{SENETECS 0F HYBRSDSZAHON AND
ENCC‘MPAEBELETY BETWEEN iSOLATES
0F SORDAREA HMECOLA

“5239333 for the Degree of M. S.
MECE'EEGAN STATE UNIVERSiTY
MIKE-ZN LEE KLOMPARENS
1973

 

 

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Michigan S ‘tc
University

 

      
  

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ABSTRACT

GENETICS OF HYBRIDIZATION AND INCOMPATIBILITY
BETWEEN ISOLATES OF SORDARIA FIMICOLA

BY

Karen Lee Klomparens

Incompatibility and compatibility (hybridization) between

isolates of Sordaria fimicola (Rob.) Cesati and DeNotaris were

 

investigated. Eight wild-type isolates of g, fimicola from seven
different geographic locations and ascospore color mutants from each
were used in this study. Map distances and limited linkage data are
presented for each color mutant and germination data for both wild-
type and mutant isolates is given.

The eight isolates were crossed in pairs in all possible
combinations, employing wild-type as one member of the pair and an
ascospore color mutant as the second member, to determine compatibil-
ity or incompatibility. Presence of hybrid perithecia, where mycelia
from the two isolates intermingled, indicated compatibility; while
incompatibility was assumed when examination of 1000 perithecia,
showed no hybridization.

Two isolates, 11 and 20, were compatible with all other isolates.
Isolates 21 and 23 were compatible with all isolates except 30.
Isolate 18 was compatible with all isolates except 19; 19 was
compatible with all isolates except 6 and 18. Isolate 6 was
compatible with all isolates except 19 and 30.

These initial data were used to determine 11 pairs of isolates

which differed in compatibility with a third isolate denoted Ix'

Karen Lee Klomparens

Isolates 18 and 20 (K8) differed in compatibility with isolate l9 and
were therefore crossed. F1 tetrads from this cross were tested for
compatibility with 1x19. Similarly, progeny from cross 11 X 19 (K12)
were tested for compatibility with Ix18. Other isolate pairs were
examined in the same manner.

F2 tetrads from these K# X Ix crosses could not be isolated for
backcrossing to each K# parent due to poor germination of spores.
Several methods were employed to overcome the reduced germination,

but without success. Poor germination was noted at the parental cross
and F1 X Ix cross levels.

On the basis of the data collected, it was obvious that some
conmon factor(s) between two isolates must be present for compatibil-
ity to be observed. The nature of the segregations from F1 X Ix
crosses indicates that a multiple-locus system must be operating to
confer incompatibility on two paired isolates.

Although a precise incompatibility mechanism cannot be proposed

from the data obtained, it appears that isolates of Sordaria fimicola

 

are evolving into species. This hypothesis correlates with Olive's
(1954, 1956, 1958) and with Esser's (1965, 1971) definition of

heterogenic incompatibility and its consequences.

GENETICS OF HYBRIDIZATION AND INCOMPATIBILITY

BETWEEN ISOLATES OF SORDARIA FIMICOLA

BY

Karen Lee Klomparens

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Botany and Plant Pathology

1973

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ACKNOWLEDGMENTS

I would like to express sincere thanks to Dr. William G. Fields
for his enthusiastic instruction which first stimulated my interest
in the fungi and especially for his encouragement, guidance and
assistance in the research and preparation of this thesis.

Appreciation is also extended to Dr. Edward K103 and Dr. Everett

S. Beneke for serving on my guidance committee.

ii

Introduction . .

Methods and Materials.

Res‘flts. O O O 0
Discussion . . .
Summary. . . . .

Literature Cited

TABLE OF

CONTENTS

iii

Page

24
33

35

LI ST OF TABLES

Table Page

I Source and geographical location of each wild-
type isolate . . . . . . . . . . . . . . . . . . . . . 4

II Linkage data and map distances of each ascospore
color mutant from the centromere of its linkage
group O O O O C O O O O O O O O O O I O O O O O O O O O 9

III Germination of ascospores from wild-type and
ascospore color mutant isolates. . . . . . . . . . . . 10

IV Results of crosses of all isolates in pairwise
combinations . . . . . . . . . . . . . . . . . . . . . 12

V Compatibility and incompatibility of F1 tetrads
from cross 18 X 20 with 1x19 . . . . . . . . . . . . . 16

VI Compatibility and incompatibility of F1 tetrads
from cross 11 X 19 with 1x18 . . . . . . . . . . . . . 18

VII Compatibility and incompatibility of F1 tetrads
from cross 18 X 23 with 1x19 . . . . . . . . . . . . . 20

VIII Res lts of the cross of K8 (18 X 20) tetrads X

19y (st) to determine germinability and
subsequent growth of spores. . . . . . . . . . . . . . 22

iv

INTRODUCTION

Sordaria fimicola (Rob.) Cesati and DeNotaris is a homothallic

 

