Mycologia, 87(6), 1995, pp. 805-820. © 1995, by The New York Botanical Garden, Bronx, N Y 10458-5126 Sexuality and somatic incompatibility in Phellinus gilvus David M. Rizzo 1 Rita M. Rentmeester Harold H. Burdsall, Jr. Center for Forest Mycology Research, USDA Forest
Products Laboratory, One Gifford Pinchot Drive,
Madison, Wisconsin 53705
Abstract: Phellinus gilvus was found to have an outcrossing, bifactorial (tetrapolar) mating system. Two
types of heterokaryon formation were evident in in­
trabasidiome homokaryon-homokaryon pairings: 1)
bidirectional nuclear migration resulting in the for­
mation of a single heterokaryotic thallus, and 2) re­
stricted nuclear migration with a heterokaryon formed
only in the interaction zone between the paired homo­
karyotic colonies. The mating system of P. gilvus is
multiallelic; pairings between homokaryotic isolates
from different basidiomes were always compatible with
bidirectional nuclear migration. One heterozygous lo­
cus had major effects on somatic incompatibility re­
actions among progeny from two studied fruiting bod­
ies. This was supported by the observation that pair­
ings among sets of heterokaryons composed of one
common nonsibling nucleus and the other sib-related
resulted in two somatic incompatibility groups, while
pairings among siblingheterokaryons resulted in three
groups. The sexual compatibility loci were separate
from the somatic incompatibility loci and no linkage
was found between them.
Key Words: heterothallism, mating systems, Phel­
linus gilvus, somatic incompatibility
INTRODUCTION
To adequately assess gene flow within and between
fungal populations in natural ecosystems, a basic un­
derstanding of sexual and somatic compatibility sys­
tems is necessary (Rayner, 1990, 1991). In the hymen­
omycetes, mating systems have been most thoroughly
examined in Schizophyllum and Coprinus (Kües and
Casselton, 1992). More research, however, is needed
Accepted for publication July 28, 1995.
1 Current address of corresponding author: Department of Plant
Pathology, University of California, Davis, CA 95616.
to elucidate the genetics behind mating in other spe­
cies that do not always follow the same mating and
nuclear migration patterns as the model systems (Ray­
ner, 1990, 1991). Mating systems in the genus Phellinus
have been difficult to establish because both homo­
karyons and heterokaryons often have multinucleate
cells, and heterokaryons lack clamp connections (Hansen, 1979; Fischer, 1987; Hennon and Hansen, 1987).
Because of this lack of distinguishing microscopic
characters, mating studies have relied on macroscopic
culture morphology to determine heterokaryon for­
mation in compatible crosses (Fischer, 1987, 1994;
Fischer and Bresinsky, 1992; Angwin and Hansen, 1993;
Larsen et al., 1994). Nuclear DNA content and iso­
zymes have also been utilized to distinguish between
homokaryons and heterokaryons (Fischer and Bresin­
sky, 1992; Dreisbach and Hansen, 1994). In the few
mating studies that have been conducted with Phelli­
nus, and the closely related genus Inonotus (Fischer
1987, 1994; Fischer and Bresinsky, 1992; Angwin and
Hansen 1993; Larsen et al., 1994), most species have
been determined to be heterothallic with a unifactorial
(bipolar) mating system, although several species are
considered to be homothallic (Fischer, 1987; Goldstein
and Gilbertson, 1981).
Somatic incompatibility systems operate in the rec­
ognition of nonself genes. In the hymenomycetes, so­
matic incompatibilityis mostly observed in interactions
between heterokaryotic secondary mycelia. However,
somatic incompatibility may be expressed between
homokaryons and override of the somatic incompat­
ibility system may be necessary for completion of the
sexual cycle (Rayner, 1990, 1991). Somatic incompati­
bility does not restrict outbreeding and, in most cases,
is thought to represent a separate genetic system from
the mating system. The genetic basis of somatic in­
compatibility is incompletely known for basidiomy­
cetes, although indications are that it may be polygenic
andcomplex (Rayner, 1991; Hansenetal., 1993b, 1994;
Malik and Vilgalys, 1994). The genetic basis of somatic
incompatibility in the genus Phellinus has been ex­
amined only with P. weirii (Murr.) Gilb. (Hansen et
al.,1994).
Phellinus gilvus (Schw.) Pat. is a cosmopolitan spe­
cies and is especially common in tropical regions. Bas­
idiomes of P. gilvus exhibit tremendous macroscopic
variation, leading to the fungus having been described
805 MYCOLOGIA
806
TABLE I. Collection numbers, number of single basidio­
spore isolates, location of origin and host substrate of Phel­
linus gilvus isolates used in mating studies
Collection
no.
No.
spores
H H B 11806
10
Florida
NO 7661
FP 133214
RMR 8
10
11
15
Guatemala
Oregon
Wisconsin
RMR 12
RMR 46
17
12
Wisconsin
Wisconsin
Location
Host
Liquidambar
styraciflua L.
Hardwood
Alnus sp.
Betula papyrifera
Marsh.
Betula papyrifera
Betula papyrifera
at least 30 different times and having over 100 syn­
onyms (Larsen and Cobb-Poulle, 1990). As part of our
investigations into intraspecific variation in P. gilvus,
we initiated studies to determine a genetic basis for
sexuality and somatic incompatibility in this species.
