mycological research 113 (2009) 583–590
journal homepage: www.elsevier.com/locate/mycres
Trikaryon formation and nuclear selection in pairings
between heterokaryons and homokaryons of the root
rot pathogen Heterobasidion parviporum
Timothy Y. JAMESa,b,*, Stina B. K. JOHANSSONa,b, Hanna JOHANNESSONa
a
Department of Evolutionary Biology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18D, 752 36 Uppsala, Sweden
Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, 750 07 Uppsala, Sweden
b
article info
abstract
Article history:
Pairings between heterokaryons and homokaryons of Agaricomycete fungi (he-ho pairings)
Received 29 July 2008
can lead to either heterokaryotization of the homokaryon or displacement of the homo-
Received in revised form
karyotic nucleus through migration of nuclei from the heterokaryon into the homokaryon.
20 January 2009
In species of Agaricomycetes with multinucleate cells (>2 nuclei per cell), he-ho pairings
Accepted 30 January 2009
could result in the stable or transient formation of a hypha with three genetically different
Published online 7 February 2009
nuclei (trikaryons). In this study, he-ho pairings were conducted using the multinucleate
Corresponding Editor: Gregory S May
Agaricomycete Heterobasidion parviporum to determine whether trikaryons can be formed
in the laboratory and whether nuclear genotype affects migration and heterokaryon forma-
Keywords:
tion. Nuclei were tracked by genotyping the heterokaryotic mycelium using nucleus-spe-
Butt rot
cific microsatellite markers. The data indicated that certain nuclear combinations were
Dikaryon
favored, and that nuclei from some strains had a higher rate of migration. A high percent-
Heterobasidion annosum
age of trikaryons (19 %) displaying three microsatellite alleles per locus were identified
Nuclear migration
among subcultures of the he-ho pairings. Using hyphal tip and conidial isolation, we ver-
Plurinucleate
ified that nuclei of three different mating types can inhabit the same mycelium, and one of
the trikaryotic strains was judged to be semi-stable over multiple sub-culturing steps, with
some hyphal tips that retained three alleles and others that reduced to two alleles per locus. These results demonstrate that nuclear competition and selection are possible outcomes of heterokaryon-homokaryon interactions in H. parviporum and confirm that
ratios of component nuclei in heterokaryons are not strictly 1:1. The high rate of trikaryon
formation in this study suggests that fungi with multinucleate cells may have the potential
for greater genetic diversity and recombination relative to dikaryotic fungi.
ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction
The life cycle of most sexual Agaricomycete fungi alternates
between homokaryotic and heterokaryotic stages. The homokaryon is a genetically haploid mycelium that contains only
a single type of nucleus, and the heterokaryon is a mycelium
containing two types of nuclei produced through the mating
of homokaryons. The heterokaryon is genetically diploid, but
the nuclei contributed by the homokaryons remain in an
unfused state until immediately before meiosis and spore production. The heterokaryons of many Basidiomycetes have
cells with exactly two nuclei per cell, one from each mating
* Corresponding author. Department of Ecology and Evolution, University of Michigan, Ann Arbor, MI 48109, USA. Tel.: þ1 734 615 7753;
fax: þ1 734 763 0544.
E-mail address: [email protected]
0953-7562/$ – see front matter ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.mycres.2009.01.006
584
partner, and are referred to as dikaryons. In nature, interactions between heterokaryotic and homokaryotic mycelia are
also possible and may result in a number of outcomes, such
as heterokaryotization of the homokaryotic mycelium (i.e.,
the Buller phenomenon) or displacement of the homokaryotic
nuclei from their resident mycelium (Buller 1930; Coates &
Rayner 1985; Crowe 1960; May 1988; Nogami et al. 2002;
Tanesaka et al. 1994). In cases of heterokaryotization, nuclei
from the heterokaryon migrate into the recipient homokaryotic mycelium to fertilize it and form a heterokaryon of novel
nuclear constitution. Heterokaryon-homokaryon interactions
can be partially or fully compatible depending on whether one
or both of the nuclei of the heterokaryon share a mating type
with the homokaryon.
