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The symbiotic relationship between roots and fungi (mycorrhizas) is one of the most prevalent associations in all ecosystems. Ectomycorrhizal (EM) fungi are prominent mutualistic partners with temperate trees and they facilitate the trees’ uptake of mineral and organic nutrients and water, in exchange for photosynthates from their host (Smith & Read, 1997). Despite their obvious ecological importance, little is still known about the population biology of most EM species, which is intrinsically tied to how forest ecosystems function and evolve. For example, we still do not understand many of the fundamental life history processes that can shape EM populations such as the importance of mating vs vegetative growth, the prevalence of colonization by monokaryotic or dikaryotic mycelia, the spread of EM fungi across landscapes, and the spatial and temporal scales at which root colonization occurs. The study of EM population genetics can provide insights into these important concepts of EM fungi, especially with the modern tools of molecular ecology and studies which have focussed on specific hypotheses concerning EM fungal biology.
The focus of most population studies of EM fungi has been on an examination of genets in local populations. Several studies have evaluated the number and size of genets (see Dahlberg, 2001; Redecker et al., 2001). These studies have demonstrated that the size of the genets is highly variable, depending on the EM taxon sampled. For example, Bonello et al. (1998) used single stranded conformation polymorphisms (SSCP) in a population of Suillus pungens and found that a single genet covered an area of at least 300 m2. In contrast, Redecker et al. (2001) used amplified fragment length polymorphic (AFLP) markers to estimate the genet size of Lactarius xanthogalactus, Russula cremoricolorandAmanitafracheti and found these to be relatively small (1.1–9.3 m2).
The size and density of genets may provide important insights into the reproductive biology of EM fungi. Repetitive sampling of genets in patches directly underneath fruitbodies have demonstrated that the genets of several EM fungi are short-lived, including Suillus grevillei, Hebeloma cylindrosporum and Laccaria amethystina (Gryta et al., 1997; Gherbi et al., 1999; Fiore-Donno & Martin, 2001; Zhou et al., 2001b). Small, short-lived genets are indicative of EM populations that reproduce primarily by sexual reproduction via meispores (see Dahlberg, 2001; Redecker et al., 2001). At the other extreme, long-lived perennial genets are characteristic of EM fungi that can spread via mycelial expansion from point sources. EM fungi that exhibit long-lived perennial genets include Leccinum duriusculum, Suillus spp. and Xerocomus pruinatus (Dahlberg, 2001; Fiore-Donno & Martin, 2001; Selosse, 2003). The high frequency of genets in local populations of R. brevipes and the presence of several basidiocarps formed by a common below-ground mycelium has been observed in different stands (Bergemann & Miller, 2002). One question that has not been addressed is of whether the allele frequencies in R. brevipes are correlated between successive fruiting seasons.
To date, relatively few studies have been published that have estimated genetic differentiation or compared the correlation between genetic differentiation and the distance between EM populations. In Rhizopogon vinicolor, Suillus grevillei, Xerocomus chrysenteron and Laccaria amethystina, no evidence of genetic structure between locations was found at geographic scales ranging from tens to thousands of meters (Gherbi et al., 1999; Kretzer et al., 2000, 2005; von Melzer & Rothe, 2000; Zhou et al., 2001a), with moderate levels detected across distances around 5 km in R. vesiculosus (Kretzer et al., 2005). In contrast, it has been shown that the geographic isolation of Tricholoma spp. and R. brevipes has led to a significant genetic differentiation across global and continental scales (Bergemann & Miller, 2002; Chapela & Garbelotto, 2004). For R. brevipes sampled in Douglas-fir, lodgepole pine and Sitka spruce stands in western North America, high estimates of genetic differentiation based on θST and FST suggested that gene flow between subpopulations was limited (Bergemann & Miller, 2002; Bergemann, 2002). It was also concluded that hosts do not appear to act as significant barriers to gene flow; rather, populations are geographically structured across western North America.
The objective of this study was to sample subpopulations of R. brevipes in more localized oak/pine woodlands to attempt to resolve the distance at which gene flow could occur. Specifically, we wanted to test for population differentiation among subpopulations isolated by distances of 230–1090 m, and to make inferences on their reproductive biology based on analyses of microsatellite loci.
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In previous studies of population differentiation among populations of R. brevipes from the west coast and Rocky Mountains, high estimates of genetic differentiation suggested that geographic barriers or distance between populations in western North America may have led to substantial genetic differentiation among populations; however, the absence of shared alleles indicated that populations from Wyoming and California may be cryptic species (Bergemann & Miller, 2002). In the current study, we dramatically decreased the spatial scale at which sampling was conducted and found that no isolation occurred between fungal subpopulations; rather, spore dispersal between sites has likely led to the observed homogeneity among the three locations separated by distances of 230–1090 m. This is also supported by the Hardy–Weinberg equilibrium model and by the recovery of new genotypes at each location coupled with a lack of allelic variation between years at each sampling subpopulation.
