexual Stages of Development

1. zygorhynchus moelleri

The zygophoric hyphae in Zygorhynchus were produced laterally from
erect vegetative mycelium (Fig. 96). As the main zygophoric hyphae
developed a second lateral zygophoric branch was produced near its base.
This new branch was then delimited by a septum from the parent filament
(Figs. 97, 98). As the lateral branches elongated they curved backward
to meet the main hyphal axis (Figs. 96, 97). At the point of contact
the main hyphal branch pushed out to delimit the gametangia (Fig. 98).
At this time the lateral suspensor initiated swelling indicating hetero-
gametangic copulation. Gametangia were eventually delimited from
suspensers by formation of transverse septa (Fig. 101).

As the zygospore matured the opposing suspensors were pushed apart
with subsequent tearing of the primary zygospore wall (Fig. 99).
Continued removal of this primary wall resulted in the appearance of a
secondary warty layer (Fig. 100). As zygospore growth continued the
primary layer was torn and sloughed off although remnants of this wall
remained attached to spines of mature zygospores (Fig. 102). The mature
zygospores displayed a characteristic appearance of the main hyphae

extending beyond the zygospore as a slender projection (Fig. 103).

21

2- Mm
Ap§1g13.coerulea
Absidia glauca

The initial stages of zygosporogenesis were similar for all species
studied. In each species, opposed hyphae of each compatabile strain
grew toward and contacted each other (Fig. 104). Soon after contact the
formation of atransverse wall delimited the gametangia (Fig. 104, 105).
In Absidia ramosa breakdown of the striated primary wall exposed a
relatively smooth layer (Fig. 105) which on growth developed a roughened
interwoven appearance (Fig. 106). The zygospore in this species was
borne between equal suspensors devoid of any appendages (Fig. 107).
The zygospore wall of A, glgggg_was similar in appearance to that of A,
r§Ap§§_(Fig. 108). In A, glAggg_enlargement of the zngSpore was
accompanied by initiation of appendage formation by abrasive swellings
on suspensor walls (Fig. 108). Appendages developed on one suspensor
only and were borne either singly or in pairs (Fig. 109). At maturity
the suspensors possessed long finger-like, curled or looped appendages
which covered the zygospores (Fig. 110). Although A, coerulea also
produced appendages on suspensors they were shorter, more rigid and
closer to the zygospore than those observed in A, gigggg_(Fig. 111).
As the zygospore expanded the coarse secondary wall separated exposing
the smooth layer of the zygospore wall (Figs. 110, 111).

Numerous mucoraceous type zygospores characterized by roughened
to course, warty projections suspended between equal and unequal
appendages were observed in Mycotypha africana (Fig. 75). Ontogeny of

zygospore fbrmation was not stadied in this species.

22

Figs. 1-12. Circinella umbellata. Scanning electron micrographs of
sporangialdevelopment.

Fig. 1. Aerial mycelium originating in mycelial mat (lOOOx).
Fig. 2. Rogust hyphal apex (lOOOx).

Fig. 3. Unwinding of sympodial sporangiophore illustrating rogust
apical area and membrane sheath (lOOOx).

Fig. 4. Unwinding of young sporganium. Rogust area still visible
(2000x).

23

 

Plate 1

24

Fig. 5. Unwinding of terminal sporangium (1600x).

Fig. 6. Enlargement of sporangium and further unwinding. Arrow
indicates depressions in sporangial wall (2000x).

Fig. 7. Separation of sporangium from sporangiophore (2000x).

Fig. 8. Characteristic ridged appearance of developing sporangium}

Arrow indicates remnants of membrane (1600x).

25

 

Plate 2

ll‘llll It, [II I'll! \I' I all, ‘r.‘|

26

Fig. 9. Transparent sporangial wall of mature sporangium.
Sporangiospore outline is visible. (2000x)

Fig. 10. Brgken sporangium with globose sporangiospores being released
l600x .

Fig. 11. Ruptured sporan ium, ovoid and globose sporangiospores and
indented columella (2000x).

Fig. 12. Columella with collar characteristic remaining after spore
dispersal (2000x).

 

Plate 3

i}il>l||llll’"|l|lllllll’l'.llllu[II['Ill 1|. ’1‘

28

Figs. 13-20. 2 orhn chus moelleri. Scanning electron micrographs
of sporanglal development.

Fig. 13. Young sporangia (2000x).

Fig. 14. Sporan ium with typical spines. Arrow indicates spineless
columella (1600x).

Fig. 15. Sporangial spikes breaking at widened bases and sloughing
primary wall (lOOOOx).

Fig. 16. Sporangium with spines (1600x).

29

 

Plate 4

30

Fig. 17. Sloughing of primary sporangial wa11(2000x).
Fig. 18. Breaking of secondary sporangial wall (2000x).