These compatibility systems were tested using both
sibling and nonsibling isolates in a series of homokar­
yon-homokaryon,
heterokaryon-homokavon,
and
heterokaryon-heterokaryon pairings. Our results in­
dicate that P. gilvus is heterothallic with two, multial­
lelic mating factors, and that one heterozygous locus
had major effects on somatic incompatibility reactions.
The sexual and somatic incompatibility system were
also found to be controlled from separate nonlinked
loci.
METHODS
Isolates. -Theorigin and numbers of Phellinus gilvus
collections from which single basidiospore isolates were
derived are listed in TABLE I. Single spore isolates were
collected in all cases by placing a portion of the hy­
menophore on the underside of a petri dish lid sus­
pended over 1% malt extract agar (MEA). Following
a spore shower, germinated basidiospores were ob­
served under 40 × magnification and individual spores
were transferred to separate MEA plates. Single spore
isolates are designated with a collection number fol­
lowed by a hyphen and the spore number (e.g., RMR
12-1, 12-2, 12-3, etc.).
Working stocks were kept at room temperature; long
term storage was at 4 C. All voucher cultures and
specimens are deposited in the Center for Forest My­
cology Research (CFMR) culture collection and her­
barium at the USDA Forest Products Laboratory.
Madison, Wisconsin.
Pairings.-All pairings were done on 3% MEA and
incubated at 20-25 C. Mycelial plugs were placed 5­
10 mm apart and allowed to grow together for at least
4 wk before examination, except where specifically
noted. Preliminary homokaryon-homokaryon, heter­
okaryon-homokaryon, and heterokaryon-heterokar­
yon pairings were made between the six collections to
observe the morphological responses between paired
mycelia. For these pairings, we assumed that single
basidiospore isolates were homokaryotic, and isolates
originating from context tissue and decayed wood were
heterokaryotic. Based on the morphologicalresponses
observed in these initial pairings, a series of experi­
ments were designed to determine the genetic basis
of sexuality and somatic incompatibility. Microscopic
observations could not be used to test sexual com­
patibility in P. gilvus; clamp connections are not pres­
ent, and both primary and secondary mycelia are bi­
nucleate (Fischer and Bresinsky, 1992; authors, per­
sonal observations). In the heterokaryon-homokaryon
and heterokaryon-heterokaryon pairings, microscopic
observations of hyphal anastomosis and lysis were made
within the barrage zone area.
Brief descriptions of individualexperiments are pre­
sented below; additional details of the experiments are
provided in the RESULTS section.
Intrabasidiome homokaryon-homokaryon crosses.-Intra­
basidiome matings were conducted by crossing 8-18
single basidiospore isolates from five of the P. gilvus
collections (RMR 12, RMR 46, FP 133214, HHB
11806, NO 7661) in all combinations. After 4 wk,
subcultures were excised from 1 cm behind the original
plug and from the interaction zone between the two
isolates (FIGS. 1 , 3, 5-8). Subcultures were made using
40× magnification and transferred to 1% MEA. All
subcultures were examined for sectoring, particularly
those from the interaction zone. In the few instances
where sectoring was observed, samples from each of
the observed sectors were used in further pairings.
After 7-10 da, subcultures were paired with the non­
sibling homokaryon, RMR 8-1. Pairings were evalu­
ated for culture morphology after 4 wk.
Heterokaryon-homokaryon pairings. -Nuclearmigration
in heterokaryon-homokaryon matings was investigated
in a time-course pairing with isolates of known nuclear
composition. A synthesized heterokaryon (RMR 12-5
× RMR 12-10) was mated with single basidiospore
isolate RMR 46-4. Subcultures (1 × 1-mm plugs) were
excised weekly over 4 wk from the advancing margins
of the two colonies, from locations within the center
of each colony and at locations within and along the
margins of the interaction zone (FIGS. 9-11). New
subcultures were taken directly adjacent to previous
locations during the 4-wk period. To determine the
nuclear composition of the subcultures, they were
paired with the original homokaryotic isolates 12-5,
12-10, and 46-4, the synthesized heterokaryon (12-5
Rizzo
ET AL.:
PHELLINUS
GILVUS
807
MATING
FIGS. 1-4. Compatible homokaryon-homokaryon matings. All photographs were taken at 4 wk after pairing. 1. Original
pairing plate showing single heterokaryotic thallus formed by bidirectional nuclear migration; letters (A, B, C) indicate positions
of subcultures shown in FIG. 2. 2. Subcultures from FIG. 1 paired with a nonsib homokaryon; all show double pigmented
lines. 3. Original pairing plate showing raised heterokaryotic mycelium in the interaction zone formed due to restricted
nuclear migration; letters (D, E, F) indicate positions of subcultures shown in FIG. 4. 4. Subcultures from FIG. 3 paired with
a nonsib homokaryon; only center plate with double pigmented lines.
× 12-10) and a nonsibling homokaryon RMR 8-1.
Colony morphology was observed after 4 wk. Nuclear
migration was followed in a similar manner in pairings
(RMR 12-1 × RMR 12-5) × RMR 46-5, and (RMR
12-5 × RMR 12-10) × RMR 12-1.