pyrenomycete belonging to the order Sphaeriales. Members of the
Sphaeriales are characterized by the production of dark, pear-shaped,
ostiolate perithecia which contain unitunicate asci and paraphyses
arising from a hymenial layer (Alexopoulos, 1962). S, fimicola
reproduces solely by sexual reproduction resulting in ascospores; the
ability to produce conidia having been lost or perhaps never existent.
Eight ascospores are arranged in an orderly, linear manner within
each ascus. Normal, wild-type ascospores are dark in color; however,
ascospore color mutants can be obtained through ultraviolet radiation
treatment. Thus, the ascospore color mutants provide a simple, visual
tool for identification of hybrid perithecia (Olive, 1956). These
characteristics, in addition to the short generation time of eight
days, make Sordaria fimicola a good subject for genetic analysis.
Recently, the subject of heterogenic incompatibility and
hybridization in homothallic species of fungi has come under a great
deal of review and discussion (Esser, 1971). The best known example
of heterogenic incompatibility, described by Karl Esser, is in
Podogpora anserina. Indeed, the terms heterogenic and homogenic, as
applied to incompatibility were introduced by Esser (1959).
Heterogenic incompatibility is defined as the prevent of

karyogamy and zygote formation when the two nuclei carry different

incompatibility factors. Heterogenic incompatibility is observed when
different geographical races of the same species are crossed. This
mechanism may be operating in g, fimicola. Although a homothallic
ascomycete, geographical races of g, fimicola, when crossed, fre-
quently produce hybrid perithecia (Fields, 1970). In some crosses,
however, no hybrid perithecia are formed. This observation suggests
the presence of an incompatibility mechanism.

It is this phenomenon that is the subject of this research. What
is the nature of the incompatibility mechanism? What processes are
affected by hybridization? How many loci may be involved in
incompatibility? Is a heterogenic incompatibility mechanism

operating? If so, what are the consequences?

METHODS AND MATERIALS

Cultures

Eight single-spore isolates of Sordaria fimicola were used in

 

this study: 6, 11, 18, 19, 20, 21, 23, and 30. These wild-type
isolates and ascospore color mutants for each were obtained from
Dr. William G. Fields at Michigan State University. Table I gives
the source and geographical location of the wild-type isolates.

Olive (1956) showed that his isolates A-1 and C-7 of g, fimicola
were compatible with certain of his isolates but not with others.

Dr. William G. Fields (personal communication) indicates that of 40
isolates of g, fimicola in the Michigan State University collection,
two distinct groups are found: those compatible with A-1 and those
compatible with C-7. The two groups are not intercompatible. All

of the isolates used in this study were of the C-7 group and therefore,
all were compatible with C-7.

Each ascospore color mutant was backcrossed with its corre-
sponding wild-type for at least three generations to eliminate any
structural aberrations that may have been present. Ascospore color
mutants are designated as follows: br = brown, 9 = gray, h = hyaline,
t - tan, and y = yellow. Ascospore color mutant 309 was not easily
used because of its very slow growth rate as compared with all other
isolates. 19y1(st) is a sterile mutant. It produces no perithecia

when grown alone; however, when crossed with a compatible isolate,

TABLE I

Source and geographical location of each wild-type isolate.

 

 

Isolate number Source Geographical location
6 Cow dung Pellston, Michigan
11 Rabbit dung Big Bend National Park, Texas
18 Raccoon dung Douglas Lake; Michigan
19 Deer dung Vanderbilt, Michigan
20 Deer dung Baldwin, Michigan
21 Deer dung Baldwin, Michigan
23 Horse dung Mackinac Is1and, Michigan

30 Cow dung Paint Rock, Texas

mutant as well as hybrid perithecia are formed. The ascospores in
the hybrid perithecia exhibit approximately 70% germination.
Stock cultures were maintained on corn meal + agar slants at

5°C.

Lassa

Isolates were crossed on B agar: 17g corn meal agar, 29
dextrose, 39 sucrose, lg yeast extract, and 1000 ml distilled water.
Acetate agar (Bretzloff, 1956) was used as germination medium: 179
corn meal agar, lg yeast extract, 79 sodium acetate, and 1000 ml
distilled water. Dissecting agar was used during micromanipulation:
lg corn meal agar, 39 plain agar, 39 sucrose, 29 dextrose, 0.1g yeast
extract, 0.79 sodium acetate, and 100 ml distilled water. Stock
cultures were maintained on corn meal + agar slants: 17g corn meal

agar, 29 glucose, lg yeast extract, and 1000 ml distilled water.

Isolation of ascospore color mutants

Because of the poor germination of some of the ascospore color
mutants, several attempts were made to secure new color mutants. An
additional mutant, 11h, was obtained from isolate number 11 using the
following method:

Isolates were inoculated onto B agar and allowed to grow for 24
hours. Cultures were then incubated at 5°C for 24 hours before
irradiation. Directly after removal from 5°C, the mycelia were
irradiated for 5 minutes using a General Electric, 15 watt germicidal
lamp at a distance of 5 inches. Plates were left at room temperature

for 4 hours at which time mycelia were irradiated a second time for

5 minutes. Petri dishes were kept in the dark between each
irradiation period and for 4-6 hours after the second irradiation to
prevent photoreactivation and subsequent repair.

At maturity, perithecia were examined singly until one was found
which had an approximate 1:1 segregation of mutant to wild-type
ascospores. Several mutant spores were dissected out with the aid of
a micromanipulator and allowed to germinate and grow. If no sterility
was observed, one ascospore isolate was selected and backcrossed to its
corresponding wild-type. The new mutant was then checked for adequate
germination.

S-bromouracil and hydroxylamine were also used in other attempts
to increase the incidence of ascospore color mutants; however,
results were negative. A potentially less laborious method of
securing mutant ascospores was also attempted. This involved allowing
spores from the irradiated mycelia to discharge onto plain agar (no
sodium acetate added) to recover mutant spores, which can germinate
without the presence of acetate. However, no mutant ascospores could

be isolated using this technique.