Fig. 19. Removal of persistent sporangial wall along patterns caused
by breaking of spines (3000x).

Fig. 20. Applanate columella with adhering sporangiospores (4000x).

31

 

Plate 5

32

Figs. 21-27. Mucor ramannianus. Scanning electron micrographs of

Fig.
Fig.

Fig.

Fig.

sporangial development.
21. Early sporangial development (3000x).

22. Formation of ridges on developing sporangium thought to be
caused by developing sporangiospores (3000x).

23. Development of geometric sporangial wall pattern. Arrow
indicates delimitation of columella (4000x).

24. Breaking of sporangial wall along geometric ridges (3000x).

33

 

Plate 6

Fig.

Fig.
Fig.
Fig.

34

25. Breaking of sporangial wall and dissemination of geometric
spores (3000x).

26. Polyhedral pattern of freeze-etched sporangiospores (25000x).
27. Polyhedral pattern of freeze-etched sporangiospores (25000x).

28. Aggregation of geometric sporangiospores on aerial
sporangiosphores (5000x) .

35

 

Plate 7

Fig.
Fig.

Fig.

Fig.

36

29. Sporangiospores adhering to columella (7000x).

30. Transversly sectioned freeze-etched sporangiospore illustrating
hexagonal pattern of spore wall (21000x).

31. Transversly sectioned freeze-etched sporangiospore (21000x).
Note how the ridged pattern of the spore is continuous with the
rest of the spore wall.

32. Plasma membrane of sporangiospore (25000x).

37

 

Plate 8

Figs. 33-42. Absidia glauca and A. coerulea. Scanning electron
micrographs of sporang a development.

Fig. 33. Breaking of sporangial wall in A. gla__u_c_a_ (lOOOx).

Fig. 34. Further sloughing of sporangial wall in A. 91253.3. (1600x).
Fig. 35. Rhizoids of A. gi_a_uc_a (700x).

Fig. 36. Sporangium of A. 9111153. Arrow indicates apophyses (2000x).

39

 

Plate 9

Fig.

Fig.

Fig.

Fig.

40

37. Globose sporangiospores attached to columella. Arrow
indicates remnants of sporangial wall. A. glauca (lOOOx).

38. Globose sporangiospores attached to columella.* Arrow
indicates internal mucilage material. A, coerulea (1600x).

39. Sporangiospores adhering to ovoid columella. A. glauca
(2000x).

40. Sporangiospores adhering to pyriform columella. A,

coerulea (lOOOx).

41

 

Plate 10

42

Fig. 41. Pyriform columella with projection. A, coerulea (3000x).

Fig. 42. Ovoid columella with projection.’ A, glauca (1600x).

43

 

Plate 11

44

Figs. 43-50. Thamnidium ele ans. Scanning electrong micrographs of
sporangia and sporangiolar development.

Figs. 50-53. Thamnidium elegans. Freeze-etch micrographs of
sporangioles.

Fig. 43. Young sporangiom (1600x). Note thickness of sporangial wall
and undeveloped sporangiospores.

Fig. 44. Breakage of thin sporangial wall (lOOOx).
Fig. 45. Spines of sporangial wall (lOOOOx).

Fig. 46. Remnants of broken sporangial wall on sporangiospores (700x).
Arrow indicates spikeless columella.

45

 

Plate 12

46

Fig. 47. Breaking of primary wall of sporangioles exposing multispored
sporangioles (400x).

Fig. 48. Enlargement of Fig. 47. Note course outer wall of spore
(7000x).

Fig. 49. Dichotomously branched sporangioles with spores visible (400x).

Fig. 50. Mature spores of sporangioles. Arrow indicates reduced
sporangiolar columella (700x).

47

 

Plate 13

Fig.

Fig.

Fig.

Fig.

48

51. Freeze-etch micrograph of spore surface. Note rough spiked
surface of outer cell wall (6900x).

52. Fractured cell with plasma membrane (PM) and spore wall (SW)
12500x .

53. Triple layered cell wall (W1, W2, W3) and plasma membrane
(PM) (21000x).

54. Germinating sporangiospore, and fractured cells with
organelles. Mitochondria (m), nucleus (n), vacuole (v) and
golgi comples (G) (8100x).

 

Plate 14

50

Figs. 55-61. Cokeromyces poitrasii. Scanning electron micrographs of
sporangiole development.

Fig. 55. Swollen apex of sporangiophore (2000x).
Fig. 56. Oval apex with sterigmata initials (2000x).

Fig. 57. Elongation of sterigmata and early sporangiole
development (2000x).

Fig. 58. Elliptical development of sporangioles (2000x).

51

 

Plate 15

52

Fig. 59. Late sporangiolar development. Arrow indicates hexagonal
pattern surrounding broken sterigmata (1600x).