Somatic incompatibility pairings. Somatic incompati­
bility in P. gilvus was examined by pairing heterokar­
yons of known nuclear composition. Heterokaryons
with a common nonsibling related nucleus were com­
pared (Hansen et al., 1993b). Collections of sibling
single spore isolates were paired with the nonsibling
homokaryon RMR 8-1. After 2 wk, the isolates had
grown together to form a single thallus and subcul­
tures were taken approximately 1 cm from the plugs
on the side away from RMR 8-1. Subcultures were
excised from this point to maintain a single mito­
chondrial type within the subcultures. Mitochondria
do not migrate along with nuclei in matings between
hymenomycetes (May and Taylor, 1988; Hintz et al.,
1988; Ainsworth et al., 1990; Smith et al., 1990; Rizzo
and May, 1994); therefore, this subculturing protocol
minimized the potential influence of cytoplasmic fac­
tors in somatic incompatibility reactions. The common
nucleus heterokaryons formed from this initial pairing
were then paired in all combinations.
Sib-related heterokaryons formed in the intrabasi­
diome crosses of RMR 12 and FP 1332 14 were paired
in all combinations. The center subculture in the
homokaryon-homokaryon mating experiments (see
above) was the source for the heterokaryons.
RESULTS
Preliminary observations. -Allpairings between non­
sibling single basidiospore isolates resulted in the for­
mation of a single thallus with no obvious demarcation
lines (FIG. 1). In general, single basidiospore isolates
808
MYCOLOGIA
FIGS. 5-8. Examples of incompatible homokaryon-homokaryon matings. Pairing morphology ranged from complete in­
termingling of hyphae with no pigment formation (FIG. 5), slight sparse zone formation between paired mycelia with little or
no pigment (FIG. 6), slight pigment formation in the interaction zone (FIG. 7), to very distinct pigment formation within the
agar (FIG. 8).
of P. gilvus appear white in culture, while isolates from
context tissue or wood decay (putative heterokaryons)
are yellow-brown or dark brown; the complete inter­
mingling of hyphae in compatible homokaryon-homo­
karyon pairings was usually, but not always, accom­
panied by a change in the color of the mycelial mat
from white to brown.
Heterokaryon-homokaryon and heterokaryon-het­
erokaryon pairings using non-sibling isolates always
resulted in two dark lines within the agar with a sparse
zone of reduced aerial mycelium between the paired
isolates (FIGS. 11, 12). Mycelia in these interactions
initially grew together with no apparent morphological
reactions; barrage formation (sparse zone delimited
by pigmented lines) began within 2 wk. Microscopic
examination of the zone between paired isolates in­
dicated anastomosis of hyphae during the initial stages
of the pairings. This was followed by lysis of hyphae
leading to the formation of the sparse zone. Mycelia
in heterokaryon-heterokaryon self-pairings always
completely intermingled, both macroscopically and
microscopically, to form a single thallus.
The observation that a pairing between a hetero­
karyon and a nonsibling homokaryon resulted in a
strong barrage reaction, while pairings between un­
related homokaryons resulted in little or no barrage
reaction, was similar to the results of crosses with Phel­
linus weirii (Angwin and Hansen, 1993). Consequently,
by pairing subcultures from a homokaryon-homokar­
yon cross with a nonsibling homokaryon, we could
RIZZO
TABLE II.
10
PHELLINUS
GILVUS
809
MATING
Intrabasidiome mating of single basidiospore isolates for Phellinus gilvus collection RMR 12
A1B1
1
ET AL.:
4
A1B2
18
20
8
19
A2B1
6
11
12
A2B2
?
14
17
3
5
7
9
16
a Symbols: /, self pairing; + , heterokaryon formation with complete nuclear migration (FIG. 1); × , heterokaryon formation
with restricted nuclear migration (FIG. 3); -, negative pairing, no heterokaryon formed (FIG. 5).
determine if heterokaryon formation had occurred in
the original homokaryotic thallus. Homokaryotic sub­
cultures (i.e., heterokaryon formation did not occur
in the original pairing) would completely intermingle
with the tester homokaryon to form a single thallus
while heterokaryotic subcultures would form double
pigmented lines with the tester (Angwin and Hansen,
1993).
Intrabasidiome homokaryon-homokaryon crosses. -conIn
trast to pairings between nonsibling homokaryons,
pairings among sibling single basidiospore isolates re­
sulted in a range of interactions from complete inter­
mingling of mycelia to a number of different barrage
reactions within the interaction zone between the in­
oculum plugs (FIGS. 1, 3, 5-8).
Two types of heterokaryon formation were evident
in compatible pairings following pairing of subcultures
with RMR 8-1. Bidirectional nuclear migration re­
sulted in the formation of a single heterokaryotic thal­
lus (FIG. 1). All subcultures taken from these crosses
formed double pigmented lines with the homokaryotic
tester to indicate heterokaryon formation throughout
the thallus (FIG. 2). A heterokaryon formed only in
the interaction zone between paired homokaryotic my­
celia (FIG. 3) was morphologically similar to the het­
erokaryotic “crossing mycelium” described by Fischer
(1987,1994) and Fischer and Bresinsky (1992) for other
species of Phellinus. Interaction zone heterokaryons
apparently resulted from restricted nuclear migration
and consisted of a line of raised, pigmented, woolly
mycelium between the paired mycelia and was not
associated with pigment formation in the agar (FIG.