Crossing of isolates

Since S, fimicola is homothallic, mycelia derived from single
ascosporic cultures will produce perithecia with asci homozygous for
any genetic factor. However, different isolates may be crossed by
placing inocula near one another on agar plates. Mycelia from each
inoculum will produce abundant perithecia, but in the area where
mycelia from the inocula intermingle, some hybrid perithecia may be

produced. That is, if the inocula were different genetically, some

perithecia would be produced which contain asci that are heterozygous
(Bistis and Olive, 1954).

In this study, each wild-type isolate was crossed with each
ascospore color mutant by placing inocula of each approximately 1 cm
apart in the center of a plate of B agar. The plates were incubated
at 25°C for 8-9 days. At maturity, 50-1,000 perithecia in the area
where the mycelia intermingled were examined for hybrid asci. The
number examined depended on how quickly hybrid perithecia were
discovered. After 1,000 perithecia were examined (the number chosen
arbitrarily) and no hybrid perithecia found, incompatibility between
the two isolates of the cross was assumed.

Occasionally, two isolates inhibited each other by restricting
growth, halting maturation of perithecia or by forming bands of dark
hyphae at the intersection of the isolates in which there were no
perithecia. Olive (1956) suggested that this inhibition may be
overcome by inoculating the two isolates at the same point on an agar
plate. However, this was not successful and subsequent inhibition

was interpreted as incompatibility.

RESULTS

The map distance of each ascospore color mutant from the
centromere of its linkage group as well as limited linkage data are
given in Table II. Each mutant was crossed with its corresponding wild-
type and asci from hybrid perithecia were examined. This table shows
that the distance of each ascospore color mutant from the centromere
of its linkage group, with the exceptions of 11y, llbr, l9y1(st), and
20t, approaches the limit of resolution for mapping one gene as deter-
mined by tetrad analysis. Hence, the distances given in map units
are minimum; in reality, the locus for the color mutant may be much
further away from the centromere.

From the limited linkage data, it appears that none of the
mutants are linked, with the exception of 11y and llbr. Only parental
ditype asci were observed in this cross (no tetratypes) which concurs
with the fact that both mutants map at approximately the same distance
from the centromere. However, one mutant is apparently modified in
hybrid asci, becoming darker in color, but not as dark as wild-type
spores. Crosses were also made of l8br X 11h, l8br X 199, 199 X 11h,
199 X 20t, and 199 X 21t, but no hybrid perithecia were found (300-
400 perithecia were examined). Hence the linkage data on these
crosses could not be obtained.

Germination percentages for each wild-type isolate and its

corresponding color mutant(s) are given in Table III. Germination

TABLE II

Linkage data and map distances of each ascospore color mutant from
the centromere of its linkage group.

 

 

Frequency
Ascospore lst division 2nd division 2nd division Map
color mutant segregation segregation Total segregation distance
6h 58 101 159 63% 31.5
11y 219 141 360 39% 19.5
llbr 131 88 219 40% 20.0
11h 109 142 251 62% 31.0
18y 109 152 261 58% 29.0
18br 115 172 281 61% 30.5
19y2 181 126 307 41% 20. 5
199 95 116 211 54% 27.0
19y1 (st) 285 o 285 0% o. o
20t 244 3 247 1.2% 0.6
21t 131 146 277 52% 26.0
23y 115 116 231 50% 25.0
309 79 99 178 56% 28.0
Linkage data:
Parental Nonparental
Cross ditype ditype Tetratype Conclusion
11y X 11h 66 36 67 unlinked
llbr X 11h 58 51 62 unlinked
llbr X 11y 76 0 O linked
18y X 18br 31 31 59 unlinked
19y X 199 19 20 34 unlinked
18br X 20t 24 20 68 unlinked

10

TABLE III

Germination of ascospores from wild-type and ascospore color mutant
isolates.

 

Germination
Isolate percentage

6 43
11g 93
11y 50
llbr 18
11$ 37
18 83
18y *
18br 55
19 2 94
19y 32
193 82
20 92
20t 70
21+ 83
21t 83
23+ 82
23y 48
30+ 94
309 85

*Not tested, ascospores coalesced in homozygous asci.

11

percentages for all isolates were based on approximately 300 spores.
Acetate agar was used as the germination medium.

Crosses of all isolates in pairwise combinations were made to
determine whether hybrid perithecia were produced. Table IV shows
the results of these crosses. The occurrence of hybrid perithecia
indicated that crosses were compatible and the absence of hybrid
perithecia indicated incompatibility. These data show that most of
the paired isolates were compatible.

Generally, in compatible crosses, only 10% or less of the
perithecia along the intersecting isolate lines were found to be
hybrid. The only notable exceptions to this were crosses of each
mutant with its corresponding wild-type isolate. Here, 20-30% of all
perithecia produced were hybrid. On close examination of compatible
crosses, it was noted that hybrid perithecia from all crosses
(except 18 X 21) were observed only along the intersecting isolate
lines. Thus, the extent of nuclear migration was spatially limited
in all crosses, except that same-isolate crosses produced more hybrid
perithecia. Since S, fimicola produces no antheridia, heterokaryotic
mycelium resulting in hybrid perithecia probably originated through
hyphal anastomoses (Carr and Olive, 1958). It is not known whether
incompatibility between isolates selected for this study is due to
failure to anastomose or to unknown factors occuring in some later
stage of ascogonial development.

