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dissertation entitled

ZYGOSPOROGENESIS IN ZYGORHYNCHUS HE'I'EROGAMUS:
AND ZYGOSPOROGENESIS AND SPOROANGIOSPOROGENESIS
IN MYCUI'YPHA AFRICANA

 

presented by

RICHARD EWARD EDELMANN

has been accepted towards fulfillment
of the requirements for

Ph .D. degree in Botany

 

 

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Major professor

Date 12/8/93

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TO AVOID FINES rotum on or before date duo.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU to An Nflrmottvo Action/Equal Opportunity Institution
mm:

 

 

ZYGOSPOROGENESIS IN ZYGORHYNCHUS HETEROGAMUS, AND
ZYGOSPOROGENESIS AND SPORANGIOSPOROGENESIS
IN MYCOTYPHA AFRICANA

By

Richard Edward Edelmann

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

1994

ABSTRACT

ZYGOSPOROGENESIS IN ZYGORHYNCHUS HETEROGAMUS, AND
ZYGOSPOROGENESIS AND SPORANGIOSPOROGENESIS
IN MYCOTYPHA AFRICANA

By

Richard Edward Edelmann

The ultrastructure of zygosporogenesis in Zygorhynchus heterogamus was studied
with scanning and transmission electron microscopy (SEM and TEM), using conventional
chemical fixation (CF) and the cryopreservation techniques of low temperature SEM
(LTSEM) and freeze-substitution (FS). Detailed observations of origin and development
of the walls of the entire sexual apparatus are reported. Gametangial septa contained
plasmodesmata from initial formation through full maturation of the zygospore. Wart
formation was initiated with the appearance of vesiculate bodies between the
zygosporangium wall and plasma membrane, and developed through deposition of a
stacked series of electron-opaque disks. Rupture of the zygosporangium wall by the
developing zygospore was concurrent with the formation of a contiguous warty layer.
The first clear TEM observations of the multiple wall layers of the fully mature zygospore
are presented. A detailed historical discussion on, and a proposal for standardized

structural nomenclature are presented.

Sexual and asexual reproduction in Mycotypha africana was observed with

LTSEM. Sexual apparati were initiated by the contact of two zygophoric hyphae. Sub-
apical contact resulted in formation of progametangia at the site of contact. Zygophoric
hyphal elongation continued, forming additional sexual contacts. Progametangia enlarged,
and gametangial septa were formed, isolating the gametangia which then fused, resulting
in the formation of the zygosporangium. Zygospores developed internally, rupturing
through the zygosporangium wall with maturation of the wart layer.
Sporangiosporogenesis commenced with the formation of specialized aerial
sporangiophores extending up to 5,000 ,um. Apical regions of sporangiophores enlarged
forming vesicles 300-400 m long. Pedicels formed synchronously on vesicles, and
ballooned outward forming sporangia. Morphologically distinct, spherical- and elongate-
sporangia formed, exhibiting a highly regular spatial arrangement, alternating between the

two sporangial types.

LTSEM provided excellent preservation of fragile reproductive structures free from
dehydration artifacts common with CF. Nine FS fixation protocols following either
propane jet freezing or high pressure freezing (HPF) of Z. heterogamus were compared.
Both methods of freezing offered acceptable results, but HPF provided more well-frozen
material. Comparison of FS with CF revealed identification of cytoplasmic ultrastructure
was better with CF. FS resulted in poor membrane preservation providing few

identifiable organelles, however wall ultrastructure was better than with CF.

To Dr. Terrence M. Hammill,
For introducing me to the fungi and sex in the Zygomycetes and

For allowing a sophmore to take apart his TEM.

To Dr. Karen L. Klomparens,
For being a true Mentor in every possible sense

And for allowing me to discover who and what I am.

And to
Lori A. Bridgers, BA, MA, MSEd
My Best Friend,

And all of the consequences.

iv

Cryopreservation .................................... 30

Acknowledgements ....................................... 31
Literature Cited .......................................... 32
CHAPTER 2: LOW TEMPERATURE SCANNING ELECTRON

MICROSCOPY OF THE ULTRASTRUCTURAL DEVELOPMENT OF
ZYGOSPORES AND SPORANGIOSPORES IN MYCOTYPHA
AFRICANA, AND THE EFFECTS OF CULTURAL CONDITIONS ON

SEXUAL VERSUS ASEXUAL REPRODUCTION ................. 38
Abstract ............................................... 38
Introduction ............................................ 39
Materials and Methods ..................................... 41
Cultural Conditions .................................. 41

Low Temperature Scanning Electron Microscopy (LTSEM) ...... 42
Results ................................................ 42
Cultural observations ................................. 42

Low temperature scanning electron microscopy ............... 45
Zygosporogenesis. .............................. 45
Sporangiosporogenesis ........................... 50

Discussion ............................................. 57
Zygosporogenesis ................................... 57
Sporangiosporogenesis ................................ 64
Literature Cited .......................................... 66
SUMMARY ................................................. 69

vi

LIST OF TABLES

Table 1. Solvent and fixative combinations used for freeze-substitution ........ 79

viii

LIST OF FIGURES

CHAPTER 1.

Figure 1. Diagrammatic illustration of structural nomenclature used in this study

of zygosporogenesis in Z. heterogamus ......................... 4
Figure 2. LTSEM of initial contact between zygophore and sexual hypha ...... 9
Figure 3. SEM of initial formation of micro-progametangium .............. 9
Figure 4. LTSEM of advanced development of the progametangia .......... 9
Figure 5. LTSEM of the transition from progametangia to discrete gametangia . . 9
Figure 6. TEM of the fusion wall .................................. 9
Figure 7. TEM of the macro-gametangial septum ....................... 9
Figure 8 a-e. TEM of developmental maturation of the gametangial septa ...... 13

Figure 9. LTSEM of zygosporangium formation: fusion wall removal and

gametangial plasmogamy ................................... 13
Figure 10. SEM of initial development of the zygospore warty layer internal to

the zygosporangium wall ................................... 13
Figure 11. TEM of fusion wall removal and gametangial plasmogamy ........ 13
Figure 12. TEM of wart initiating centers ............................ 15
Figure 13. TEM of wart initials ................................... 15

Figure 14. SEM of the developing wart layer and enlargement of macro-
suspensor .............................................. 15

Figure 15. SEM of the advancement of the rupture of the developing zygospore
wall through the zygosporangium wall .......................... 15

ix

Figure 16. TEM of specialized stacked endoplasmic reticulum within the

developing zygosporangium/zygospore ..........................
Figure 17. TEM of wart layer rupture of zygosporangium wall .............
Figure 18. TEM of the advanced development of the wart layer .............
Figure 19. LTSEM of a nearly mature zygospore .......................
Figure 20. SEM of two fully mature sexual apparati on a single sexual hypha . . .
Figure 21. TEM of a nearly mature zygospore wart layer .................
Figure 22. TEM of a fully mature zygospore wall ......................

Figure 23. TEM of a fully mature zygospore wall ......................

CHAPTER 2.

Figure 1. Initial contact between two zygophores .......................

Figure 2. Following contact, adhesion of the two zygophores resulting in the
formation of the fusion wall, and swelling of the progametangia

occurred ...............................................

Figure 3. After an initial period of swelling, internal gametangial septa were
formed delimiting two gametangia of approximately equal size, and the

suspensors .............................................

Figure 4. Fusion of the two gametangia occurred with the removal of the fusion
wall and was observed as a smoothing or flattening of the junction

between the two gametangia .................................

Figure 5. Shortly after formation of the zygosporangium, internal wart initials

became externally visible in the region closest to the former fusion wall . .

Figure 6. The development of the warts and swelling of the internal zygospore

wall ruptured through the outer zygosporangium wall ...............

Figure 7. Further swelling resulted in a spherical appearance to the developing
zygosporangium/zygospore complex, with the internal zygospore warts

visible through the zygosporangium wall ........................

15

17

17

17

17

46

46

46

46

49

Figure 8. Maturation resulted in the complete replacement of the zygosporangium
by the zygospore, with the contiguous, external warty layer of the
zygospore clearly visible ................................... 49

Figure 9. At maturity of the zygospore, the warts exhibited an irregular conical
shape, and an interdigitation of their bases ....................... 49

Figure 10a. Convoluted sexual hyphae showing the randomness of sexual
apparatus formation ....................................... 49

Figure 10b. Diagram of fig. 10a defining the individual sexual hyphae ........ 49

Figure 11. The sporangiophore, a straight, unbranched aerial hypha, extended
undifferentiated for 2,000-5,000 pm above the agar ................. 51

Figure 12. A 100-200 ,um apical region of the sporangiophore enlarged, forming
a cylindrical vesicle of often irregular diameter initially .............. 51

Figure 13. Shortly after the initial enlargement, the vesicle diameter became more
uniform with a slight tapering at either end ...................... 51

Figure 14. Enlargement of the vesicle surface showed the uniform two-
dimensional spatial arrangement of the newly-formed pedicel initials ..... 52

Figure 15. The pedicels enlarged into two distinct, alternating types, and the
sporangial initials formed at their apices ......................... 52

Figure 16. An enlargement of the vesicle surface shows the arrangement of the
shorter pedicels supporting the S-sporangia and the larger pedicels
supporting the E-sporangia .................................. 52

Figure 17. At a slightly later stage, the S-sporangia and the E-sporangia were
clearly visible and appeared generally uniform in their development ..... 52

Figure 18. At maturity the entire surface of the vesicle was covered with
regularly spaced unispored sporangia including the apical region ........ 54

Figure 19. Enlargement of the vesicle surface shows the high degree of spatial
ordering of the sporangia ................................... 54

Figure 20. Due to their orientation, perpendicular to the vesicle, the E-sporangia

were found to be more susceptible to being dislodged at maturity than the
S-sporangia ............................................. 54

xi

Figure 21. At maturity the sporangiophore, and the vesicle remain erect for

several months, with a clearly observable hollow interior ............. 56

Figure 22. At maturity all the unispored sporangia had fallen from their

pedicels ...............................................

APENDIX A.

Figure 1. Illustration of RMC three-welled aluminum freeze-substitution

chamber ...............................................

Figure 2. Fragile initial contact of the zygophore with the sexual hypha .......

Figure 3. Expansion and delimitation of the gametangia by the formation of

gametangial septa ........................................

Figure 4. Preservation of a mucoidal layer and remnants of the zygosporangial

wall after rupture by the developing warty layer ...................

Figure 5. Fracture of an early post-cleavage sporangium showing preservation of

internal matrix supporting the spore protoplasts ....................

Figure 6. Fixation with 2% 0804, and 0.5% uranyl acetate in acetone (Protocol

1), following high pressure freezing, resulted in little freezing damage . . . .

Figure 7. Fixation with 2% OsO‘, and 0.5% uranyl acetate in acetone (protocol

I), following high pressure freezing, resulted in little freezing damage . . . .

Figure 8. Fixation with 2% OsO4 in methanol (protocol 11), following propane jet

freezing, with embedment in Spurr’s resin .......................

Figure 9. Fixation with 2% 0504 in methanol (protocol II), following propane jet

freezing, with embedment in Spurr’s resin .......................

Figure 10. Fixation with 1% 0304, and 0.25% uranyl acetate in methanol,
following propane jet freezing, with embedment in Spurr’s resin resulted
in membrane preservation, but membranes appeared highly wrinkled and

distorted ...............................................

xii

56

81

83

83

83

83

83

83

86

86

86

Figure 11. Fixation with 0.5% 0504 in acetone (protocol IV), following high
pressure freezing, with embedment in Quetol resulted in a finely granular
cytoplasm with uniform fixation, and high contrast between organelles and
the cytoplasm ........................................... 86

Figure 12. Fixation with 0.1% 0804 in acetone (protocol V), following propane
jet freezing, with embedment in Quetol resin resulted in poorer fixation
than that of higher concentrations of OsO,I as seen in lower cytoplasmic
density, and a general distorted and "fuzzy" appearance of the
organelles .............................................. 86

Figure 13. Fixation with 0.025% OsO4 in acetone (Protocol VI), following
propane jet freezing, with embedment in Quetol again resulted in very poor
fixation and low contrast within the cytoplasm, and the cytoplasm in
general exhibited a "splotchy" nature ........................... 86

Figure 14. Fixation with 1.7% glutaraldehyde in acetone (protocol VII), following
propane jet freezing, with embedment in Quetol produced good fixation in
general, high contrast, numerous identifiable organelles, and some
membrane preservation ..................................... 86

Figure 15. Fixation with 0.5% Uranyl acetate methanol (protocol VIII), following
high pressure freezing, with embedment in Quetol resin resulted in very
low contrast in the cytoplasm and a general fuzziness in appearance ..... 90

Figure 16. Conventional immersion fixation with aqueous 1% formaldehyde and
2% glutaraldehyde in 0.05M sodium cacodylate buffer followed by
1%OsO4 and embedment in Spurr’s resin resulted in generally good
fixation, with good contrast, and clearly defined membranes ........... 9O

xiii

CHAPTER 1: ZYGOSPOROGENESIS IN
ZYGORHYNCHUS HETEROGAMUS VUILLEMIN (MUCORALES),

WITH A PROPOSAL FOR STANDARDIZATION OF STRUCTURAL NOMENCLATURE

ABSTRACT

The ultrastructural details of zygosporogenesis in Zygorhynchus heterogamus
Vuillemin, 1903, were observed with scanning and transmission electron microscopy
(SEM and TEM), using both conventional chemical fixation and the cryopreservation
techniques of low temperature SEM (LTSEM) and freeze-substitution for TEM. Detailed
observations of origin and development of the walls of the entire sexual apparatus are
reported. Maturation of both gametangial septa was observed to be identical and
plasmodesmata were observed to penetrate the septa from their initial formation through
full maturation of the zygospore. Wart formation was initiated with the appearance of
vesiculate bodies closely oppressed to the zygosporangium wall, external to the plasma
membrane. Wart development progressed through the deposition of a stacked series of

electron-opaque disks. Rupture of the zygosporangium wall by the developing zygospore

2

was concurrent with the formation of a contiguous warty layer. For the first time, clear
TEM observations of the multiple wall layers of the fully mature zygospore are presented.
A detailed historical discussion on, and a proposal for standardized structural
nomenclature is presented. Comparisons of conventional chemical fixation and cryo-
preservation is made, including the advantages of LTSEM in the preservation of delicate
reproductive structures, and the benefits and failings of freeze-substitution in the

preservation of mature zygospores.

Key Words: Cryo-Fixation, Freeze-substitution, High-Pressure freezing, Mucorales,

Propane Jet Freezing, Ultrastructure, Zygosporangium, Zygospore.

INTRODUCTION

By definition the key characteristic of the class Zygomycetes is the formation of
zygospores, and yet surprisingly little detail is known about this form of sexual
reproduction. Numerous studies have reported on the physiology, mating type behaviors
and certain limited morphological aspects such as wall structure, wart morphology, and
septa] structure. To date, limited studies have been published that correlate contemporary
ultrastructural techniques of scanning electron microscopy, transmission electron
microscopy and/or light microscopy of the developmental morphology of

zygosporogenesis as a whole (Hawker and Beckett, 1971, O’Donnell, et al., 1976 & 1977,

3
Moss and Lichtwardt, 1977, Ansell and Young, 1983).

Terminology.—The terminology used throughout this paper for designating the various
structures of zygosporogenesis in Zygorhynchus heterogamus Vuill. is detailed
diagrammatically in Figure 1, and is described as follows. The term sexual apparatus was
used to include all the discrete components of a single sexual reproductive complex
leading to the formation of a single zygospore regardless of the developmental stage, with
the exclusion of the sexual hyphae. Sexual hyphae were defined as the central,
specialized aerial hyphae upon which the sexual apparati developed. Zygophores were
designated as the branches arising from the sexual hypha, prior to contact and fusion, and
eventual formation of the gametangia and suspensors (Blakeslee, 1904). Upon contact
and adhesion of the zygophores with a sexual hypha, the zygophores were designated as
progametangia (Blakeslee, 1904). After formation of the gametangial septa, the apical
regions of the progametangia were defined as gametangia, and the basipetal regions as
suspensors. After plasmogamy of the two gametangia the resulting structure was defined
as a single zygosporangium. Zygospore was used to refer to the structure which formed
de novo, within the zygosporangium. The prefixes macro- and micro- were used to
distinguish the two gametangia, and their associated structures, based on size and
origination point on the sexual hyphae.

The ultrastructural details of zygosporogenesis have been revealed very slowly
partly because of the difficulty in preparing mature zygospores. The following study of
zygosporogenesis in Z. heterogamus fulfills two goals: first, it is a comprehensive

ultrastructural study correlating scanning and transmission electron microscopy using

Figure 1. Diagrammatic illustration of structural nomenclature used in this study of
zygosporogenesis in Z. heterogamus.

   

       
   

 

 

 

 

 

 

 

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5

some of the most modern techniques available. Secondly, when combined with previous
research on Sporangiosporogenesis (Edelmann and Klomparens, 1994), it provides an

extensive study of the ultrastructural development of reproduction in Z. heterogamus.

MATERIALS AND METHODS

Cultural Conditions—A culture of Zygorhynchus heterogamus Vuillemin was obtained
from E. S. Beneke and A. L. Rogers, Michigan State University. Species identification
was verified using Zycha et al. (1969), Hesseltine et al. (1959), and Domsch et al.
(1980). Subcultures were grown and maintained on a 1.8 % V-8 juice agar (177 ml V-8
juice, 2 g calcium carbonate, 20 g Bacto-Agar, 823 ml deionized water), incubated at 22 -

24 C, with 12 hr light/ 12 hr dark. Cultures were grown 3 - 21 days and were prepared

for observation.

Electron microscopy.—-Specimens for electron microscopy were prepared for each
specific technique as detailed below. Scanning electron microscopy (SEM) was
performed using a JEOL 35CF scanning electron microscope at 10 kV. Approximately
450 - 500 sexual apparati in the course of 12 different experimental preparations were
examined with SEM. Transmission electron microscopy (TEM) was performed using a
JEOL 100 CX II transmission electron microscope at 80 kV. Approximately 50 sexual

apparati in the course of 27 different experimental preparations were examined with TEM.

6

Conventional immersion fixation. Specimens for TEM and SEM were prepared
as follows: blocks of agar were cut from areas of growing colonies containing the desired
stages of zygospore development. These specimens were fixed in 2.5% glutaraldehyde
and 1% paraformaldehyde solution buffered with 0.05M sodium cacodylate (pH 7.2) and
1% sucrose, rinsed in buffer (6X @ 20 min), secondary fixation with buffered 1.0%
osmium tetroxide, and rinsed in water (4X @ 20 min). TEM samples were en bloc
stained with 0.5% uranyl acetate. All samples were dehydrated with an ethanol series
(25%, 50%, 75%, 85%, 95%, 100%, 100%, 100% @ 30 min). The SEM samples were
critical point dried, mounted on aluminum stubs, electrically grounded with silver paint,
sputter coated with 21 nm of gold and observed. The TEM samples were infiltrated and
embedded with Spurr’s epoxy resin (Spurr, 1969) or Quetol/nadic methyl anhydride
(Kushida, 1975), ultrathin sectioned, and stained with uranyl acetate and Reynold’s (1963)

lead citrate (See Edelmann and Klomparens, 1994).