3). Only subcultures taken from the raised mycelium
reacted with the tester homokaryon to indicate het­
erokaryon formation; subcultures from the original
homokaryotic isolates completely intermingled with
the tester (FIG. 4). After 15 wk in the intrabasidiome
cross with RMR 12, additional subcultures were taken
from the original homokaryotic parents to determine
if invasive growth by the restricted heterokaryon had
occurred. Nuclear migration into the original homo­
karyotic parents was not detected and both isolates
remained homokaryotic (data not shown). Unilateral
nuclear migration was observed in a very small per­
centage of the crosses and was characterized by only
one of the homokaryotic parents being converted to
a heterokaryon.
Heterokaryon stability was examined by taking 10­
15 hyphal tips from heterokaryons formed by restrict­
ed or complete bidirectional nuclear migration. All
hyphal tip cultures formed two dark lines when paired
with RMR 8-1. No evidence of a breakdown in the
heterokaryotic state to homokaryotic progenitors was
detected.
Incompatible crosses (i.e., no heterokaryon for­
mation) displayed a variety of pairing morphologies,
ranging from the near absence of demarcation lines
to the formation of a single dark line within the me­
dium (FIG. 5-8). All subcultures excised from these
pairings intermingled with the tester homokaryon to
form a single thallus (not shown).Incompatible crosses
810
MYCOLOGIA
TABLE III.
Intrabasidiome mating of single basidiospore isolates for Phellinus gilvus collection FP 133214
A3B3
A3B4
1
9
10
A403
11
17
3
15
A4B4
5
4
12
13
a Symbols: /, self pairing; +, heterokaryon formation with complete nuclear migration (FIG. 1); × , heterokaryon formation
with restricted nuclear migration (FIG. 3); -, negative pairing, no heterokaryon formed (FIG. 5).
with bidirectional nuclear migration. The mating fac­
tors for each spore family were therefore numbered
separately (TABLES II, III, IV).
were scored for the intensity of the negative reaction
to use for comparison with the results of the somatic
incompatibility pairings (see below).
Based on heterokaryon formation and nuclear mi­
gration patterns (bidirectional and restricted), the re­
sults of crosses with RMR 12 and FP 133214 were
consistent with a bifactorial (tetrapolar) system in which
four different mating groups were detected (TABLES
11, 111). Crosses with collections RMR 46 (TABLE IV),
HHB 11806 (data not shown), and NO 7661 (data not
shown) resulted in the formation of heterokaryons
mostly by restricted nuclear migration; bidirectional
nuclear migration was observed in a very small per­
centage of the crosses and only two mating groups
could be assigned. Interbasidiome crosses, with iso­
lates representing all of the mating types from each
of the different spore families, were all compatible
TABLE IV.
several weeks many cultures
Fruiting in culture. -After
of P. gilvus formed basidiomes on the agar surface in
which setae and basidiospores similar to field collected
specimens were formed. In matings with bidirectional
nuclear migration, basidiomes were scattered across
the agar surface. Single spore isolates were collected
from these basidiomes, and, when paired in all com­
binations, resulted in the recovery of the same four
mating types as the parental strains. Both bidirectional
and restricted nuclear migration were observed in these
second generation crosses.
Basidiomes were formed on the raised mycelium in
the interaction zone of matings with restricted nuclear
Intrabasidiome mating of single basidiospore isolates for Phellinus gilvus collection RMR 46
A5B5?
1
8
9
12
A6B5? 4
10
11
13
3
5
6
7
a Symbols: /, self pairing; +, heterokaryon formation with complete nuclear migration (FIG. 1); × , heterokaryon formation
with restricted nuclear migration (FIG. 3); -, negative pairing, no heterokaryon formed (FIG. 5).
RIZZO
ET
AL.:
PHELLINUS
migration. Single spore isolates were collected from
these basidiomes and, when paired in all combinations,
resulted in the recovery of only two mating types. All
heterokaryons formed in these pairings were the result
of restricted nuclear migration.
Many homokaryotic isolates formed basidiomes
without being paired with a compatible homokaryon.
In addition, we observed fruiting at the edges of petri
dishes in compatible matings with restricted nuclear
migration. Microscopic examination of these basi­
diomes revealed very few spores present and mostly
aborted basidia, or basidia with only two sterigmata.
Although spores were difficult to find in microscopic
mounts, we were able to collect spore showers. All
crosses among single basidiospore isolates collected
from these basidiomes resulted in complete intermin­
gling of mycelia, as would be seen in a self pairing,
and reacted as homokaryons when paired with non­
sibling homokaryons. In addition, colony morphology
of all single spore isolates from homokaryotic basi­
diomes were identical compared to a range of colony
morphologies observed for single spore isolates col­
lected from heterokaryotic basidiomes.
Heterokaryon-homokaryon pairings. -Myceliafrom (RMR
12-5 × RMR 12-10) and 46-4 had grown together by
1 wk; very little pigment was evident in the interaction
zone and hyphae from both progenitors intermingled
(FIG. 9). By approximately 2 wk, a distinct sparse zone
was visible and pigment had formed within the agar
at the margin of the interaction zone (FIG. 10). By 4­
6 wk the barrage reaction was very strong (FIG. 11)
and identical to that noted in heterokaryon-hetero­
karyon pairings (FIG. 12).
All subcultures (FIG. 9-11, subcultures D through
J) excised over the 4-wk period from the original
homokaryotic thallus (RMR 46-4) formed double lines
when paired with homokaryons RMR 12-1 0 and RMR
8-1, and heterokaryon (RMR 12-5 × RMR 12-10).