Table IV shows that isolates 11 and 20 have similar compatibility
patterns as do isolates 21 and 23. Other pairs of isolates differed

from one another in compatibility with one or more isolates. Isolates

 

 

 

12

TABLE IV

Results of crosses of all isolates in pairwise combinations.

+ denotes hybrid perithecia formed (compatibility)

- denotes no hybrid perithecia formed (incompatibility)

 

Isolate
number Isolate number
6 11 18 19 20 21 23
6 + + + — + + +
11 + + + + + + +
18 + + + — + + +
19 - + - + + + +
20 + + + + + + +
21 + + + + + + +
23 + + + + + + +

30 - + + + + - -

30

13

18 and 20 differed from one another in that 20 was compatible with 19,
but 18 and 19 were incompatible. The same result was found with 11
and 18. 11 was compatible with 19, but 18 was not. In these examples,
18 and 20 were compatible as were 11 and 18. Thus, in terms of
incompatibility, pair 18 and 20 as well as pair 11 and 18, differed
from one another in their reactions with only a single isolate,
number 19 (denoted as Ix)' Likewise, pair 11 and 19 and pair 19 and
20 differed in compatibility with one isolate, number 18 (also denoted
as Ix)'

Other isolates differed in compatibility with two isolates.
Pair 18 and 21 differed in compatibility with both isolates 19 and 30,
as did pair 18 and 23. In these cases, 18 was not compatible with
isolate 19, but was compatible with 30, while both 21 and 23 were
compatible with 19, but not with 30. Isolate 6 was compatible with
all isolates except 19 and 30.

For five pairs of isolates which differed in compatibility
with a third isolate, Ix’ 18 and 20, 18 and 21, 18 and 23, 11 and 19,

and 6 and 20, F tetrads were isolated with little difficulty.

1
However, for pairs 11 and 21, 11 and 23, 11 and 18, 19 and 20, 19 and
21, and 19 and 23, complete tetrads could not be recovered. In each
case, when the isolates were crossed, hybrid perithecia were produced.
When hybrid asci were examined, however, one, two, three, or all

four members of the tetrad (spore pairs) failed to germinate. There
was no discernible pattern as to which ascospore, mutant or wild-type,

would not germinate and segregation of non-germination was not

consistent from tetrad to tetrad within each cross. This problem was

14

especially perplexing in a cross such as 19 X 20, where each isolate
alone had at least 70% germination of ascospores.

Olive (1956) reported a similar observation when crossing
ascospore color mutants of S, fimicola with wild-type isolates. The
mutant spores had a "remarkable decrease" in germination when isolated
from heterozygous asci.

Since S, fimicola is a homothallic fungus and is therefore not
usually crossed with other isolates, the granular appearance of mutant
ascospores and the germination failure of both mutant and wild-type
ascospores in heterozygous asci may have been due to some cytoplasmic
lethality factor and/or to an unknown genetic mechanism. Structural
aberrations did not appear to be the cause of the poor germination
since first and second division segregations in hybrid asci were in
proportion to those numbers obtained in the original mapping of the
isolates in all cases studied escept in the cross of 18 X 21.

In this cross, each wild-type member in each recovered tetrad,
when grown out alone on B agar, produced numerous hybrid perithecia
containing aberrant 6:2 asci. Most of these asci were 6+:2br, although
6br:2+ appeared as well. Contrary to the pattern of all other
crosses where hybrid perithecia were found only along intersecting
mycelial lines, these hybrid perithecia containing the aberrant asci
were found all over the plate. Original stock cultures of 18+, 21+,
18br, and 21t were then grown out on B agar, but only normal
homozygous asci were observed. It appears, therefore, that the cross
of 18 X 21, regardless of which isolate was the wild-type parent,

results in some chromosomal (probably structual) aberration.

15

This phenomenon resulted in some difficulties when the tetrads
were crossed with the Ix' Asci appearing to be from hybrid perithecia
had to be studied closely in order to discern the segregation in the
asci. This extreme care was necessary to distinguish aberrant asci
from those which may actually have been present in hybrid perithecia
from the cross of F1 tetrads X Ix' The difficulties encountered led
to the abandonment of this cross, 18 X 21, as a source of information
in this study.

Pairs of isolates which differed in compatibility patterns by
one isolate (denoted as Ix in each case) which one of the pair was not
compatible with, such as pair 18 and 20, where Ix = 19, were analyzed
genetically. Each pair with this characteristic was crossed and 9-10
F tetrads from hybrid perithecia were isolated with the aid of a

1

micromanipulator. The F1 tetrads were analyzed by crossing each spore
pair of the tetrad with the differing isolate, Ix' and noting the
segregation of compatibility and incompatibility with that isolate.
The results of these crosses are given in Tables VBVII. A tetrad
member was classified as incompatible with the Ix isolate only after

a minimum of 1000 perithecia were examined.