Low temmrature SEM (LTSEM). Samples were prepared using the procedures

detailed in appendix A according to the following general protocol. Specimens were cut
from the agar plates, 7 mm x 20 mm x 1 mm, mounted on to an Emscope SP 2000
cryostage, with Tissue-Tel: II O.C.T. cryo-embedding medium (Lab-Tel: Products, Miles
Laboratories, Inc.) mixed with powdered graphite, and plunge frozen in a liquid nitrogen
slush. Surface water crystals were viewed and sublimated away (10-15 min) at
approximately ~60 C in a JEOL 35 CF scanning electron microscope with the Emscope
cryostage attachment. Specimens were then re-cooled and sputter coated with 40 nm of

gold at -190 C, and observed at -80 to -90 C.

7

Freeze-Substitution for TEM. Specimens were selected and teased off the agar
plates using a macroscope and dissecting needles. Desired samples were ultrarapidly
frozen with either an RMC MF 7200 propane jet freezer or a Balzer’s HPM 010 high
pressure freezer. Frozen samples were freeze substituted with the following general
protocol adapted from C‘zymmek (Czymmek and Klomparens, 1992), and specifically
detailed in appendix A. Specimens were freeze-substituted with 1-2% osmium tetroxide
in acetone at approx. -80 C for 3 - 4 days. Samples were then warmed to room

temperature, rinsed with 100% acetone, embedded and sectioned as above.

RESULTS

Sexual hyphae arose from below the agar surface as specialized aerial hyphae,
initially indistinguishable from vegetative hyphae, 5 - 8 pm in diameter at first, enlarging
up to 8 - 10 tan with maturation. Sexual apparati were never observed to develop
amongst lateral hyphae growing along the agar surfaces. Upon reaching a height of 80 -

100 pm, lateral branches, the macro-zygophores, emerged subapically from the sexual
hyphae. The sexual hyphae typically continued elongation ranging up to approximately
150 pm or more. Both the sexual hyphae and the macro-zygophores characteristically
exhibited a greater degree of curvature than vegetative hyphae or sporangiophores which
were typically straight. All hyphal surfaces, except actively growing regions, such as
hyphal apices and zygosporangia, were covered with crystalline-like echinulations which
enlarged with maturation of the hypha. These echinulations were observed with both

conventional SEM and LTSEM, and were determined to be true features and not

8

preparation artifacts. It was noted that these spines appeared very sharply pointed in

LTSEM but rounded or eroded after conventional fixation.

The zygophore elongated up to 80 - 100 am until contacting either the same or
a different sexual hypha from which it initially arose (Fig. 2). Contact between a specific
zygophore and a sexual hypha was apparently random. Within regions of low aerial
hyphal density, i.e. colony peripheries, the zygophores typically contacted the same sexual
hypha from which they arose. Within denser, older growth regions, contact was often
made with a sexual hypha different than that from which the zygophore arose. When
contact was made with the parent sexual hypha, contact was always made further apically
along the sexual hypha from the point of origination of the zygophore. The point of
contact on the sexual hypha was always observed at least 10 am from the apex and
usually at a greater distance. The point of contact on the zygophore ranged from apically
to 10 pm subapically, rarely further away. The number of sexual apparati per sexual
hyphae typically ranged from two to five. The initial point of contact was extremely
fragile and typically was dislodged during conventional fixation procedures, and was only

observed with LTSEM.

Shortly after initial contact between the zygophore and the sexual hypha, the
micro-progametangium arose from the sexual hypha at the point of contact (Fig. 3). Due
to the rarity of observation, it was surmised that this stage was extremely short in
duration. After the initial formation of the micro-progametangium, both the micro- and

the macro-progametangia ballooned outward (Fig. 4). The zygophore/macro-suspensor

Figs. 2-7. Initial formation of sexual apparatus in Z. heterogamus. 2. LTSEM of initial
contact between zygophore (Zp) and sexual hypha (SH);Bar = 10am. 3. SEM of initial
formation of micro-progametangium (mPG). At this stage the zygophore was
redesignated as the macro-progametangium (MPG); bar = 10am. 4. LTSEM of advanced
development of the progametangia. The zone of contact between the two progametangia
had enlarged and was firmly established as the fusion wall (FW). The apices of both
progametangia were noted to be free of crystalline-like echinulations; bar = 10am. 5.
LTSEM of the transition from progametangia to discrete gametangia. With the formation
of internal gametangial septa, eight distinct structures were designated: the sexual hypha,
SH; the micro-suspensor, mS; the micro-gametangial septum, mGS; the micro-
gametangium, mG; the fusion wall, FW; the macro-gametangium, M6; the macro-
gametangial septum, M68; and the macro-suspensor, MS. The relative size alone was
the only significant difference between those structures designated micro and macro; bar
= 10,um. 6. TEM of the fusion wall. The fusion wall (FW) appeared to be contiguous
with the exterior walls of the micro-gametangium(mG) and the macro-gametangium
(MG), with no discemable structural differences. The edges of the fusion wall where it
divided to form the gametangial walls appeared to be tattered (arrow), indicative of being
stressed by the expansion of the gametangia; bar = Lam. 7. TEM of the macro-
gametangial septum. The micro-gametangial septum and the macro-gametangial septum
(MGS) appeared identical except for size. The gametangial septa consisted of two
electron-translucent layers separated by an electron-transparent layer. With maturation
an electron-opaque layer was laid down on the suspensor (MS) side of the septa; bar =

0.5,um.

 

 

10

appeared to increase in diameter with maturation, ranging up to 12 - 14 pm at full
maturation (as determined by SEM and LTSEM). The macro-progametangium increased
in volume at a greater rate than the micro-progametangium, and maintained a volume
three to four times that of the micro-progametangium throughout maturation. The surface
of both progametangia remained relatively free of the crystalline-like echinulations,
although a few progametangia were observed to have a few echinulations. At this stage
in development, the region of contact between the two progametangia had increased in
area and was noted to be firmly established and was not dislodgable by the physical
forces associated with specimen preparation. With the establishment of this adhesion

between the two progametangia, the region of contact was designated as the fusion wall

(Fig. 4, FW).

Following the initial swelling of the two progametangia, the formation of internal
gametangial septa were identifiable with SEM observation (Fig. 5, GS). With the
formation of these septa, eight distinct structures were designated, as shown in Figure 5:
the sexual hypha, SH; the micro-suspensor, mS; the micro-gametangial septum, mGS; the
micro-gametangium, mG; the fusion wall, FW; the macro-gametangium, MG; the macro-
gametangial septum, MGS; and the macro-suspensor, MS. The relative size alone was
the only significant difference between those structures designated micro and macro.
Though the actual sizes of individual sexual apparati differed over a wide range,
particularly when correlated with colony age, the relative size ratios between specific
components remained fairly consistent. It was noted, however, that the micro-suspensor

was often so small as to be unobservable in the SEM.

11

Sectioning through this developmental stage revealed the continuous nature of the
shared fusion wall (Fig. 6). No distinction was observed between the portion of the
fusion wall provided by the micro-gametangium and the macro-gametangium. The fusion
wall appeared to be contiguous with the exterior walls of the gametangia, with no
structural differences discernable with TEM. It was noted that as the gametangia
ballooned outward, the edges of the fusion wall where it divided to form the gametangial
walls appeared to be tattered, indicative of being stressed by the expansion of the
gametangia (Fig. 6, arrow). No significant difference between the cytoplasmic contents
of the two gametangia was observable. At this stage, both gametangia contained typical
zygomycetous cytoplasmic structures with the exception of an increased concentration of
nuclei (relative to vegetative hyphae), very few lipid bodies, and very small amounts of

endoplasmic reticulum.

The micro- and the macro-gametangial septa were noted to be identical in structure
except that the macro-gametangial septa were approximately twice the diameter of the
micro-gametangial septa and therefore had greater surface area. Both gametangial septa
joined to the exterior wall in a ’T’ shaped junction, and marked the division between the
exterior wall of the gametangia and the suspensors (Fig. 7). Shortly after the initial
formation of the gametangial septa there were no structural differences between the
gametangial walls and the suspensor walls. Figure 8 shows a series of micrographs
revealing the development of the gametangial septa from its initial formation to just prior

to complete zygospore maturation.

12

From the earliest stages of the development of the gametangial septa, numerous
plasmodesmata were observed to perforate the septa (Figs. 7, 8a - e). As the gametangial
septa developed these plasmodesmata were always observable, and apparently elongated
as the septa thickened. The gametangial septa appeared structurally different from the
exterior walls (Figs. 7, 8 a - e). Proceeding from the gametangium to the suspensor, the
gametangial septa appeared to be composed of a light electron-opaque layer, an electron
transparent layer, a second light electron-opaque layer, and a dark electron-opaque layer
(Figs. 7, 8 c). At the earliest stages of development, only two electron-opaque layers with
a central electron-transparent layer were observable (Fig. 8 a, b). At the latest stages of
development, a darker electron-opaque layer was distinguishable on the interior of the
gametangial side of the septa and was contiguous with zygospore wall layers in later
developmental stages (Fig. 8 c, d, e). The darkest electron-opaque layer on the suspensor
side extended beyond the gametangial septum itself and along the interior of the

suspensor for 2 - 3 am at later developmental stages.

Following formation of the gametangial septa, the next developmental stage was
marked by the removal of the fusion wall and plasmogamy. Fusion wall removal was
distinguishable in SEM observation by a transition from a distinct union of the two
discrete progametangia and gametangia (Figs. 4, 5) to a slight depression indicative of the
formerly separate gametangia (Fig. 9, arrows). The removal of the fusion wall was
designated as a transition from the two gametangia to a single zygosporangium.
Concurrent with removal of the fusion wall was a continued enlargement of the former

gametangial regions, and the rounding of the zygosporangium between the two

13

Figs. 8-11. Gametangial septa and fusion wall. 8 a-e. TEM of developmental maturation
of the gametangial septa. In all figures, a-e, the zygosporangium/zygospore is on the left
and the suspensor is on the right. No differences were noted between micro- and macro-
gametangial septa. a. Earliest observed stage of gametangial septum formation, with the
presence of plasmodesmata (arrowheads). b. Thickening of the septa. c. Appearance of
the electron opaque layer on the interior of the suspensor side of the septa (P). d.
Elongation of the plasmodesmata with increased thickening of the septa (freeze-
substituted). e. The gametangial septa] region of a fully matured zygospore, with the
presence of plasmodesmata (arrowheads) penetrating the outer most zygospore wall (*;
freeze-substituted). Standard bar = 0.2m for a-c; 1am for d; 0.5am for e. 9. LTSEM of
zygosporangium formation: fusion wall removal and gametangial plasmogamy. Prior to
completion of fusion wall removal a steady decrease in the distinguishablity of the
junction of the two gametangia, the fusion wall itself (arrows), was observable; bar =
10m. 10. SEM of initial development of the zygospore warty layer internal to the
zygosporangium wall. This developing warty layer of the zygospore was observed
externally by the appearance of shallow indentations (arrowheads). A thin line of scarring
(arrows) marked the previous position of the fusion wall; bar = 10m. 11. TEM of fusion
wall removal and gametangial plasmogamy. No specific internal features were noted
associated with fusion wall removal. At this stage the cytoplasm of the zygosporangium
generally matched that of vegetative hyphae with the inclusion of numerous nuclei (N),
mitochondria (Mt), few lipids (Lp), and endoplasmic reticulum (arrowheads); bar = 1m.

 

14

gametangial septa. At this stage it was noted that the zygosporangium was markedly
larger than the suspensors. The zygosporangium continued to enlarge, forming a smooth
exterior with only a fine line of scarring marking the former location of the fusion wall
(Fig. 10, arrows). The surface of the zygosporangium was covered with numerous
shallow depressions indicating the interior location of developing wart initials (Fig. 10,
arrow heads). These shallow depressions initially appeared closest to the former fusion
wall location, on the surface of the former macro-gametangial region, and advanced

towards both gametangial septa with maturation.

Observations of the interior of the zygosporangium revealed a progressive removal
of the fusion wall beginning from the center and proceeding towards the exterior walls
(Fig. 11). From a point shortly after fusion wall removal and throughout later
development, multiple observations revealed a steady increase in the number of lipid
bodies present in the zygosporangium. Concurrent with the removal of the fusion wall
was the appearance of regularly-spaced membrane-containing or vesiculate bodies
appressed to the interior of the zygosporangium wall external to the plasma membrane
(Fig. 12). These vesiculate bodies initially appeared close to the remnants of the fusion
wall within the former macro-gametangium. Concurrent with the completion of fusion
wall removal, these vesiculate bodies appeared generally evenly spaced throughout the
entire zygosporangium (Fig. 12). Shortly after their initial appearance these vesiculate
bodies were no longer observed, rather disks of electron-opaque material were noted
closely appressed to the zygosporangium wall, external to the plasma membrane,

exhibiting approximately the same spacing as the former vesiculate bodies (Fig. 13).

15

Figs. 12-16. Early development of zygospore. 12. TEM of wart initiating centers.
Concurrent with the last stages of fusion wall removal, numerous regularly spaced
vesiculate bodies (arrows) appeared between the zygosporangium wall and its plasma
membrane; bar = 1m, inset bar = 0.2m. 13. TEM of wart initials. The wart initials
(WI) appeared as regularly spaced electron-opaque disks appressed to the zygosporangium
wall external to the plasma membrane; bar: 1am. 14. SEM of the developing wart layer
and enlargement of macro-suspensor. External observation of early wart layer
development revealed the warts as hemispherical bulges of the zygosporangium wall, with
some rupture of the zygosporangium wall, initially appearing on the former macro-
gametangial side of the fusion wall scar (arrowhead); bar = 10m. 15. SEM of the
advancement of the rupture of the developing zygospore wall through the zygosporangium
wall. The developing wart layer progressed from the location of the former fusion wall
(arrow heads) towards the gametangial septa; bar = 10m. 16. TEM of specialized stacked
endoplasmic reticulum within the developing zygosporangium/zygospore. Concurrent
with fusion wall removal multiple-layered, endoplasmic reticula were observed within the
regions closest to both gametangial septa within the zygosporangium, and rarely within
the central regions; bar = 1am.

 

16

These electron-opaque disks were designated as wart initials.

External observation of the zygosporangia, shortly after internal formation of the
wart initials, revealed the developing warts erumpant through the zygosporangium wall
(Fig. 14). The rupture of the warts was first noted on the former macro-gametangial side
of the fusion wall scar. Following their original appearance, the rupture of the warts
proceeded from the fusion wall scar towards the gametangial septa (Fig. 15). Remnants
of the zygosporangium wall were commonly noted adhering to the apices of the warts
(Fig. 15, arrows). Concurrent with the external appearance of the warts, the macro-
suspensors continued to enlarge immediately behind the macro-gametangial septa,
resulting in the formation of a globose structure, with a diameter exceeding that of the
zygosporangium (Fig. 14, 15). This swelling seen with the macro-suspensors was not
observed to occur with the micro-suspensors. Within the developing zygosporangium in
regions near to the gametangial septa, unique, multi-layered membranous structures were
observed (Fig. 16). These structures appeared to be composed of closely appressed,

sheet-like expanses of 6 - 20 membrane layers, similar to ER in appearance.

Sections through zygosporangia at this stage revealed that the maturing warts had
formed a contiguous layer between the zygosporangium wall and the plasma membrane
(Fig. 17, arrow). The formation of this wart layer was designated as marking the
transition from zygosporangium to zygospore. The structure of the warts themselves
appeared to be a stacked series of electron-opaque disks, increasing in diameter from the

apex of the warts to their bases, resulting in individual warts having an elongated, conical

17

Figs. 17-22. Maturation of zygospore wall layers. 17. TEM of wart layer rupture of
zygosporangium wall. The warts (W) of the outer layer of the zygospore developed as
a stacked series of electron opaque disks. Enlargement of these warts apparently resulted
in the rupture of the external zygosporangium wall which appeared as remnants (R)
adherent to the wart apices. Coinciding with rupture of the zygosporangium wall, thin
regions of wall material were observed connecting the bases of individual warts (arrows);
bar = 0.5m. 18. TEM of the advanced development of the wart layer. With maturation,
the individual warts obtained a length of 2.5-3am. The stacked electron opaque layers
were still clearly observable, as well as the thin regions of wall material connecting the
individual warts (arrows). Remnants (R) of the zygosporangium wall were commonly
observed attached to the wart apices. This specimen was freeze-substituted; bar = 1m.
19. LTSEM of a nearly mature zygospore. The outer wart layer of the zygospore formed
a continuous wall between both gametangial septa (arrows) with the only notable
remnants of the zygosporangium in the regions closest to the gametangial septa. The
micro-suspensor (m8) is clearly shown to be free of the crystalline-like spines observable
on the macro-suspensor (MS); bar = 10m. 20. SEM of two fully mature sexual apparati
on a single sexual hypha. At maturity the zygospores obtained a nearly spherical shape
18-25am in diameter. Arrangement of multiple sexual apparati on a single sexual hypha
was observed to be apparently random with the exception that the micro-suspensors were
always noted to be acropetal to the macro-suspensors when both originated from the same
sexual hypha; bar = 10m. 21. TEM of a nearly mature zygospore wart layer. The
material of the outer wart layer of the zygospore wall increased in density particularly in
the regions between individual warts (arrow). At this stage initial development of the
inner most electron translucent zygospore wall layer was observed (arrowheads). Freeze-
substituted specimen; bar = Iran. 22. TEM of a fully mature zygospore wall. The
zygospore wall consisted of three major layers: (1) an outer electron opaque warty layer
(W), approximately 0.6;rrn thick between the warts (arrow), (II) a middle laminate layer
approximately 0.3;rm thick (L), and (III) an internal electron translucent layer 0.75-0.8am
thick. The inset contains a zygospore wall fractured during specimen preparation. The
arrowheads show the fracture of the inner wall layer confirming that the wall layer is not
an artifact of preparation. Freeze-substituted specimen; bars = 1am.

 

18

shape at maturity (Fig. 17, 18). The edges of the electron-opaque disks, comprising the
warts, appeared extremely ragged, and without a solidly defined profile (Fig. 16, 17). The
electron density differences shown within the warts in Figure 18 were believed to be
artifacts of preparation and not attributable to compositional differences. The initial
rupture of the warts through the zygosporangium wall occurred prior to a stable
connection between individual warts. The initial contiguous wart layer was fragile and

often ruptured during TEM embedment procedures, presumably due to osmotic pressures.

Coinciding with extension and maturation of the wart layer, the zygospores
enlarged and rounded up, forming a globose to spherical shape at maturity (Fig. 20). The
diameter of the fully mature zygospores generally exceeded that of the macro-suspensors
by approximately 25 - 30%, ranging from 18 - 25 ,um. Figure 20 demonstrates an
example of two sexual apparati on a single sexual hypha and the diversity of sizes of
sexual apparati. No pattern of arrangement of the zygospores on the sexual hypha was
determined, with exception of the micro-/macro-suspensor arrangement when occurring

on a single sexual hypha as mentioned previously.

Upon completion of the outer warty layer of the zygospore, inner wall layers were
observed in the final developmental stages of zygospore maturation (Fig. 21). At
maturation, the zygospore wall appeared to be composed of three structurally distinct
regions: an electron-opaque outer warty layer, 0.6m thick between warts (Fig. 22 & 23,
W); an intermediate 0.3m thick, laminate layer (Fig. 22 & 23, L); and an inner, 0.75-

0.8;rm, electron-transparent layer (Fig. 22 & 23, I). These separate wall layers appeared

19

Fig. 23. TEM of a fully mature zygospore wall. The individual warty (W), laminate (L),
and inner (I) layers appeared to be tightly adherent to each other and no separation of the
wall layers was observed. The laminate layer was composed of individual electron-
transparent sheets of material embedded in a matrix of electron-opaque material. The
inner layer appeared to be composed of homogenous electron-transparent material; bar =

0.1m.