The same subcultures completely intermingled to form
an apparent single thallus when paired with homo­
karyons RMR 46-4 and RMR 12-5. These results sug­
gest migration of the RMR 12-5 nucleus into the
homokaryotic thallus to form a new heterokaryotic
association with RMR 46-4. No migration of the RMR
46-4 nucleus was evident into the thallus of (RMR
12-5 × RMR 12-10) (FIG. 9, subculture A). Results
similar to the above pairing were obtained in heter-
F IGS. 9-11. Time course heterokaryon-homokaryon
pairing RMR 46-4 × (RMR 12-5 × 12-10). Letters on the
figures represent subculture points (see TABLE V). Culture
morphology at: 1 wk (FIG. 9); 2 wk (FIG. 10); 4 wk (FIG. 11).
GILVUS
MATING
81 1
812
MYCOLOGIA
okaryon-homokaryon pairings (RMR 12-1 × RMR 12­
5) × RMR 46-5, and (RMR 12-5 × RMR 12-10) ×
RMR 12-1 (data not shown).
Sectoring in the original homokaryotic thallus (an
indication of migration of both nuclei from the het­
erokaryon) was observed in less than 1% of the hun­
dreds of heterokaryon-homokaryon matings made
during the course of this study. Generally only one
nucleus was accepted into the homokaryotic thallus
during heterokaryon-homokaryon matings.
Somatic incompatibility pairings. -Two
experiments uti­
lizing synthesized heterokaryons were conducted: 1)
pairings among sets of heterokaryons composed of
one common nonsibling nucleus and the other sibrelated and 2) heterokaryons in which all nuclei were
sib-related.
Compatible pairings were scored as those in which
the mycelia from the paired isolates intermingled with
no demarcation lines to form an apparent single thal­
lus (FIG. 13). Mycelia in self-pairings always completely
intermingled to form a single thallus. The incompat­
ible reaction observed was the same as that observed
in pairings between non-sibling heterokaryons; two
pigmented lines were formed within the agar with a
distinct sparse zone between them (FIG. 12, see Pre­
liminary observations).
In the pairings in which all nuclei were sibling re­
lated, we observed a small number of pairings where
the paired mycelia did not merge smoothly to form a
single thallus, but only limited amount of pigment,
usually in a single line rather than a double line, was
formed within the agar (FIG. 14). This reaction dif­
fered from the usual somatic incompatibility reaction
in that the isolates never appeared to grow together
and then die back to form the sparse zone. The in­
termediate reaction was most often observed in pair­
ings that were predicted to be somatically compatible.
Two somatically compatible (SC) groups were de­
tected within the set of RMR 12 sibling isolates when
heterokaryons with the nucleus from nonsibling RMR
8-1 in common were paired in all possible combina­
tions (TABLE V). The two SC groups were SCl (1, 3,
4, 5, 6, 8, 14, 16) and SC2 (7, 9, 10, 11, 12, 13, 17).
The two SC groups delineated in this experiment were
not correlated with the four different mating groups
(see TABLE 11). The pairings were very easy to score
and very few questionable interactions were noted.
FIGS. 12-14. Examples of heterokaryon-heterokaryon
pairings demonstrating somatic incompatibility. Photographs were taken at 4 wk after pairing. 12. Incompatible
reaction with double pigmented lines. 13. Compatible pairing with no barrage zone. 14. Intermediate reaction with
single pigmented lines. This reaction was observed only with
sibling heterokaryon-heterokaryonpairings.
R IZZO
ET
AL.:
P HELLINUS
GILVUS
MATING
813
T ABLE V. Results of pairings among heterokaryons with a common nucleus. Numbers represent single spore isolates from
RMR 12 which were mated with RMR 8-1. For mating types, see TABLE II
SC1
1
3
4
5
SC2
6
8
14
16
7
9
10
11
12
13
17
a
Symbols: +, completely compatible, mycelia intermingle with no demarcation lines; =, double pigmented lines formed
between mycelia.
Forty-two sibling-related heterokaryons, resulting
from intrabasidiome matings with collection RMR 12
(including those formed by bidirectional nuclear mi­
gration and restricted nuclear migration), were paired
among themselves in all combinations. Using the for­
mation of double pigmented lines as our primary cri­
terion of somatic incompatibility, three somatically
compatible groups were delineated (TABLE VI). This
result is consistent with three heterokaryotic geno­
types SCl SC1, SCl SC2, and SC2 SC2; these geno­
types are based on the groups determined by pairing
heterokaryons with the common nonsibling nucleus
RMR 8-1 (TABLE V). Therefore, for RMR 12, the over­
all results between the two pairing experiments were
consistent (i.e., the same two groups of isolates were
delineated). The intermediate reactions did not fall
into a set pattern that would allow isolates to be as­
signed to specific groups.
Two SC groups were also detected within the set of
FP 133214 sibling spores when synthesized hetero­
karyons, with the nucleus from nonsibling RMR 8-1
in common, were paired in all possible combinations
(TABLE VII). The two groups were SC3 (FP 133214­
1, 4, 9, 10, 11, 15, 16, 17) and SC4 (FP 133214-3, 5,
12, 13). Reaction intensities were strong and very few
questionable pairings were noted. The two SC groups
were not correlated with the four different mating
groups, although all of the isolates with the A3 allele
were in the same SC group (see TABLE 111).