Table V shows that tetrads from the cross 18 X 20, when crossed
with Ix19, demonstrate every conceivable combination of compatibility
and incompatibility, i.e. tetrads had from 0 to all 4 members
compatible with isolate 19. This was not the case when tetrads from

the cross 11 X 19 were crossed with 1x18. Here, as Table VI shows,

tetrads had from 0 to 3 members compatible with isolate 18. The

16

 

 

 

TABLE V
Compatibility and incompatibility of F1 tetrads from cross 18 X 20
with I 19.
x
Compatibility (+) or
Tetrad number Tetrad spore pair incompatibility if) with I
IL
1 1 +
2 +
3 _
4 +
2 1 +
2 +
3 +
4 +
3 1 +
2 +
3 +
4 +
4 l -
2 _
3 -
4 _
5 l +
2 -
3 .-
4 .—
6 l -
2 +
3 +
4 +
7 1 -
2 +
3 _
4 +
8 -

bump
+

17

Table V (Cont'd)

Compatibility (+) or

 

 

 

Tetrad number Tetrad spore pair incompatibility (-) witthn
9 1 +
2 +
3 ..
4 -
10

bUNH
I + + +

18

 

 

 

TABLE VI
Compatibility and incompatibility of F1 tetrads from cross 11 X 19
with I 18.
x
Compatibility (+) or
Tetrad number Tetrad spore pair incompatibilityi(-) with I“
1 l -
2 +
3 _
4 _
2 l +
2 +
3 -
4 _
3 1 -
2 ..
3 +
4 -
4 1 +
2 .-
3 +
4 +
5 l -
2 +
3 .—
4 ..
6 1 +
2 -
3 -
4 .—
7 l -
2 _
3 _
4 -
8 l -
2 _
3 .-
4 +
9 .-

bWNl-J
I

19

fewer number of different ascal classes recovered from this cross may
simple be due to the fact that not enough tetrads were analyzed.

In Table VII, tetrads from cross 18 X 23, when crossed with
1x19, had only 0, 1 or 3 members compatible with isolate 19. The
ascospore color mutant for isolate 23, 23y, had relatively poor
germination (48%) and since no other mutant could be isolated; this
cross was abandoned as a source of further information so that data
are incomplete for this table.

Ideally, tetrads were to be isolated from each cross of K#-Fl
tetrads X Ix' However, the phenomenon of reduced germination in
heterozygous asci was again encountered (as with some of the original
K# crosses). This second group of tetrads was to be backcrossed to

each of the original F parents to secure a more accurate picture of

1
the number of loci involved in the incompatibility mechanism and
whether or not these loci are linked or unlinked.

In an attempt to overcome the problem of reduced germination in
the K#-Fl X Ix crosses, several methods were employed. For each
method, the cross 18 X 20 (K8) was used as the test cross. Since
dissecting agar, which contains sodium acetate, was used in all cases
of tetrad isolation, absence of acetate was ruled out as a cause of
the poor germination.

The first method involved the use of a sterile mutant, l9yl(st)
as the mutant Ix isolate. Although 19y1(st) produces no perithecia
when grown homozygously, when paired with a compatible isolate,

mutant ascospores are produced in heterozygous asci. Wild-type members

of the F1 tetrads of the cross 18 X 20 were paired with 19y1(st) and

20

TABLE VII

Compatibility and incompatibility of F tetrads from cross 18 X 23

 

 

 

with I 19. 1
x
Compatibility (+) or
Tetrad number Tetrad spore_pair incompatibility (-) with I“
1 1 -
2 +
3 +
4 +
2 1
2 -
3
4 .—
3 l -
2 _
3 .—
4 _
4 1 -
2 +
3 .—
4 .-
5 1 -
2 _
3 _
4 _
6 l -
2 ..
3 .—
4 -
7 l +
2 ..
3 .—
4 .—
8 l -
2 -
3 _
4 _
9 .—

bWNH
1

21

the resultant hybrid asci dissected and spores examined for
germinability.

With this method, germination of all spores was increased
considerably as is shown in Table VIII. Three to four pairs of spores
in each resulting tetrad germinated. However, in many cases, only
one or two members of the tetrad would continue to grow while the
remaining two or more aborted after growth of only a few millimeters.
As the table shows, there seems to be no discernible pattern to pre-
dict which spores would germinate and abort and which would continue
to grow.

A second attempt to overcome reduced germination involved the
use of a new isolate which was employed at the parental, K# cross
level. Several new mutant isolates were tested in order to find one
with a compatibility pattern similar to isolate 18. Isolate 6h was
selected, although it did not have an identical pattern (see Table IV)
and its germination percentage was quite low (43). 6h and 20 were

paired and resultant hybrid asci dissected. These F tetrads were

1
then paired with 1819. Progeny from this cross were isolated, with
the same result as all other cross (except those with 19y1(st) as the
mutant Ix isolate). Only one or two members of each tetrad germinated
and grew. There was no germination and subsequent abortion as with

K8 progeny X l9y1(st). The cross, 6h X 20, was therefore abandoned

as means of securing more data on incompatibility between the

isolates chosen for this study. With all the methods attempted
failing to eliminate or reduce the germination problem, the F

2

progeny X each parent phase of genetic analysis was omitted.

22

TABLE VIII

8 1 .
Results of the cross of K (18 X 20) tetrads X 19y (st) to determine
germinability and subsequent growth of spores.

