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Within forest soils, that host a large and diverse community of microbes that compete and interact with each other and with plant roots, ectomycorrhizal fungi are almost ubiquitous (Taylor et al., 2000). The mutualistic ectomycorrhizal symbiosis allows trees to grow efficiently in suboptimal environments in boreal, temperate and some subtropical forest ecosystems (Smith & Read, 1997).
In a forest ecosystem, roots are exposed to an assemblage of several different species of ectomycorrhizal fungi (Taylor et al., 2000; Dahlberg, 2001). Each species exists as a population of many individuals between which there is almost invariably some genetic variation (Debaud et al., 1995). The fact that the host plants are exposed to mycobiont populations which are genetically diverse is a crucial consideration in forest ecology. Individual symbionts vary in their ability to colonize different genotypes of host-plant, their capacity to utilize mineral and organic soil nutrients, and in their adaptation to abiotic factors, such as soil pH and drought. Each of these abiotic and biotic factors might affect the course of a beneficial symbiosis and the establishment and subsequent dissemination of an individual fungal symbiont. Questions about the temporal and spatial structures of communities and populations of ectomycorrhizal fungi, together with the origin and maintenance of their genetic variation, are therefore critical when trying to understand how populations of these symbiotic fungi evolve and disappear at different stages of development of forest ecosystems (Egger, 1994; Dahlberg & Stenlid, 1995; Dahlberg, 2001). By implication, such a knowledge can help in the understanding of biogeochimical cycles in forest ecosystems (Taylor et al., 2000).
When and how genetic individuals of a given species (also referred to as genets) arrive at a site and how interactions between them evolve with maturation of fungal and host-plant populations are key questions in the assessment of dynamics in populations of ectomycorrhizal fungi. To understand the dispersal, establishment, and evolution of ectomycorrhizal populations, it is necessary to analyse these populations over several seasons and compare them at different successional stages of forest ecosystems at different habitats. The source and maintenance of genetic diversity within these ectomycorrhizal populations have been questioned in previous studies (Dahlberg & Stenlid, 1990, 1994; De La Bastide et al., 1994; Gryta et al., 1997, 2000; Bonello et al., 1998; Selosse et al., 1998, 1999; Gherbi et al., 1999; Sawyer et al., 1999; Zhou et al., 1999, 2001; Guidot et al., 2001; Redecker et al., 2001). From these investigations, conclusions about the relative role of vegetative spreads vs spore establishment of ectomycorrhizal basidiomycete can be drawn (Egger, 1994; Dahlberg, 2001).
In open forest communities, genet recruitment from spore germination occurs massively during a short period early in the development of the population, leading to the development of numerous small genetically different individuals. Many early colonizers, such as Hebeloma cylindrosporum and Laccaria spp., appear to have relatively small nonpersistent genets (De La Bastide et al., 1994; Gryta et al., 1997, 2000; Selosse et al., 1998). For some Suillus species, it is believed that no further genet recruitment from spores occurs after the initial colonizing phase and vegetative growth through hyphal networks appears to dominate in closed fungal communities (i.e. older forests). These communities are claimed to be resistant to genet establishment from spores, unless small disturbances in favourable sites create ‘windows of opportunity’ for local new genet recruitment (Dahlberg & Stenlid, 1995). Once thought to be dominated by a few, large (20–40 m) and long lasting genets, undisturbed closed forests can host populations of ectomycorrhizal basidiomycetes having high levels of genetic diversity characterized by small, short life-span genets (Gherbi et al., 1999; Redecker et al., 2001). Some ectomycorrhizal fungal populations found in undisturbed mature forest settings could therefore propagate by intense fruiting and spore germination. Whether population structure is related to microsite heterogeneity (Zhou et al., 1999, 2001; Guidot et al., 2001) or the biological/genetic features of the species is unknown. A way to validate the above mentioned models on the processes driving the evolution of genetic structure in populations of ectomycorrhizal fungi is to determine the genetic structure of a given species in different habitats differing by their ecological characteristics (Redecker et al., 2001).