Twenty-four heterokaryons resulting from intra­
basidiome matings with collection FP 133214 were
paired among themselves in all combinations. Because
all isolates with mating locus A3 were in the same SC
group in the pairing with a common nonsibling nu­
cleus (TABLE VII) , only two heterokaryotic genotypes
were predicted, SC3 SC3 and SC3 SC4. Based on
double line formation, most of the pairings fell into
the predicted two groups (TABLE VIII). The inter­
mediate reactions did not fall into a set pattern that
would allow isolates to be assigned to specific groups;
however, pairings involving the heterokaryons (11 ×
3) and (11 × 12) showed a high proportion of inter­
mediate reactions with both SC3 SC3 and SC3 SC4
genotypes (TABLE VIII).
A number of other somatic incompatibility pairings
were also conducted (data not shown) using single
spores and various combinations of nonsibling isolates
as a common nucleus. In all cases, only two somatically
compatible groups were detected and the reactions
were very easy to score.
As part of our observations of somatic incompati­
bility in P. gilvus, the intensity of the incompatible
reaction was scored in intrabasidiome homokaryon­
homokaryon crosses with RMR 12 and FP 133214
(TABLES IX, X). This rating was done prior to the
observation of the results of the heterokaryon-heter­
okaryon somatic incompatibility pairings. Pairings were
scored on a scale of 0 to 3: the 0 score represented
complete intermingling of hyphae with no pigment
formation, 1 indicated a slight sparse zone between
paired mycelia with little or no pigment formation, 2
indicated some pigment formation in the interaction
zone, and a 3 rating indicated very distinct pigment
formation within the agar (FIGS. 5-8). Very little pig-
MYCOLOGIA
814
TABLE VI Results of parrings between synthesized sibling heterkaryons from collection RMR 12. See Table II for results
of original pairings
SC1SC1
1×
3
5
6
SC1SC2 4×
14
16
5
14
8×
16
3
5
6
10× 14
16
3
5
6
14
16 a Symbols: +, compatible, mycelia merge with no demarcation lines; =, double pigmented lines within agar; -, mycelia did
not merge smoothly, often a single pigmented line formed in agar.
ment formation was noted in crosses between homokaryons that were sexually incompatible but within the
same SC group, while barrage formation was much
more intense between sexually incompatible homo-
karyons that were in different SC groups (TABLES IX,
X). Pairings between sexually compatible isolates did
not result in pigment formation within the agar; all of
these pairings were scored as 0.
RIZZO
ET AL.:
PHELLINUS
GILVUS
815
MATING
TABLE VI. Results of pairings between synthesized sibling heterokaryons from collection RMR 12. See TABLE II for results
of original pairings (Extended)
SC1SC2
1×
7
9
11
SC2SC2
4×
12
13
17
7
9
11
8×
12
13
17
DISCUSSION
The results of the homokaryon-homokaryon pairings
demonstrate that P. gilvus has an outcrossing breeding
system that tentatively may be considered bifactorial.
We have observed compatible matings in both intrabasidiome and interbasidiome crosses, and P. gilvus
has completed its life cycle following such crosses. By
7
9
11
10×
12
13
17
7
9
11
12
13
17
pairing subcultures taken from homokaryon-homo­
karyon crosses with a nonsibling homokaryon, we were
able to detect heterokaryon formation and determine
nuclear migration patterns. A genetic basis for the
pairing test was confirmed in a time-course heterokaryon-homokaryon pairing experiment. Genetic con­
trol of somatic incompatibility reactions was found to
be complex; it was determined, however, that sexual
816
MYCOLOGIA
T ABLE VII. Results of pairings among heterokaryons with
a common nucleus. Numbers represent single spore isolates
from FP 133214 which were mated with RMR 8-1. For
mating types, see TABLE III
SC3
1
4
9
10
(Hirt, 1928). This report was based on the formation
of basidiomes in culture by single basidiospore iso­
lates. Fruiting of homokaryons, however, does not
necessarily indicate a nonoutcrossing mating system
(Collins, 1979; Rayner, 1990). A number of our single
basidiospore isolates also formed basidiomes in cul­
ture; however, single spore isolates obtained from such
basidiomes had identical colony morphologyand, when
paired in all combinations among themselves, all iso­
lates from a single basidiome intermingled as in self
pairings. Additionally, these isolates behaved as hom­
okaryons when paired with nonsibling homokaryons,
which would rule out a predominantly secondary ho­
mothallic mating system. We believe these fruiting
bodies are simply the result of homokaryotic fruiting
similar to what has been described for other hymen­
omycetes (Leslie and Leonard, 1979).
Two types of heterokaryon formation were evident
in intrabasidiome pairings of P. gilvus: bidirectional
nuclear migration resulting in the formation of a single
heterokaryotic thallus and restricted nuclear migra­
tion with a heterokaryon formed only in the interac­
tion zone between the paired homokaryotic colonies.
Even though 50 % of random sibling matings resulted
in stable heterokaryon formation, nuclear migration
patterns indicate two factors or loci may be involved
SC4
11
15
17
3
5
12
13
Symbols: + , completely compatible, mycelia intermingle
with no demarcation lines; =, double pigmented lines formed
between mycelia.
a
and somatic incompatibility in P. gilvus are controlled
by separate nonlinked loci.