Fig. 60. Sporangiolar dispersal (700x).

53

 

Plate 16

54

Figs. 61-74. Aycotypha afriCana and Aycotypha'microspora. Scanning
electron micrographs ofimonosporic sporangiolardevelopment.

Fig. 61. Development of aerial hyphae A. micrOSpora (400x).

 

Fig. 62. Swelling and darkening of aerial hyphae A. microspora (lOOOx).

 

Fig. 63. Protrusion of primary sterigmata A. microspora (lOOOx).

Fig. g4. Diggonal pattern of young primary sterigmata A. microspora
7000x .

55

 

Plate 17

56

Fig. 65. Early development of secondary steri mata. Note absence of
sterigmata at tip. A, microspora (2000x1

Fig. 66. Dimorphic helical pattern of sterigmata A, microspora (3000x).

 

Fig. 67. Early sporangiolar development A, microspora (3000x).

 

Fig. 68. Mature dimorphic sporangioles M. microspora (1600x).

57

 

Plate 18

Fig.

Fig.
Fig.

Fig.

58

69. Release of sporangioles allowing observation of dimorphic
sterigmata. A, microspora (4000x).

70. Release of sporangioles. ‘A, africana (5000x).

71. Release of sporangioles in groups of two or three. A,

africana (7000x).

72. Operculate-like opening of ampullae A, africana (3000x).

59

 

Plate 19

60

Fig. 73. Broken sterigmata after spore dispersal. A. africana (3000x).
Fig. 74. Atypical ampullae formation. A, microspora (lOOOx).
Fig. 75. Zygospores of A, africana (lOOOx).

Fig. 76. Freeze-etched spherical spore of A. microspora (21000x).
Note area of sterigmata breakage.

 

61

 

Plate 20

62

Fig. 77. Freeze-etched spherical spore of A. microspora (21000x).

Fig. 78. Freeze-etched oval spore of A. microspora (40000x). Note area
of sterigmata attachment.

63

 

Plate 21

64

Figs. 79-86. Cunninghamella sp. Scanning electron micrographs of
monosporic sporangiolar development.

Fig. 79. Cymose branched sporangiophores. g, echinulata (400x).
Fig. 80. Terminal and sub-terminal vesicles of o, elegans (400x).

Fig. 81. Sporangioles at different stages of development from same
sporangiophore. g, echinulata (400x). '

Fig. 82. Abnormal sporangiolar germination. Q, echinulata (2000x).

 

Plate 22

66

Fig. 83. Abnormal double vesical formation 9, echinulata (lOOOx).

 

Fig. 84. Spiney sporangioles of Q, echinulata (1600x).

Fig. 85. Enlargement of sporangiole. Note hexagonal plate area of
spine attachment (7000x).

Fig. 86. Germinating sporangioles.‘ Note abscence of spines (2000x).

67

 

Plate 23

Figs. 87-95. Syncephalastrum racemosum. Scanning electron micrographs
of merosporangia development.

Figs.(87. Swollen apex of sporangiophore with merosporangia initials
2000x .

Fig. 88. Developing merosporangia (1600x).
Fig. 89. Developing merosporangia (lOOOx).

Fig. 90. Transverse septa laid down to delimit spores in
merosporangia (800x).

 

 

Plate 24

70

Fig. 91. Delimited spores in merosporangia (2000x).
Fig. 92. Roughened nature of spore wall (21000x).
Fig. 93. Interwoven pattern of spore wall (25000x).
Fig. 94. Plasma membrane of spore (16000x).

71

 

Plate 25

72

Fig. 95. Spore wall (Sw) and plasma membrane (Pm) of spore. Note
three layers of Sw and cytoplasmic connections indicated by
arrow (40000x). '

 

73

 

P1ate 26

74

Figs. 96-103. Zygorgynchus moelleri. Scanning electron micrographs of
zygosporogenes s. .

Fig. 96. Formation of lateral zygosporic hyphae from vegetative
mycelium (lOOOx).

Fig. 97. Termination of lateral hyphae by septum (lOOOx).

Fig. 98. Delimitation of gametangium. Arrows indicate septal
, formation (1600x).

Fig. 99. Delimitation of large gametangium and disintegration of
primary zygosporic wall (2000x).

 

Plate 27

76

Fig. 100. Desintegration of primary zygospore wall. Arrows indicate

similar ornamentation of gametangium and zygospore walls (2000x).

Fig. 101. Further zygospore development. Arrows indicate transverse
septa delimiting gametangium (lOOOx).

Fig. 102. S iked zygospore with remnants of primary sporangial wall
(1600x).
Fig. 103. Mature zygospores showing characteristic appearance of main
?yphae)projecting beyond the zngSpore as a slender elongation
000x .