Laccaria amethystina is found in both recently established plantations and mature forests. We have investigated the genetic structure of L. amethystina populations in a 150-yr-old-monospecific beech (Fagus sylvatica) forest in the Vosges range in north-eastern France (Gherbi et al., 1999). Surprisingly, a high density (> 5000 genets per ha) of small size, short life-span genets was found indicating an important role for sexual reproduction in this mature forest ecosystem. To assess whether this feature can be influenced by the habitat, we used the random amplified polymorphic DNA (RAPD) technique to estimate DNA variation within a population of L. amethystina in the mixed forest of La Chanéaz (Swiss Mittelland). Xerocomus chrysenteron and X. pruinatus, two boletoid species were also analysed for comparative analysis.
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Genet size in ectomycorrhizal fungi can reveal features of colonization strategy and growth. Fruiting body and ectomycorrhiza surveys have shown that the size of the genets from ectomycorrhizal fungi may vary with the forest age. It is widely believed that the presence of many small genets indicates multiple spore establishment events and large genets reflects predominant vegetative spread (Dahlberg & Stenlid, 1990, 1995; Dahlberg, 2001). Estimated densities of populations range from 30 to 5200 genets ha−1 depending on the species and forest age (Gryta et al., 1997; Gherbi et al., 1999; Dahlberg, 2001; Redecker et al., 2001). Populations of Suillus bovinus and S. variegatus in old conifer forests consist mostly of discrete, large (up to 27 m) and old (> 150 yr) genets which do not seem to intermingle (Dahlberg & Stenlid, 1994, 1995; Dahlberg, 2001). One of the largest genet of ectomycorrhizal fungi to date (300 m2) has been found in Suillus pungens in a Bishop pine forest in coastal California (Bonello et al., 1998). It was thus thought that vegetative growth and dissemination over long periods dominate over spore colonization in established forests typified by canopy closure, lack of young trees, and organic N-rich litter (Dahlberg & Stenlid, 1995). However, recent studies (Gherbi et al., 1999; Redecker et al., 2001) have demonstrated that undisturbed mature forests may host fungal populations characterized by numerous, short life-span genets relying mainly on spore dissemination for local colonization. Initial genet recruitment from spores followed by vegetative spread (large, old genets) and repeated recruitment from spores (small, short life-span genets) may be considered as two extremes of a continuum rather than as two clearly distinct recruitment patterns in ectomycorrhizal basidiomycetes.
As spatial structuration of populations of ectomycorrhizal fungi differs between taxa (Dahlberg, 2001), we have analysed populations of three ectomycorrhizal basidiomycetes, Laccaria amethystina, Xerocomus chrysenteron, and X. pruinatus in the undisturbed mixed forest of La Chanéaz. The investigated stands comprised 40-yr-old and 140-yr-old conifer and hardwood species, together with a few young, mainly endomycorrhizal, trees. Molecular typing of sampled basidiocarps revealed two constrasting genetic structures for these species. Both X. chrysenteron and X. pruinatus populations consisted of a discrete, large genet. Assuming a mean growth rate of 0.5 m per year for the underground mycelium of ectomycorrhizal fungi (Dahlberg & Stenlid, 1994; Bonello et al., 1998; Selosse et al., 1998), the founder of the 110 m-large X. chrysenteron clone probably associated with the older Picea abies when they established 140-yr-ago. The closely related species X. pruinatus also formed large clones (40 m2) in the same plot. X. chrysenteron collected in the beech forest of Aubure (Vosges) (Gherbi et al., 1999) also pertained to a single large genet (> 100 m2) (H. Gherbi & F. Martin, unpublished). These results were consistent with the findings of Dahlberg & Stenlid (1994, 1995) showing that, although they produce a considerable number of spores, many boletoids extend mostly by vegetative growth and the genet establishment from spore germination progressively decreased in the ageing population. Their capacity to develop extensive hyphal cords probably enables the large genets to visit the roots of more trees and to pool more total C than species which are more common, but produce smaller genets (Bonello et al., 1998).