 

20

to be tightly adherent or fused to each other and no separations of the wall layers, due to
specimen preparation, i.e. osmotic pressures, etc., were observed (Fig. 23). The laminate
layer was composed of individual electron-transparent sheets of material embedded in a
matrix of electron-opaque material. The inner layer appeared to be composed of
homogenous electron-transparent material. The compositional nature of each individual

zygospore wall layer was not determined in this study.

DISCUSSION

The prefixes micro- and macro- are suggested as preferable to the prejudicial terms
‘male’ and ‘female’ used by early workers (Gruber, 1912, Atkinson, 1912, and Green,
1927). Blakeslee (1904) establishes clear arguments against the use of the terms ‘male’
and ‘female’ for both heterothallic strains of zygosporic cultures (for which he suggested
the use of + and -), and the "zygophoric branches" of homothallic species. Blakeslee
(1904) specifically argued for the use of smaller and larger to correspondingly replace the
terms ‘male’ and ‘female’ in describing gametangia of Z. heterogamus. I propose that
the more formal prefixes micro- and macro- be substituted for ‘smaller’ and ‘larger’ due
to ( 1) their better applicability to syntactical modification of the terms progametangium,
gametangium and suspensor, and (2) their specificity in distinguishing the different

developmental pathways of each.

21

Zygophore and Sexual Hypha.—I suggest the terminology sexual hypha in referring to
the central hypha supporting the sexual apparati because it is a distinct aerial hypha upon
which all the other structures associated with zygosporogenesis develop. Blakeslee (1904)
defined the term zygophores as the initial hyphae which arise, are chemotactically
attracted to each other, contact, fuse, and transform into the progametangia. In
heterothallic species these zygophoric hypha arise separate and distinct from each other,

and are justifiably called zygophores.

In some homothallic fungi, i.e. Rhizopus sexualis (Smith) Callen, these zygophores
arise due to dichotomous branching of a single aerial hypha (Hawker and Beckett, 1971).
These branches are easily distinguishable from the originating hyphal trunk, do not re-
contact the originating hyphal trunk, generally appear very similar to each other, and
transform into progametangia. For these reasons, the use of the term zygophores is again
clear and justifiable. However, no specialized term has been used in the literature to

specifically define this "originating hyphal trunk" as I have described it.

In homothallic species of Mucor, Blakeslee (1904) described that "the zygophores
generally arise from comparatively distant parts of the mycelium, and yet so far as has
been observed, zygospores are never formed between branches of a single aerial hypha."
Blakeslee also stated that this was in "direct contrast" to Zygorhynchus wherein the "two
zygophoric branches" usually do originate from a single aerial hypha. Furthermore
Blakeslee (1904 & 1913) describes very different developmental pathways for these two

heterogenous zygophoric branches, but does not suggest a specific nomenclatural

22

difference in terminology for these structures.

In the case of Z. heterogamus and other members of the genus, i.e. Z. exponens
Burgeff, Z. moelleri Vuill., there is only one hyphal branch which arises from, and is
distinguishable from, the original aerial hypha or hyphal trunk as described above. This
zygophoric branch does eventually transform into one of the progametangia. The second
”mating hypha", or zygophore, which develops or transforms into the second
progametangium, and later gametangium and suspensor in other members of the
zygomycetes does not form in Z. heterogamus. The "originating hyphal trunk" itself gives
rise, de novo, to the second progametangium. This second progametangium, however,
is formed only after contact by a zygophore and does not exhibit chemotactic growth.
I propose that the term sexual hypha be used to describe this "originating hyphal trunk",
as this distinguishes it from the zygophore. Further, I support Blakeslee’s definition of

zygophore as that structure which directly transforms into a progametangium.

Some researchers have designated the sexual hypha as the second zygophore in
Zygorhynchus (Blakeslee, 1904 & 1913; Hesseltine, et al., 1959; O’Donnell, et al., 1978a),
describing it as uncurving, smaller, diminutive, slender, and/or weaker. In each case their
designation of the sexual hypha as the second zygophore is based on the observation of
the formation of a septum either directly above, or at some distance acropetally from, the
first or macro-zygophore branching, which thus distinguishes two morphologically
different zygophores. My research suggests that this definition is inadequate because it

only holds true for terminal sexual apparati, and does not provide a good descriptor for

23

the more typical case where multiple sexual apparati are found on a single aerial hypha
(Fig. 20). In all other instances the zygophore is a terminal structure and does not branch
to form multiple sexual apparati. The sexual hypha as I define it is analogous to an

ascogenous hypha in its formation of multiple sexual apparati.

Zygosmrangium and Zygosmrer—The terms zygosporangium and zygospore have been

used seemingly indiscriminately, even interchangeably, in the literature for over a century
in referring to a variety of different structures. In fact, neither term is included in
Ainsworth & Bisby’s Dictionary of the Fungi (Hawksworth, et al., 1983)! Various
authors have used a variety of other terms such as prozygospore (O’Donnell, et al., 1977),
exospore, endospore (Benjamin, 1959, Hesseltine, et al., 1959), perispore, mesospore and
fusion cell to describe either the same or differing aspects, layers and structures of the
later stages of zygosporogenesis. I propose the adoption of a standardization of the terms,
as originally used by Blakeslee (1904), and others (O’Donnell, et al., 1978b), in the
following manner. Zygosporangium refers to the entire initial structure, between the two
gametangial septa, which arises from the fusion of the two gametangia following removal
of the fusion wall {Blakeslee actually used the term zygote and zygospore. Following
current mycological conventions I have changed zygote to zygosporangium, similar to the
changes proposed by Dangeard (1906a) of Blakeslee’s (1904) progametes and gametes
to progametangium and gametangium, respectively, which are now generally accepted}.
The external wall of this zygosporangium is contiguous with that of the suspensors as
described here with Z. heterogamus, and was originally the same wall which was the

external wall of the progametangia. Further examples of this have been reported in R.

24
sexualis (Hawker and Gooday, 1967, 1968, and Hawker and Beckett, 1971), Phycomyces

blakesleeanus (Kunze) Burgeff (O’Donnell et al., 1976), P. nitens Fries (O’Donnell et al.,

1978b), and Mucor mucedo L.:Fr. (Edelmann and Hammill, unpublished).

The zygospore refers to the entire structure which forms de novo, internal to the
zygosporangial wall. Regardless of the number of wall layers which may be
distinguishable by various authors in various species, the zygospore shall describe the
entire collection of layers which form internal to the zygosporangium wall. In most cases
of zygosporogenesis and azygosporogenesis, the initial, outer most wall layer of the
zygospore consists of a heavily pigmented, often verrucosely ornamented layer which
forms de novo internal to the zygosporangium, as reported here with Z. heterogamus.
Additionally, this de novo wall formation internal to the zygosporangium has been
reported in R. sexualis (Hawker and Gooday, 1967, 1968, and Hawker and Beckett, 1971),
P. blakesleeanus (O’Donnell et al., 1976), P. nitens (O’Donnell et al., 1978b), and in
other species by Blakeslee (1904), Vuillemin (1904), Dangeard (1906b), and Ling-young
(1930). The use of the terms zygosporangium and zygospore as described here are
directly analogous to the asexual structures sporangium and sporangiospore, particularly
as found in those members of the Zygomycetes producing unispored sporangia,
Mycotypha Fenner and Cunninghamella Matruchot, as described by Khan and Talbot

(1975).

The term fusion wall is preferable to the term fusion septa, as used by some

authors (O’Donnell, et al.,1977, 1978a, and 1978b) due to the origin of the wall. Initially,

25

a fusion wall is a portion or region of the exterior wall of the zygophore, progametangia
and gametangia. In contrast, septa are internal partitions formed de novo within an
existing walled structure, i.e. hyphae, spore, or conidium, subdividing that structure. The
fusion wall, as in Z. heterogamus, is an external wall which only becomes "internalized"
due to the appression of the two progametangia/gametangia, thereby forming the interface
between these two discrete structures. The fusion wall’s role developmentally is thus
directly opposite that of a septum. With development, the fusion wall is removed,
allowing for the union of two separate structures, the gametangia, and the formation of

a single structure, the zygosporangium.

Initiation of plasmogamy of the two gametangia in this study of Z. heterogamus
is described as occurring with fusion wall removal, and specifically not ‘fusion wall
dissolution’ as is generally described by most authors. Fusion wall dissolution in R.
sexualis (Hawker and Gooday, 1969; Hawker and Beckett, 1971), P. blakesleeanus
(Sassen, 1962), P. nitens (O’Donnell, et al., 1978b), and Gilbertella persicaria (Eddy)
Hesseltine (O’Donnell, et al., 1977) is reported to have occurred through the actions of
various vesicles, and microbodies associated with the fusion wall and through a putatively
enzymatic degradation. In Z. heterogamus no observations of similar structures associated
with fusion wall removal were made, and no specific proposals for the actual methods of
fusion wall removal, such as an enzymatic centripetal degradation or fusion wall
dissolution, can be justified at this time. Observations made of the junction between the

two gametangia (Figs. 5, 6, 10 & 11) indicate that this peripheral area of the fusion wall

26

is under stress, presumably due to the expansion of the gametangia and zygosporangium,
prior to and post plasmogamy respectively. Similar indications of physical stress were
also reported in Mycotypha africana Novak & Backus (Chapter 2), with fully hydrated
specimens (discounting them as an artifact of dehydration). O’Donnell, et al. (19783),
reported similar observations in P. blakesleeanus. Such observations support the notion
that in addition to putative, internal enzymatic activity, the fusion wall may actually

undergo an evagination, similar to that proposed for Mac. mucedo (Hammill, 1987).

Associated with the rupture of the zygosporangium wall by the developing warts
was an apparent slime covering as observed with LTSEM in both Z. heterogamus (Fig.
19) and Myc. africana (Chapter 2). The observation of this slime covering seems to
correlate with the gelatinized ‘inner primary wall layer’ reported in P. nitens (O’Donnell,
1978b), and in R. sexualis (Hawker and Beckett, 1971). In both P. nitens and R. sexualis
this gelatinized wall layer was observed with TEM, occurring between the ruptured outer
zygosporangium wall and the developing outer wart layer of the zygospore. O’Donnell,
et al. (1978b), and Hawker and Beckett (1971) proposed this layer as arising from a
gelatinization of the secondary wall layer of the zygosporangium. The descriptive
differences between ‘slime’ and ‘gelatinized’ may be due to the nature of the methods of
observation, fully hydrated and unfixed in the former, and fixed, dehydrated, and
embedded in the latter, or simply a matter of semantics. I propose that the inner wall
layer of the zygosporangium is softened or degraded by enzymes allowing for the
expansion of the developing warts of the zygospore through the zygosporangium wall,

with the end products of zygosporangial wall degradation appearing as a slimy film, or

27

gelatinous layer.

Blakeslee (1904) noted a distinct positioning of the micro-progametangium
forming further apically on the sexual hypha from the branching point of the macro-
progametangium in Z. heterogamus. This observation was confirmed in this current
study. Since the micro-gametangium does not form until contact by the zygophore on the
sexual hypha, this specific positioning of the micro-Imacro-progametangia is likely due
to some type of negative-geotropism of the zygophore, similar to that of the sexual hypha

and the sporangiophores.

The cylindrical developmental nature of the warts as reported here in Z.
heterogamus, was very different from the conical or ”flower pot" development reported
in R. sexualis (Hawker & Gooday, 1968, and Hawker & Beckett 1971), Mac. mucedo
(Hammill, 1987), Mac. hiemalis Wehmer, and Syzygites megalocarpus (Ehrenberg) Fries,
[= Sporodinia grandis (Link) Wallroth],(Ling-Young, 1931). My study of Z.
heterogamus, reveals that the warts develop as stacks of electron-opaque disks, forming
stacked columns of material. O’Donnell, et al. (1977), reports that the warts of G.
persicaria develop as electron-opaque, stratified deposits. However his stratified layers
run parallel to the long axis of the warts, and very definitely appear to be sectioning
artifacts. In P. blakesleeanus, O’Donnell, et al. (1976), reports that the warts appear as
stacks of fibrillar material, with the stacks perpendicular to the long axis of the warts.
In this case it would appear that the warts of Z. heterogamus and P. blakesleeanus

develop very similarly. Although, O’Donnell, et al. (1978b) report on zygosporogenesis

28

in P. nitens, it is unclear as to how wart development occurs in this species.

The structure of nearly mature and fully mature zygospore walls has been reported
with mechanically broken and cryo-fractured zygospores prepared for conventional SEM
in R. sexualis (Hawker & Beckett, 1971), P. blakesleeanus (O’Donnell, et al., 1976), G.
persicaria (O’Donnell, et al. 1977), P. nitens (O’Donnell, et al., 1978b), and members of
the genus Zygorhynchus (O’Donnell, et al., 1978a), although it is hard to distinguish the
correct relationship of the various wall layers due to their crushed nature, poor fixation
and significant dehydration artifacts. These authors and others agree that concurrent with
development of the warty layer of the zygospore, penetration of fixatives and embedding
resin through the wall is significantly inhibited, and very poor material is available for
TEM observation following ultrathin sectioning. The use of freeze-substitution in my
research has allowed the presentation of the first ultrastructurally detailed TEM
observation of zygospore wall development following the formation of a contiguous wart

layer.

It is clear that in Z. heterogamus two subsequent layers form underneath the
exterior wart layer. The intermediate layer is an apparently laminate layer composed of
numerous discrete sub-layers. Observations of this laminate layer indicate that the
individual electron-transparent sub-layers are not continuous around the entire
circumference of the zygospore but rather that these sub-layers appear individually to
cover a smaller portion of the roughly spherical surface area of the zygospore and are

embedded in a matrix of electron-opaque material. The structure of this laminate layer

29

has the appearance very similar to that of engineered laminate wooden construction
material, and it is important to note that such laminate structure is extremely strong. The
inner most layer is a fairly thick, electron-transparent layer. No information regarding the
chemical composition of these layers can be provided at this time. Future studies will
hopefully provide this information as well as show the roles of these wall layers during

zygospore germination.

It is possible that Z. heterogamus is pseudo-homothallic. Edelmann and
Klomparens (1994) reported that the sporangiospores of Z. heterogamus were almost
exclusively binucleate. It is possible that these binucleate sporangiospores are
heterokaryotic for mating type. Germination and mycelial growth from such
heterokaryotic sporangiospores would yield a thallus which is composed of heterokaryotic
mycelium and apparently homothallic due to the presence of both mating type nuclei.
Such an occurrence would be very similar to that described in P. blakesleeanus by
Blakeslee (1906), Burgeff (1912, 1915, and 1925) and in Pilobolus crystallinus (T ode)
Fries as described by Krafeyzk (1935). These authors report that during zygospore
germination and germsporangium development incomplete ‘sex’ (mating type) segregation
occurs through the formation of either heterokaryotic, binucleate sporangiospores, or
sporangiospores containing diploid nuclei. As a result of this, germination of these
sporangiospores yield heterokaryotic or ‘bisexual’ mycelium, which, in P. blakesleeanus,
produces homothallic zygospores (Blakeslee, 1906, and Burgeff, 1912). These
homothallic isolates are apparently unstable and break down within a few sexual

reproductive generations (Cutter, 1942). Homothallism in Z. heterogamus may represent

30

a stable occurrence of this heterokaryotic or bisexual condition.

Cgopreservation.—The use of cryopreservation techniques in this study of Z.
heterogamus has afforded new insights into zygospore development. The use of LTSEM
allowed for the preservation of the delicate initial contact of the zygophore with the
sexual hypha, as well as excellent preservation of the early stages of progametangia] and
gametangial formation. In observations of Z. heterogamus it was noted that conventional
immersion fixation and critical point drying of the specimens for SEM usually resulted
in disruption of the initial zygophore contact, and dehydrational collapse of the early
stages of the progametangia and gametangia. Due to the dehydration of the specimens
in conventional SEM, it is always questionable as to what extent the specimens have
shrunk (generally considered to range from 5-25% in a good preparation) in comparison
to their natural, living state. Since LTSEM specimens are fully hydrated, the only
reduction in size that may occur is an effect of thermal contraction, however, from +20
C to -90 C there is less than a 1x10“% change in volume of amorphous pure water
(Weast, 1984). Additionally, because of the minimal specimen treatment associated with
LTSEM as compared to conventional SEM preparation, better preservation of surface
material, i.e. mucoid layers, crystalline ornaments, is possible. I believe LTSEM offers
the best specimen preservation currently available, and should be considered to most

accurately represent the living state.

In comparison of conventional chemical immersion fixation and freeze-substitution

31

for TEM, numerous authors have shown the advantages of freeze-substitution for
members of the Ascomycetes (Czymmek and Klomparens, 1992), and Basidiomycetes
(Mims, et al., 1988). In this study of a member of the Zygomycetes, preservation of the
cytoplasmic details of Z. heterogamus by a variety of freeze-substitution protocols was
less than optimal (for a detailed discussion of these results see appendix A). In particular,
poor preservation of membranes was noted, limiting organelle identification and
determination of cytoplasmic events. These results were very similar to those reported
by Heath and Rethoret (1982), in their observation of mitotic microtubule preservation in
Z. moelleri via freeze-substitution. However, freeze-substitution did provide excellent
preservation of the wall material in Z. heterogamus. In this study freeze-substitution has,
for the first time, provided TEM observation of the details of the multilayered wall of

fully matured zygospores.

ACKNOWLEDGEMENTS

I would like to acknowledge Connie Bricker and Laura Sadowski at Miami
University, Oxford OH, for their assistance with the Balzers high pressure freezing unit,
and Martha Powell and Allen Allenspach for their generosity in allowing my use of the

Balzers (NSF DIR 88-20387 grant to MP and AA).

32

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Blakeslee, A. F. 1906. Zygospore gerrninations in the Mucorineae. Ann. Mycol. 4: 1-

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Blakeslee, A. F. 1913. Conjugation in the heterothallic genus Zygorhynchus.

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Burgeff, H. 1912. Uber Sexualitiit, Variabilitiit und Vererbung bei Phycomyces nitens.

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Burgeff, H. 1915. Untersuchen fiber Variabilitat, Sexualitat und Erblichkeit bei

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Burgeff, H. 1925. Uber Arten und Artkreuzung in der Gattung Phycomyces Kunze.

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Cutter, V. M., Jr. 1942. Nuclear behavior in the Mucorales II. The Rhizopus,

Phycomyces, and Sporodinia patterns. Bull. Torrey. Bot. Club. 69: 592-616.

Czymmek, K. J., and K.L. Klomparens. 1992. The ultrastructure of ascosporogenesis
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Dangeard, P. A. 1906a. La fécondation nucléaire chez les Mucorine’es. Compt. rend.

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Dangeard, P. A. 1906b. Les Mucorinées. Chapitre IV of Recherches sur le dévelopment

du pe’rithéce chez les Ascomycetes. Le Botaniste 9: 227-253, pls. 9-14.

Domsch, K.H., W. Gains, and Traute-Heldl Anderson. 1980. Compendium of soil

fungi. Academic Press, London, England.

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Green, Ethel. 1927. The life history of Zygorhynchus moelleri. Ann. Bot. (London). 41:

419-435.

Gruber, E. 1912. Einige Beobachtungen uber den Befruchtungsvorgang bei

Zygorhynchus moelleri Vuill. Ber. Deuts. Bot. Ges. 30: 126-133.