—-——"-—‘-—~——_ w—Iw‘

77

 

 

Plate 28

78

Figs. 104-111. Absidia sp. Scanning electron micrographs of

Fig.

Fig.

Fig.

Fig.

zygosporogenes1s.

104. Initial contact of opposed zygosporic hyphae. A, ramosa.
(lOOOx).

105. Disintegration of primary gametangial wall. A, ramosa.
(lOOOx).

106. Coarsel roughened zygospore between equal suspensors. A,
ramosa (700x).

107. Mature zygospore. ‘A, ramosa (700x).

79

 

P1ate 29

Fig.

Fig.
Fig.

Fig.

80

108. Developing zygospore. Note abrasive swellings (A) of
appendage fonmation on suspensor wall (5). A, galuca (700x).

109. Early appendage development. A, glauca (lOOOx).

110. Fin erlike appendages surrounding zygospore. Note tertiary
layer (T . ‘A, glauca (400x). .

111. Fingerlike appendages in A, coerulea. Arrow indicates
tertiary layer. (400x).

81

 

Plate 30

DISCUSSION

A. Asexual Stages of Development

The taxonomy of the fungi is based primarily on the morphology of
the various types of asexual structures. Current classification within
the Mucorales (Zycha, 1935; Hesseltine, 1955) is based almost entirely
on observations at the light microscopic level. A number of papers
have dealt with spore development and spore structure of selected
species of Mucorales (Christenberry, 1940; Hesseltine, 1955; Benjamin,
1959, 1966; Ingold, 1965, Zycha gt_gl,, 1969; Dykstra, 1974). The
transmission electron microscope has increased magnification over light
microscopy for further spore studies (Bartnicki-Garcia, 1962, 1968;
Bracker, 1968; Bracker gt_gl,, 1971; Fletcher, 1973a, 1973b; Grove, 1975).
With the development of SEM techniques a new interest was stimulated in
spore surface characteristics (Ellis g;_21,, 1970; Bland and Couch, 1973;
'Cole and Aldrich, 1971a, 1971b; Cole, 1973a, 1973b, 1973c, 1974; Grove,
1975). A greater depth of field of the SEM combined with its high degree
of resolution identifies new cellular details not previously visible
with the light microscope. The discovery of the critical point drying
technique (see Hayat and Zerkin, 1973) further increased the reliability
of SEM observations by reducing shrinkage and distortion of fragile
fungal tissues. When combined with previous observations from light
and TEM microscopy the scanning electron microscope can greatly increase
the understanding of structural and functional development in fungi.

82

83

Further information on spore surface and interanl structure is
evident with freeze-etch techniques. This technique allows us to
examine a structure within a 2 nm region of the plane of the membrane
whereas sectioning methods provide information about the structUre in a
40-60 nm area of the membrane.

Although many investigators have studied biochemical and morphologi-
cal changes associated with spore germination (Bartnicki-Garcia and,
Nickerson, 1962: Bartnicki-Garcia and Rogers, 1964; Bartnicki-Garcia
gt_gl,, 1968; Bracker and Halderson, 1971; Fletcher, 1973a; Dykstra,
1974), studies on spore ontogeny are limited to TEM papers on
Gilbertella persicaria (Bracker, 1968) and Thamnidium elegans (Fletcher,
1963b) and light microscopy studies by Ingold (1965) and Dobbs (1939).'
The purpose of this investigation was to study spore ontogeny of the
major types of asexual and sexual propagules in the Mucorales utilizing
SEM and freeze-etch techniques.

1. Columellate Sporangia Development

The Mucoraceae is the largest family in the Mucorales. Sporangial
ontogeny and spore structure were investigated in the present series of
studies in a number of genera from this family. Previous TEM studies
of sporangiospore development have been at the pre-cleavage stage of
spore development at sporangial maturity (Bracker, 1968). Light micro-
scopic diagrams illustrate early and late stages of sporangial develop-
ment but fail to show full sequences of sporangial ontogeny (Ingold,
1965).

Rugosity of terminal hyphal apices as observed in this study of
Circinella umbellata has not been previously reported. It appears that

this rugosity proceeded and perhaps even initiated swelling of hyphal

 

A .lllulllllln‘l

I11 ‘1.

84

apices and eventual sporangial formation. Since the most obvious

change during sporangial development are transformations in cytomembranes
(Bracker, 1968), apical rugosity may indicate membrane changes to
accommodate expansion of hyphal tips.

Fletcher (1973a), mentions the difficulty in accurately correlating
internal cytoplasmic events with external morphological changes since
sporangial size is not indicative of internal developmental events.
Although this is certainly true, the relatively thin, transparent mem-
brane of species such as Circinella umbellata allows some observations
of internal development. SEM micrographs of spores during their develop-
ment indicate definite changes in the sporangial wall during sporangio-
spore maturation. Further biochemical and TEM studies must be performed
to identify these changes.

