Genetic evidence for somatic haploidization in developing fruitbodies of Armillaria tabescens
Grillo R, Korhonen K, Hantula J, Hietala AM. 2000.Fungal Genetics and Biology30: 135–145.
Access Fungal Genetics & Biology at: http://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 Cnidarians 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 (Esser & Kuehnen, 1967). 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’; Esser & Kuehnen, 1967). 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). Individuals originating from fusion of neighbouring genets are already reported in Ascomycetes (e.g. in lichens; Murtagh et al., 2000). In Homobasidiomycetes, 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 in Homobasidiomycetes 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 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.).
What about dikaryotic subhymenial hyphae (Fig. 1c,d)? Grillo and co-workers (2000) obtained diploid cultures of A. tabescens by crossing known haploid isolates. They 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, development 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 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, several meiotic events occur 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. In the future, Armillaria mosaics may be suitable models for looking for genes that favour cheating strategies.
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.
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.