In fully compatible matings in the lab between heterokaryons and homokaryons (hereafter referred to as he-ho
pairings), it has often been observed that one of the nuclei in
the heterokaryon preferentially fertilizes the homokaryon
(Raper 1966). These results have suggested inter-nuclear
selection acts during the process of heterokaryotization
(Anderson & Kohn 2007). In the species Coprinus macrorhizus,
Psilocybe coprophila (Kimura 1958), Schizophyllum commune
(Crowe 1963), and Stereum hirsutum (Coates & Rayner 1985),
nuclear selection generally appears to favor heterokaryon
formation between nuclei that are genetically less similar.
The selective advantage of genetically dissimilar nuclei could
be explained by heterosis or by the phenomenon where
genetic heterogeneity between nuclei leads to a more effective
override of the self-non-self rejection response that accompanies all hyphal interactions (Coates & Rayner 1985).
Another possible way to explain the biases that determine
which nucleus of a heterokaryon will fertilize a homokaryon
posits an interaction between the nuclei and the mitochondria/cytoplasm. Nuclei, but not mitochondria, undergo migration during mating in Agaricomycetes, and this difference in
mode of inheritance increases the potential for genomic conflict between nuclear and mitochondrial genomes (Aanen et al.
2004). This hypothesis has been suggested to explain the observations in many Agaricomycete species, wherein, mating
between fully compatible homokaryons results in unidirectional migration of nuclei into only one of the homokaryotic
mycelia (May & Taylor 1988). In he-ho interactions, genomic
conflict between cytoplasmic and nuclear genes in the heterokaryon could influence nuclear migration and fertilization of
the homokaryotic mycelium. The effects of the cytoplasm
on nuclear selection in he-ho mating are largely unknown.
Yet another possible explanation for nuclear selection in
he-ho pairings is an effect of the MAT-B mating-type locus
(Ellingboe & Raper 1962). MAT-B is known to encode pheromones and pheromone receptors that are involved in controlling nuclear migration; the lipopeptide pheromones control
nuclear migration by stimulation of compatible receptors,
whereas the receptors are hypothesized to control acceptance
of immigrating nuclei (Brown & Casselton 2001; Kothe et al.
2003). Because not all pheromones are capable of stimulating every receptor allele (Fowler et al. 2004), nuclear selection could act
to favor combinations in which the MAT-B alleles of the nuclei
encode highly compatible receptor-pheromone combinations.
Many studies of he-ho pairings have observed that both nuclei of the heterokaryon migrate into the homokaryon (Ellingboe
T. Y. James et al.
& Raper 1962; Ramsdale 1999), suggesting that during the fertilization or displacement process, hyphae or mycelial sectors containing three genetically different nuclei (trikaryotic hyphae)
may be formed. Whether these trikaryons form is uncertain,
nor is it certain whether they could proliferate and be stably
maintained through conjugate nuclear division and clamp cell
formation as dictated by the mating-type genes (Brown & Casselton 2001; Raper 1966). In this paper, the term trikaryon is used
sensu de Serres & Brockman (1968) and refers to a heterokaryon
that is composed of three types of genetically different nuclei
and does not refer to the exact number of nuclei per cell. Despite
the knowledge of the precise cellular organization, the existence
of more than two haploid genome equivalents in a single heterokaryotic mycelium has been demonstrated several times in Basidiomycetes. In dikaryotic Basidiomycetes species, genetic
triploids have been observed, wherein a diploid nucleus undergoes conjugate division with a haploid nucleus, a 2N þ N nuclear condition (Koltin & Raper 1968; Lin et al. 2007). In a diploid
Agaricomycete genus (Armillaria spp.), diploid-haploid pairings
revealed mating products consistent with triploidy (Carvalho
et al. 1995) or 2N þ N (Rizzo & May 1994). Finally, in Heterobasidion
annosum, one heterokaryon isolated from a stump was shown to
possess four genetically different nuclei (Johannesson & Stenlid
2004). All of these observations challenge the notion that heterokaryons of most Agaricomycete fungi are comprised of two nuclear genomes.
The formation and maintenance of trikaryotic hyphae or
hyphae of even greater nuclear numbers are likely to be promoted in species that have more than two nuclei per cell
(i.e., multinucleate or plurinucleate species), a prerequisite
satisfied by many species of Agaricomycetes (Boidin 1971).