M = mutant spore pair

WT = wild-type spore pair

____indicates germination

* indicates abortion (germination but no subsequent growth)

Cross Tetrads

8 l

K -6-3 X 19y (st) M} M WT W_T_
11* WT 1 1

8 1

K -2-2 X 19y (st) S; g_ WZf HE?
WT E* 11* M
1* 1* 11* 11
1* 1* 1T. 11*

8 1

K -2-1 X 19y (st) W'_I‘_* WT _M_ M}
M M WT 33*
WT WT £4. 5*

8 1

K -3-2 X 19y (st) Sf M WT} sz
11* 11* 1 1*
1* 1 11* 11*
11* 11 1* 1
11* WT 1 1
1* 1 WT WT
11* 11* 1* 1

8 l

K -l-4 X 19y (st) g?‘ g? WT WT
1* 1 WT m
1* 1* 11* 11
11* 11 1* 1*

8 1

K -10-3 X 19y (st) S_ S. WT SI}
11 11 1* 1*
11 fl“ 1 1
1* 1* 11 11

23

To collect further concrete evidence for an incompatibility

scheme, Fl tetrads of both K8 (18 X 20) and K12 (11 X 19) were
backcrossed to each respective parent. Every member of every tetrad
was compatible with both parents. Furthermore, members of each tetrad
were intercompatible as well as being compatible with members of

other tetrads in the same cross.

DISCUSSION

From the initial compatibility data in Table IV, it appears
that among the eight geographical isolates chosen for this study,
there are six "incompatibility groups." One such group includes
isolates 21 and 23, another 11 and 20, while 6, 18, 19, and 30 each
compose one group. These "incompatibility groups" have been referred
to as geographical races by Esser (1971) and as subspecies by Caten
and Jinks (1966). Although there is some differential compatibility
between isolates when paired, incompatibility does not mean that the
isolates do not belong to the same species.

When all the data are considered, it appears that for
compatibility (hybridization) to be observed, at least one common
allele, between two isolates, is required. Considering the nature of
S, fimicola as a homothallic fungus and therefore self-fertile, if a
genetic system is indeed operating to confer compatibility, it follows
that like alleles would be required for compatibility and unlike
alleles (at one or more loci) would determine incompatibility. Esser
(1965) stated this in the following way: "Since the (homothallic)
fungus is self-compatible, the inter-race incompatibility may be due
to heterogeneity" (parentheses mine). Data from crosses of F1 progeny
of both K8 and K12 to each respective parent and amongst themselves
illustrate the hypothesis that some common factor(s) was inherited by

each member of each tetrad resulting from a compatible cross.

24

25

Several observations can be made when the data are viewed
collectively. First, the "relative heterothallism" noted by

Pontecorvo (1953) in Aspergillus nidulans, in which more than 50%

 

of the perithecia from a single cross were hybrid, was not noted in
this research. In fact, less than 10% of all perithecia from any one
cross were hybrid except those between a wild-type isolate and its
ascospore color mutant(s). This suggests that there may be some
possible selection against hybridization at some point in the sexual
cycle, perhaps in limiting nuclear migration. Cross 18 X 21, however,
clearly exhibited a different set of characteristics.

In this cross, hybrid perithecia were found in abundance (although
not 50%) all over the plate. There was no limitation to nuclear
migration. However, there appeared to be a structural aberration
which produced 6:2 asci almost exclusively in hybrid perithecia.

These phenomena occurred only in this cross. Thus, isolates 18 and 21
appear to be only semi-compatible, producing hybrid perithecia but
with aberrant asci.

An analysis of Tables VEVII shows that at least 5 different ascal
classes result from these crosses. Cross 18 X 20 (Table V) produced

all 5 classes, i.e. F tetrads were produced with every combination

1
of compatible and incompatible (with 1x19) pairs of spores. These
various combinations (of 0-4 compatible spores pairs in each tetrad)
and the fact that there is not a 1:1 segregation of the compatibility
factor indicate that more than 1 or 2 genes are probably involved.

Also obvious in the data presented are the germination difficul-

ties observed in spores of hybrid asci. These difficulties may be

26

related or due to a number of factors. Cytoplasmic lethality factors,
the phenomenon of hybridization in a homothallic fungus, or one or
more nuclear genetic factors including those which may be involved in
incompatibility may be a cause of reduced germination. As noted
previously, there does not appear to be any pattern as to which type
of spore, mutant or wild-type, is subject to reduced germination.

The phenomenon of germ-tube production and subsequent abortion

and cessation of growth as observed in F progeny of 18 X 20 (K8) X

l
19y1(st) (Table VIII) is particularly unique. Although other crosses
exhibit this sequence of events occasionally, it is most noticeable in
this cross. A possible cause to be considered here is that enough
chromosomes are paired to permit development and germination of spores;
however, the nucleus may cease to divide after germetube production,
thereby curtailing any further growth. The block halting the
completion of the sexual cycle in this cross seems to affect the
growth of mycelium following germination, although germination itself
appears to proceed normally. This is unlike other crosses where

spore maturation as well as germination are primarily affected at

both the parental cross and F X Ix cross levels.

1

Germination difficulties made complete data collection impossible.
Further crossing could have shown how many loci may be involved in
incompatibility, whetherithese loci are linked or unlinked and whether
they are indeed separatefloci or simply alleles at one locus.

However, on the basis of the data that were obtained, several

mechanisms for incompatibility can be disCussed.

27

It is obvious from Tables VBVII, that there is not a 1:1
segregation of the incompatibility locus (loci). Also apparent is
the fact that some common factor(s) must be present for compatibility
to be conferred on two isolates and that this factor(s) is inherited
by all progeny of a compatible cross. Any proposed mechanism for
incompatibility must take into consideration these facts.