Sexuality in Phellinus gilvus.-Our
results contrast with
a previous study suggesting P. gilvus is homothallic
TABLE VIII. Results of pairings between synthesized sibling heterokaryons from collection FP 133214. See TABLE III for
results of original pairings
SC3SC3
1×
4
9×
15
4
15
11×
4
15
SC3SC4
17×
4
15
3
1×
12
13
3
9×
12
13
3
11×
12
13
17×
3
12 13
a Symbols: +, compatible, mycelia merge with no demarcation lines; =, double pigmented lines within agar; -, mycelia did
not merge smoothly, occasionally a single pigmented line formed in agar.
RIZZO
TABLE IX.
ET AL.:
PHELLINVS
GILVUS
MATING
817
Phenotypes of incompatible matings in intrabasidiome cross with single basidiospore isolates from RMR 12
AlBn
A2Bn
SC2
SC1
1
4
8
10
SC2
SC1
3
6
5
14
16
7
9
11
12
13
17
Ratings: 0, complete intermingling of mycelia; 1, sparse zone formation, very little or no pigment formation; 2, distinct
reaction between isolates with some pigment formation; 3, very strong pigment formation.
a
thallus. These results indicate that the mating system
of P. gilvus is multiallelic at both mating factors.
The mating system observed here for P. gilvus is
similar to that described for outcrossing isolates of
Coprinus patouillardii Quél. (Kemp, 1980). The secondary mycelium of C. patouillardii also lacks clamp
connections and B= matings result in stable, fertile
heterokaryons formed only at the interaction zone. In
other hymenomycetes (e.g., Schizophyllum commune Fr.,
in mating. A=/ , B =/ matings resulted in heterokaryon
formation via bidirectional nuclear migration, and
A =/ , B= matings resulted in heterokaryon formation
with restricted nuclear migration. Common A heterokaryons were not observed in any of the crosses.
Restricted nuclear migration did not occur in pairings
among single spore isolates from different basidiomes.
All interbasidiome pairings had bidirectional nuclear
migration and resulted in the formation of a single
TABLE X. Phenotypes of incompatible matings in intrabasidiome cross with single basidiospore isolates from FP 133214.
Ratings as in TABLE IX
A4Bn
A3Bn
SC3
1
a
9
See TABLE IX, footnote
10
a
5c4
SC3
11
17
for explanation of ratings.
4
15
16
3
5
12
13
818
MYCOLOGIA
Coprinus cinereus (Fr.) S. F. Gray), the A locus has been
implicated in clamp cell formation and synchronized
nuclear division, while the B locus is thought to reg­
ulate nuclear migration and septal breakdown follow­
ing hyphal fusion (Kües and Casselton, 1992). Common
B matings in clamped hymenomycetes will often result
in an unstable heterokaryon or heterokaryons unable
to complete the life cycle (Kües and Casselton, 1992).
For C. patouillardii, Kemp (1980) considered one of
the mating factors to control nuclear migration while
the other to control fertility. As noted above, however,
fruiting in P. gilvus appears to be controlled by a
separate genetic system; fruiting has been observed in
both homokaryons and heterokaryons.
Basidiomes formed as a result of B= matings in P.
gilvus produce spores of only two mating types (e.g.,
A1B1 and A2B1) and crosses among these sibling sin­
gle spore progeny will not have nuclear migration. This
was determined in crosses with single spore progeny
derived from in vitro fruiting that were the result of
common B matings. In our crosses with spores from
field collected fruiting bodies RMR 46, HHB 11806,
and NO 7661, almost all heterokaryons were formed
by restricted nuclear migration. In these collections,
the parental heterokaryons possibly were homozygous
for the B mating factor. Alternatively, the low number
of spores available for the pairing experiments may
not have included all four mating types.
In terms of nuclear migration, P. gilvus appears to
be intermediate between other Phellinus and Inonotus
species for which mating studies have been conducted.
Restricted nuclear migration has been previously de­
scribed for 15 heterothallic, bipolar Phellinus and In­
onotus species, including pairings among both sibling
or nonsibling isolates (Fischer, 1987, 1994; Fischer and
Bresinsky, 1992). These species have potentially lost
the mating factor that controls nuclear migration, or
this factor has become fixed in a homozygous condi­
tion in certain populations. In contrast, bidirectional
nuclear migration was detected in 95% of compatible
matings in intrabasidiome pairings with P. weirii (Ang­
win and Hansen, 1993). The genetic structure of the
mating locus of P. weirii may be fundamentally dif­
ferent from P. gilvus to control both nuclear migration
and heterokaryon stability.
In each of the P. gilvus spore collections, several
crosses were observed that did not fit expected nuclear
migration patterns, e.g., crosses that resulted in re­
stricted nuclear migration rather than bidirectional
nuclear migration. These exceptions may be due to
recombination within the mating loci. The mating loci
of P. gilvus may be as complex as those described for
S. commune and C. cinereus in consisting of several
linked genes (Day, 1960; Kües and Casselton, 1992; May
et al., 1991). More detailed genetic analysis would be
necessary to determine the structure and recombi­
nation rates of the mating factors in P. gilvus, as well
as other possible genetic explanations (e.g., modifier
genes, Raper and Raper, 1964).