The root rot pathogen Heterobasidion annosum has a highly multinucleate mycelium with as many as 80 nuclei per cell (Stenlid
& Rayner 1991). A lack of strong control of nuclear pairing and
conjugate division is suggested in H. annosum, where nuclear
ratios may become imbalanced towards one or the other mating-type nucleus (James et al. 2008; Ramsdale & Rayner 1996).
These observed nuclear ratio imbalances have been largely
attributed to nuclear competition within a heterokaryon.
In this paper we perform he-ho pairings with isolates of
H. parviporum (¼H. annosum European S-intersterility group) to
test if nuclear selection occurs and is influenced by mitochondrial
genotype. Furthermore, we investigated if trikaryotic heterokaryons are frequently formed, and whether they are transient
or stable. Four homokaryotic strains were used to construct
heterokaryons, and molecular markers were used to track the
individual nuclei in subcultured mycelia. A high degree of nuclear
selection, nuclear displacement, and the formation of trikaryons
were observed in subcultures from these pairings. Evidence is
presented to show that the hyphae can be truly trikaryotic, i.e.
nuclei of three different mating types can inhabit the same cell,
and are semi-stable over multiple rounds of subculturing.
Materials and methods
Strains and culture conditions
Four homokaryotic strains of H. parviporum were used (Table 1):
Br-518_c2 (originating from Brynge, Sweden), Ult1.5 (Ultuna,
Trikaryon formation and nuclear selection in Heterobasidion
Table 1 – Outcome of the he-ho pairings comparing
behavior of homokaryotic strains as nuclear recipients
(female or cytoplasmic parent) and potential nuclear
donors (male parent). For the nuclear donors, shown are
the percentage of times a given nucleus migrated into
and heterokaryotized the recipient mycelium (either to
form a two or three component heterokaryon), the
percentage of times the nucleus did not migrate into the
homokaryon, and the percentage of times the nucleus
displaced the recipient homokaryotic nucleus, including
the instances when both nuclei of the paternal
heterokaryon displaced the recipient homokaryon
Homokaryotic strain as nuclear recipient
% heterokaryotization
% female
sterile
% displaced
40.7
71.4
56.5
5.3
40.7
11.9
37.0
94.7
18.5
16.7
6.5
0.0
87074/1
95191
Br-518_c2
Ult1.5
Homokaryotic strain as nuclear donor
87074/1
95191
Br-518_c2
Ult1.5
% nucleus
fertilized
homokaryon
% nucleus did
not emigrate
% nuclear
displacement
17.5
15.7
48.1
54.8
82.5
70.6
40.4
28.6
0.0
13.7
11.5
16.7
Sweden), 87074/1 (Vicenza, Italy), and 95191 (Ural, Russia).
These strains were determined to all be of different mating
types based on pairings. Strains were routinely cultured on
weak malt extract agar (M/10: 0.2 % malt extract; 2 % agar),
and mating and incompatibility assays were carried out on
rich malt extract agar (MEA: 2 % malt extract; 2 % agar). All culturing was carried out on 90 mm Petri dishes. Growth rates
were determined as described in James et al. (2008).
Heterokaryon synthesis
The four homokaryons were crossed in all possible combinations by pairings on MEA to produce the heterokaryons used
in these experiments. Strains were inoculated w2 cm apart
from each other on Petri dishes, and heterokaryons were isolated by subculturing mating interactions onto M/10 after 11 d
of co-culture. Heterokaryons with identical nuclear genotypes
but variable cytoplasmic genotypes were isolated by subculturing from both sides of the interaction zone, at w1 cm distance. Nuclear migration in H. annosum sensu lato is not as
fast as many other Agaricomycetes (Stenlid & Rayner 1991),
and therefore subculturing was repeated for strains that had
not successfully mated at 17 and 32 d post-inoculation. These
crosses resulted in the synthesis of 6 different heterokaryons
each in two cytoplasmic backgrounds, for a total of 12 strains
for investigation in he-ho interactions.