Young (1968a) observed the presence of spines on spores of AAggg_
plumbeus, Syzgites Aggalocarpus, Choanephora hesseltina, Q, brifieldeii,
Q, circubitarum, and g, trispgra. Since the structure and location of
the spines varied considerably, Young concluded that little taxonOmic
significance should be given to such structures. Hawker gt_gl, (1970)
gave the spines taxonomic significance by suggesting that the outer
walls of sporangia, sporangiola and conidia resemble each other. In
addition, the conidium was homologous to the outer wall of the
sporangium wall. Studies by Jones 93 31, (1976) further supported
Hawkers' observation by showing that both spines of sporangia in
Agggr_plumbeus and conidia in Cunninghamella echinulata contain calcium
oxalate dehydrate. Further analytical studies of various asexual spores
must be performed before taxonomic significance can be given to such

structures.

85

In the previous studies little mention was made of the possible
function of spines. Micrographs of sporangia inZygorhynchus moelleri

and Thamnidium elegans indicate that the spines break at basal swellings

 

leaving small openings in the sporangiolar wall. Further disintegration
of the sporangial walls seems to follow the pattern of broken spine
bases. Khan and Talbert (1975) stated that spines in the sporangiolum
of Cunninghamella echinulata are hollow and arise from the outer spore
wall. These studies indicate that on spore germination, spines are
either completely lacking or drastically reduced in size on surfaces
of sporangioles in Cunninghamella echinulata. Since spines are
extensions of the outer sporangiolar wall, it seems feasible that they
may have similiar relationships in sporangia. If so, this continuity
would support the idea that spines aid in cell wall desintegration.

The structure of asexual propagules has a definite influence on
the mechanism of spore dispersal (Ingold, 1965). SEM studies on
sporangia ontogeny may clarify mechanisms of spore dispersal. Agggg,
ramannianus exhibits morphological characteristics found only in one
other mucroaceous fungus, Mucor petrinsularis. The polyhedral spore
shape of Mucor ramannianus has been previously reported by Ingold
(1965) but in his descriptions he stated "that following irregular
ruptures in the sporangial wall, spores of A, ramannianus are released
in a spore drop". In this study scanning electron micrographs of
developing sporangia indicate formation of-definite polyhedral ridges
in the surface of sporangial walls in A, ramannianus. Sporangial
dehiscence follows the definite pattern of these polyhedral ridges re-
sulting in dissemination of spore drops.

Persistent sporangial wall fragments and spore dehiscence are

86

taxonomic and morphological characteristics. Hesseltine and Ellis (1973)
and Hesseltine and Fennel (1955) described' Circinella as possessing
persistent walls while Ingold (1965) described the sporangia as dehiscent.
A better understanding of morphological features associated with both
types of sporangia was achieved in scanning electron microscopic studies.
In this study it was possible to accurately determine the method of
spore dispersal by observing sporangia at different stages of develop-
ment we are able to indicate transitions in sporangial wall morphology.
Characteristic columella collars formed from remnants of the sporangial
wall are clearly visible in this study.

A great variation exists in fungal spore structure according to
species (Hawker and Madelin, 1976). The taxonomic value of surface
structure of dormant spores has been shown in the delimitation of
species of Elaphomyces (Hawker, 1968) and in the relationships between
groups of hypogeous Gasteromycetes (Hawker, 1971). However, spore
ornamentation may not always have taxonomic value. Many spores of the
same species differ widely in surface structures (Payak, 1962).
Additionally many spores of one species may resemble spores of other
unrelated species as is observed in the teliospores of smuts (Khanna
gt 31,, 1966) and basidiospores of Homobasidiomycetes (Perreau, 1969).
Although it may be difficult to attribute any taxonomic significance to
spore morphology in Circinella, Absidia and zygorhynchus, the geometric
shape exhibited in Mucor ramannianus definitely has taxonomic

significance. Young (l968a) described spores of A, ramannianus as being

 

slightly wrinkled while Ingold (1965) described the polyhedral condition
but failed to illustrate his descriptions. As previously stated this

type of spore is unusual in mucoraceous fungi. Freeze-etch micrographs

87

help illustrate the many geometric patterns. Fractured cells clearly
demonstrate that the ridges are continuous with the outer sporangio-
spore wall. Ridges form on spores during early sporangial development
and gradually adhere to and displace the sporangia wall aiding wall
dehiscence. This ridged pattern may be an evolutionary adaptation to
better utilize available sporangial space. The close packing of
sporangiospores in spore drops (Fig. 29) demonstrates the efficiency of
this arrangement.

