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Peabody RB, Peabody DC, Sicard KM. 2000. A genetic
mosaic in the fruiting stage of Armillaria gallica. Fungal
Genetics and Biology 29: 72–80.
Grillo R, Korhonen K, Hantula J, Hietala AM. 2000. Genetic
evidence for somatic haploidization in developing fruitbodies
of Armillaria tabescens. Fungal Genetics and Biology 30:
135–145.
Access Fungal Genetics & Biology at: www.academicpress.com/fgb
A genetic individual (a ‘genet’) can be fragmented in several
physical and physiological units, due to asexual reproduction
or clonal growth followed by separation of subunits – as in
garden plants propagated by cuttings. The opposite phenomenon
(i.e. a physical individual made of several genets) is more
rarely observed: it is a mosaic (or a chimera). Mosaics may
result from mutations within a clone, such as some plants
with green and white sectors, but also from fusion between
two or more genets (e.g. in colonial animals (such as Cnidrians
and Chordates; Rinkevich & Weissman, 1987)) and amoebae
(Dao et al., 2000). Such mosaics are predicted to exist in higher
fungi too – but recent works published in Fungal Genetics &
Biology (Grillo et al., 2000; Peabody et al., 2000) now demonstrate
a new type of mosaic in Homobasidiomycetes.
Sexual mosaics in Homobasidiomycetes
Homobasidiomycetes include various well-known and
ecologically important fungal groups, such as gilled fungi,
forming their meiotic spores on a supporting cell called a
basidium. The basidiospores germinate to form haploid, septate
hyphae. Haplonts are self-sterile and only those carrying different
alleles at the one or two mating-type loci can mate. The fusion
of compatible haploid hyphae is followed by a bidirectional
nuclear invasion: the haploid nuclei migrate in the other
thallus, divide and leave a copy in each recipient cell (Casselton
& Economou, 1985). However, the nuclear fusion is delayed
and, when migration is completed, each hyphal cell harbours
two divergent haploid nuclei that divide synchronously, often
forming clamped hyphae (Fig. 1a). Such binucleate cells are
called dikaryons. Since they retain haploid nuclei, dikaryons can
still dikaryotize a haploid mycelium by giving it a compatible
nucleus (the ‘Buller phenomenon’). Dikaryotic hyphae are
the vegetative state of Homobasidiomycetes and, in favourable conditions, form fruitbodies, fleshy structures made of
aggregated hyphae and bearing basidia. During basidial
development, the haploid nuclei fuse and immediately
undergo meiosis, generating four haploid nuclei that migrate
separately in the four basidiospores (Fig. 1a).
Whenever nuclear migration is unilateral during mating
(e.g. in the Buller phenomenon) or incomplete, a nuclear
mosaic is formed (de la Bastide et al., 1995). This, or a secondary loss of one nucleus in some cells, could produce the
haploid hyphae recovered in dikaryotic cultures of some species, such as Heterobasidion annosum (Hansen et al., 1993). In
addition, since mitochondria do not follow the migrant nuclei
(Barroso & Labarère, 1997), the newly formed dikaryon
always retains the cytotype of the previously haploid hyphae,
giving rise to a temporary mitochondrial mosaic (Casselton &
Economou, 1985). Mitochondrial and nuclear mosaics have
been observed in vitro, but differential growth can canceal
all but one sector during further development (Barraso &
Labarère, 1997), maybe explaining why such mosaics have
never been observed in the field to date.
Somatic mosaics in homobasidiomycetes
Mosaics can also arise from somatic fusions between dikaryons
or between sexually incompatible haplonts (i.e. with identical
mating-type alleles). Such vegetative fusions are controlled by
a variable number of loci: a death of the fusion compartment
occurs when genets carry different alleles ( Worrall, 1997),
preventing the spread of diseases and resource spoilage by
potential competitors. But closely related genets, that bear
identical alleles at vegetative incompatibility loci, can fuse
through hyphal anastomoses and share space and resources.
Strikingly, a similar requirement for identity at several loci
governs fusion/rejection in animal mosaics (Rinkevich &
Weissman, 1987). Those kin-restricted fungal mosaics are
thought to allow physiological exchanges and co-existence of
nuclei of various origins – but their existence has not been
demonstrated in nature.
Nuclear fusions can occur in multinucleate cells of fusion
mosaics: the resulting diploid vegetative nuclei undergo mitotic
crossing-over and divide spontaneously into haploid recombinant nuclei (Marmeisse, 1991), probably through aneuploid
states. This parasexual recombination creates, in turn, new
mosaics (Hansen et al., 1993) but, again, the occurrence of
such mosaics in field populations remains to be demonstrated
(Anderson & Kohn, 1998).