Nuclear selection in heterokaryon-homokaryon pairings
Each test heterokaryon was completely compatible with two
of the other homokaryons because no nuclear types were
585
shared. Crosses between each heterokaryon and these two
compatible homokaryons were carried out to determine
whether the homokaryon (female or recipient mycelium)
would show preference in heterokaryon formation of one or
the other nucleus. For each he-ho combination, six replicate
pairings were established in the following manner: small
w5 5 mm subcultures were inoculated w2 cm distant to
each other in the middle of a Petri dish containing MEA. The
he-ho pairings were allowed to develop for 22 d, after which
an interaction zone associated with pigment and a lack of aerial hyphal growth developed. In order to detect the introgression of nuclei into the homokaryon, the subculturing method
described by Coates & Rayner (1985) was employed. A
4 20 mm strip of agar was cut from the homokaryotic side
of the interaction w4 mm behind the original homokaryotic
inoculum. The agar strips were transferred onto fresh MEA
and incubated for 15–16 d after which the outgrowing mycelia
were examined for the presence of visible sectors indicating
multiple genetically different mycelia.
The plates with the agar strips were then subcultured onto
Petri dishes containing M/10. When the plates with agar strips
had visible sectors, the mycelia from each of the sectors were
subject to further analysis. The subcultures were investigated
for clamp connections indicating heterokaryosis. After 1 week
of growth, and regardless of whether cultures displayed clamp
connections, the plates were again subcultured to Petri dishes
containing M/10 overlain with sterile cellophane. After another week of growth the mycelia were harvested from the
surface of the cellophane and DNA techniques were used to
estimate the nuclear content of the mycelia.
DNA techniques
Microsatellite markers were used to track individual nuclei of
subcultures produced by the he-ho matings. By knowing the
alleles at the microsatellite loci for each homokaryon, the
microsatellite profiles of the subcultures could be analyzed
and nuclear content assigned. Protocols for DNA isolation
and microsatellite PCR are given in James et al. (2008). Samples
were analyzed on a MegaBACE 1000 (Amersham) capillary sequencer by using fluorophore labeled reverse primers, and
electropherograms were analyzed with the software Genetic
Profiler (Amersham). Two microsatellite markers were used
to determine the nuclear composition of the subcultures isolated from he-ho pairings (Ha-ms1 and Ha-ms10; Johannesson
& Stenlid 2004). Alleles (size in bp) for homokaryotic strains at
locus Ha-ms1 were: 87074/1 (179), 95191 (157), Br-518_c2 (161),
Ult1.5 (157). Alleles at locus Ha-ms10 were: 87074/1 (295),
95191 (324), Br-518_c2 (293), Ult1.5 (289). An additional locus
(Ha-ms6) was used to genotype putative trikaryotic hyphal
tip isolates, with alleles: 87074/1 (269), 95191 (275), Br-518_c2
(275).
Formation and stability of trikaryons
Subcultures of the he-ho pairings produced 25 putative
trikaryons based on molecular markers. Two of these were investigated further to determine if they were truly trikaryotic,
defined here to be containing three different nuclei in the
same mycelium or cell, and whether they were stable over
586
T. Y. James et al.
multiple cell divisions. Single conidiospore isolates of the trikaryons were obtained by dilution plating on M/10, and single
hyphal tips were isolated following the method of Hansen
et al. (1993). For one trikaryon a second round of hyphal tip
isolation was accomplished. Single conidial and hyphal tip
isolates were grown in 5 ml of M/10 broth, the mycelium harvested after 10 d, and DNA was extracted from mycelia and
genotypes determined using microsatellite markers.
Results
Nuclear migration and selection in he-ho matings
The four homokaryons were each confronted with 3–6 heterokaryons with which they were fully compatible. After
co-culture, a strip of agar was taken from the initially homokaryotic side of the interaction so that sectors of genetically
different mycelia could be detected (Coates & Rayner 1985).