Edelmann, R. E., and Klomparens, K. L. 1994. The ultrastructural development of
sporangiospores in multispored sporangia of Zygorhynchus heterogamus.

Mycologia 86(1): (in press).

Hammill, T. M. 1988. The ultrastructure of zygosporogenesis in Mucor mucedo.

Mycol. Soc. Amer. Newslett. 39: 30-31.

Hawker, L. E. and M. A. Gooday. 1967. Delimitation of the gametangia of Rhizopus
sexualis (Smith) Callen: an electron microscope study of septum formation. J.

Gen. Microbiol. 49: 371-376.

Hawker, L. E. and M. A. Gooday. 1968. Development of the zygospore wall in

Rhizopus sexualis (Smith) Callen. J. Gen. Microbiol. 54: 13-20.

Hawker, L. E., and M. A. Gooday. 1969. Fusion, subsequent swelling and final
dissolution of the apical walls of the progametangia of Rhizopus sexualis (Smith)

Callen: an electron microscope study. New Phytol. 68: 133-140.

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Hawker, L. E., and A. Beckett. 1971. Fine structure and development of the zygospore

of Rhizopus sexualis (Smith) Callen. Phil. Trans. R. Soc. London. B. Biol. Sci.

263: 71-100.

Hawksworth, D. L., B. C. Sutton, and G. C. Anisworth. 1983. Anisworth & Bisby’s

dictionary of the fungi. 7th ed. Commonwealth Mycological Instit., Kew, Surrey.

Heath, 1. B., and K. Rethoret. 1982. Mitosis in the fungus Zygorhynchus moelleri:
evidence for stage specific enhancement of microtubule preservation by freeze

substitution. Europ. J. Cell Biol. 28: 180-189.

Hesseltine, C. W., C. R. Benjamin and B. S. Mehrotra. 1959. The genus

Zygorhynchus. Mycologia 51: 173-194.

Khan, S. R., and P. H. B. Talbot. 1975. Monosporous sporangiola in Mycotypha and

Cunninghamella. Trans. Brit. Mycol. Soc. 65: 29-39.

Krafczyk, H. 1935. Die bildung und Keimung der Zygosporen von Philobolus

crystallinus und sein heterokaryotisch Myzel. Beitr. Biol. Pflanzen 23: 349-396.

Kushida, H. 1975. Hardness control of the quetol 651 cured block. J. Elect. Micro. 24:

299.

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Ling-Young, M. 1930-1931. Etude biologique des phénoménes de la sexualité chez les
Mucorinées. Rev. Gén. Bot. 42: 144-158, 205-218, 283-296, 348-365, 409-428,

491-504, 535-552, 618-639, 681-704, 722-752; 43: 30-43.

Mims, C. W., R. W. Roberson, and E. A. Richardson. 1988. Ultrastructure of freeze-
substituted and chemically fixed basidiospores of Gymnosporangium juniperi-

virginianae. Mycologia 80: 356-364.

Moss, S. T., and Lichtwardt, R. W. 1977. Zygospores of the Harpellales: An

ultrastructural study. Can. J. Bot. 55: 3099-3110.

O’Donnell, K. L., Hooper, G., and Fields, W. G. 1976. Zygosporogenesis in

Phycomyces blakesleeanus. Can J. Bot. 54: 2573-2586.

O’Donnell, K. L., J. J. Ellis, C. W. Hesseltine, and G. R. Hooper. 1977.

Zygosporogenesis in Gilbertella persicaria. Can. J. Bot. 55: 662- 675.

O’Donnell, K. L., S. L. Flegler, J. J. Ellis, and C. W. Hesseltine. 1978a. The

Zygorhynchus zygosporangium and zygospore. Can. J. Bot. 56: 1061-1073.

O’Donnell, K. L., Flegler, S. L., and Hooper, G. R. 1978b. Zygosporangium and

zygospore formation in Phycomyces nitens. Can. J. Bot. 56: 91-100.

37

Reynolds, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in

electron microscopy. J. Cell Biol. 17: 208-212.

Sassen, M. M. A. 1962. Breakdown of cell wall in zygote formation of Phycomyces

blakesleeanus. Proc. K. Ned. Akad. Wet. 65: 447-452.

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microscopy. J. Ultrastructural Research 26: 31- 43.

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Inc., Boca Raton, Florida.

Vuillemin, P. 1904. Recherches morphologiques et morphogénique sur la membrane des

zygospores. Annales Mycologici 2: 483-506.

Zycha, H., R. Siepmann, G. Linnemann. 1969. Mucorales: eine Beschreibung aller

Gattungen und Arten dieser Pilzgruppe. J. Cramer, Lehre, Germany.

CHAPTER 2: Low TEMPERATURE SCANNING ELECTRON MICROSCOPY OF
THE UL'I‘RASTRUCTURAL DEVELOPMENT OF ZYGOSPORES
AND SPORANGIOSPORES IN M YCOTYPHA AFRICANA, AND THE EFFECTS

OF CULTURAL CONDITIONS ON SEXUAL VERSUS ASEXUAL REPRODUCTION.

ABSTRACT

The ultrastructural details of sexual and asexual reproduction in Mycotypha
africana were observed with low temperature scanning electron microscopy. Sexual
reproduction was enhanced over asexual reproduction with a reduction of incubation
temperature, the absence of light, and through the use of a minimal amount of inoculation
material. Repeated subculturing of actively growing zygosporic cultures resulted in a loss
of zygosporic competence, which was reversible following 2-3 months storage of these
cultures at 14-18°C, 24 h dark. Sexual apparati were initiated by the contact of two
zygophoric hyphae. Subapical contact resulted in formation of progametangia at the site
of contact. Continued elongation of the zygophoric hyphae and additional, subsequent
sexual contacts were observed. If sexual contact was apical, the apical region of the
zygophoric hypha converted into a progametangium, stopping elongation. The

progametangia enlarged, and gametangial septa were formed, isolating the gametangia,

38

39

which fused, resulting in the formation of the zygosporangium. Zygospores developed
internally and were observed externally by the rupture through the zygosporangium wall
of the developing wart layer of the zygospore proper. Sporangiosporogenesis commenced
with the formation of specialized aerial sporangiophores extending up to 5,000 pm. The
apical regions of the sporangiophores enlarged, forming vesicles 300-400 um long.
Pedicels formed synchronously on the surface of the vesicles, the tips of which ballooned
outward forming the sporangia. Two morphologically distinct types of sporangia were
formed: spherical- and elongate-sporangia. These sporangia were formed in a highly

regular spatial arrangement, alternating between the two sporangial types.

Keywords: Unispored sporangia, Zygosporangium, Zygospores

INTRODUCTION

The fungal class Zygomycetes exhibits a wide variety of sexual and asexual
reproductive structures including homo- and heterothallic zygospores, azygospores,
multispored sporangia, few-spored sporangiola, unispored sporangia, and merosporangia.
Within these defined developmental structures, the Zygomycetes exhibit a tremendous
variety of unique and beautiful morphologies the development of which is mostly poorly
understood or unknown. In her original description of the genus Mycotypha, E. A. Fenner

(1932) stated that "the unusual nature of [this] fungus and its many variations make it an

40

interesting object for study from the stand point of mycology." Furthermore, the highly
uniform spatial arrangement of the alternating unispored sporangia on the vesicles of M.
africana Novak & Backus suggest an excellent model organism for the study of the

fundamental aspects of subcellular structural organization.

Asexual reproduction in M. africana is initiated with the development of a long
sporangiophore supporting an elongate vesicle covered with two distinct types of
unispored sporangia. The similarity of this structure to the plant genus Typha L., or
cattail, is the derivation of the name (i.e. Mycotypha = fungal cattail). M. afiicana also
forms apparently homothallic zygospores. Conversion between the two forms of
reproduction readily occurred with either the exposure to or the exclusion of light,
respectively. The descriptive terminologies of zygosporogenesis used in this study are

those which were detailed in chapter 1.

The advanced technique of low temperature scanning electron microscopy
(LTSEM) was used to best preserve the extremely delicate ultrastructural details of
reproduction in this fungus. Due to a minimum of specimen treatment, and the possible
observation of fully hydrated specimens, LTSEM provides one of the best methods of
observing fungi in their ’natural state’. LTSEM avoids the loss of aqueous or alcohol

soluble surface materials, and the artifacts of specimen shrinkage due to dehydration.

This study of M. africana was undertaken for comparison with previous research

on Zygorhynchus heterogamus Vuill. (Chapter 1) of homothallic zygosporogenesis in a

41

second genus of the order Mucorales. Additionally, the genus Mycotypha is in the family
Thamnidiaceae, and Zygorhynchus is in the family Mucoraceae, allowing for a broader
perspective on zygosporogenesis in general towards the future goal of establishing a
general pattern for ultrastructural development of sexual reproduction in the class
Zygomycetes. Thirdly, the mixture of two morphologically distinct unispored sporangia
on a single vesicle is unique in the Zygomycetes. Fourthly, due to the length and
delicacy of the aerial asexual reproductive structures, M. africana was a good test
specimen for the technique of LTSEM. And finally, M. africana was studied simply

because of its deemed beauty.

MATERIALS AND METHODS

Cultural Conditions

A culture of Mycotypha africana Novak & Backus was obtained from E. S. Beneke and
A. L. Rogers, Michigan State University. Species identification was verified using Zycha,
et al. (1969), and Hesseltine, et al. (1959), and has been submitted to the American Type-
Culture Collection. Subcultures were grown and maintained on a 1.8 % V-8 juice agar
(177 ml V-8 juice, 2 g calcium carbonate, 20 g Bacto-Agar, 833 ml deionized water),
incubated at 14-18 °C, with 12 h light/ 12 h dark or 24 h dark. Cultures were grown 3-

21 days and were prepared at the various stages of Sporangiosporogenesis and

42

zygosporogenesis for observation.

Low Temperature Scanning Electron Microscopy (LTSEM)

Specimens were cut from the agar plates, 7 mm x 20 mm x 1 mm, mounted on to an
Emscope SP 2000 cryostage, with Tissue-Tek O.C.T. cryo-adhesive (Lab-Tek Products,
Miles Laboratories, inc.) mixed with powdered graphite, and plunge frozen in a liquid
nitrogen slush. Surface water crystals were viewed and sublimated away (10-15 min) at
approximately -60 °C in a JEOL 35CF SEM with the Emscope cryostage attachment.
Specimens were then recooled and sputter coated with 40 nm of gold at -190 °C, and
observed at -80 to -90 °C at 10 kV. Approximately 280 sporangial apparati and 350

zygosporic apparati were observed during the course of this research.

RESULTS

Cultural observations

Inoculation of cultures with a small agar plug, approximately 2 mm x 2 mm, from
previously grown cultures, and grown at 20-22 °C, with 12 h light/ 12 h dark, resulted
primarily in normal sporangial reproduction, with the colony surfaces appearing dark grey

by four days. Light macro- and microscopic observations revealed that immature vesicles

43

and sporangial initials were hyaline, and mature sporangia were dark grey to black in
color. At 10-14 days regions of zygospore formation were noted in these cultures, and
generally comprised approximately 5% of the total colony. It was noted that zygospore
production was not uniform throughout the colony, but rather was confined to specific
regions within the colony which were almost exclusively zygosporic with few or no
sporangiophores. Light microscopic observations of developing zygospores showed that
the zygospores developed a dark amber coloring with maturation, becoming nearly black

at maturity.

Plug-inoculated cultures never covered the entire surface of the agar plate. Colony
expansion dramatically slowed after 14 days of growth, with an average colony diameter
of 30-40 mm. Colony expansion appeared to stop after 8-12 weeks achieving a maximum

diameter of 50-70 mm.

Cultures grown at 14-18 °C with 24 h dark, following agar plug inoculation,
produced enhanced zygospore production comprising up to 25% of the total colony.
Regardless of age, colonies grown in complete darkness remained hyaline to light grey
in color. Apparently normal sporangial reproduction was also present, though the
sporangial heads did not develop the darker pigmentation observed in cultures grown in
the presence of light. Light microscopic observation of the germination of these light
colored single spored sporangia indicated that they were viabile. Transfer of these dark-

grown cultures to light resulted in the production of darker pigmented sporangia.

44
Zygospore production in 40-70% of the total colony was obtained by altering the

inoculation method. Inoculation was made by wetting a flame-sterilized, stiff-wire
inoculating needle in a fresh agar plate, gently passing this needle through the aerial
mycelium of a 7-10 day old dark-grown culture, and then inoculating a fresh agar plate
by dragging this needle in a series of parallel lines across the agar plate, approximately
1 mm below the agar surface. This inoculation method was designated few-spored
inoculation. These plates were then grown at 14-18 °C in 24 h dark. This inoculation
and colony transfer method became the standard procedure used throughout the rest of
this study. Increased colony growth per plate was obtainable by performing a second
inoculation of the plate oriented perpendicular to the first set of inoculation lines.
Attempts were made to further enhance zygospore production by use of two separate
colonies for each of the two series of perpendicular inoculations. No apparently
significant change in zygospore production was noted in these dually inoculated plates,

specifically where the lines of the two inoculation sources crossed on the agar surface.

Zygospore production was noted to decrease with repeated transfers. Zygospore
production remained high, 40-70%, for 3-4 successive transfers, 10-14 days apart. By
transfers 5-6, zygospore production dropped off to approximately 20% of the total colony.
By transfers 7-8, zygospore production dropped to less than 1%. Allowing the cultures
to remain undisturbed at 14-18 °C and 24 h dark for 2-3 months, and then re-transferring
to fresh agar plates, zygosporic ’competence’ was reestablished. However, following
reestablishing zygosporic competence the same pattern of decreasing zygospore

production with repeated transfers was observed. This procedure of loss of and

45

reestablishment of zygosporic competence was repeated seven times over the course of
three years. No studies were undertaken to determine the specific minimum period

required for reestablishment of zygosporic competence.

Low temperature scanning electron microscopy

Zygosporogenesis. Initial contact between two zygophores occurred apically to 65 um
subapically on either or both zygophores (Fig. 1). No morphologically distinguishable
differences were noted between the two zygophores. Zygophores ranged 6 to 8 ,um in
diameter, and were never branched though any one zygophore formed up to eight sexual
apparati. The zygophores extended up to approximately 500 ,um in total length, and 15
to 100 um between sexual apparati. Shortly after contact between the zygophores, the
two zygophores became firmly adherent to each other and this region of contact was
designated the fusion wall (Fig. 2, FW). Concurrent with the establishment of the fusion
wall the regions immediately behind the fusion wall enlarged, elongating and ballooning
outward (Fig. 2). The establishment of the fusion wall and this enlargement marked the
initial formation of the progametangia. If contact and progametangia formation was
subapical, continued elongation of the zygophore and additional sexual contacts occurred.
If contact was made at the apex of a zygophore, no further elongation of that zygophore

occurred.

Swelling of the progametangia continued, widening and flattening the fusion wall

46

Figs. 1-10. LTSEM of sexual reproduction in M. africana. Fig. 1. Initial contact
between two zygophores. Fig. 2. Following contact, adhesion of the two zygophores
resulting in the formation of the fusion wall (FW), and swelling of the progametangia
occurred. When the contact point was subapical, the zygophore apices continued to
elongate, forming later, additional contacts with the same or different zygophores. Fig.
3. After an initial period of swelling, internal gametangial septa (arrows) were formed
delimiting two gametangia (G) of approximately equal size, and the suspensors (Sp). Fig.
4. Fusion of the two gametangia occurred with the removal of the fusion wall and was
observed as a smoothing or flattening of the junction between the two gametangia (large
arrowheads). Plasmogamy resulted in the formation of a single zygosporangium between
the two gametangial septa (arrows). Fig. 5. Shortly after formation of the
zygosporangium, internal wart initials (arrowheads) became externally visible in the
region closest to the former fusion wall. Fig. 6. The development of the warts and
swelling of the internal zygospore wall ruptured through the outer zygosporangium wall.
Standard bar = 10 um in figs. 1-6.

 

47

region, however, the discrete nature of the two progametangia was clearly observable
(Fig. 3). No morphological differences were noted that distinguished the two
progametangia. The progametangia obtained a maximum diameter of 15-18 um, at which
time internal gametangial septa formed 11-20 pm behind the fusion wall within both
progametangia (Fig. 3, arrows). Formation of these gametangial septa marked the
transition from progametangia to apical gametangia (Fig.3, G) and distal suspensors (Fig.
3, Sp). Apparently concurrent with completion of the gametangial septa, initiation of
fusion wall removal occurred. Fusion wall removal was identified from external
observation by a transition from the closely oppressed but discrete gametangia (Fig. 3)
to a more continuous profile at the fusion wall (Fig. 4, arrowheads). Initiation of fusion
wall removal and plasmogamy of the two gametangia marked the formation of the
zygosporangium, which was designated as the region between the gametangial septa (Fig.

4).

Following formation of the zygosporangium, shallow depressions were observed
in the zygosporangium wall adjacent to the site of the former fusion wall. These shallow
depressions indicated the internal formation of the wart initials (Fig. 5, arrowheads). Wart
development progressed from the site of the fusion wall towards both gametangial septa
simultaneously. As development of the wart initials continued they enlarged, and
ruptured through the zygosporangium wall (Fig. 6). Displacement of the zygosporangium
wall with the internal development of a contiguous wart layer marked the development
of the zygospore separate from and internal to the zygosporangium. Remnants of the

zygosporangium wall were adherent to the warts at all further stages of zygospore

48

maturation. LTSEM observation of the ruptured zygosporangium wall and the surface of
the developing external wart layer of the zygospore revealed the presence of a slimy or

mucilaginous material covering the zygosporangium/zygospore complex.

Continuously from fusion wall removal, the zygosporangium and zygospore
enlarged, smoothing out the depression resultant from the former fusion wall, initially
forming a cylindrical zygosporangium/zygospore complex (Fig. 6) and subsequently a
globose to spherical zygospore (Figs. 7-9). No major swelling was noted in the

suspensors themselves. At maturity the zygospores obtained a diameter of 38-50 ,um.

Development of the warts continued towards the gametangial septa until a
continuous, external wart layer of the zygospore was formed between the septa (Fig. 8).
Maturation of the wart layer continued with an increase in the height and diameter of the
warts themselves. At maturity the warts exhibited an irregular conical shape, with a
maximum height of 6-8 [1111, and a diameter of approximately 8-12 pm (Fig. 9). The

surfaces of the warts were crenulate with an interdigitating of the bases of adjacent warts.

No pattern of sexual apparati formation was determinable, with the exception that
sexual apparati were never observed forming on sporangiophores. Figure 10 shows a
typical example of the apparently random nature of sexual apparatus formation, with four

zygophores forming six sexual apparati.

49

Fig. 7. Further swelling resulted in a spherical appearance to the developing
zygosporangium/zygospore complex, with the internal zygospore warts visible through the
zygosporangium wall (arrowheads). Fig. 8. Maturation resulted in the complete
replacement of the zygosporangium by the zygospore, with the contiguous, external warty
layer of the zygospore clearly visible. Remnants of the zygosporangium wall were noted
adhering to the apices of the warts (arrowheads). Fig. 9. At maturity of the zygospore,
the warts exhibited an irregular conical shape, and an interdigitation of their bases.
Standard bar = 10 pm in figs.7-9. Fig. 10a. Convoluted sexual hyphae showing the
randomness of sexual apparatus formation; bar = 50 um. Fig. 10b. Diagram of fig. 10a
defining the individual sexual hyphae.