Freeze-etching was used in this study to elucidate fine structure
of spores not visible in the SEM. One of the major advantages of the
freeze-etch technique is that cells remain viable throughout the entire
freeze-fracture process allowing examination of replicas of living
specimens. As a result, artifacts that might be produced during chemical
fixation procedures are substantially reduced.

Cole (1973a, 1973b, 1974) utilized the SEM and freeze-etch technique
to study ontogeny of conidia in Penicillium. Cole stated that rodlet
patterns, very similar to those in the cell wall of Aycotyphya sp. and
Syncephalastrum racemosum, are associated with physical changes of the
cell. In addition rodlet patterns can be used to determine stages of
spore development. Although Cole admitted that a much larger sample of
conidia must be studied to determine the taxonomic value of freeze-etch-
ing techniques, the advantages derived from utilizing SEM and freeze-
etching together are evident. One organism representing each type of
asexual spore was selected for freeze-etch study. The invaginations of the
plasma membrane were visible by this technique. Campbell (1970)
suggested that the invaginations in Alternaria brassicicola were

associated with melanin synthesis and Allen gt_gl, (1971) found that

88

- invaginations were associated with numerous Amultienzyme complexes?
particles. In this study no attempt was made to correlate such
hypothesized activities with observed membrane patterns. Rather the
patterns were compared to see if they constituted valid taxonomic
information.

In the Mucoraceae the usually conspicuous columella persisted
after the shedding of spores. The columella is typically more or less
globose, ovoid or pyriform. Different interpretations of columella
function have been presented. Jones gt_gl, (1976) attribute spore
dispersal in Agggr_plumbeus to bursting of the sporangium caused by
enlargement of the columella. Ingold (1965) believed that internal
sporangial mucilage material obtains moisture through the columella

to gradually expand and break the sporangial wall. In Gibertella

 

persicaria Bracker (1968) showed that when the sporogenesis region

is in mid-cleavage, the columella cleavage is almost complete with only
a few protoplasma connections to the rest of the sporangia. Bracker

did not report any columella enlargement prior to spore dispersal. As
previously mentioned it is difficult when interpreting SEM photomicro-
graphs to correlate internal cytoplasmic events to external morphologi-
cal features. Accurate interpretation of columella function is most
difficult. Columellate sporangia examined in this thesis indicate
columella were usually not delimited until sporangia had reached maximum
. growth or at least near maximum growth. Internal mucilage material
(Ingold, 1965) was not as evident as previously described except in
5p§1g13_§p, This descrepancy may be due to changes that occurred during
dehydration and critical point drying. O'Donnefl‘s (1976) refined method

of cryofracturing apothecia in Peziza quelepiodotia illustrate the

 

89

advantages of this method to other methods in resolving tissue types

in apothecia. Perhaps similar studies with mucoraceous fungi could

add important information as to columella function. Features such as
apophysis development in'AQsigjg,§p, are more visible when viewed in
the SEM. Apophysis development was shown complete at the time of spore
maturation. Earlier stages exhibited little swelling of the sporangio-
phore apex.

These studies indicate that columella formation is probably com-
plete at the time of spore release. Although some enlargement of the
columella may take place after the spores reach maturity, this increase
seems negligible. These studies on columellate sporangia, especially
Mucor ramannianus (Figs. 21-25) indicate that enlargement of the spores,
other than the columella result in breakage of the sporangial wall.
This breakage may also be accompanied by expansion of internal mucilage
due to water absorbance.

2. Columellate Sporangia and Sporangioles.

The differences between sporangia and sporangioles may not be as
distinct as was previously believed. The presence of columella in
sporangioles of Helicostylum has been reported by Lythgoe (1958) and
Benjamin (1959). Their findings contradict conventional definitions
(Ainsworth 1971). Columellate sporangia and sporangioles have also
been reported in Thamnidium elegans (Fletcher, 1973a). This research
supports Fletcher's views. Fletcher showed similar methods of
columella delimitation in both sporangia and sporangioles indicating
that perhaps both are basically columellate. Minor variations in
position of the cleavage apparatus may result in delimiting columella
at different positions. The observation that both a terminal

colunellate mucoraceous sporangium as well as colunellate sporangioles

9O

occur on the same sporophore supports the theory of sporangiole
development from sporangioles. Benjamin (1958) believes that the
presence or absence of a columella as an absolute character in dis-
tinguishing sporangia and sporangioles is more apparent than real.
The evidence presented herein supports this view.

Although the difference between the persistent sporangial walls
and fugacious sporangiolar walls is evident in SEM photomicrographs the
taxonomic significance of such a difference has not been reported.
Benjamin (1958) and Hawker §t_al, (1970) both argue that the reduction
of the many spored sporangium to a single spored state occurred along
several lines of evolution with the Mucorales. Hesseltine and Anderson
(1956) showed a similarity in structure in the presence of small
sporangia and spores suggestive of sporangioles found in larger
sporangia. This feature along with similarity in columella formation
and the size and structure of spores indicate a gradual transition from
large columellate sporangium to smaller columellate sporangioles.