Of 105 he-ho matings that were subcultured, 34 displayed
indications of sectoring upon subculture. No more than two
A
sectors were ever identified and sectoring was typically not
associated with a strong rejection response, such as heavy
pigmentation or a barrage. Twenty-nine of these sectored
plates were comprised of genetically distinct mycelia based
on the genotyping results. Genotyping the 134 independent
subcultures revealed 40.3 % homokaryons, 40.3 % heterokaryons of two nuclear types, and 19.4 % heterokaryons with
three nuclear types (Fig 1). Pairings with three of the homokaryons (87074/1, 95191, Br-518_c2) lead to a high degree of
heterokaryotization, however pairings with Ult1.5 were
mostly female sterile, i.e. did not accept nuclei (Table 1). However, as a component of a heterokaryon, the Ult1.5 nucleus
was very successful at migrating into the recipient homokaryon (Table 1, Fig 1). Nuclei of strains Ult1.5 and Br-518_c2
displayed a similar high ability to access homokaryons in
he-ho pairings, whereas nuclei of strains 87074/1 and 95191
showed a lower relative ability to migrate (Table 1).
The two parental nuclei of a heterokaryon displayed differences in the ability to fertilize the homokaryon, and these differences were investigated for evidence of nuclear selection
(Fig 1). Pairings in which a single nucleus fertilized the
C
het.
nucleus1
87
homokaryon
95
Ult
Ult
87
87
Br
Br
87
Ult
*
Ult
95
Br
het.
nucleus2 (C)
Ult
Ult
95
87
87
Ult
95
87
subculture
B
D
87
Br
95
87
95
87
Br
95
Br
Br
87
95
*
Ult
95
Br
Ult
87
Br
95
Ult
87
95
Ult
Ult
87
Br
95
Ult
95
Br
Ult
87
95
Br
Br
Ult
Fig 1 – Nuclear genotypes recovered from subcultures of he-ho pairings. In the large circles are displayed the paired strains
in he-ho interactions. The homokaryon (nuclear recipient) is shown as the right half of the circle. The two nuclei of
the heterokaryon are shown in the left half of the circle, separated by a dashed line (het. nucleus1 & het. nucleus 2 (C)).
The cytoplasmic parent of the heterokaryon (het. nucleus2 (C)) is displayed in the southwestern quadrant. The smaller circles
connected by arrows show the nuclear genotypes of the subcultured he-ho interactions. When the subcultures displayed
sectoring on MEA, the two sectors were individually genotyped, and the sectors are displayed as two circles connected by a
dashed line. (A) he-ho interactions with homokaryon Ult1.5. (B) homokaryon 95191. (C) homokaryon 87074/1. (D) homokaryon Br-518_c2. Asterisks indicate putative trikaryons that were subjected to additional analyses.
Trikaryon formation and nuclear selection in Heterobasidion
recipient homokaryon (uninuclear fertilization) were compared for the three female fertile homokaryotic strains. For
strain 95191, pairings with heterokaryon (87074/1 þ Br518_c2) led to fertilization by Br-518_c2 6 out of the 8 times
that uninuclear fertilization occurred. Pairings with
(Ult1.5 þ Br-518_c2) led to uninuclear fertilization only one
time (by Ult1.5). Pairings with (87074/1 þ Ult1.5) led to uninuclear fertilization 8 times, all by Ult1.5 (Fig 1B). These data suggest that for recipient strain 95191, Ult1.5 and Br-518_c2 have
similar ability to fertilize and are both more male fertile than
87074/1. For strain 87074/1, pairings with heterokaryotic
strains (Ult1.5 þ Br-518_c2 & Ult1.5 þ 95191) led to uninuclear
fertilization in only three instances (all by Ult1.5). Instead,
pairings with homokaryon 87074/1 led to a high rate of binuclear migration and a high rate of nuclear displacement
(Fig 1C & Table 1). For strain Br-518_c2 pairings with
(95191 þ 87074/1) led to three uninuclear fertilizations, all
by strain 87074/1. In pairings with heterokaryotic strains
containing the Ult1.5 nucleus, the primary outcome was uninuclear fertilization, all of which involved the fertilization by
the nuclei from Ult1.5 (n ¼ 18; Fig 1D).
The effect of cytoplasmic background on nuclear migration
The test heterokaryons were isolated in the cytoplasmic backgrounds of both progenitor homokaryons, allowing us to test
the effect of cytoplasmic origin and mitochondrial genotype
on nuclear migration during he-ho matings. Nine pairs of heterokaryons were investigated for differences using Fisher’s
exact test. For only one of the pairs a significant effect of cytoplasmic background was observed, the he-ho pairing between
homokaryon 87074/1 and heterokaryon Ult1.5 þ Br-518_c2
(P < 0.01). In pairings in which the heterokaryon was in the
Br-518_c2 cytoplasmic background, very little heterokaryon
formation was observed on the homokaryotic side, whereas
in pairings with the Ult1.5 cytoplasmic background, a high
frequency of three component heterokaryons were formed
(Fig 1C). Altogether, these data demonstrate that origin of
cytoplasm did not have a strong effect on nuclear migration
from heterokaryons into recipient homokaryons.