A one-locus, 2-a11e1e (or more) system obviously could not
explain the results. The alleles would need to be identical in order
for initial compatibility to occur between selected parent isolates:
all progeny would then inherit this common allele and incompatibility
between one parent with a third isolate could not be explained. A
2-locus-2-a11eles at each locus scheme would not explain the data for
similar reasons. In keeping with the premise that at least one factor
must be in common for compatibility to occur between 2 isolates, one
allele at at least 1 locus must be the same in the chosen parent
isolates. The result would be that the second locus, if segregating
independently, must be different which would give only a 1:1 segrega-
tion in the progeny for compatibility at this second locus. This can
be better illustrated using the example of cross 18 X 20. Recall that
18 and 20 are compatible with each other; however, 18 is not
compatible with 19, while 20 is compatible with 19. Two loci, A and
B, with alleles x and y at each locus are chosen with a common allele,
x, at the A locus conferring compatibility between 18 and 20. This
would fulfill requirements of common alleles for compatibility,
however, the cross of 18 X 20 would yield a 1:1 segregation at the B

locus (if each parent possessed a different allele at this locus) which

3

pr - I‘m—“nu

28

clearly is not represented in the data of the F progeny of 18 X 20

1
when paired with 1x19.

These arguments can be applied to many schemes involving more
than 2 loci. However, as one proposes more and more unlinked loci,
one is bound to discover a scheme that will fit the data collected.

For instance, a 5 locus system with 2 alleles at each locus and common
alleles at either loci A and B or at C and D or at E, where 18 has

a genotype of ABCDE, 20 of abch, and 19 abode would fit the data
given in Table V. Although this scheme may fit the data collected, it
is not feasible to propose it because of the lack of data (due to
germination difficulties in backcrossing) to confirm it. It is also
unknown as to when in the sexual cycle these incompatibility loci act,
whether in pre- or post-plasmogamy.

Although a precise mechanism cannot be proposed, it would appear
from the data collected that 1) several loci are involved, 2) like
alleles at one or more of these loci confer compatibility, and
3) incompatibility is a function of one or more unlike alleles at
major incompatibility loci. This latter condition is, in essence.
the basis of a heterogenic incompatibility mechanism. The significance
of heterogenic incompatibility in homothallic fungi, such as
S, fimicola, has been discussed by Mather (1942), Olive (1958), Esser
and Kuenen (1965), Caten and Jinks (1966), and Esser (1966, 1971).

The homothallic fungus, already self-fertile, is prevented from
breeding with unlike genotypes in some instances, thereby reducing
future variability within the species. With heterogenic incompatibility,
an effective inbreeding mechanism, races or "incompatibility groups"

become reproductively isolated from other isolates within the same

29

species which have differing genotypes at the incompatibility loci.
According to Esser (1965), it is these races, not the species, which
are actually the smallest units of evolution. It can be argued, then,
that through geographical separation, races, fitting all the physical
and morphological descriptions of the species, may be evolving into
new species by the process of reproductive isolation.

Darwin's theory, which explains evolution and survival of the most
fit through advantageous selection of small variations in the genetic
complement, seems directly opposed to any evolutionary advantages of
an inbreeding scheme which would limit variations. What, then, are
the advantages and disadvantages of heterogenic incompatibility and
homothallism for a fungus like S, fimicola?

A factor to be considered here is that S, fimicola has no asexual
reproductive mechanism to produce numerous copies of the genetic
complement which seems presently well-adapted. Homothallism
accomplishes this through self-fertility, allowing variations only
through mutation and perhaps through hybridization with compatible
isolates (although the extent of hybridization in nature is not
known). Through heterogenic incompatibility, present adaptation to
the environment is good, but future adaptations may be limited because
of little variation due to a high degree of homozygosity (Mather,
1942). However, as Olive (1954) pointed out, homozygous asci, of
either parent, in hybrid perithecia are scarce or absent, indicating
that mutant and wild-type nuclei may be attracted to each other oftener
than 2 mutant or 2 wild-type nuclei in the same perithecium. This

would tend to increase the genetic variation of a given isolate.

30

Two major theories have been advanced on homothallism and its
position in the evolutionary scale in relation to heterothallism.
Since compatibility between isolates in this study can be considered
"unbalanced heterothallism" (Olive, 1958), a short discussion of
these theories in pertinent although concrete evidence for both is
lacking.

One theory, advanced by Whitehouse (1949), views homothallism
as more highly evolved than heterothallism. He argues that a
heterothallic fungus is involved in more outbreeding, therefore more
evolutionary change through increased variation. The loss or
modification of mating-type loci could develop a homothallic species
(homothallic forms having a comparatively fixed genotype). Whitehouse
also suggests that homothallism is perhaps more favored by natural
selection because it is a scheme for certain sexual reproduction which
does not depend on close proximity of spores or hyphae of compatible
mating types. He does not, however, consider the reduced variability
of homothallism due to inbreeding as a liability.

Opposed to this idea is Olive's theory (1958) which views
compound loci, i.e. recombinable groups of loci closely related in
function as well as on the chromosome, as the most feasible basis
for an explanation of the development of balanced heterothallism from
homothallism. Recombination between 2 pseudoallelic mutations,
double mutations within a compound locus which separate into homologous
chromosomes, or chromosomal deletions may result in self-sterility
of normal self-fertile fungi. The cross-fertility then required for

sexual reproduction would resemble l-locus heterothallism. Also

31

considered in this theory is the fact that only 10% of the Ascomycetes
are heterothallic while as high as 65% of Basidiomycetes, generally
considered to be more highly evolved, are heterothallic.