Besides sexuality and fusion, the accumulation of mutations
in a growing clone can lead to a mosaic of sectors differing by
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Fig. 1 Karyological and life-cycle of
Homobasidiomycetes (modified from
Guillaumin et al., 1991). (a) A typical lifecycle with dikaryotic vegetative hyphae (an
arrow indicates the clamp connection usually
formed in dikaryotic hyphae). (b) A typical
Armillaria life-cycle with diploid vegetative
hyphae, after nuclear fusion in early
dikaryons (e.g. Armillaria mellea). (c) A
variation of the Armillaria life-cycle with
dikaryotic subhymenial hyphae, after an
ill-defined reversion to dikaryon (e.g. A.
tabescens, analysed in Grillo et al., 2000).
(d) Life-cycle of the Armillaria gallica genets
analysed by Peabody et al. (2000).
their molecular fingerprints. For example, polymorphic (and
probably highly mutable) loci have been shown to vary among
isolates of a forest clone of the ectomycorrhizal Hebeloma
cylindrosporum (Gryta et al., 2000). Whether such genets still
form a single thallus with physiological links – a true mosaic
– is not known. To summarize, although mosaics may very
well occur in vegetative Homobasidiomycetes, their existence
in nature has not yet been demonstrated.
Haploid mosaics in Armillaria fruitbodies
There are now reports of mosaic fruitbodies in Armillaria,
a genus of several lignivorous species that fruit profusely and
propagate through strand-like aggregations of hyphae called
rhizomorphs (Smith et al., 1992). A unique characteristic of
the Armillaria life-cycle is that clamped dikaryotic hyphae
quickly become diploid after mating (Anderson & Ullrich,
1982; Guillaumin et al., 1991). Vegetative hyphal cells have a
single, diploid nucleus and cease forming clamp connections
(Fig. 1b) in both the vegetative mycelium and the fruitbody.
In some species, such as Armillaria gallica, A. otoyae and A.
tabescens, the subhymenial layers, which sustain the basidia,
contain dikaryotic, clamped hyphae (Fig. 1c; Guillaumin
et al., 1991), suggesting an ill-understood reversion to the
dikaryotic state during fruitbody formation. More curiously,
photometric studies of DAPI-stained material suggested that
uninucleate cells of A. gallica fruitbodies are not diploid, but
haploid (Peabody & Peabody, 1987).
The latter observation was first met with skepticism, but
Diane and Robert Peabody persevered. They convincingly
demonstrated that field-collected A. gallica fruitbodies
contain genetically different haploid hyphae (Peabody et al.,
2000). By culturing hyphae from the fruitbody’s stipe, they
generated cultures derived from single tip cells (STC). They
then compared the genotype of fruitbody pieces with those
of STC cultures at 17 loci (isozyme loci, mating types and
ribosomal DNA). In all five fruitbodies investigated, the
pieces shared the same genotype, with six heterozygous loci,
which suggested that they all derived from the same diploid
genet. However, cultures from STC always had a single allele
at each polymorphic locus, therefore suggesting that the stipe
hyphae were haploid.
The fruitbodies were thus mosaics whose apparently diploid
genetic pattern was actually a combination of various haploid
patterns. No heterozygous STC culture was found, substantiating that no dikaryotic or diploid (or even aneuploid)
hyphae were present. What is then the origin of this mosaic?
Haploid genets living in the substrate could have aggregated
to form fruitbodies. But, in all fruitbodies investigated, the
total set of alleles does not exceed two at each locus (i.e. the
set of a diploid). This favours the hypothesis of a haploidization
from a diploid mycelium (Fig. 1d) carrying the various alleles
recovered. Since different haploid genets (up to nine per
fruitbody) were recovered, the haploidization would include
recombination, as does meiosis.
The mosaic state observed in 1986 was not demonstrated
between 1989 and 1998. Sporophores collected after 1988
carried only a single allele at all loci and, thus, prevented the
demonstration of mosaicism. Recent unpublished analyses of
ribosomal DNA showed the existence of mosaic A. gallica
fruitbodies at a nearby site (R. B. and D. C. Peabody, pers. comm.).
Dikaryotic mosaics
What about dikaryotic subhymenial hyphae (Fig. 1c,d)?
Grillo and co-workers (2000) obtained diploid cultures of A.