The formation and stability of trikaryons
He-ho pairings in this study led to a high frequency of formation of heterokaryons with three nuclear types; approximately
1/3 of all of the heterokaryons formed contained three nuclei
as determined by the molecular markers. Further experiments
were conducted on two of these three nucleus heterokaryons
to determine whether they were truly trikaryotic or were mixtures of two or more heterokaryotic individuals. Subcultures
of the putative trikaryons were obtained by isolating single
conidia or single hyphal tips. For one of the isolates, the heterokaryon formed by the pairing of the homokaryon 87074/1
and the heterokaryon (Ult1.5 þ Br-518_c2), both hyphal tips
(n ¼ 3) and conidial isolates (n ¼ 17) contained either the
Ult1.5 or the Br-518_c2 nucleus, or both. The 87074/1 nucleus
was not observed, suggesting this nucleus had been lost
from the heterokaryon(s) during the subculturing process.
The second heterokaryotic strain investigated (tri1),
formed by the pairing of the homokaryon 95191 and the
587
heterokaryon (Br-518_c2 þ 87074/1), demonstrated a more
complex recovery of nuclei in subcultures (Fig 2) and definitive
proof of trikaryosis. Single conidial isolates from this heterokaryon displayed a diversity of genotypes. Most were genotypically homokaryotic for one of the three nuclear types,
and the heterokaryotic genotypes (95191 þ Br-518_c2) and
(95191 þ 87074/1) were also observed. Three hyphal tips were
isolated from this heterokaryon in a first round of subculture.
Genotyping revealed that one isolate was comprised of nuclei
from 95191 and Br-518_c2. A second hyphal tip isolate was
comprised of markers for all three isolates, but only two
alleles at any locus: (95191 þ 87074/1) for Ha-ms1 and (87074/1
þ Br-518_c2) for Ha-ms6 and Ha-ms10. The final hyphal tip
isolate (ht5) contained all three component nuclei as assessed
by three marker loci. The stability of the trikaryon isolated
from ht5 was further assessed by additional subculturing
and another round of hyphal tip isolation (Fig 2). Half of the
hyphal tip subcultures from isolate ht5 were also trikaryotic
and half of the subcultures were dikaryotic. One of the subcultures displayed only two alleles at one of the marker loci
(Ha-ms1), but three alleles were observed at the other two
loci (Fig 2). These results are consistent with loss of heterozygosity due to somatic recombination (Anderson & Kohn 2007).
The linear growth rate of tri1 was also estimated on 0.5 %
malt extract agar and found to be 0.12 mm/hr with a 95 % confidence interval of 0.09–0.16 mm/hr. This value is lower than
all of the homokaryons (n ¼ 10) and all but one of the heterokaryons (n ¼ 40) reported in James et al. (2008).
Discussion
With these experiments we have explored nuclear selection,
replacement, and trikaryon formation (i.e., heterokaryons
composed of three genetically different types of nuclei) during
he-ho pairings in H. parviporum. By using a set of heterokaryons synthesized in a complete crossing design, and molecular markers to distinguish and track each nuclear type,
the experiments allowed us to examine the ability of specific
nuclei to both emigrate from a heterokaryotic mycelium and
to discriminate among potential immigrating nuclei as a maternal homokaryon. Further, by using multiple replicate pairings, we were able to test the strength of nuclear selection/
competition interactions relative to environmental/epigenetic
factors. Finally, the experiments allowed for a high sensitivity
to detect tri-nucleate heterokaryons in this species known to
have imbalanced nuclear ratios in the mycelium (James et al.
2008; Ramsdale & Rayner 1996).