Heterogenic incompatibility between two isolates (or geographical
races) of a species resulting in unbalanced heterothallism or even the
balanced heterothallism observed by Pontecorvo suggests a possible
intermediate step between homothallism and heterothallism. Further-
more, the incompatibility between selected isolates, reduced
germination of ascospores in hybrid asci, and the character of the

segregations of F progeny when crossed with Ix suggest a genetic

1
mechanism for incompatibility that fits the definition of heterogenic
incompatibility. If heterogenic incompatibility is indeed operating,
the possibility that races may be evolving into separate species
cannot be overlooked in S, fimicola.

However, this evolution in S, fimicola may not be strictly
separated geographically as is the case with higher plants and
animals. Tables I and IV illustrate this point. The source of
isolates 11 and 30 is Texas, while the remaining isolates were
obtained in Michigan. It is interesting to note that, although from
sources 1500 miles apart, 11 is compatible with all Michigan isolates
as well as isolate 30, and has a compatibility pattern identical to
isolate 20. Thus, a geographical barrier may not produce the
profoundly different changes in this fungus that may be noted in a
higher organism. Also noted in these tables is the fact that although
both isolates 20 and 21 were isolated from deer dung near Baldwin,

Michigan, their compatibility patterns differ with one isolate,

number 30. Here, no geographical barrier has separated the isolates,

32

yet they differ in compatibility with other isolates. With further
study and a solution to the problem of reduced germination involved in
hybrid asci and backcrossing, a more precise picture of the

incompatibility mechanism operating between isolates of S, fimicola

may be elucidated.

SUMMARY

1. Thirteen ascospore color mutants of Sordaria fimicola were mapped

 

in relation to their centromeres and limited linkage data was
collected. Germination data for each wild-type isolate and its
ascospore color mutant(s) were also obtained.

2. Each of 8 wild-type isolates was crossed with each ascospore color
mutant in pairwise combinations and compatibility (hybridization) and
incompatibility were noted.

3. On the basis of the compatibility data, 6 "incompatibility groups"
were defined.

4. Pairs of isolates differing in compatibility with a single isolate
(Ix) were crossed and 9-10 tetrads for each pair were recovered.
These F1 tetrads were then crossed with the differing isolate (Ix)
and compatibility and incompatibility noted.

5. Germination difficulties were encountered in both parental crosses
and in F1 X Ix crosses. No solution was found.

6. One cross, 18 X 21, gave aberrant asci in many hybrid perithecia,
apparently due to a structural abnormality.

7. Data indicates that some common factor(s) between two isolates must
be present for compatibility to be observed. Also indicated is that

a multiple locus system must be operating in the incompatibility

mechanism.

33

34

8. Although a precise incompatibility mechanism cannot be proposed
from the data given, it appears that isolates of S, Simicola are

evolving into species.

LITERATURE CITED

LITERATURE CITED

Alexopoulos, C.J. 1962. Introductory Mycology. John Wiley and Sons,
Inc. New York, N.Y. 613 pp.

Bistis, G. and L.S. Olive. 1954. Ascomycete spore mutants and their
use in genetic studies. Science 120:105-106.

Bretzloff, C.W. 1954. The growth and fruiting of Sordaria fimicola.
Amer. J. Bot. 41:58-67.

Carr, A.J.H. and L.S. Olive. 1958. Genetics of Sordaria fimicola.
II. Cytology. Amer. J. Bot. 45:142-150.

Caten, C.E. and J.L. Jinks. 1966. Heterokaryosis: Its significance
in wild homothallic Ascomycetes and Fungi Imperfecti. Trans. Br.
Mycol. Soc. 49:81-93.

Esser, K. 1959. Die incompatibilitatsbeziehungen zwischen
geographischen rassen von Podospora anserina (Ces.) Rehm. II.
Die wirkungweise der semi-incompatibilitats gene. Z. Vererbungst.
390:29-52.

. 1965. Heterogenic incompatibility. Incompatibility in
fungi: A symposium. K. Esser and J.R. Raper, editors. pp. 6-13.

. 1971. Breeding systems in fungi and their significance for
genetic recombination. Molec. Gen. Genetics 110:86-100.

and R. Kuenen. 1965. Genetics of fungi. Springer-Verlag.
New York, N.Y. 479 pp.

Fields, W.G. 1970. An introduction to the genus Sordaria. Neurospora
Newsletter 16:14-17.

Mather, K. 1943. Polygenic inheritance and natural selection. Biol.
Revs. Camb. Phil. Soc. 18:32-64. '

Olive, L.S. 1954. Cross-karyogamy and segregation in a homothallic
fungus. Bull. Torrey Bot. Club. 81:95-97.

. 1956. Genetics of Sordaria fimicola. I. Ascospore color
mutants. Amer. J. Bot. 43:97-107.

 

. 1958. On the evolution of heterothallism in fungi. Amer.
Natur. 42:233-251.

 

35

36

Pontecorvo, G. 1953. The genetics of Aspergillus nidulans. Adv. in
Genetics 5:141-238.

 

Whitehouse, H.L.K. 1949. Heterothallism and sex in the fungi. Proc.
Nat. Aca. Sci. U.S. 24:411-445.

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