 

50

Sporangiosporogenesis. Sporangiosporogenesis initiation was observed as the formation
of a straight, unbranched, undifferentiated, aerial sporangiophore, a uniform 8-10 um in
diameter extending SOD-5,000 pm from the agar surface (Fig. 11). The apical regions of
the sporangiophores expanded outward forming a cylindrical vesicle (Fig. 12).
Observation of intermediate stages of vesicle expansion indicate that expansion occurred
uniformly within a 100-200 [an apical region. At the earliest stages of vesicle expansion,
slight variations in diameter were noted (Fig. 12), but these discrepancies in diameter
were not observable by the time distinct pedicels formation was seen (Fig. 13). The
earliest indications of pedicel initiation were observed as a series of slightly raised,
regularly spaced, latitudinal ridges on the vesicle surface (Fig. 12, arrows). Shortly after
this stage, pedicel initials were clearly observable as distinct individual projections from
the surface of the vesicle (Fig. 13, arrows). At this point in development the vesicles
themselves exhibited a slight tapering at either end. The vesicles continued to expand in
both length and diameter throughout sporangial initiation, obtaining a maximum size of

350-400 um in length and 20-24 pm in diameter at maturity.

The pedicel initials exhibited a high degree of spacial order in their arrangement
on the vesicle surface (Fig. 14). Pedicel development appeared to be generally
synchronous over the entire surface of the vesicle with a slight developmental delay on
the order of 1-2 h at the extreme vesicular ends. The extreme apical regions generally

remained without pedicels until late in development (Fig. 15, *). Following formation of

51

Figs. 11-22. LTSEM of asexual reproduction in M. afi'icana. Fig. 11. The
sporangiophore, a straight, unbranched aerial hypha, extended undifferentiated for 2,000-
5,000 ,urn above the agar. Fig. 12. A 100-200 um apical region of the sporangiophore
(Sph) enlarged, forming a cylindrical vesicle (V) of often irregular diameter initially. The
surface of the vesicle was covered with a series of regularly Spaced ridges (arrows). Fig.
13. Shortly after the initial enlargement, the vesicle (V) diameter became more uniform
with a slight tapering at either end. Regularly arranged pedicel initials were visible on
the vesicle surface (arrows). Standard bar = 50 um in figs. 11-13.

 

 

 

 

 

52

Fig. 14. Enlargement of the vesicle surface showed the uniform two-dimensional spatial
arrangement of the newly-forrned pedicel initials; bar = 10 um. Fig. 15. The pedicels
enlarged into two distinct, alternating types, and the sporangial initials formed at their
apices. The spherical sporangia (S-sporangia) formed on the shorter pedicels, and the
elongated sporangia (E-sporangia) formed on the large pedicels. An approximately 7 pm
region of the vesicle apex remained free of sporangial initials (*); bar = 10 um. Fig. 16.
An enlargement of the vesicle surface shows the arrangement of the shorter pedicels
supporting the S-sporangia (S) and the larger pedicels supporting the E-sporangia (E).
The pedicels at this point appeared to be generally conical in shape. It was noted that
there was some Slight, regional asynchronous development of the pedicel-sporangia
structures; bar = 5 m. Fig. 17. At a slightly later stage, the S-sporangia (S) and the E-
sporangia (E) were clearly visible and appeared generally uniform in their development;
bar = 2.5 pm.

 

53

the pedicel initials the pedicels enlarged and elongated into two distinct sizes. The two
sizes of pedicels alternated in their spacial arrangement such that no two similarly sized
pedicels were adjacent to each other. Shortly after elongation of the larger pedicels above

the height of the smaller pedicels, sporangial initials formed at their apices (Fig. 15).

The formation of the sporangial initials was, again, generally synchronous over the
entire surface of the vesicle (Fig. 15), although slight differences in developmental
progression indicated by differences in sporangial sizes were observable at the earliest
stages of sporangial formation (Fig. 16). In addition to the apparent slight differences of
developmental progress, two different sporangial types were observable. These sporangial
types were designated elongate-sporangia (E-sporangia, Fig. 16, E) and spherical-
sporangia (S-sporangia, Fig. 16, S) based on their morphologies at maturity. At this stage
it was determined that the E-sporangia developed on the longer pedicels and the S-
sporangia developed on the shorter pedicels. The developing sporangia continued to
enlarge, becoming more uniform in size and shape (Fig. 17). The slight developmental

differences within each sporangial type noted previously were no longer observable.

Maturation of the sporangia was synchronous over the entire vesicle (Fig. 18). At
maturity the vesicle apical region, formerly observed to be free of sporangia, also held
mature sporangia. Mature elongate sporangia were 4.5 x 1.9-2.2 [1111, and mature spherical
sporangia were 2.8-3.2 am in diameter. The sporangia maintained their uniform Spacing,
of alternating between S- and E-sporangia, over the entire vesicle with the exception that

the last 2 to 3 rows of sporangia at the base of the vesicles were generally only

54

Fig. 18. At maturity the entire surface of the vesicle was covered with regularly spaced
unispored sporangia including the apical region; bar = 100 ,um. Fig. 19. Enlargement of
the vesicle surface shows the high degree of spatial ordering of the sporangia. The S-
sporangia (S) fit neatly below and between the E-sporangia (E), which were supported
further above the vesicle surface on the longer pedicels; bar = 10 um. Fig. 20. Due to
their orientation, perpendicular to the vesicle, the E-sporangia were found to be more
susceptible to being dislodged at maturity than the S-sporangia. Upon removal of the E-
sporangia, the arrangement of the S-sporangia between the longer pedicels (larger
arrowheads) of the E-sporangia, was more easily observed. (smaller arrowheads show the
smaller pedicels from which S-sporangia were dislodged); bar = 10 ,um.

. a t .(t-\..&.‘\ \ III.
wfiu........«..........,......u..>_«a..... , ,
‘0.oaovo e... cnv\oc a I... of ‘0’ OI aft.

1 :- sent-'1. 0’ I.\ossf lllllllllllll o,
. c I no.4 -.«.\.-\.I u..‘.. . o

‘0 .0.
. . “as... P .
3...»...HWQ.

 

55
E-sporangia (Fig. 19). The longer pedicels of the E-sporangia supported these sporangia

above the S-sporangia on their shorter pedicels (Fig. 19). At maturity the E-sporangia
were generally noted to be dislodged from the vesicle before the S-sporangia (Fig. 20).
This allowed confirmation of the arrangement of the S-sporangia on the shorter pedicels,

and the spacial regularity of the pedicels and their associated sporangia (Fig. 20).

Sporangial dissemination appeared to occur due to pedicel abscision, and sporangia
were easily dislodged from the vesicles by few slight disturbances of the culture plates.
At sporangial dissemination it was noted that the apices of the vesicles collapsed,
revealing the hollow cylindrical nature of the mature vesicles (Fig. 21). Examination of
the pedicels following sporangial dissemination revealed small pores at their apices,
indicative of an empty interior (Fig. 22). An apparent rigidity to the sporangiophore and
vesicle walls was noted due to the fact that both structures remained upright for several

months following sporangial maturity.

56

Fig. 21. At maturity the sporangiophore, and the vesicle remain erect for several months,
with a clearly observable hollow interior (arrow); bar = 100 um. Fig. 22. At maturity all
the unispored sporangia had fallen from their pedicels. The apparent empty nature of the
pedicels with clearly visible orifices at their apices was noted; bar = 10 um.

 

57

DISCUSSION

Zygosporogenesis. Low temperature SEM proved to be an excellent technique for the
observation of zygosporogenesis in M. afi'icana for several reasons. Due to the capability
of observing fully-hydrated specimens, dehydrational artifacts associated with critical
point drying in conventional SEM preparation, such as a 5-25% shrinkage (Flegler et al.,
1993) and specimen distortions commonly found with fungal specimens, were completely
avoided. The earliest stages of sexual contact and progametangial formation are generally
fragile, and easily damaged by the physical forces associated with conventional chemical
immersion fixation/preparation techniques (personal observations). The limited specimen
treatment of LTSEM avoids this physical damage of the specimens, and allows for the
preservation of surface materials, i.e. crystalline-like omamentations and mucoid layers,
which might be washed away with conventional techniques. The only negative aspect of
the observation of fully-hydrated specimens with LTSEM is in the observation of septa,

which are generally more apparent with the shrinkage associated with dehydration.

The variability observed in M. africana in the number of sexual apparati formed
by any single zygophoric hypha of from one to eight can be explained developmentally.

Sexual apparati were often noted to arise generally wherever two zygophoric hyphae

58

randomly came into contact, without any specific arrangement, save that no single
zygophoric hypha was ever observed to produce a sexual apparatus following contact with
itself. No pattern of maturation, i.e. either an increase or decrease in maturation with
proximity to the apex, was seen. However, observations of M. afi'icana Showed that if
sexual contact was made at the apex of a zygophore, further elongation of that zygophore
apparently did not occur. At no time during the observation of approximately 350 sexual
apparati was there anything indicative of the formation of a branch from a zygophore

following apical contact.

It has been established that hyphal tip elongation occurs in association with some
type of apical organizing center (Bartnicki-Garcia, 1990, and Lopez-Franco & Bracker,
1993). I propose that sexual contact at the apex of a zygophore may have resulted in the
disruption or replacement of its apical elongation center with the
progametangial/gametangial/zygosporangial organizing architecture, and no further
elongation of that zygophore was possible. When sexual contact occurred subapically,
this elongation organizing center continued to function, allowing further elongation of the
zygophore and additional sexual contacts. The zygophores of M. africana often continued
to elongate and develop additional sexual apparati. It is important to keep in mind that
the continued existence and development of zygophores in M. africana is unlike other
members of the Mucorales, i.e. Mucor mucedo L.:Fr. (Personal observations) and
Rhizopus sexualis (Smith) Callen (Hawker & Beckett, 1971), in which, after contact and

formation of the progametangia, further development of the zygophores stops.

59

Initiation of plasmogamy of the two gametangia is described as occurring
concurrent with fusion wall removal, and specifically not ‘fusion wall dissolution’ as is
generally described by most authors. Fusion wall dissolution in R. sexualis (Hawker &
Gooday, 1969; Hawker & Beckett, 1971), Phycomyces blakesleeanus (Kunze) Burgeff
(Sassen, 1962), P. nitens Fries (O’Donnell, et al., 1978b), and Gilbertella persicaria
(Eddy) Hesseltine (O’Donnell, et al., 1977) is reported to occur through the actions of
various vesicles, and microbodies associated with the fusion wall and through a putatively
enzymatic degradation. Since no internal ultrastructural observation of processes involved
with fusion wall removal were made, no proposals for the actual methods of fusion wall
removal, such as an enzymatic centripetal degradation or fusion wall dissolution, can be
made. Observations made of the junction between the two gametangia (Fig. 3) indicate
that this peripheral area of the fusion wall is under stress. These observations of a
stretching or stressing of the external fusion wall region were repeatedly made. Since
LTSEM allows for the observation of fully hydrated specimens, and extreme care was
taken to ensure that significant, noticeable dehydration of the specimens did not occur,
these observations are not believed to be sample preparation artifacts. Similar indications
of physical stress were also reported in Z. heterogamus (Chapter 1), and observed in P.
blakesleeanus (O’Donnell, 1976). Such observations support the notion that in addition
to putative, internal enzymatic activity, the fusion wall may actually undergo an

evagination, similar to that proposed for Muc. mucedo (Hammill, 1987).

Typically in zygosporogenesis, the wart initials first appear at the former site of

the fusion wall, and progress toward the gametangial septa as seen here in M. africana,

60

and also many members of the Mucorales, i.e. Z. heterogamus (Chapter 1), Muc. mucedo
(Edelmann & Hammill, unpublished), G. persicaria, (O’Donnell, et al., 1977), and R.
sexualis (Hawker & Gooday, 1968; Hawker & Beckett, 1971). It is possible that this
phenomenon may be associated with an organizing center associated with elongation and
development of the progametangia and gametangia, similar to those apical organizing
centers associated with hyphal elongation (Bartnicki-Garcia, 1990, and Lopez-Franco &

Bracket, 1993).

Hyphal tips of the Zygomycetes do not contain specifically organized structures
such as the Spitzenkdrper found in members of the Basidiomycetes and Ascomycetes. The
hyphal tips do have regions of high concentrations of vesicles and microbodies which
have been proposed to function similarly. Similar regions of concentrations of vesicles
and microbodies have been reported by Hawker & Gooday (1969), and Hawker & Beckett
(1971) in the apical regions of the progametangia and gametangia of R. sexualis. Time-
course light microscopic studies of living cultures of Z. moelleri Vuill. (Blakeslee, 1913,
and Green, 1927), Z. heterogamus (Blakeslee, 1913), Cunninghamella echinulata Thaxter,
Muc. mucedo, and a second species of Mucor [= Mucor v.] (Blakeslee, 1904) Show that
elongation/enlargement of the progametangia and gametangia specifically occurs apically.
I believe it is reasonable to conclude that very similar processes and mechanisms are
responsible for the apical elongation of both vegetative hyphae and progametangia and

gametangia.

The apices of the progametangia and gametangia actually become the fusion wall.

61

Similar organizations of vesicles and microbodies have been reported in regions
associated with the fusion wall of R. sexualis (Hawker & Gooday, 1969; Hawker &
Beckett, 1971), P. blakesleeanus (Sasson, 1962), P. nitens (O’Donnell, et al., 1978b), and
G. persicaria (O’Donnell, et al., 1977). Following removal or dissolution of the fusion
wall, these organelles would still be situated in the region where initial formation of the
warts are first observed to appear in these same fungi. The mechanism by which hyphal
elongation occurs has been proposed to be a combination of lysis and synthesis
(Bartnicki-Garcia, 1972). I propose that these same basic structures are associated in
sequence with (I) adhesion of the two progametangia and formation of the fusion wall,
(11) elongation of the progametangia and gametangia, (III) wall dissolution associated with
fusion wall removal, and (IV) formation of the wart layer and subsequent layers of the
zygosporangial wall, with a genetic regulation of the balance between lysis and synthesis.
This could also explain the cessation of elongation of the zygophoric hyphae following
apical sexual contact discussed above, due to genetic redirection of the apical organizing

center of the zygophoric hyphae from elongation to sexual apparatus formation.

Associated with the rupture of the zygosporangium wall by the developing warts
was an apparent slime covering as observed with LTSEM in both M. africana (Figs. 6-8)
and Z. heterogamus (Chapter 1). The observation of this slime covering seems to
correlate with the gelatinized ‘inner primary wall layer’ reported in P. nitens (O’Donnell,
1978b), and in R. sexualis (Hawker & Gooday, 1968; Hawker & Beckett, 1971). In both
P. nitens and R. sexualis this gelatinized wall layer was observed with TEM, occurring

between the ruptured outer zygosporangium wall and the developing outer wart layer of

62
the zygospore. O’Donnell, et al. (1978b), and Hawker & Beckett (1971), proposed this

layer as arising from a gelatinization of the secondary wall layer of the zygosporangium.
The descriptive differences between ‘slime’ and ‘gelatinized’ may be due to the nature
of the methods of observation, fully hydrated and unfixed in the former, and fixed,
dehydrated, and embedded in the latter, or simply a matter of semantics. I propose that
the slime layer observed in M. africana is indicative of an enzymatic process occurring
and associated with penetration of the zygosporangium wall by the developing warts, with
the end products of a softening or degradation of the zygosporangial wall appearing as
a slimy, mucoid film or a gelatinous layer external to the warts as seen in P. nitens, and

R. sexualis.

In the description of the effects of various environmental conditions and observed
cultural growth the terminology ‘zygosporic competence’ was used to describe cultures
which produced zygospores. Four factors observed to affect sexual reproduction in M.
afi’icana were: 1) temperature, 2) light, 3) quantity of inoculum, and 4) rate of
subculturing. The enhancement of sexual reproduction versus asexual reproduction by the
reduction of incubation temperature is quite common in the Mucorales, although to date
no specific regulatory mechanism for this effect has been reported. The conversion from
sexual to asexual reproduction following exposure to light indicates the presence of a
photosensitive regulatory mechanism. Both the quantity and rate of subculturing effects
indicate the possible presence of some physiologically active compound also regulating
sexual versus asexual reproduction. A possible mechanism that can be proposed is that

actively, rapidly growing cultures of M. afi'icana develop some ‘anti-sexual reproduction’

63

compound. Repeated transfers of such actively growing cultures to fresh media every 10-
14 days allows for the buildup of this putative compound. AS concentration levels of this
compound build it results in suppression of zygosporic competence. After 7 successive
transfers this compound reaches concentration levels where almost complete suppression
of sexual reproduction occurs. During continued incubation (i.e storage) of these sexually
suppressed cultures, at 14-18 °C and 24 h darkness, allows for a reduction of the levels
of this putative compound, possibly due to a drop in nutrient concentration in the media,
and a regaining of zygosporic competence, as was observed in this study. These factors
observed affecting the loss of zygosporic competence, i.e. repeated subculturing, exposure
to light, or heavy inoculation, provide a fertile area for research into the physiological or
genetic controlling mechanisms triggering zygosporogenesis. However, such research was

beyond the scope of the present developmental study.

It is possible that sexual reproduction in M. africana is pseudo-homothallic. It can
be conceived that each of the two types of sporangia, E- and S-sporangia, are
homokaryotic for a single mating-type. Germination of a single Sporangiospore would
lead to the development of hyphae containing a single mating-type. Since each sporangial
type matures and disperses through pedicle dehiscence simultaneously, and are inoculated,
generally, simultaneously and in the same location, it is possible that following
germination of each, anastomosis occurs between the single mating-type hyphae allowing
for the development of hyphae heterokaryotic for both mating types, and thus a pseudo-
homothallism. Determination of this could be done with single-spore culturing, and

subsequent mating crosses, but such culture techniques were beyond the scope of this

current study.

Sporangiosporogenesis. Low temperature SEM was found to be an excellent technique
for the preservation and observation of the asexual reproductive structures of M. africana.
The unispored sporangia were found to be extremely fragile and very easily dislodged
from the vesicles during chemical immersion fixation and preparation (unreported
observations). The sporangiophores were also found to be susceptible to physical damage
due to their length. This physical damage was greatly reduced with the minimal specimen

preparation associated with LTSEM.

The most striking aspects of Sporangiosporogenesis in M. africana is in its
simultaneous production of two morphologically distinct types of unispored sporangia on
a single vesicle and the high degree of spatial organization of these sporangia. Within the
zygomycetes these reproductive features are unique to the members of this genus. Of
additional note is my observations of the intermediate stages of early vesicle enlargement,
which indicate that the diameter increase is simultaneous. However, due to the static
nature of LTSEM observation no evidence was collected determining whether vesicle

elongation with maturation occurs acropetal or basipetally or both.

AS a result of these cultural and ultrastructural studies, I believe that both sexual
and asexual reproduction in Mycotypha africana offer a number of distinctive

characteristics for future study, specifically with regards to regulatory mechanisms. The

65

possible regulatory mechanisms associated with sexual reproduction of (1) low incubation
temperature, (2) light sensitivity, and (3) the loss of zygosporic competence with repeated
subculturing, would provide a choice organism for molecular and genetic developmental
study. (4) The simultaneous formation of the two distinctly morphologically different
unispored sporangia, spherical (S-sporangia) and elongate (E-sporangia), allow for a
comparison of ultrastructural development and possible genetic control mechanisms. (5)
Extremely regular structural organization in the alternation of the S- and E-sporangia, is
analogous to the determinate developmental organization of members of the
Laboulbeniales, although M. afi'icana is easier to culture. Thus, asexual reproduction in
Mycotypha provides a good subject for the study of subcellular developmental

organization and spatial recognition.