Our results confirmed a number of possible outcomes can
result from he-ho pairings in H. parviporum. Three types of interactions besides Buller phenomenon mating, female sterility, nuclear displacement, and trikaryon formation were all
frequently observed (Table 1). The nuclei of each homokaryotic strain tended to have its own intrinsic behavior in he-ho
pairings. Strain Ult1.5 displayed the highest male fertility as
a nuclear donor but was nearly female sterile (i.e., it did only
rarely accept nuclei as a homokaryon). Conversely, strain
95191 showed the highest female fertility but the lowest
male fertility. Strains Ult1.5 and Br-518_c2 showed similar
ability to behave as nuclear donors, and in pairings with the
588
T. Y. James et al.
Initial he-ho pairing
x
95191
bulk hyphal transfer
87074/1
single conidial isolation
Br-518_c2
hyphal tip isolation
not genotyped
strip taken from ho side
recombinant genotype
ms10
ms10
ms6
ms6
ms1
ms1
ht5
Fig 2 – The genealogy of subcultures obtained from trikaryotic isolate (tri1) and their genotypes. tri1 was initially obtained by
mating homokaryon 95191 and heterokaryon Br-518_c2 3 87074/1 (Br-518_c2 cytoplasm). Each arrowed line represents
a subculturing step. Subcultures were obtained by bulk hyphal transfer (solid arrows), single conidial isolation (dotted
arrows), and single hyphal tip isolation (dashed arrows). Bulk hyphal transfer involves the transfer of a w9 mm2 piece of
agar from an old to a new Petri dish. Subcultures that were genotyped are represented by circles colored to indicate nuclear
origin of the alleles. Isolates represented as a connection of three points are recombinant strains with different genotypes at
the three microsatellite loci (Ha-ms1, Ha-ms6, and Ha-ms10).
Br-518_c2 strain as the maternal homokaryon, the nucleus
from the Ult1.5 strain was preferentially the heterokaryotizing
nucleus. These results suggest that the Ult1.5 and Br-518_c2
nuclei have a high degree of compatibility and that the stability of the heterokaryon formed is high. These two strains are
both from Sweden, and thus this compatibility may stem
from their genetic similarity relative to the other two strains.
This observation would contradict the typical observation that
nuclear selection favors more genetically distant loci in he-ho
pairings (Anderson & Kohn 2007), but does support the previous demonstration that heterokaryons of H. annosum from
sympatric populations show lower evidence of genomic conflict as measured through recovery of nuclear types in conidial
isolates (Ramsdale & Rayner 1996). Another possibility is that
the strains may possess a pair of mating-types that are highly
compatible. Unfortunately, no data exist on the nuclear ratio
of this heterokaryon, but it may be predicted that the nuclear
ratio is not highly imbalanced as has been observed in many
lab-synthesized heterokaryons (James et al. 2008; Ramsdale
& Rayner 1996).
These observations of nuclear selection in H. parviporum
confirm previous studies indicating that nuclear selection is
the rule rather than exception in he-ho pairings of Agaricomycetes (Coates & Rayner 1985; Crowe 1963; Ellingboe & Raper
1962). In he-ho pairings, the resulting heterokaryons can be
thought of as the product of selection by the maternal nucleus, the ability of the paternal nucleus to fertilize/invade,
or selection on the newly formed heterokaryon. In H. parviporum, we observed a relationship between fertilization success
(paternal trait) and resilience to nuclear displacement (female
trait; Table 1), suggesting that the ability to invade a mycelium
and the resistance to invasion/displacement are linked traits
that differ between nuclei and promoting the idea that nuclear
frequencies are a product of nuclear competition within
a common mycelium. Studies with Schizophyllum commune
have definitively shown that both nuclei migrate into the
homokaryon at an equal rate but that the onset of the migration process may determine nuclear selection (Ellingboe 1964).
As previously mentioned, one mechanism by which selection
by the maternal nucleus could occur is through selection for
compatible pheromones and receptors encoded by the
MAT-B locus (Vaillancourt et al. 1997). Septal breakdown in
the recipient could be slowed during ingress of the nonfavored nucleus encoding a less compatible pheromone.
Nonetheless, nuclear selection in he-ho pairings has been
attributed to genetic factors both inside and outside of the
mating-type loci (Raper 1966), and nothing is known of the
structure of the mating-type locus in H. parviporum. The use
Trikaryon formation and nuclear selection in Heterobasidion
of breeding experiments to create pedigreed strains of varying
relatedness could help clarify the genetic basis of differential
nuclear migration in he-ho pairings of H. parviporum.
