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The extent of gene dispersal is a fundamental factor in population biology. Dispersal determines the degree of connectivity among subpopulations, and thus affects population evolutionary potential (Slatkin, 1987). From an ecological perspective, dispersal allows species to colonize new environments. For many plant-symbiotic fungi, such as ectomycorrhizal (ECM) or plant-pathogenic fungi, dispersal propagules are spores that are often dispersed by wind. The finite lifespan of the hosts or the host tissues colonized by these fungi necessitates dispersal. Therefore, dispersal is critical for these fungal species to persist from one leaf/root to another, or from one generation of host to another.
With fungi, dispersal has traditionally been estimated by direct methods, such as spore trapping, or by monitoring the spatiotemporal progression of the symptoms/signs of colonization (McCartney et al., 2006). These direct methods are subject to the following technical limitations: first, spore traps are ineffective at low spore concentrations, as is the case when traps are far (> 100 m) from the inoculum source (Aylor, 2003), and, second, monitoring the progression of colonization at a field scale does not accommodate dispersal at the landscape scale. Indirect, genetic methods can be used to overcome such limitations. The theory of isolation by distance (IBD; Wright, 1943) posits that genetic similarity between neighboring individuals is higher than between more distant individuals. For a fungus with a limited capacity for spore dispersal, we would thus expect to find a nonrandom distribution of genotypes (i.e. spatial genetic structure) within the spatial scale of local dispersal. To estimate the spatial scale of local dispersal, spatial autocorrelation methods (Hardy & Vekemans, 1999; Smouse & Peakall, 1999) have provided indirect estimates of gene dispersal that are consistent with direct estimates of propagule dispersal in several plant species (Vekemans & Hardy, 2004). The assessment of dispersal and demographic connectivity among individuals is of fundamental importance to define the management unit for a threatened species or to guide strategies aimed at controlling an invasive or pathogenic species.
Contemporary spore dispersal in fungi has recently been characterized using neutral genetic markers and individual-based methods, and, in a few cases, spore dispersal has been suggested to be spatially limited. For instance, using spatial autocorrelation analysis, Carriconde et al. (2008) revealed that neighboring genotypes of the ECM fungus Tricholoma scalpturatum were more closely related than expected under the assumption of a random spatial distribution, suggesting that spore dispersal was spatially limited in this wind-dispersed species. Similarly, the distribution of genotypes of the Pacific golden chanterelle (Cantharellus formosus) were shown to be nonrandom at distances up to 400 m, suggesting restricted spore dispersal at such spatial scales (Dunham et al., 2006). By contrast, a random distribution of genotypes of Rhizopogon vinicolor and R. vesiculosis within study sites of 50 m × 100 m suggested that spore dispersal, perhaps facilitated by small mammal mycophagy, occurred at spatial scales larger than the boundaries of the study sites in these two species (Kretzer et al., 2005). This latter study illustrates the more common finding of an absence of spatial genetic structure in ECM fungi (reviewed by Carriconde et al., 2008), suggesting extensive spore dispersal. Indirect estimates of gene dispersal have contributed to a better understanding of fungal population structures.
Species of the fungal genus Armillaria are important components of many natural forests and timber plantations of temperate, boreal and tropical regions of the world (Baumgartner et al., 2011). Some Armillaria spp. function mainly as saprotrophs (Kile et al., 1991) and play key roles in carbon and mineral cycling in forest ecosystems. Other species, such as A. ostoyae and A. mellea, are best known as virulent plant pathogens that are the causal agents of Armillaria root disease (Morrison et al., 1991). Armillaria ostoyae is one of two pathogens responsible for the majority of coniferous timber losses in the world (Kile et al., 1991). Similarly, A. mellea is an aggressive pathogen of numerous horticultural crops (e.g. Vitis, Juglans, Citrus, Prunus) and ornamentals, and is known to infect > 500 species (Raabe, 1962).
For the more aggressive species (e.g. A. mellea), the fungus colonizes living woody roots, kills them and uses them as a substrate. It can persist saprotrophically in the form of a mycelium within roots after the host tree dies or is cut down. These colonized roots serve as an inoculum source when the roots of replanted trees grow into direct contact and are colonized by this resident mycelium. Mycelium can also spread below ground within the form of a specialized structure, known as a rhizomorph. Rhizomorph growth and the growth of mycelium through susceptible roots have long been considered as the two main modes of infection and spread of Armillaria root disease (Redfern & Filip, 1991). Indeed, the potential for such subterranean expansion of an individual genotype over many hectares has been documented among different Armillaria species in different forest ecosystems around the world (Adams, 1974; Shaw & Roth, 1976; Korhonen, 1978; Anderson & Ullrich, 1979; Kile, 1983; Legrand et al., 1996; Prospero et al., 2003). In one study, in vitro growth rates were used to estimate the age of a genotype of A. gallica that covered 15 ha of forest, and this genotype is considered to be one of the oldest organisms on Earth (Smith et al., 1992).