66

LITERATURE CITED

Bartnicki-Garcia, S. 1972. The bursting tendency of hyphal tips of fungi: presumptive

evidence for a delicate balance between wall synthesis and wall lysis in apical

growth. J. Gen. Microbiol. 73: 487-500.

Bartnicki-Garcia, S. 1990. Role of vesicles in apical growth and new mathematical
model of hyphal morphogenesis. Pp. 211-232. In: Tip Growth in Plant and

Fungal Cells. Ed., I. B. Heath. Academic Press, San Diego.

Blakeslee, Albert Francis. 1904. Sexual reproduction in the Mucorineae. Pro. Am.

Acad. Arts Sci. 40: 205-319.

Blakeslee, Albert Francis. 1913. Conjugation in the heterothallic genus Zygorhynchus.

Mycologisches Centralblatt. 2: 241-246, 2 pls.

Fenner, E. A. 1932. Mycotypha microspora, a new genus of the Mucoraceae.

Mycologia 24: 186-199.

67
Flegler, S. L., J. W. Heckman, and K. L. Klomparens. 1993. Scanning and

transmission electron microscopy: an introduction. W. H. Freeman and Company,

New York.

Green, Ethel. 1927. The life history of Zygorhynchus moelleri. Ann. Bot. (London) 41:

419-435.

Hammill, T. M. 1988. The ultrastructure of zygosporogenesis in Mucor mucedo.

Mycol. Soc. Amer. Newslett. 39: 30-31.

Hawker, L. E., and Gooday, M. A. 1968. Development of the zygospore wall in

Rhizopus sexualis (Smith) Callen. J. Gen. Microbiol. 54: 13-20.

Hawker, L. E., and M. A. Gooday. 1969. Fussion, subsequent swelling and final
dissolution of the apical walls of the progametangia of Rhizopus sexualis (Smith)

Callen: an electron microscope study. New Phytol. 68: 133-140.

Hawker, L. E., and Beckett, A. 1971. Fine structure and development of the zygospore
of Rhizopus sexualis (Smith) Callen. Phil. Trans. R. Soc. London. B. Biol. Sci.

263: 71-100.

Hesseltine, C.W., C. R. Benjamin, and B. S. Mehrotra. 1959. The genus

Zygorhynchus. Mycologia 51: 173-194.

68
Lopez-Franco, R. and C. E. Bracker. 1993. Morphological variation of the

Spitzenképer in growing hyphal tips. Mycol. Soc. Amer. Newslett. 44: 47.

O’Donnell, K. L., G. Hooper, and W. G. Fields. 1976. Zygosporogenesis in

Phycomyces blakesleeanus. Can J. Bot. 54: 2573-2586.

O’Donnell, K. L., J. J. Elis, C. W. Hesseltine, and G. R. Hooper. 1977.

Zygosporogenesis in Gilbertella persicaria. Can. J. Bot. 55: 662-675.

O’Donnell, K. L., S. L. Flegler, J. J. Ellis, and C. W. Hesseltine. 19783. The

Zygorhynchus zygosporangium and zygospore. Can. J. Bot. 56: 1061-1073.

O’Donnell, K. L., S. L. Flegler, and G. R. Hooper 1978b. Zygosporangium and

zygospore formation in Phycomyces nitens. Can. J. Bot. 56: 91-100.

Sassen, M. M. A. 1962. Breakdown of cell wall in zygote formation of Phycomyces

blakesleanus. Kan. Akad. v. Wet. Amsterdam C65: 447-452.

Zycha, H., R. Siepmann, and G. Linnemann. 1969. Die Mucorales: Eine
beschreibung aller gattungen und arten dieser pilzgruppe, mit einem beitrag zur
gattung Mortierella. Verlag Von J. Cramer, Germany. 355pp, 155 ill. (English
translation: Keys to the families, genera and species of the Mucorales. Richard

T. Hanlin. 1973. Verlag Von J. Cramer.)

SUMMARY

The ultrastructural details of zygosporogenesis in Zygorhynchus heterogamus
Vuillemin were observed with scanning and transmission electron microscopy
(SEM and TEM), using both conventional chemical fixation and the
cryopreservation techniques of low temperature SEM (LTSEM) and freeze-
substitution for TEM. Detailed observations of origin and development of the

walls of the entire sexual apparatus were made.

In Z. heterogamus, maturation of both gametangial septa is apparently identical
and plasmodesmata were observed to penetrate the septa from their initial

formation through full maturation of the zygospore.

In Z. heterogamus, wart formation was initiated with the appearance of vesiculate
bodies closely appressed to the zygosporangium wall, external to the plasma
membrane. Wart development progressed through the deposition of stacked series
of electron-opaque disks. Rupture of the zygosporangium wall by the developing

zygospore was concurrent with the formation of a contiguous warty layer.

69

70

For the first time, clear TEM observations of the multiple wall layers of the fully
mature zygospore were observed. Three major layers were clearly distinguishable:
(I) an outer, electron-opaque wart layer, (II) an intermediate, electron-translucent
laminate layer, composed of closely appressed sub-layers, and (III) an internal

electron-transparent layer.

Following a historical review of structural nomenclature used for

zygosporogenesis, I propose a standardized terminology:

Zygophore — Specialized sexual reproductive hyphae prior to contact and fusion,
exhibiting chemotactic, pheromonal growth responses.

Progametangia — a specific hyphal structure which develops following sexual
contact and fusion, and differentiates into a gametangium and suspensor.

Sexual Hypha — a central, specialized aerial hypha upon which the sexual apparati
developed.

Sexual Apparatus - all the discrete components of a single sexual reproductive
complex leading to the formation of a single zygospore regardless of the
developmental stage, with the exclusion of the sexual hyphae.

Fusion Wall — the portion or region of the exterior wall of the zygophore which,
shortly after sexual contact, fuses the progametangia, and subsequently the
gametangia together, becoming a morphologically internal wall.

Zygosporangium — the single cell directly resulting from the fusion and

plasmogamy of the two discrete gametangia.

71

Zygospore — the thick walled resting spore which forms de novo within the
zygosporangium.

Macro- and Micro- - prefixes used to designate the larger and smaller
progametangia, gametangia, gametangial septa, and suspensors of Z.

heterogamus, based on their morphology and developmental pathways.

Cultural experimentation with Mycotypha africana revealed that reproduction was
convertible from asexual to sexual by reducing the incubation temperature from
22°C to 14-18°C and switching from 12 h light/ 12 h dark to 24 h dark. The
reduction in temperature for sexual reproduction is common within the
zygomycetes. However, the photosensitivity is uncommon and provides a subject
for future study of environmentally inducible genetic regulation of developmental

pathways within the Zygomycetes.

Further experimentation on enhancing zygospore production in M. afiicana
revealed two additional features: (I) few-spore inoculation resulted in greater
zygospore production than plug-inoculation or high spore concentrations
inoculation, and (II) zygosporic competence was lost following repeated
subculturing of actively growing cultures, but was reversible following a minimum
of 2-3 months of storage at 14-18°C and 24 h darkness. This is believed to be
indicative of some type of physiologic regulator/suppressor of sexual reproduction,

which builds up in actively growing cultures.

10.

11.

72

LTSEM of M. africana revealed strong similarities to homothallic
zygosporogenesis in Z. heterogamus, particularly in the formation of multiple

zygospores arising from an individual zygosporic hypha.

In M. africana, sexual apparati were initiated by the contact of two zygophoric
hyphae. Subapical contact resulted in formation of progametangia at the site of
contact, and continued elongation of zygophoric hypha with the formation of
additional, subsequent sexual contacts. If sexual contact was apical the apical
region of the zygophoric hypha converted into a progametangium, stopping

elongation.

In M. africana, the progametangia enlarged, and gametangial septa were formed,
isolating the gametangia, which fused, resulting in the formation of the
zygosporangium. Zygospores developed internal to the zygosporangium and were
observed externally by the rupture through the zygosporangium wall of the

developing wart layer of the zygospore proper.

In M. africana, sporangiosporogenesis commenced with the formation of
specialized aerial sporangiophores extending up to 5,000 pm. The apical regions
of the sporangiophores enlarged, forming vesicles 300-400 um long. Pedicels
formed synchronously over the surface of the vesicles, the tips of which ballooned
outward to form the sporangia. Two morphologically distinct types of unispored

sporangia were formed and designated spherical- and elongate-sporangia (S- and

12.

13.

14.

73
E-sporangia, respectively). These sporangia were formed in a highly regular

spatial arrangement, alternating between the two sporangial types.

The simultaneous formation of the S-sporangia and E-sporangia, and their highly
regular spatial arrangement on a single vesicle is unique in the Zygomycetes. This
pattern of development allows for a comparison of ultrastructural development and
possible genetic control mechanisms. The extremely regular structural
organization in the alternation of the S- and E-sporangia is analogous to the
determinate developmental organization of members of the Laboulbeniales -
though easier to grow. Thus Mycotypha provides a good subject for the study of

subcellular organization and spatial recognition.

LTSEM provided excellent preservation of fragile reproductive structures free
from the dehydration artifacts common with conventional chemical fixation of

fungal structures.

Z. heterogamus was preserved using nine different freeze-substitution fixation
protocols for transmission electron microscopy (TEM) observation. Specimens
were frozen with either propane jet freezing or high pressure freezing. Both
methods of freezing offered acceptable results, but a greater quantity of well
frozen material was obtained with high pressure freezing. Comparison of freeze
substituted material with conventionally fixed material revealed that the

observation of cytoplasmic ultrastructural details was better with conventional

15.

74

fixation. In general, freeze-substitution provided poor membrane preservation
providing few identifiable organelles. The only exception was found in the
observation of the multiple wall layers of fully matured zygospores, which prior

to this work had never been reported.

A bibliography of material on zygosporogenesis since the first report of
zygospores by Ehrenberg, 1829, was compiled. This bibliography contains
references to all known morphological and developmental studies on sexual
reproduction in the Zygomycetes, sensu lato, though some references are more
oriented towards physiology. Additionally, where known, author’s full names
were used in order to present an historical perspective to the development of

mycology.

APPENDIX A: CRYOPRESERVATION AND FREEZE-SUBSTITUTION

0F MUCORACEOUS FUNGI

ABSTRACT

Direct observation of fully hydrated specimens of Zygorhynchus heterogamus was
made using low temperature scanning electron microscopy (LTSEM). LTSEM provided
excellent preservation of fragile reproductive structures free from the dehydration artifacts
common with conventional chemical fixation of fungal structures. Fungal material was
preserved with freeze-substitution using nine different fixation protocols for transmission
electron microscopy (TEM) observation. Specimens for freeze-substitution were frozen
with either propane jet freezing or high pressure freezing. Both methods of freezing
offered acceptable results, but a greater quantity of well frozen material was obtained with
high pressure freezing. Comparison of freeze substituted material with conventionally
fixed material revealed that the observation of cytoplasmic ultrastructural details was
better with conventional fixation. In general, freeze-substitution provided poor membrane

preservation and few identifiable organelles. The only exception was found in the

75

76

observation of the multiple wall layers of fully matured zygospores, which prior to this

work had never been reported.

INTRODUCTION

During the past decade, the use of cryopreservation techniques for both the
observation of frozen hydrated samples using low temperature scanning electron
microscopy (LTSEM), and the techniques of ultrarapid freezing followed by freeze-
substitution for the preparation of biological materials for transmission electron
microscopy (TEM) have gained wide acceptance (Gilkey & Staelin, 1986; Menco, 1986;
Dahl & Staelin, 1989). Whereas excellent results for some samples have been obtained,
the techniques are still being perfected for broader applications. The variety of techniques
and protocols reported to date has been limited, even though widely diverse samples have
been examined. The diversity of specimen preparation protocols used for conventional
chemical fixation will be required for cryopreservation as well. A "standard" technique
and set of methods for all specimens will not suffice for cryopreservation, just as they

have not been adequate for conventional fixation.

Since cyropreservation and freeze-substitution were introduced for fungi (Howard
& Aist, 1978), a concentration of osmium tetroxide (1-2%) in acetone is the almost
exclusive substitution fixative reported. For a number of tissue types this protocol

produces very good results in the fungal classes Ascomycetes (Czymmek & Klomparens,

77

1992), and Basidiomycetes (Mims, et al., 1988). When the same protocol was followed
for Zygorhynchus heterogamus, a member of the fungal class Zygomycetes, however, the
results were far from optimal. As I began to explore a diverse set of protocols for
cryopreserving and freeze-substituting these fungi, I gained some new insights into freeze-
substitution and report them here to provide a basis for continuing avenues of

experimentation and further development of cryopreservation techniques.

MATERIALS AND METHODS

Fungal Cultures

Zygorhynchus heterogamus Vuillemin was cultured on 1.8% V-8 juice agar at 21-

24°C with 12 hr light and 12 hr dark for 3-5 days.

Low Temperature-SEM

Blocks of agar, 20 x 8 x 3mm, containing the desired stage or stages of growth
were cut from actively growing fungal cultures. These blocks of agar were mounted on
the Emscope SP 2000 cryo-unit specimen stage using Tissue-Tek II O.C.T. cryo-
embedding (Lab-Tek Products, Miles Laboratories, inc.) medium mixed with powdered
graphite. The specimens were plunge frozen in a freshly thawed liquid nitrogen slurry,

and placed under vacuum. Samples were either fractured or kept whole, transferred to

78
a JEOL 35CF SEM with the Emsc0pe SP 2000 cryo-stage, and sublimated at -60°C under

high vacuum (1045 torr) to remove surface ice. Etched specimens were transferred back

to the Emscope cryo-unit, sputter coated with gold, and returned to the SEM. All
observations were made with a specimen temperature of -84 to -110°C, and an

accelerating voltage of 10 kV.

Freeze-substitution for TEM

Specimens were prepared according to the following general protocol. A variety
of freezing and solvent/fixative combinations were used (Table 1):

(1) Specimens were ultra-rapidly frozen with either an RMC MF 7200 Propane
Jet Freezing Unit or a Balzers HPM 010 High Pressure Freezing unit.

(2) Samples were incubated in a solvent/fixative solution at -85°C for
approximately 72 hours.

(3) Samples were rinsed with fresh substitution solvent at -85°C prior to warming
to room temperature.

(4) Samples were warmed to room temperature (-20°C, -4°C, +4°C, +22°C in 0.5
hour steps) and rinsed with the substitution solvent to remove excess fixative (1 hour).
Some samples were then en bloc stained with 0.5% uranyl acetate (W:V) in the fixation
solvent.

(5) Samples were infiltrated (IO-25% steps, fixation solventzresin) with either
Spurr’s hard (1967) or Quetol 651/NMA (Kushida, 1975) hard resin and polymerized.

(6) Polymerized specimen blocks were sectioned and post-sectioned stained with

79

aqueous 0.5% uranyl acetate and Reynolds’ (1963) lead citrate.

Table 1. Solvent and fixative combinations used for freeze-substitution.

 

 

 

    

 

 

 

 

 

 

 

 

 

J E __
RMC MF 7200 Balzers HPM 010
Freeze-substitution Propane Jet High Pressure
l Solvent and Fixative(s) Freezing Unit Freezing Unit1
(I) 2% OsO4 and 0.05% uranyl acetate Figs. 65 & 7°
in acetone
(H) 2% OsO4 in methanol Figs. 85 & 95
(III) 1.0% OsO4 and 0.25% uranyl Fig. 105
acetate in methanol II
(IV) 0.5% 0504 in acetone Fig. 11°
(V) 0.1% 0304 in acetone Fig. 12°
(VI) 0.025% 0504 in acetone Fig. 13°
(VII) 1.7% glutaraldehyde in acetone Fig. 14°
(VIII) 0.5% uranyl acetate in methanol Fig. 15°
(IX) 0.5% barium permanganate in Data not
acetone presented
E

 

 

 

 

 

 

s - Embeddment in Spurr’s hard
0 - Embeddment in Quetol 651/NMA

The specimen holder for the RMC MF 7200 Propane Jet Freezing Unit measured
a total volume of 0.05mm deep and 1mm in diameter, and the specimen holder for the
Balzers HPM 010 High Pressure Freezing Unit measured a total volume of 0.6mm deep

and 1 mm in diameter.

Preparation of the solvent/fixative was conducted by adding the fixative to

‘ This was carried out at Miami University, Oxford, Ohio.

80

precooled (-85°C) solvent. Substitution was carried out in a precooled (-85°C) RMC
three-welled aluminum substitution chamber (Figure 1). One chamber was filled with a
mixture of the solvent and a desiccant (oven-dried Drierite or calcium sulfate) as a
moisture scavenger (Czymmek and Klomparens, 1992). The second chamber was half
filled with the solvent/fixative solution and the samples. The third chamber was either
filled with additional samples for substitution or additional dehydrant mixture depending

on the number of samples.

81

Fig. 1. Illustration of RMC three-welled aluminum freeze-substitution chamber.

.4.’ "'
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y W i
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.4?- .~.' 0 K

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b
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Specimen
Chamber

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82

RESULTS

Low Temperature Scanning Electron Microscopy

There are numerous developmental features during reproduction in Zygorhynchus
heterogamus which are extremely fragile or ethereal and are easily lost or destroyed by
the physical stresses of immersion fixation, and critical point drying. Low temperature-
SEM was used to better preserve these fragile structures and provide an overview to the
morphological development of sexual reproduction in the fungus Z. heterogamus. The
general developmental scheme for sexual reproduction is highly complex and involves a
number of morphological changes which are detailed in Chapter 1. Low temperature-

SEM provided excellent preservation at all stages of development.

Preservation of the thin walled progametangia and the highly delicate initial
contact between the two progametangia was clearly observed (Fig. 2). The enlarged
gametangia, and indications of the internal gametangial walls and the fusion wall were
observed with little signs of dehydration collapse, as the walls remained smooth and
unwrinkled (Fig. 3). A nearly mature zygospore whose walls had ruptured through the
exterior zygosporangium wall appeared to be well preserved (Fig. 4). Remnants of the

zygosporangium wall were found still attached to the warty omamentations of the

83

Figs. 2-16. Details of specimen preparation techniques used in the study of
zygosporogenesis in Z. heterogamus.

Figs. 2-5. Low temperature scanning electron microscopy; bars = 10 um. Fig. 2. Fragile
initial contact of the zygophore (Zp) with the sexual hypha (SI-I). Fig. 3. Expansion and
delimitation of the gametangia by the formation of gametangial septa. Fig. 4.
Preservation of a mucoidal layer and remnants of the zygosporangial wall after rupture
by the developing warty layer. Fig. 5. Fracture of an early post-cleavage sporangium
showing preservation of internal matrix supporting the spore protoplasts.

Figs. 6-15. TEM of freeze-substituted preparations following either propane-jet freezing
or high-pressure freezing. Figs. 6 & 7. Fixation with 2% 0504, and 0.5% uranyl acetate
in acetone (protocol I), following high pressure freezing, resulted in little freezing
damage; bars = 1 Ian.

 

84

zygospore wall, as well as indications of the presence of a mucilaginous material. At all
observed stages of development preservation of fine crystalline-like spines covering the
fungal surfaces was noted. With deep-etching of the specimens it was determined that

these spines were real structures and not ice crystal artifacts.