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tabescens by crossing known haploid isolates, and isolated
diploid STC cultures to ensure that no remnant haploid
hyphae were present, and let them fruit in vitro. In fruitbody
primordia showing clamped subhymenium, dikaryons were
recovered and submitted to experimental dedikaryotization
by protoplast formation or micromanipulation. The resulting haploid cultures were genotyped (for the mating types
and random-amplified markers): they were shown to be
recombinant, differing from the haploid progenitors of the
diploid culture. In A. tabescens fruitbodies, a haploidization
including recombination, therefore, leads to a mosaic dikaryotic subhymenium.
In Armillaria species, dikaryotic, clamped hyphae form
temporarily after mating, before diploidization. A simple
explanation of dikaryotic subhymenial hyphae in A. gallica
would be a secondary mating among haploid hyphae from
the fruitbody flesh. The same could apply for A. tabescens,
although evidence for haploid hyphae are lacking in this species. If so, what prevents mating from taking place earlier, for
example in the stipe? Do such haploid and dikaryotic mosaics
exist in all Armillaria species with clamped subhymenium
(Fig. 1c)? Alternatively, they may be idiosyncratic to some
genets (e.g. mutated in genes governing the cell cycle). Interestingly, Grillo and co-workers (2000) noted that some A.
tabescens isolates had both diploid and dikaryotic subhymenium, in the same or different fruitbodies; similarly,
appearance of dikaryotic hyphae in the subhymenium of
Armillaria ostoyae depends on environmental conditions for
a given genet (Guillaumin et al., 1991). The determinism of
mosaics by haploidization awaits further studies.
In other Homobasidiomycetes, fruitbody hyphae are supposed to be dikaryotic: they are clamped (at least in species
with clamped dikaryons) and isolations lead to dikaryotic
cultures. But what about possible haploid hyphae, in small
amounts, that would be outcompeted during in vitro isolations? Due to the isolation screens (clamped hyphae and/or
identity at all loci with the whole fruitbody) and the use of
fruitbody pieces in molecular ecology, mosaic fruitbodies,
if they exist, are likely to remain hidden, as they were in A.
gallica populations (Smith et al., 1992).
Haploidization as a source of mosaics
The exact timing of haploidization, before or during
fruitbody formation, is unknown. In absence of STC cultures
from the rhizomorphs, their diploid or mosaic state remains
to be assessed. This would be a major source of somatic
variation and adaptability – however, populational analyses
rather favour the picture of stable, long-living genets (Smith
et al., 1992).
The mechanism of haploidization also remains open.
Several nuclei probably undergo independent haploidization,
leading to the various recombinants. Is this similar to the
parasexual haploidization already mentioned for vegetative
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diploid nuclei in other Homobasidiomycetes? There are only
rare examples of vegetative haploidization in diploid Armillaria mycelia (Anderson & Ullrich, 1982). Since no evidence
for aneuploid nuclei was found, an alternative view is a true
somatic meiosis, creating haploid hyphae after segregation
(or degenerescence of some) of the haploid nuclei. Somatic
meiosis exists in some algae (van den Hoek et al. (1995): in
the red alga Porphyra, meiosis occurs after the settlement of
a diploid spore, leading to a mosaic of four haploid sectors;
in the green alga Prasiola, it occurs during the growth of a
diploid thallus, forming a mosaic of haploid sectors on the
original diploid thallus.
In Armillaria, caryogamy takes place in vegetative cells, earlier than in the other Homobasidiomycete life-cycle. Meiosis
itself could be also anticipated, as a result of a general heterochrony of the caryological cycle in some Armillaria species.
But the typical meiosis still occurs in basidia (Fig. 1c,d),
meaning a second haploidization event. What selected such a
redundancy? Note that, if subhymenial hyphae differ from
the diploid parental genotype, the basidial meiosis is not
strictly redundant. A recovery of meiotic tetrads on gills or
genetic fingerprinting of the subhymenial dikaryons would
help clarification.
Co-operating or cheating genets?
Regardless of their origin, the various genets in Armillaria
fruitbodies co-operate to form a reproductive organ that
contains both fertile and sterile parts (stipe and pileus). But
coexistence and co-ordinated morphogenesis does not necessarily
mean co-operation. In a single-genet fruitbody, the sacrifice of
cells in the sterile parts is selected since they contribute to the
survival of their own genes, transmitted to the basidiospores.
In a mosaic fruitbody, any genet unscrupulously investing
less in sterile parts than in producing basidia would have a
selective advantage. A similar phenomenon, called germ-line
parasitism, occurs in mosaics formed by fusion in animals
and in the colonial amoebae Dictyostelium discoideum. In the
latter case, a mosaic sporophore is formed after aggregation of
vegetative amoebal cells, eventually genetically different. Some
strains do not contribute to the stalk, a sterile structure favouring
sporal dissemination, and are over-represented among spores
(Dao et al., 2000).