Spines were also observed on the outer sporangial walls of
Thamnidium glggags, Although they appeared more short and blunt than
those previously described in Zygorhynchus moelleri, they appeared to
have the same function.

3. Monosporic Sporangiola

The development of single-spared sporangioles directly from the
surface of terminal vesicular enlargements is clearly illustrated in
SEM photomicrographs in this study (Figs. 55-86). While all other

species of Cokeromyces possess multipsored sporangioles,Cokermoyces

 

poitrasii exhibits further evolutionary advancement in only single-

spored sporangiola. Although development of monosporic Sporangiola

91

have been recently studied at the ultrastructure level in Mycotypha and
Cunnigghamella (Khan and Talbert, 1975), no similar studies have been
performed on Cokeromyces. The presence of multi-spored sporangioles

in other species of Cokeromyces supports the presence of monosporic
sporangiola in CokeromyCes poitrasii.

In general, developmental sequences as in Cokeromyces'poitrasii
viewed in the SEM support early light microscopic descriptions of
Shanor gt al, (1950). However, the presence of hexagonal plates at the
base of pediculate sporangioles is reported here for the first time.
These divisions in the outer vesicular wall are similar to the hexa-
gonal patterns observed at the base of spines in Cunninghamella
echinulata. Zycha (1969) indicated that spores were disseminated by
either breaking of the appendage at the sterigmata base or by breaking
at the sterigmata apex close to the sporangiole. The only method
observed was breaking of the sterigmata near the vesicle. The long
sterigmata of Cokeromyces poitrasii and the single spored sporangiola
indicate that this species may represent an intermediate stage in the
development of the monosporic sporangiola present in Aycotypha and
Cunninghamella from the multispored sporangium.

Until recently many monosporic sporangioles were considered
conidia (Ingold and Zoberi, 1963, Ingold, 1965; Hawker gt_a1,, 1970;
and Dykstra, 1974). Findings blehan and Talbert (1975) showed that
sporangiolar walls and spore walls are of different origins and com-
parable with the sporangiolor walls and sporangiospore walls of multi-
sporic sporangia. The presence of conidia in mucoracous fungi thus
seems highly questionable.

Initial studies placed Aycotypha in the Mucoraceae (Fenner, 1932).

92

Bessey (1950) also placed Mycotypha in the Choanaphoraceae because of
its mucoraceous thallus. Hesseltine (1952) however placed Mycotypha

in the Cunninghamellaceae along with'Cunninghamella and Thamnocephalis

 

Many authors followed this classification (Hesseltine, 1955; Benjamin,
1959; Milko, 1967). Young (1969) suggested that Mycotypha be moved
from the Cunninghamellacea to the Thamnidiaceae because of the
separation of the sporangiolar wall from the sporangiole. Benney
(1973) agreed with this interpretation. Recent findings (Khan and
Talbert, 1975) that the spores in Cunninghamella and Mycotypha consist
of a protoplast surrounded by a two-layered spore wall inside a
sporangiolar wall justifies the placement of this genera in the
Cunninghamellacea.

Scanning and freeze-etch photomicrographs in this study support
the findings of Khan and Talbert (1975) that detachments of the
sporangiola in Mycotypha is accomplished by a circumsissile break at
the base of the sterigmata. This is an important feature in differen-
tiating the conidia from the monosporic sporangiolum since the septum
laid down during conidium ontogeny fbrms a part of the conidial wall
while the septum of the monosporic sporangiolum is not related to spore
wall formation. The dimorphic nature of both sterigmata and
sporangioles in Aycotypha sp, is clearly evident in SEM photomicrographs.

Spores of Mypotypha africana and A. microspora may be clearly
differentiated by their shape and mode of dissemination. By following
ontogeny of sporangioles in the SEM this difference can be further
clarified.

The ampullae in Mycotypha has been shown to have a large central
vacuole at maturity (Khan and Talbert, 1975). Fractured ampullae

93

viewed in the SEM verify the presence of large vacuoles as well as
showing the presence bf dimorphic apices.

Development of spores in Cunninghamella is similar to Mycotypha
except that the wall layer of the sterigmata and ampulla is continuous
with the inner layer of Mycotypha spores and the outer layer of
Cunninghamella spores. The presence of spines in the outer wall layer
of Cunninghamella (Fig. 85) is another significant difference. The
similarity of structure of Cunninghamella spines with those of
sporangia in other mucoraceous fungi has been discussed in an earlier

section. Spores of Cunninghamella lost their spiny appearance becoming

 

relatively smooth on germination.