Fracturing of frozen aerial fungal structures was obtained (Fig. 5). These
specimens, due to their aerial nature, extending up to 8 mm from the culture surface, had
to be embedded in the Tissue-Tek mounting media for support and then frozen. This
procedure revealed the arrangement of internal developing asexual spores within the
developing asexual sporangium. Preservation of a matrix material supporting the

immature spores as well as organelles within the spores themselves was observed.

Freeze-substitution For TEM

A diverse series of freeze-substitution techniques and fixation protocols were used
in an attempt to optimize ultrastructural preservation for TEM with a variety of results.
Fixation with 2% 0804, and 0.5% uranyl acetate in acetone (protocol 1, Table 1),
following high pressure freezing, resulted in little freezing damage (Figs. 6 & 7).
Embedment in Spurr’s resin (Fig. 6) or Quetol 661 (Fig. 7) was compared. The fixation
preserved a generally finely granular matrix. Very few regions of membrane preservation

were observed, rather regions of electron-transparency, where membranes were assumed

85

to have been located, were seen. Ribosomes were clearly identifiable, as were nuclei due
to the staining of chromatin material. Mitochondria were identified by an arrangement
which appeared to once have been lamella with the absence of membranes. Likewise,
endoplasmic reticulum was identified as ribbon-like electron-transparent regions. Lipids
were stained but appeared as electron-transparent regions due to exposure correction for
the cytoplasmic density. No other organelles were identifiable. The nearly mature
zygospore walls exhibited excellent preservation, and darkly stained multiple wall layers
were observed. Minimal separation between the walls and the plasmalemma was noted.
The Quetol resin (Fig. 7) offered improved contrast over the Spurr’s resin (Fig. 6), and

showed fewer separations between the walls and plasma membranes.

Fixation with 2% 080‘ in methanol (protocol 11), following propane jet freezing,
with embedment in Spurr’s resin, failed to preserve the majority of the interior of fully
mature zygospores and was presumed to be due to inadequate ultrarapid freezing of the
large zygospores (Figs. 8 & 9). Some of the lipid material was inadequately fixed and
leached out of the sections and appeared as uniformly distributed contaminations on the
surface of the sections. However, some of the lipid material, closer to the periphery of
the zygospores, was preserved and exhibited staining typical of conventional fixation.
What appeared to be separation of the plasmalemma and the zygospore wall (Fig. 8) was
determined to be a previously undescribed interior, electron-translucent wall layer, present
in fully mature zygospores. Few membrane regions were preserved and few clearly

identifiable organelles were noted. Excellent preservation and staining of the multi-

layered walls was observed (Fig. 9).

86

Figs. 8 & 9. Fixation with 2% 0504 in methanol (protocol 11), following propane jet
freezing, with embedment in Spurr’s resin. Fig. 8. This method failed to preserve the
majority of the interior of fully mature zygospores. This lack of preservation was
presumed to be due to inadequate ultrarapid freezing of the large zygospores; bar = 10
um. Fig. 9. Excellent preservation of the wall ultrastructure; bar = 0.5 pm. Fig. 10.
Fixation with 1% 0304, and 0.25% uranyl acetate in methanol (protocol III), following
propane jet freezing, with embedment in Spurr’s resin resulted in membrane preservation,
but membranes appeared highly wrinkled and distorted; bar = 1pm. Fig. 11. Fixation with
0.5% OsO4 in acetone (protocol IV), following high pressure freezing, with embedment
in Quetol resulted in a finely granular cytoplasm with uniform fixation, and high contrast
between organelles and the cytoplasm; bar = 2 am. Fig. 12. Fixation with 0.1% 0804 in
acetone (protocol V), following propane jet freezing, with embedment in Quetol resin
resulted in poorer fixation than that of higher concentrations of 0804 as seen in lower
cytoplasmic density, and a general distorted and "fuzzy" appearance of the organelles; bar
= 2 am. Fig. 13. Fixation with 0.025% OsO4 in acetone (Protocol VI), following propane
jet freezing, with embedment in Quetol again resulted in very poor fixation and low
contrast within the cytoplasm, and the cytoplasm in general exhibited a "splotchy" nature;
bar = 1 ,um. Fig. 14. Fixation with 1.7% glutaraldehyde in acetone (protocol VII),
following propane jet freezing, with embedment in Quetol produced good fixation in
general, high contrast, numerous identifiable organelles, and some membrane preservation;
bar = 2m.

t:
Mm W
F .Ill

nit

 

87

Fixation with 1% OsO,, and 0.25% uranyl acetate in methanol (protocol III),
following propane jet freezing, with embedment in Spurr’s resin resulted in membrane
preservation, but membranes appeared highly wrinkled and distorted (Fig. 10). In general,
the cytoplasm exhibited gross distortion of ultrastructural morphology with very few
identifiable organelles. Lipids, in general, were identifiable by their uniform electron-
translucent staining comparable to conventional fixation, but some lipids did exhibit non-
uniform fixation. Wall preservation was good, with excellent preservation of the

plasmodesmata perforating the gametangial septa.

Fixation with 0.5% 0904 in acetone (protocol IV), following high pressure
freezing, with embedment in Quetol resulted in a finely granular cytoplasm with uniform
fixation, and high contrast between organelles and the cytoplasm (Fig. 11). Membranes
were not observed, although regions where their presence was expected were seen as
unstained, electron-transparent areas. Areas identified as endoplasmic reticulum were
explicitly noted. Nuclei were presumptively identified by their size and number as were
mitochondria. Lipids appeared as highly electron-transparent areas. Few separations

between the plasmalemma (cytoplasm) and the walls were observed.

Fixation with 0.1 % 0804 in acetone (protocol V), following propane jet freezing,
with embedment in Quetol resin resulted in poorer fixation than that of higher
concentrations of OsO, (protocols I-IV) (Fig. 12). Membranes were not fixed, and the

definition between organelles and the surrounding cytoplasm was indistinct. Lipids were

88

unstained, and nuclei were questionably identified by their size and staining pattems
which appeared to distinguish chromatin material. Based on size and staining
characteristics, a diverse number of additional organelles were apparent, though were not
specifically identifiable. All organelles appeared to be slightly distorted in shape. Walls

were darkly stained, but few details, such as multiple layers, were observed.

Fixation with 0.025% 080, in acetone (Protocol VI), following propane jet
freezing, with embedment in Quetol again resulted in very poor fixation and low contrast
within the cytoplasm, and the cytoplasm in general exhibited a "splotchy" nature (Fig.
13). Lipids were identifiable and had a uniform electron-translucence comparable to

conventional fixation. No other organelles were identifiable.

Fixation with 1.7 % glutaraldehyde in acetone (protocol VII), following propane
jet freezing, with embedment in Quetol produced good fixation in general (Fig. 14). The
cytoplasm had high contrast, and numerous organelles were identifiable. Some regions
of membranes were preserved, most notably within areas of endoplasmic reticulum.
Nuclei were identifiable, and exhibited staining of nucleoli and chromatin material.
Lipids were identifiable and showed a uniform electron-translucency comparable with
conventional fixation. Few separations between the plasmalemma and the walls were

noted.

Fixation with 0.5% Uranyl acetate methanol (protocol VIII), following high

pressure freezing, with embedment in Quetol resin resulted in very low contrast in the

89

cytolplasm and a general fuzziness in appearance (Fig. 15). Membranes appeared to be
fixed, but were indistinct. Putative endoplasmic reticulum was identifiable, but the
tripartite structure of their membranes was not observable. Nuclei were identifiable by
the staining of their chromatin material and size. Lipids were also identifiable and had
a generally uniform electron-translucency comparable to conventional fixation. The
organelles were apparently distorted in their size, shape and overall appearance, and, other
than those mentioned above, could not be conclusively identified. Walls were darkly
stained but revealed little structural details. Few separations between plasmalemma and

the walls were noted.

Fixation with 0.5% barium permanganate in acetone (protocol IX), following
propane jet freezing, with embedment in Quetol resin was carried out, but data are not
shown. No ultrastructural details were observable. Walls were identifiable, however no
specific organelles were distinguishable. Gross distortions of the cytoplasm were
observed, with no membranes visible. Contrast was low within the tissue. Sectioning of
this material was poor, and numerous electron-opaque contaminations were noted in the

sectioned material (data not shown).

In comparison, conventional immersion fixation with aqueous 1%
formaldehyde and 2% glutaraldehyde in 0.05M sodium cacodylate buffer followed
by 1%OsO4 and embedment in Spurr’s resin resulted in generally good fixation, with
good contrast, and clearly defined membranes which exhibited a tripartite structure (Fig.

16). Membranes did exhibit a slightly wrinkled appearance, and organelles were not as

90

Fig. 15. Fixation with 0.5% Uranyl acetate methanol (protocol VIII), following high
pressure freezing, with embedment in Quetol resin resulted in very low contrast in the
cytoplasm and a general fuzziness in appearance; bar = 1am. Fig. 16. Conventional
immersion fixation with aqueous 1% formaldehyde and 2% glutaraldehyde in 0.05M
sodium cacodylate buffer followed by 1%OsO4 and embedment in Spurr’s resin resulted
in generally good fixation, with good contrast, and clearly defined membranes; bar = 1am.

 

91

smooth in circumference as those observed in the best freeze-substituted material. Nuclei
containing chromatin material, mitochondria and their lamella, endoplasmic reticulum,
numerous vesicles, and microtubules were positively identifiable. Lipids were identified
and observed to frequently contain a non-uniformly distributed, darkly staining granular
material in the early to mid- stages of zygospore development. Few separations between
the plasmalemma and the walls were noted. Stages beyond the early zygospore wall

formation exhibited poor internal fixation, and poor infiltration of the embedding resin.

DISCUSSION

In working with the freeze-substitution of our fungal samples I recalled the
statement that is taught to all beginning biological microscopists: the initial steps in
sample preparation are first ’killing’ and, second, fixation. These two steps are often
combined both practically and intellectually when experiments use chemical immersion
techniques. With the use of some combination of an aldehyde and/or osmium tetroxide
in the initial preparation step both ’killing’ and fixing occur somewhat simultaneously
during conventional liquid chemical fixation. However, when using ultra-rapid freezing
followed by freeze-substitution, this is not always the case. ’Killing’ and fixation may
occur separately. Consequently there are two distinct sources of artifact: ice-crystal

damage and fixation artifact.

In freeze-substitution, ’killing’ occurs with the cessation of all metabolic processes

92

and relies on thermal kinetics with the removal of enough heat energy from the system
to stop all the biological functions of the cells. Under these conditions the term ’killing’
is a misnomer, as can be shown in the examples of long term cryogenic storage of cell
and tissue cultures. The use of the term ’immobilization’ far better describes this process.
Since immobilization occurs ultrarapidly (104 seconds, Gilkey and Staehlin, 1986) in
relation to biological processes, even with high pressure freezing (10’1 seconds), ultra-
rapid freezing provides one of the best methods for the preservation of biological
specimens. Further, the immobilization itself results in no ultrastructural artifacts, since
it involves only energies and not physical matter (i.e. changes in any atomic or molecular
structures). The main artifact associated with this freeze-immobilization is the formation
of ice crystals, which is related to the method and rate of freezing. These artifacts have
been thoroughly covered in numerous published papers and shall not be duplicated here

(for reviews see Gilkey and Staehlin, 1986, Menco, 1986, and Dahl and Staehlin, 1989).

The second major artifact associated with freeze-immobilization occurs during
handling and loading of the specimens into the holders for various ultrarapid freezing
devices. The samples can easily suffer physical damage and distortions prior to
immobilization. It has been our observation that the best freezing of the samples occurred
when the samples were tightly packed in the sample holders, eliminating all the gas
pockets. Unavoidably, this tight packing may cause some physical damage and distortion

to the specimens.

The primary set of artifacts of concern in this paper are those associated with the

93

fixation of the specimens. As with chemical immersion fixation, there is no universal
fixation protocol for freeze-substitution of all samples. Each sample type and
experimental condition, due to its unique physiological and biochemical composition, can
require a reconfiguration of solvent, fixative(s), fixative(s) concentration and duration of

-85°C incubation in order to obtain the optimal preservation.

Prior to the use of the enclosed RMC three-welled aluminum substitution chamber,
individual polypropylene vials were used. Holes were made in the lids of these vials in
order to accommodate pressure changes within the vials with changes in temperature.
These vials were fitted into an aluminum block in which appropriate sized holes were
drilled. The aluminum block was used in order to facilitate transport of the samples to
and from the -85°C freezer, and to act as a heat sink during transport. Filling of the vials
with fixative and samples was done in a dry-ice (solid carbon dioxide) filled styrofoam

box in order to maintain temperature.

Two major problems were encountered with the use of the vials for freeze-
substitution. Firstly, too much water was absorbed by the substitution fluid during freeze-
substitution, resulting in poor sample dehydration. Secondly, too much carbon dioxide
(from the dry-ice) was absorbed by the substitution fluid which resulted in effervescence
of the fixation fluid to the point of boiling over and/or spitting through vent holes as the
samples were warmed to room temperature. Both of these conditions were considered
undesirable and/or dangerous. Use of the larger aluminum substitution chamber with the

additional desiccant alleviated these problems, and was found to be far easier to work

94

with.

Comparisons of Spurr’s hard resin with that of Quetol 651/NMA resin showed
slightly better results with the Quetol. The Quetol resin has a lower viscosity than the
Spurr’s resin and thus provided improved ease of infiltration with less structural damage,
i.e. fewer separations of the cytoplasm from the surrounding wall material. The Quetol
resin showed very low background electron density, good staining qualities and provided
excellent specimen contrast. Although the Quetol 651/NMA resin was found to be
slightly more brittle than the Spurr’s hard resin, no problems with trimming or sectioning
were encountered. With the exception of cost, the Quetol 651 resin was believed to be

superior to the Spurr’s resin for the preparation of Zygorhynchus heterogamus.

To date only one paper has been published in which freeze-substitution of a
member of the zygomycetes has been reported (Heath & Rethoret, 1982). This work dealt
with the preservation of microtubules associated with mitosis in the fungus, Z. moelleri.
Freeze-substitution in this work was done via plunge freezing in liquid-nitrogen-cooled
liquid propane followed by substitution at -77°C with 2.5% osmium tetroxide and 0.1%
uranyl acetate in acetone. Specimens were then rinsed with acetone and embedded in
Epon. This work reports that freeze-substitution provides better preservation of
microtubules when compared with conventional immersion fixation, but that little else was
preserved by freeze-substitution including nucleus-associated organelles (NAOs) and
nuclear envelopes. These authors conclude that for mitotic studies in " any organism" both

conventional fixation and freeze-substitution should be used.

95

The work reported by Heath and Rethoret correlates very well with the present
study of Z. heterogamus. The preservation of walls via freeze-substitution was believed
to be superior to that of conventional fixation. With conventional fixation, during the
later stages of zygosporogenesis the development of an extremely thick wall limited the
penetration of fixatives and/or embedding resin yielding poor specimens for observation.
The freeze-substitution of Z. heterogamus in this work has revealed for the first time the

details of the multi-layered wall structure of fully mature zygospores (Chapter 1).

Lipids were well preserved, having an appearance and electron-opacity very
similar to those found in conventionally fixed specimens. However, they appear lighter
in the micrographs presented here as an artifact of photographic enlargement and contrast.
Due to the overall density and low contrast of the cytoplasm preserved with freeze-
substitution, the micrographs shown here have been printed lighter and with high contrast
in order to show general cytoplasmic details, thus artificially lightening the lipids and
giving them the appearance of being electron-transparent or unstained. It is important to
note that lipids were indeed preserved, but that selective printing to highlight cytoplasmic

details forfeits the obvious staining of the lipids.

In comparison of the freezing methods used, high pressure freezing yielded larger
quantities of well-frozen material with less preparation damage than did propane jet
freezing. Using either technique, it was found that the best preservation of material was
obtained when the total volume of the sample holders was completely filled with material.

Leaving air spaces within the sample matrix appeared to cause damage to the samples due

96

to vibration of the specimens during freezing, resulting in broken frozen tissue. Whereas
high pressure freezing provided the largest quantity of frozen material, propane jet
freezing did yield quite acceptable results. Due to the high equipment cost of high
pressure freezing (approx. $150 k), propane jet freezing ($35 k) should be considered as

a viable alternative.

A comparison of the freeze substitution techniques used with conventional
immersion fixed material shows that ultrastructural details of the cytoplasmic material are
best preserved with conventional fixation, with the exception of the wall layers. The
question that remains is, why? In similar comparisons of specimens from the fungal
classes Ascomycetes, Basidiomycetes, Chytridiomycetes, and Oomycetes freeze-
substitution yields generally better preservation than conventional fixation. In general,
for all tissue structures examined in Z. heterogamus, including asexual, sexual and
vegetative hyphae and structures, conventional fixation yielded superior usable
ultrastructural cytoplasmic details. Freeze-substitution did give smooth organelle profiles,
but, poor membrane preservation with freeze-substitution limited organelle identification
and the subsequent determination of cytoplasmic events. Very similar results have been
found with several members of the genus Caemansia (K. L. Klomparens, personal

communications).

The only possible explanation that can be offered for the preservational differences
between the Zygomycetes and the other fungal classes is some difference in physiological

makeup of members of the Mucorales from species in other fungal classes. However, no

97

direct evidence can be presented to support this proposal. It is necessary to keep this in
mind when using freeze-substitution for any new specimens, and that various permutations

on fixation protocols that have worked for similar samples may need to be tried.

Of the freeze-substitution protocols tried, the best results were obtained with 2%
osmium tetroxide, 0.05% uranyl acetate in acetone with embeddment in Quetol 65 UN MA
resin for the preservation of Z. heterogamus. However, I would hesitate to say that these
are the best results possible. In order to seek a better fixation protocol, I would suggest
that other fixatives be sought out and tried. And I suggest that "fixatives" other than
those commonly associated with electron microscopy be experimented with, even
chemicals not generally considered to be fixatives. There is still debate within the
literature as to how and when "fixation" takes place with freeze-substitution, and in
considering a new fixative it would be absurd to accept a foregone conclusion of "well,

that can not possibly work.”

98

ACKNOWLEDGEMENTS

I would like to acknowledge Connie Bricker and Laura Sadowski at Miami
University, Oxford, OH, for their assistance with the Balzers high pressure freezing unit,

and Martha Powell and Allen Allenspach for their generosity in allowing my use of the

Balzers (NSF DIR 88-20387 grant to MP and AA).

LITERATURE CITED

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ascospore initial development. Can. J. Bot. 70: 1669-1683.

Dahl, R., and L. A. Staelin. 1989. High-pressure freezing for the preservation of

biological structure: theory and practice. J. Electron Microsc. Tech. 13: 165-174.

Gilkey, J. C., and L. A. Staelin. 1986. Advances in ultrarapid freezing for the

preservation of cellular ultrastructure. J. Electron Microsc. Tech. 3: 177-210.

99
Heath, I. B., and K. Rethoret. 1982. Mitosis in the fungus Zygorhynchus moelleri:

evidence for stage specific enhancement of microtubule preservation by freeze

substitution. Europ. J. Cell Biol. 28: 180-189.

Howard R. J., and J. R. Aist. 1978. Hyphal tip cell ultrastructure of the fungus
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224-234.

Kushida, H. 1975. Hardness control of the quetol 651 cured block. J. Electron

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Menco, B. P. M. 1986. A survey of ultra-rapid cryofixation methods with particular
emphasis on applications to freeze-fracturing, freeze-etching, and freeze-

substitution. J. Electron Microsc. Tech. 4: 177-240.

Mims, C. W., R. W. Roberson, and E. A. Richardson. 1988. Ultrastructure of freeze-
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Reynolds, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in

electron microscopy. J. Cell Biol. 17: 208-212.