Allelic segregation occurred among basidiospores germinated by Peabody et al. (2000) as well as among those
obtained by Grillo et al. (2000) but the number investigated
does not allow the detection of segregation bias. Similarly, the
low number of cultures recovered from the fruitbody hyphae
does not conclusively show any over-represented genet.
‘Cheating genes’, if they exist, could even determine the
haploidization, that allow to select for themselves – at least,
they could make profit of it. In the future, Armillaria mosaics
may be suitable models for looking for genes that favour
cheating strategies.
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Summary
There is now evidence of mosaicism for some Homobasidiomycetes. Although mosaics have long been predicted to
occur at the vegetative stage, they were unexpectedly observed
in fruitbodies of Armillaria species as a result of a haploidization process. For A. gallica , Peabody and co-workers (2000)
demonstrated that stipe flesh is a mosaic of haploid hyphae
with differing genotypes; for A. tabescens, Grillo et al. (2000)
demonstrated that the subhymenium is a mosaic of dikaryotic
hyphae harbouring recombinant, haploid nuclei. Further work
will clarify the determinism and mechanism of haploidization,
as well as its genetic consequences on basidiospore production.
These data emphasize the plasticity of gene reassortment and
caryological processes in fungi: their role in shaping the
genetic structure of fungal populations, although likely to
be of major importance (Anderson & Kohn, 1998), is still
unknown territory.
Acknowledgements
I wish to thank H. Gryta (Université Paul Sabatier, Toulouse),
B. Marcais (Institut National de la Recherche Agronomique,
Nancy) and J. J. Guillaumin (Institut National de la Recherche
Agronomique, Clermont-Ferrand) for valuable comments on
this article, as well as Robert and Diane Peabody for the rich
discussions we had about their work.
M.-A. Selosse
Institut de Systématique (CNRS FR 1541), Muséum
National d’Histoire Naturelle, 43, rue Cuvier, F-75005
Paris, France
(tel +33 1 40 79 38 22; fax +33 1 40 79 38 44;
Email [email protected])
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Barroso G, Labarère J. 1997. Genetic evidence for non random sorting
of mitochondria in the basidiomycete Agrocybe aegerita. Applied and
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de la Bastide PY, Kropp BR, Piché Y. 1995. Vegetative interactions
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Casselton LA, Economou A. 1985. Dikaryon formation. In: Moore D,
Casselton LA, Wood DA, Frankland JC, eds. Developmental Biology of
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Dao DN, Kessin RH, Ennis HL. 2000. Developmental cheating
and the evolutionary biology of Dictyostelium and Myxococcus.
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Esser K, Kuehnen R. 1967. Genetics of Fungi. Heidelberg, Germany:
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Grillo R, Korhonen K, Hantula J, Hietala AM. 2000. Genetic evidence
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Gryta H, Debaud JC, Marmeisse R. 2000. Population dynamics of the
symbiotic mushroom Hebeloma cylindrosporum: mycelial persistence
and inbreeding. Heredity 84: 294 –302.
Guillaumin JJ, Anderson JB, Korhonen K. 1991. Life cycle,
interfertility and biological species. In: Shaw III CG, Kile GA,
eds. Armillaria Roots Disease. Agriculture Handbook No 691.
Washington DC, USA: USDA Forest Service, US Department
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Hansen EM, Stenlid J, Johansson M. 1993. Somatic incompatibility
and nuclear reassortement in Heterobasidion annosum. Mycological
Research 97: 1223 –1228.
Marmeisse R. 1991. Parasexual analysis in the basidiomycete
mushroom Agrocybe aegerita common-B and common-AB.
Mycological Research 95: 465– 468.
Peabody DC, Peabody RB. 1987. Haploid monokaryotic basidiocarp tissues in species of Armillaria. Canadian Journal of Botany 65:
69 – 71.
Peabody RB, Peabody DC, Sicard KM. 2000. A genetic mosaic in the
fruiting stage of Armillaria gallica. Fungal Genetics and Biology 29:
72 –80.
Rinkevich B, Weissman IL. 1987. Chimeras in colonial invertebrates:
a synergistic symbiosis or somatic- and germ-cell parasitism? Symbiosis
4: 117–134.
Smith ML, Bruhn JN, Anderson JB. 1992. The fungus Armillaria
bulbosa is among the largest and oldest living organisms. Nature
356: 428 – 431.
Tommerup IC, Broadent D. 1975. Nuclear fusion, meiosis and the
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Key words: Mosaic, Chimera, basidiomycetes, Armillaria,
fruitbody, life-cycle, haploidization, kikaryons.
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