Given the numerous studies that have demonstrated nuclei have differential fertilization success, he-ho pairings are
a clear demonstration in which selection acts at the level of
the nucleus rather than the heterokaryotic mycelium as
a whole. We hypothesized selection may also act on cytoplasmic genes to influence fertilization in he-ho pairings because
the inheritance of cytoplasm and nuclei differ (Aanen et al.
2004). Selection on the nucleus should favor genes that increase fertilization by emigration; however, cytoplasmic
genes should counter-select for mutations that prevent nuclei
from emigrating, because an adjacent (and possibly highly
competitive) heterokaryotic mycelium will be formed through
Buller phenomenon mating. In the present study, only one of
the nine investigated cases showed a significant effect of the
cytoplasmic background on nuclear migration. This particular
instance was near-male sterility of the Ult1.5 þ Br-518_c2 heterokaryon in pairings with the homokaryon 87074/1 when the
cytoplasm was from the Br-518_c2 parent. The failure to observe a larger effect in this study could be because only four
cytoplasmic and nuclear backgrounds were investigated.
Studies of additional Agaricomycete species are warranted
before concluding that the effect of cytoplasmic genotype on
nuclear selection during he-ho pairings is largely insignificant.
The term trikaryon is invoked to describe the tri-genomic
mycelia subcultured from he-ho pairings of H. parviporum in
this study. In this paper, the term is defined as a mycelium
with three nuclear types in which the precise cellular constituency is unknown (de Serres & Brockman 1968). Previous
studies have also found three genome equivalents can coexist
within a single mycelium of other Agaricomycete and Tremellomycete fungi. These studies described either 2N þ N dikaryons (Casselton 1965; Koltin & Raper 1968; Lin et al. 2007; Rizzo
& May 1994) or triploid hyphae with a single nucleus (Carvalho
et al. 1995). The actual existence of trikaryotic cells containing
three genetically distinct nuclei per cell is only possible for
fungi with multinucleate cells such as H. parviporum. The
rate of trikaryon formation observed from he-ho pairings of
H. parviporum is higher than the rate of tri-genomic mycelia
formed in diploid-haploid pairings with Armillaria spp.
(Carvalho et al. 1995; Rizzo & Harrington 1992). We hypothesize that the highly multinucleate hyphae of H. parviporum
may promote the formation and maintenance of a trikaryotic
state. The possibility that the subcultures of tri1 with three genomes were triploid or 2N þ N can be largely ruled out because
single conidial isolates mostly reduced to the haploid progenitor nuclei (Fig 2). Trikaryons of H. parviporum are suspected to
have a lower stability than most two-component heterokaryons, which generally do not show loss of heterozygosity
from repeated subculture (T. Y. James, unpublished observations). A link between the lack of stability of the trikaryon
and inter-nuclear competition may be posited based on the
evidence for a reduced fitness (i.e., growth rate) for the
trikaryon tri1.
Tri-genomic mycelia of Agaricomycetes may contribute to
the genetic diversity and evolution of natural populations of
fungi. Tri-genomic mycelia have been demonstrated to undergo recombination to exchange genes between nuclei,
589
through either completion of the sexual cycle (Kimura &
Kadoya 1962; Koltin & Raper 1968) or by shuffling of genes by
somatic recombination (Anderson & Kohn 2007). In this study,
several hyphal tips were observed in which markers of the
three genomes appeared to have been recombined into two
nuclei (Fig 2 and results not shown). Fungi that can also produce asexual conidia from heterokaryotic mycelia may also
be able to form propagules that are the product of recombination of three genomes without meiosis or fruitbody production through a parasexual cycle. This potential to recombine
genetic information from more than two nuclei to generate
novel genotypes during the life cycle is unique to fungi. These
higher-order heterokaryons could increase the adaptive potential of a species provided the negative effects of nuclear
competition are not also increased.
Acknowledgements
We thank the Wenner-Gren, C. F. Lundtröms, and Carl Tryggers Foundations for funding. Thanks also to Jan Stenlid and
Åke Olson for helpful discussions and encouragement.
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