4. Merosporangiferous Mucorales.

In Syncephalastrum racemosum large numbers of cylindrical
sporangiola (merosporangia) arise by budding from the surfaces of
terminal swollen apices. SEM observations clearly illustrate a pro-
liferation resulting in elongation of cylindrical sporangiola and the
eventual protoplasmic division to form spores. Spores that were
earlier deScribed as smooth (Benjamin, 1959) to slightly rugose (Young,
1968a) were verrucose with reticulate rodlets in freeze-etch photo-
micrographs (Figs. 92, 93). Rodlet patterns were similar to those
previously described in Aycotypha and Penicillium (Hess g§,g1,, 1968)
and Aspergillus (Hess and Stocks, 1969). The plasma membrane had
localized depressions (Fig. 94) similar to those reported in Rhizopus
arrihizus (Hess and Weber, 1973).

The presence of invaginations on plasma membranes seems to be a
more common characteristic in fungal organisms. Hess and Weber (1973)

noted this structural difference but believed that the depressions had

94

similar function to the invaginations found in other plasma membranes
(see page 87). Fungal organisms have a great diversity in the number
of cell layers spores possess. The three-layered cell wall of

Syncephalastrum racemosUm is similar to that reported in Penicillium

 

megasporum (Sassen, gt_al,, 1967) and NeurospOra (Sussman, 1966).
Warts of the outer spore wall are shown to arise from and are contin-

uous with the first wall layer.

8. Sexual Stages of Development

Previous SEM descriptions of zygospore structure are limited to a
brief description of zygospore ornamentation by Laane (1974) and a
critical survey of the taxonomic value of zygospore ornamentation in
A159; and ZygorAynchus by Schripper 9511; (1975). O'Donnell 31311.,
recently (1976) studied zygosporogenesis in Phycomyces blakesleeanus
utilizing TEM, SEM and freeze-fracturing methods.

ZygosporeS'are an important taxonomic criterium in the Mucorales.
Although ornamentation of zygospores in Zygorhynchus were studied by
Schripper gt al, (1975) events leading to zygospore formation were not
presented. In my study zygosporogenesis in Zygorhynchus was studied
because of the close relationship between the asexual stages of
' ZyggrAyggAg§_and Agggr, The presence of heterogametangia is the main
taxonomic criteria in differentiating these two genera.

Zygorhynchus moelleri is homothallic with zygosporic hyphae
arising from the same vegetative hyphae. Detailed observations of
sexual stages of Phycomyces blakesleanus (Sassen, 1962; O'Donnellgt_al,,
1976) and Rhizopus sexualis (Hawker and Beckett, 1971) have revealed

that a number of wall layers are laid down in succession within the

95

original gametangial wall, resulting in a many layered wall in the
dormant spore. The exact number of walls seem to vary from four
(O'Donnellg§_a1,, 1976) to five or more (Grove, 1975). This discrepancy
is probably due to different interpretations of similiar wall structures.

In Zygorhynchus moelleri fusion of the apical progametangia walls
results in a pushing out of the main hyphae to delimit gametangia. At
the same time swelling of secondary hyphal apices is initiated. Further
fusion results in an increase in diameter of the fusion wall similar to
Rhizopus (Hawker and Beckett, 1971). Septal formation to delimit
zygospores has been reported to occur both prior to and after the fusion
wall has dissolutioned (Hocking, 1967; O'Donnell at 21:9 1976). Septal
formation in Zygorhynchus appears to take place after dissolutionment.
Gametangia were observed delimited further down the length of the
suspensor differing from previous light microscopic observations of
Fitzpatrick (1930). Scanning electron photomicrographs allow the
observations of only two layers. These layers corresponded to the
outer primary wall and warty wall layers of O'Donnell gt_al, (1976).
Zygospores are covered with small warts which upon maturation become
spiny. These zygospores correSpond to the Group A, starfish-like orna-
mented zygospores of Schripper gt_§l, (1975).

Apsigia_sp. differ from ZygorAynchus moelleri in having equal
. gametangia, being heterothallic and having appendages surrounding
zygospores in many species. Septal formation to delimit gametangia
seems to occur before the fusion wall has dissolved. Again, it is
difficult to correlate external and internal events but photos indicate
delimitation of gametangia while zygosporic hyphae from opposite
mating types are still distinguishable. Disintegration of the primary

96

gametangial wall by penetration of the roughened secondary wall was the
same in all species studied. Developmental procedures, as well as the
apperance of a smooth quartenary layer were similar to those observed
in Phycomyces (O'Donnell _ei a_l_ ., 1976).

This study presents another viewpoint of the ontogeny of the
single-spared sporangiolum from the multispored terminal columellate
sporangium. When viewed and compared to previous light and TEM studies

a better understanding of the overall process is achieved.

11111 II.- DII‘Ill‘IlI an

1 ’0 'l'illluliill

.‘IIt

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