Heterokaryon-homokaryon interactions. -Our
results in­
dicate that nuclear migration occurs between heter­
okaryons and homokaryons of P. gilvus in the manner
described for other hymenomycetes (e.g., Buller, 1931;
Ellingboe and Raper, 1962; Rayner and Todd, 1979;
Aylmore and Todd, 1984; Coates and Rayner, 1985;
May and Taylor, 1988). Anastomosis between heter­
okaryons and homokaryons in P. gilvus initially occurs
without barrage formation. Nuclear migration pro­
ceeds rapidly and, within 1 wk, new genetic associa­
tions among nuclei have been established in the pre­
viously homokaryotic thallus. Once new nuclear as­
sociations occur, somatic incompatibility reactions de­
velop between the original heterokaryon and the newly
formed heterokaryon resulting in barrage formation
(double pigmented lines with a sparse zone between).
The demonstration of nuclear migration in hetero­
karyon-homokaryon pairings provides a genetic basis
for the pairing tests used to determine the mating
system in P. gilvus.
Similar reactions between heterokaryons and hom­
okaryons occur in P. weirii, which apparently also has
bidirectional nuclear migration (Angwin and Hansen,
1993). Barrage formation in heterokaryon-homokar­
yon pairings has been described for several Phellinus
species in which only restricted nuclear migration oc­
curs (Fischer, 1994; Fischer and Bresinsky, 1992). The
basis of barrage formation in heterokaryon-homokar­
yon crosses under these circumstances has not been
experimentally tested in these other Phellinus species.
Usually only one nucleus is selected in heterokar­
yon-homokaryon interactions with P. gilvus, which is
similar to nuclear migration patterns described for S.
commune and C. cinereus (Ellingboe and Raper, 1962;
Swiezyynski and Day, 1960). “Tracks” (Coates and Ray­
ner, 1985) of somatically incompatible genotypes re­
sulting from the migration of both nuclei of the het­
erokaryon into the homokaryotic thallus were very
rarely observed in P. gilvus.
Genetic control of somatic incompatibility. -Pairings
among synthesized heterokaryons from two P. gilvus
spore families indicated that one heterozygous locus
had major effects on somatic incompatibility. This was
supported by the delineation of the same groups of
somatically compatible isolates in several different
pairing experiments. Heterokaryons were homozy­
gous or heterozygous for this locus, but paired het­
erokaryons needed to have identical genotypes at the
locus to be fully compatible. A single locus regulating
somatic incompatibility has been suggested for P. wei-
RIZZO
ET AL.:
PHELLINUS
rii (Hansen et al., 1994). As with P. gilvus, pairings
among heterokaryons with a nonsibling common nu­
cleus in P. weirii resulted in only two compatible groups.
However, incompatibility between sibling hetero­
karyons that were predicted to be identical for the
somatic incompatibility locus suggests that these as­
sociations are complex in P. gilvus and more than one
gene or locus may be involved. Somatic incompatibility
in Pleurotus ostreatus (Jacq.:Fr.) Kummer appears to
be regulated by up to three loci, although in some
dikaryons incompatibility may be determined by a sin­
gle locus of large genetic effect (Malik and Vilgalys,
1994). Three to four loci appear to control somatic
incompatibility in Heterobasidion annosum (Fr.) Bref.;
nonsibling common nucleus crosses resulted in an av­
erage of 12% compatibility (Hansen et al., 1993b). A
more detailed genetic analysis of somatic incompati­
bility in P. gilvus, particularly at the population level,
than is presented in this paper will be necessary to
sort out inheritance patterns of somatic incompati­
bility genes. Additionally, the effects of cytoplasmic
factors on somatic incompatibility are not known for
P. gilvus. All of our experiments were designed to
maintain a single mitochondrial type in the paired
isolates. Potential mitochondrial affects on somatic in­
compatibility have been observed in C. cinereus and H.
annosum (May, 1988; Hansen et al., 1993b).
Somatic incompatibility in P. gilvus was not corre­
lated with mating type in our experiments; each SC
group contained isolates of all mating types. Because
somatic incompatibility systems operate to prevent in­
vasion of nonself genes, it has been hypothesized that
successful mating in hymenomycetes requires the
override of somatic incompatibility (Coates et al., 1985;
Rayner, 1990, 1991). Correlation of the P. gilvus SC
groups with the intensity of the negative phenotype
in homokaryon-homokaryon matings generally sup­
ports this hypothesis. Sexual compatibility is the result
of compatibility of nonself alleles rather than self al­
leles being incompatible (Casselton and Economou,
1985); therefore, the barrage reactions observed be­
tween incompatible homokaryons are most likely due
to expression of somatic incompatibility genes.
While our results confirm a genetic basis for indi­
vidualism in Phellinus gilvus, in some predictable in­
stances no obvious somatic incompatibility reactions
occurred between closely related heterokaryons. In
these pairings, we observed the formation of an ap­
parently single thallus in pairings between heterokar­
yons with three or four different (via meiosis) nuclei
that shared somatic incompatibility alleles. The genetic
consequences of intermingling isolates with four dif­
ferent nuclei is not known, i.e., we do not know if new
genetic associations are formed among the nuclei and
if the new thallus acts as a single physiological unit.
GILVUS
MATING
819
ACKNOWLEDGMENTS
Comments on the manuscript by Everett Hansen, Tina Dreis­
bach, Mike Larsen, Tom Volk, Jim Anderson, and two anon­
ymous reviewers are greatly appreciated. Paul Gieser and
Georgiana May offered helpful discussions on fungal mating
systems. The technical assistance of Fran Silver is also ac­
knowledged.
LITERATURE CITED
820
MYCOLOGIA