Another important aspect with respect to sampling is the adequacy of the sampling employed on a particular ‘population’, which will intrinsically be tied to the reproductive biology of the EM fungi studied. If the sampling effort using above-ground fruitbodies is not a reflection of the existence of diversity below-ground, then estimates of genetic structure may not reflect the population in question (Rogers & Rogers, 1999). For example, Selosse et al. (2001) evaluated how sampling could effect the interpretation of genet distribution of Laccaria spp. Since the fruiting of genets was not synchronized, it was suggested that a single sampling would have biased their estimates of genotypic diversity; therefore, an erroneous conclusion of low genet diversity would have been made, which was not the case when multiple sampling dates and years were considered (Selosse et al., 2001). In the present study, several collections were made each year within a 3–4-week period, which covered most of the fruiting period for this species in the years sampled, since fruiting is highly correlated with rainfall in this Mediterranean ecosystem and varies dramatically by year (M. Smith, pers. comm.). Nevertheless, a more intensive sampling within years and over additional years would have likely resulted in the recovery of more genotypic diversity in this system and may have reinforced the absence of a genetic structure.
Our results support the idea that gene flow is occurring in R. brevipes in this ecosystem; however, the exact mechanism of its dispersal is unclear. It is generally assumed that wind is the predominant mechanism of spore dispersal of EM epigeous fungi, whereas animals facilitate spore dispersal in hypogeous EM fungi (Johnson, 1996; Molina et al., 2001). In some studies it has been clear that spore dispersal has contributed to the high levels of genotypic diversity (Gherbi et al., 2004); however, others have maintained that the wind gusts required at ground level may be inadequate for the dispersal of Russula spores (Allen & Hipps, 1984). In the field, we have observed that most basidiocarps of R. brevipes are subepigeous and rarely mature above the litter layer, suggesting that wind may not be a primary mechanism for dispersal (Bergemann & Miller, 2002). In our present study, we found several remnants of basidiocarps that had been consumed by mammals such as small rodents or deer (S.E. Bergemann & G.W. Douhan, pers. obs.). We speculate that either animal-mediated dispersal (directly or indirectly) or wind dispersal may be factors in maintaining the absence of genetic structure in R. brevipes in localized populations. The range of foraging small mammals can approach more than 100 ha with a tendency to forage near EM trees, leading to an increased dispersal distance and likelihood of colonization (Johnson, 1996). However, further experimentation will be required to determine dispersal mechanisms in R. brevipes.
If aerial-dispersed spores are generally deposited in an exponential leptokurtic pattern whereby most spores land near the parental sporocarps (Gregory, 1945) and establish successfully, the relationship between geographic separation and the genetic distance of genets should be correlated. In this study, no correlation between the genetic and geographic separation of genets was found. Similar results were obtained in parentage analyses of Rhizopogon vinicolor and R. vesiculosus, whereby most genets sampled in immediate vicinities were unrelated (Kretzer et al., 2005). Kretzer et al. (2005) hypothesized that competition may prevent the offspring of R. vinicolor and R. vesiculosus from establishing near parental genets. An alternative hypothesis to account for the incidence of unrelated genotypes in nearby locations is that mating systems strongly favor outcrossing, whereby spores from outside the resident subpopulation have a higher likelihood of successful mating and subsequent establishment. Gryta et al. (2000) found that the inbred progeny of Hebeloma cylindrosporum were often short-lived compared to the survival success of outcrossed mycelia. Elucidating the patterns of genet distribution in stands is likely to require a better understanding of the interactions operating between genets of EM fungi, an understanding of the longevity and residence of genets and productive biology for R. brevipes.
In analyses of the population variation of EM fungi using molecular tools, the size and number of genets has been used to measure their genetic diversity (see Redecker et al., 2001). In most cases, there seems to have been a high density of EM genets sampled, with the exception of a few EM fungi that form large extensive genets including Suillus, Xerocomus and Cortinarius spp. (see Redecker et al., 2001). The power of resolving genets is likely to be dependent on the frequency of alleles within populations and the number of loci analyzed. For example, in Cantharellus formosus, the co-occurrence of genets across plots separated by large distances suggests that the frequency of microsatellite alleles was insufficient for resolving novel genets in aggregates of basidiocarps (Dunham et al., 2003). In these analyses, the expectation of encountering a genet of R. brevipes by chance was quite low because the allelic diversity was high across several loci; however, the saturation between sample size and allelic diversity was only reached in one population during a single fruiting season.
Perhaps it is not too surprising that no genetic structure was detected in the present study given the uniformity of the ecosystem studied and the relatively small spatial scale across which it was sampled. Moreover, it is also not too surprising that geographic structuring, and possibly cryptic speciation, was evident in a previous study based on a large-scale geographic sampling (Bergemann & Miller, 2002). The question that remains to be determined for populations of R. brevipes is how large are the genetic neighborhoods of this ‘species’ and or what is the distance between potentially isolated lineages or cryptic species? Populations of R. brevipes from western North America would be ideal for studying this, given that this region holds the world's greatest extent of mesic, temperate coniferous forests (Brunsfeld et al., 2001) where R. brevipes is likely to occur. Furthermore, California has more recognized subspecies of animals and plants than any other region in North America of comparable size (Myers, 1990; Myers et al., 1999). In western North America, disjunct populations between the Pacific coastal and the interior Rocky Mountain regions have been discovered in at least 60 plant, animal and fungal species (Brunsfeld et al., 2001), and the genetic spatial structuring of taxonomic groups may coincide with the age of California's mountain ranges and aridification in the regions (Calsbeek et al., 2003). A reasonable and testable hypothesis is that phylogeographic patterns of genetic structuring of R. brevipes occur in the mountains that surround the Great Central Valley of California and the coastal and interior ranges of the Pacific Northwest could be the focus of future research.