100

Spurr, A. J. 1969. A low-viscosity epoxy resin embedding medium for electron

microscopy. J. Ultrastructural Research, 26: 31-43.

APPENDIX B: BIBLIOGRAPHY OF ZYGOSPOROGENESIS LITERATURE SINCE 1820

The following is a bibliography of literature primarily covering the morphology
and development of sexual reproduction in the Zygomycetes, although some citations are
more oriented toward physiology. According to A. de Bary (1864), A. F. Blakeslee
(1904) and M. L. Keene (1914), C. G. Ehrenburg in 1820 published the first mycological
account of the observation of zygospores. Because of their better historical perspective,
rather than mine nearly a century later, I have no reason to question their accuracy.
Wherever possible, the full names of authors are included in order to present an historical

perspective of the development of mycology.

Since the illustrations of older publications were often not interleaved with the text
of the article, where known, the illustrative plates referenced by an article are included
in the citation. On a number of occasions I received copied articles from other
institutions in which the plates were not included because they were not specifically cited
in my interlibrary requests. Although I have tried to be comprehensive, I do not claim
that this list is 100% complete. This material is primarily of European and North
American origins. The years 1830-1880 are particularly problematic and may very well

be incomplete.

101

102

Alvarez, Maria Isabel, Maria Isabel Pelaez, and Arturo P. Eslava. 1980.
Recombination between ten markers in Phycomyces. Molec. Gen. Genet. 179:
445-452.

Alvarez, M. 1., and A. P. Eslava. 1983. Isogenic strains of Phycomyces blakesleeanus
suitable for genetic analysis. Genetics 105: 873-879.

Ansell, Penelope J. 1982. A simple technique for Studying the in situ development of
zygospores. Trans. Br. Mycol. Soc. 79: 554.

Ansell, P. J., and Young, T. W. K. 1983. Light and electron microscopy of Mortierella
indohii zygospores. Mycologia 75: 64-69.

Atkinson, George Francis. 1912. The morphology of Zygorhynchus and its relation to
the Ascomycetes. Science 25: 151.

Bainier, G. 1882. Etudes sur les Mucorinées. These pre’sentée a I’Ecole de Pharmacie,
Paris. 136 p., 11 pls.

Bainier, G. 1883. Observations sur les Mucorinées. Annales des sciences Naturelles
bot. 6(15): 70-104, pls. 4-6.

Bainier, G. 1883. Sur les zygospores des Mucorinées. Annales des sciences Naturelles
bot. 6(15): 342-356, pls. 17-19.

Bainier, G. 1884. Nouvelles observations sur les zygospores des Mucorinées. Annales
des Sciences Naturelles bot. 6(19): 200-216, pls. 7-10.

Bainier, G. 1989. Absidia caerulea. Bull. Soc. bot. de France 36: 184-186.

Bainier, G. 1903. Sur quelques espéces de Mucorinées nouvelles ou peu connues. Bull.
Soc. Myc. de France 19: 153-172, pls. 6-7.

Bainer, G, and A. Sartory. 1913. Etudes morphologique et biologique de Muratella
elegans n. sp. Bull. Soc. Myc. de France 29: 129-136. 3 pl.

Baird, E. A. 1924. The structure and behavior of the nucleus in the life history of
Phycomyces nitens (Agardh) Kunze and Rhizopus nigricans Ehrbg. Trans. Wisc.
Acad. Sci. Arts Lett. 21: 357-380.

Barbara Price, B. A. 1927. Recherches sur les especes élémentaries dans le genre
Mucor (Mucor hiemalis). Bull. Soc. Bot. Genéve 2(19): fasc. 1.

Barnett, Horace L. and V. G. Lilly. 1956. Factors affecting the production of
zygospores by Choanephora cucurbitarum. Mycologia 48: 617-627.

103

Barnett, H. L., V. G. Lilly, and R. F. Kraus. 1956. Increased production of carotene
by mixed + and - cultures of Choanephora cucurbitarum. Science 123: 141.

de Bary, Heinrich Anton. 1864. Beitréige zur Morphologie und Physiologic der Pilze.
I. Syzygites megalocarpus. Abhandlungen der Senckenbergischen
Naturforschenden Gesellschaft 5:74-88, pls. 7-10.

de Bary, H. A. 1864. Beitrage zur Morphologie und Physiologic der Pilze. I. Syzygites
megalocarpus. Abhandlungen der Senckenbergischen Naturforschenden
Gesellschaft 5: 210-232, pls 30-31.

de Bary, H. A. 1865. Zur Kenntnis der Mucorineen. Abhandlungen der
Senckenbergischen Naturforschenden Gesellschaft 5: 345-375.

de Bary, H. A., and Michael Stepanovitch Woronin. 1866. Zur Kenntnis der
Mucorineen. Beitrage zur Morphologie und Physiologic dcr Pilze II. p.13-34, pls.
5-7.

de Bary, H. A., and M. S. Woronin. 1890. Beitrfige zur Morphologie und Physiologic
der Pilze. Dritte Reihe. Abhandlungen der Senckenbergischen Naturforschenden
Gesellschaft, Bd. 11, pp.133.

Beckett, A., I. B. Heath, and David J. McLaughlin. 1974. An atlas of fungal
ultrastructure. Longsman, London.

Benjamin, Chester R. and Clifford W. Hesseltine. 1959. Studies on the genus
Phycomyces. Mycologia 51: 751-771.

Benjamin, Richard K. 1959. The merosporgiferous Mucorales. Alisa 4: 321-433.
Benjamin, R. K. 1961. Addenda to "the merosporgiferous Mucorales". Alisa 5: 11-19.
Benjamin, R. K. 1962. A new Basidiobolus that forms microspores. Alisa 5: 223-233.

Benjamin, R. K. 1963. Addenda to "the merosporgiferous Mucorales" II. Alisa 5: 273-
288.

Benjamin, R. K. 1965. Addenda to "the merosporgiferous Mucorales" III. Alisa 6: l-
10.

Benjamin, R. K. 1979. Zygomycetes and their spores. Pp. 573-621. In: The Whole
Fungus. The Sexual-Asexual Synthesis, Vol. 2. Bd., Bryce Kendrick. National
Museum of Natural Sciences, Ottawa, Canada.

104

Benjamin, R. K., and B. S. Mehrotra. 1963. Obligate azygospore formation in two
species of Mucor (Mucorales). Alisa 5: 235-245.

Benny, Gerald L., and Richard K. Benjamin. 1975. Observations on Thamnidiaceae
(Mucorales). New Taxa, new combinations, and notes on sellected species. Alisa
8: 301-351.

Benny, G. L., and R. K. Benjamin. 1976. Observations on Thamnidiaceae
(Mucorales). II. Chaetacladium, Cokeramyces, M ycatypha, Phascolomyces.
Alisa 8: 391-424.

Bergman, K., P. V. Burke, Enrique Cerdé-Olmedo, C. N. David, M. Delbriick, K. W.
Foster, E. W. Goodell, M. Heisenberg, G. Meissner, M. Zalokar, D. S.
Dennison, and W. Shropshire, Jr. 1969. Phycomyces. Bacterial. Rev. 33: 99-
157.

Berkeley, Miles Joseph. 1875. Notices of North American fungi. Grevillea 3: 109.

Berlese, Augusto Napoleone, and J. B. de Toni. 1888. Mucoraceae in Phycomyceteae.
In: Sylloge Fungorum. Bd., Saccardo, P. A., Vol. 7, part 1: 182-233.

Bezssonoff, N. 1920. Sur l’obtention expe’rimentale de la sexualité chez les chamignons
et sur la structure typique du plasma sexuel. Compt. Rend. Acad. Sci. (Paris) 170:
288-290.

Binding, H., and H. J. Weber. 1974. Isolation, regeneration and fussion of Phycomyces
protoplasts. Mal. & Gen. Genet. 123: 1-16.

Black, H. D. 1971. The production, characterization and genetics of mutants of Mucor
hiemalis Wehmer. Ph.D. Dissertation, Univ. Nebraska, Lincoln.

Blakeslee, Albert Francis. 1904. Sexual reproduction in the Mucorineae. Pro. Am.
Acad. Arts Sci. 40: 205-319, pls. 1-4.

Blakeslee, A. F. 1904. Zygospore formation a sexual process. Science, N. S. 19: 864-
866.

Blakeslee, A. F. 1906. Differentiation of sex in thallis, gametophyte, and sporophyte.
Bot. Gaz. 92: 161-178.

Blakeslee, A. F. 1906. Zygospore germinations in the Mucorineae. Ann. Mycol. 4: 1-
28, pls. 1-4.

Blakeslee, A. F. 1906. Zygospores and sexual strains in the bread mold. Science, MS.
24: 118-122.

105

Blakeslee, A. F. 1907. Heterothallism in bread mold, Rhizopus nigricans. Bot. Gaz. 43:
415-418.

Blakeslee, A. F. 1909. Papers on the mucors. Bot. Gaz. 47: 418-423.

Blakeslee, A. F. 1913. Conjugation in the heterothallic genus Zygorhynchus.
Mycologisches Centralblatt. 2: 241-246, 2 pls.

Blakeslee, A. F. 1913. A possible means of identifying the sex of (+) and (-) races in
the mucors. Science, MS. 37: 880-881.

Blakeslee, A. F. 1915. Sexual reactions between hermaphroditic and dioecious mucors.
Biol. Bull. (Woods Hole, Mass.) 29: 87-103.

Blakeslee, A. F. 1920. Sexuality in the mucors. Science 51: 375-382 and 403-409.
Blakeslee, A. F. 1920. Mutations in Mucors. J. Heredity 19: 278-284.

Blakeslee, A. F., and J. L. Cartledge. 1927. Sexual dimorphism in Mucorales. II.
Interspecific reactions. Bot. Gaz. (Chicago) 84: 51-57.

Blakeslee, A. F., J. L. Cartledge, and D. S. Welch. 1921. Sexual dimorphism in
Cunninghamella. Bot. Gaz. (Chicago) 72: 185-219.

Blakeslee, A. F., J. L. Cartledge, D. S. Welch, and A. D. Bergner. 1927. Sexual
dimorphism in Mucorales. I. Interspecific reactions. Bot. Gaz. (Chicago) 84: 27-
50.

Blakeslee, A. F., D. S. Welch, and J. L. Cartledge. 1921. Techniques in contrasting
Mucor. Bot. Gaz. (Chicago) 72: 162-172.

Bonfante-Fasolo, P., and S. Scannerini. 1976. The ultrastructure of the zygospore of
Endagone flammicorona Trappe & Gerdemann. Mycopathologia 59: 117-123.

Bracker, Charles E. 1967. Ultrastmcture of fungi. Annu. Rev. Phytapathol. 5: 343-374.

Brefeld, Oscar. 1872. Zygomyceten. Botanische Untersuchgen tiber Schimmelpilze,
Heft 1. Leipzig. 1: 1-64, pls. 1-6.

Brefeld, O. 1875. Uber copulirende Pilze. Situngber. der Gesellsch. nat. Fr. Berlin: 65-
88; also in Bot. Zeitung (1875) 33: 834-838, 845-853.

Brefeld, O. 1900. chr die geschlechtlichen un ungeschlechtlichen Fruchformcn bei
copulirenden Pilzen. Jahresber. Schles. Ges. f. vaterl. Kultur 78: 71-84.

106

Brefeld, O. 1908. Untersuchungen aus dcm Gesamtgebiete der Mykologie 14. In: Die
Kulture der Pilze. Heinrich SchOningh (Miinstcr, Westphalia).

Breslauer, A. 1912. A propos du dimorphisme sexuel des Mucorinées. Bull. Soc. de
Genéve 2: 288-294.

Bullock, J. D., D. Drake and D. J. Winstanley. 1972. Specificity and transformation
of the trisporic acid series of fungal sex hormones. Phytachem. 11: 2011-2018.

Burgeff, H. 1912. Uber Sexualitat, Variabilitat und Vererbung bei Phycomyces nitens.
Ber. Deut. Bot. Ges. 30: 679-685.

Burgeff, H. 1914. Untersuchen iiber Variabilitat, Sexualitiit und Erblichkeit bei
Phycomyces nitens Kunze I. Teil Flora N. F. 7: 259-316.

Burgeff, H. 1915. Untersuchen iiber Variabilitat, Sexualitfit und Erblichkeit bei
Phycomyces nitens Kunze II. Tiel Flora N. F. 8: 351-448.

Burgeff, H. 1920. Sexualitat und Parasitismus bei Mucorineen. Ber. d. deutsch. bot.
Ges. 38: 318-327.

Burgeff, H. 1920. Uebcr den Parasitismus dcs Chaetacladium und heterocaryotische
Natur der von ihm auf Mucorineen erzeugten Gallen. Z. Bot. 12: 1-35.

Burgeff, H. 1924. Untersuchgen iibcr Sexualitat und Parasitismus bei Mucorineen. I.
Botanische Abhandlungen 4: 1-35.

Burgeff, H. 1925. Uber Arten und Artkreuzung in der Gattung Phycomyces Kunze.
Flora 18-19: 40-46.

Burgeff, H. 1928. Variabilitat, Verebung und Mutation bei Phycomyces blakesleeanus
Bgff. Z. Induktive Abstammungs-u. Vererbung 49: 26-94.

Burgeff, H. and A. Seybold. 1927. Zur Frage der biochemischen Unterscheidung dcr
Geschlcchter. Zeitschr. f. Bat. 19: 497-537.

Burger, Owen F. 1919. Sexuality in Cunninghamella. Bat. Gaz. 68: 134-146.

Burkert, H. 1923. Entwicklung Untersuchgen iiber Mucor strictus Hagem. Thesis,
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Burnet, John H. 1956. Carotene and sexuality in Mucoraceae, especially Phycomyces
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107

Butler, E. E., J. M. Ogawa, and T. Shalla. 1960. Notes on Gilbertella persicaria from
California. Bull. Torrey Bat. Club. 87: 397-401.

Callen, E. O. 1940. The morphology, cytology, and sexuality of the homothallic
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Cerda-Olmedo, Enrique. 1974. Phycomyces. Pp. 341-355. In: Handbook of Genetics,
Vol. 1. Bd., R. C. King. Plenum Press, New York.

Cerda-Olmedo, E. 1975. The genetics of Phycomyces blakesleeanus. Gent. Res.,
Camb. 25: 285-296.

Chien, Chiu-Yuan, E. G. Kuhlman, and W. Gams. 1974. Zygospores in two
Mortierella species with ‘stylospores’. Mycologia 66: 114-121.

Chodat, R. 1896. Experiences relatives 5 l’action des basses temperatures sur le Mucor
mucedo. Bull. de l’Herbier Boissier 4, no. 12.

Chodat, R. and W.-H. Schopfer. 1927. Carotine ct sexualité. Compt. Rend. Séances
de la Soc. Phys. Hist. Nat. Genéve 64: 176-179.

Christenberry, G. A. 1940. A taxonomic study of the Mucorales in the southeastern
United States. J. Elisha Mitchell Sci. Soc. 56: 333-366.

Cocconi, G. 1900. Ricerche intorno ad una nuova Mucorinea del gencre Absidia. Mem.
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Cutter, V. M., Jr. 1942. Nuclear behavior in the Mucorales. II. The Rhizopus,
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Cutting, E. M. 1921. Heterothallism and similar phenomena. New Phytap. 20: 10-16.

Dangeard, Pierre-Augustin. 1894. Researches sur la reproduction sexuclle des
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108

Dangeard, P. A. 1895. Considerations sur les phénoménes de reproduction chez les
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Dangeard, P. A. 1900. La reproduction sexuelle des champignons. Le Botaniste 7: 89-
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Dauphin, J. 1905. Nouvelles recherches sur l’appareil reproducteur des Mucorinées.
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Dunn, G. A. 1921. A comparative study of the two races of Rhizopus nirigans.
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Ehrenburg, C. G. [1820]. 1829. Syzygites cine ncue Schimmelpilzgattung nebst
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Eslava, Arturo P., Maria Isabel Alvarez, Patricia V. Burke, and M. Delbriick. 1975.
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Eslava, A. P., M. I. Alvarez, and M. Delbriick. 1975. Meiosis in Phycomyces. Proc.
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Falck, R. 1901. Die Bedingungen und Bedeutung der Zygotenbildung bei Sporodinia
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Gama, W. 1977. A key to the species of Mortierella. Persoonia 9: 381-391.

Garns, W., and S. T. Williams. 1963. Heterothallism in Mortierella parvispora
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Gams, W., Chin-Yuan Chien, and K. H. Domsch. 1972. Zygospore formation by
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Gauger, Wendell. 1961. The germination of zygospores of Rhizopus stolonifer. Am J.
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Gauger, W. 1965. The germination of zygospores of Mucor hiemalis. Mycologia 57:
634-641.

Gauger, W. 1966. Sexuality in an azygosporic strain of Mucor hiemalis. I. Breakdown
of the azygosporic component. Am. J. Bot. 53: 751-755.

Gauger, W. 1975. Further studies on sexuality in azygosporic strains of Mucor
hiemalis. Trans. Br. Mycol. Soc. 64: 113-118.

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101: 211-217.

Gauger, W. 1984. Genetic analysis utilizing single germ sporangia in two species of
Rhizopus. J. Gen. Appl. Microbial. 30: 337-345.

Gauger, W., Maria Isabel Pelaez, Maria Isabel Alvarez, and Arturo P. Eslava. 1980.
Mating-type heterokaryons in Phycomyces blakesleeanus. Exp. Mycol. 4: 56-64.

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Gooday, G. W. 1969. Hormonal control of sexual reproduction in the Mucor mucedo.
New Phytol. 67: 815-821.

Gooday, G. W. 1973. Differentiation in the Mucorales. Pp. 269-293. In: Microbial
Differentiation. Symp. Soc. Gen. Microbial. Imperial College, London.

Gooday, G. W., P. Fawcett, D. Green, and G. Shaw. 1973. The formation of fungal
sporopollcnin in the zygospore wall of Mucor mucedo: a role for the sexual
carotenogencsis in the Mucorales. J. Gen. Microbiol. 74: 233-239.

Green, Ethel. 1927. The life history of Zygorhynchus moelleri. Ann. Bot. (London) 41:
419-435.

Griiber, E. 1901. Uber das Verhalten dcr Zellkeme in den Zygosporen von Sporodinia
grandis. Bericht d. Deuts. Bot. Ges. 19: 51-56.

Griiber, E. 1912. Einige Beobachtungen uber den Befruchtungsvorgang bei
Zygorhynchus moelleri Vuill. Ber. Deuts. Bot. Ges. 30: 126-133.

Grove, S. N. 1975. Form and function in Zygomycete spores. Pp. 559-592. In: The
fungal spore, farm and function. Eds., D. J. Weber and W. H. Hess. Wiley-
Interscience, New York.

Guéguen, F. 1921. Sur un nouvcl organe diffe’rcncié du thalle des Mucorinées. Compt.
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Hagem, O. 1907. Untersuchen iiber norweigische Mucorineen. I. Videnskabssclskabet
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Hagem, O. 1910. Untersuchungen iiber norweigische Mucorineen. Annales Mycologici
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Hawker, L. E., Patricia McV. Abbott, and Margaret A. Gooday. 1968. Internal
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Hawker, L. E., and A. Beckett. 1971. Fine structure and development of the zygospore
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Hawker, L. E., and M. A. Gooday. 1967. Delimitation of the gametangia of Rhizopus
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