By contrast, the L. amethystina population was made of numerous discrete genets (up to 13800 ha−1). Among the analysed genets, only 4% of the identified genets fruited the following years suggesting a considerably large mortality of genets. The genet size varied from the mere diameter of the basidiocarp (i.e. c. 2–4 cm) to 5.40 m in length. The present results are in agreement with a previous survey carried out in a different habitat, a 150-yr-old monospecific beech forest situated at an altitude of 1000 m asl (Gherbi et al., 1999). In the latter study conducted in the Vosges mountains (north-eastern France), L. amethystina populations exhibited up to 5200 short life-span, small-size genets per ha. The dramatic renewal of genets suggested a recolonization of the site by new genets each year and/or erratic fruiting of ‘cryptic’ below ground genets (Gherbi et al., 1999). The numerous L. amethystina genets are likely to have arisen from repetitive establishment from basidiospores, as suggested by their small size. Large amounts of feeder roots with high turnover rates (Vogt et al., 1986) give constant opportunities for ectomycorrhizal fungi to become established (Kranabetter & Wylie, 1998). The growth pattern of L. amethystina suggests that these new host root tips are colonized each season, the spreading of the new genets probably following the elongation of roots. In situations with strong intraspecific competition we could expect a reduced clonal diversity because fewer fitter genets would predominate. Yet, a high DNA diversity was maintained at both sites analysed (i.e. Aubure and La Chanéaz). Obviously, none of the genet produced sufficient clonal offspring to contribute to its predominance.
Colonization by numerous small-sized genets, with high fruiting rates, is reminiscent of young natural populations of ectomycorrhizal fungi (e.g. Suillus bovinus (Dahlberg & Stenlid, 1990); Laccaria bicolor (De la Bastide et al., 1994; Selosse et al., 1998, 1999) and Hebeloma cylindrosporum (Gryta et al., 1997; Guidot et al., 2001)). This population structure was postulated to arise in recently established or disturbed stands (Dahlberg & Stenlid, 1995). Recent investigations have however, shown that this ‘early stage’ population structure is also found for ectomycorrhizal basidiomycetes in closed undisturbed forest settings (Gherbi et al., 1999; Zhou et al., 1999, 2001; Redecker et al., 2001).
The population structure may depend on: the dispersal of the spores; the extent of growth of the monokaryotic mycelium (that germinated from the basidiospores of the previous seasons) and the opportunities for mating between compatible monokaryotic mycelia that thus occur; and the persistence of the below-ground mycelia. The spatial distribution of H. cylindrosporum (Guidot et al., 2001) and Suillus grevillei (Zhou et al., 2001) genets in the soil showed that the development of basidiocarps was tightly correlated with that of the extramatrical and ectomycorrhizal mycelia of the same genet. Disappearance of basidiocarps of these species at a given place was associated with the disappearance of the corresponding subterranean mycelia within 1 yr. If this behaviour is also true for L. amethystina, basidiocarp analysis appears to reflect both the genetic diversity and the spatial structure of its subterranean populations.
Microsite heterogeneity which promotes the coexistence of genets through diversifying selection has been hypothesized to account for the mechanisms underlying the maintenance of a high level of genotypic diversity in fungal species (Frankland, 1998). Because Xerocomus species have been able to maintain a clonal structure for decades, this hypothesis alone is insufficient to explain the high genetic diversity of genets in aged forest ecosystems. The fact that the investigated stands are included in a fungal reserve protected by a fence precludes any major perturbation related to large animal and human activities.
The low similarity between RAPD patterns (Fig. 2) and the low correlation between spatial distance separating basidiocarps and the RAPD similarity matrix (Mantel’s test) indicate that L. amethystina genets found on plot 59 did not arise from a common (or a few) colonization event. These results suggest that establishment of novel genets and colonization of host root tips are largely a stochastic process, with much spore dispersal occurring on a scale greater or equal to that of this study (50 m2). The results of this study are somewhat at odds with the allelic analysis on S. grevillei (Zhou et al., 2001) which has shown that the spread of alleles within and between populations might be by repeats of short-distance spore dispersal.
In conclusion, the prominent role of the sexual spore reproduction over the perennial spread of the vegetative mycelium in the soil for ectomycorrhizal species, such as L. amethystina, is not restricted to pioneer situations, but is found in stable established forest ecosystems. These species are able to establish novel genets from spores repeatedly over several decades without apparent perturbation of the sites and/or host plant genetic structure. This suggests a rapid alteration of the subterranean parts of L. amethystina genets and a constant colonization of new feeder roots. Knowing the spatial structure of the studied populations through the fruiting body location will facilitate the characterization of their genetic structure at the level of ectomycorrhizal roots and extramatrical hyphae using codominant genetic loci (Guidot et al., 2001). So far, investigations on populations of ectomycorrhizal fungi have mainly been descriptive or correlative and no experimental study has yet been performed to manipulate these populations. Identification of causal processes controlling the dynamics and structure of ectomycorrhizal populations will advance our understanding of forest ecosystems.