As a result of sexual reproduction, Armillaria species produce mushrooms (also known as sporocarps or basidiocarps) that release basidiospores. The epidemiological importance of basidiospores is thought to be low, relative to colonization by a resident mycelium. In orchards, vineyards and timber plantations, a disease center is typically inhabited by one to a few diploid individuals (i.e. resident mycelia) that originate from the previous forest stand (e.g. Rizzo et al., 1998; Baumgartner & Rizzo, 2002; Prospero et al., 2008). Furthermore, inoculation attempts with basidiospores in the field often fail (e.g. Rishbeth, 1970) and the haploid mycelia that germinate from basidiospores are rarely detected in nature (e.g. Peabody et al., 2000). By contrast, other studies suggest that basidiospores are important. First, the neutral genetic structure of Armillaria populations is probably a result of sexual reproduction (Prospero et al., 2008; Baumgartner et al., 2010). Second, in some temperate hardwood and conifer forests, a disease center among naturally established host plants is inhabited by multiple, intermingled, diploid individuals of Armillaria (Ullrich & Anderson, 1978; Rishbeth, 1988; Legrand et al., 1996). Finally, in conifer plantations of New Zealand, basidiospores of A. novae-zelandiae have been shown to colonize freshly cut wood of Pinus radiata (Hood et al., 2008).
Little is known about the spatial scale of basidiospore dispersal of Armillaria. A recent study investigated basidiospore dispersal gradients of A. novae-zelandiae in podocarp–hardwood forests of New Zealand using spore traps (Power et al., 2008). These direct estimates of spore dispersal suggest that the vast majority of basidiospores are deposited within a few meters of the sporocarps, with only a small proportion traveling up to 150 m from the forest edge (the farthest distance sampled).
In the present study, we investigated the spatial distribution of A. mellea genotypes in Golden Gate Park, a human-made, urban woodland in San Francisco, CA, USA. This study site is unique among other studies of fungal genetic structure because it represents a potentially closed system; the park is bordered by a large urban area, which is further surrounded on three sides by the Pacific Ocean. The park consists almost entirely of planted hosts that were established in the early 1870s on land covered primarily by sand dunes, with trees restricted to the eastern end of the park (the exact locations of which are unknown). According to the First Biennial Report of the San Francisco Park Commissioners (McLaren, 1872), ‘The Golden Gate Park contains c. 1000 acres (405 ha), of which 270 acres (109 ha) at the eastern end is good arable land, covered in many places with trees and shrubbery’. Throughout 730 acres (296 ha) of the park, which was referred to in the Commissioner’s 1872 Report as ‘a waste of drifting sand’, the planting of trees pre-dates the presence of A. mellea. There is no evidence that sand dunes supported woody plant hosts to harbor A. mellea, and therefore we hypothesize that genotypes recovered from hosts planted in such areas were originally established by spores. This approach is different from past Armillaria studies in orchards, vineyards and timber plantations (e.g. Rizzo et al., 1998; Baumgartner & Rizzo, 2002), in which resident genotypes spanning the root systems of multiple, adjacent hosts probably influenced subsequent colonization by spores. We used microsatellite markers (Baumgartner et al., 2009) and spatial autocorrelation analyses of kinship coefficients to evaluate the relative contributions of spores and mycelial growth to the spread of Armillaria root disease, and to test the hypothesis of spatially limited spore dispersal.
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Using a combination of microsatellite genotyping, somatic incompatibility testing and spatial coordinates, we gained insights into the colonization process of an organism that spreads by both aerial dispersal of sexual spores and subterranean, vegetative expansion of individual genotypes. Genetic similarity among neighboring isolates of A. mellea was higher than among more distant isolates. Vegetative, below-ground growth and wind dispersal of basidiospores, which, from our findings, are both spatially limited, contributed to the nonrandom distribution of diploid individuals. The high proportion of unique MLGs and physical linkage equilibrium detected among the microsatellite loci support the important role of sexual reproduction, relative to vegetative growth, in shaping the population genetic structure of A. mellea in Golden Gate Park.
Over all loci, a significant heterozygote deficit was detected, and this is in agreement with a previous study of other A. mellea subpopulations in the USA (Baumgartner et al., 2010) and analyses of other Armillaria species (Saville et al., 1996; Prospero et al., 2008). Inbreeding as a result of subdivision of the sample into multiple subpopulations that do not freely exchange migrants (i.e. Wahlund effect; Wahlund, 1928) can be ruled out, because we did not find evidence of population structure among MLGs. However, a weak population subdivision, as expected under the IBD pattern identified in this study, is not typically detected by assignment methods, such as that implemented in STRUCTURE (Waples & Gaggiotti, 2006). We hypothesize that significant heterozygote deficit may reflect inbreeding as a result of sibling matings or of somatic recombination through haploid–diploid matings, as suggested by Baumgartner et al. (2010). The likelihood of sibling matings is supported by our finding of a higher genetic similarity among neighboring MLGs relative to distant MLGs. Although this process may occur in Armillaria populations, our estimates of high genotypic richness and multilocus linkage disequilibrium are nonetheless consistent with a reproductive life history predominated by outcrossing.
In contrast with previous beliefs with regard to the spread of Armillaria root disease (Redfern & Filip, 1991), our results reveal a more important role of spore dispersal, relative to mycelial growth, in the colonization of new habitat. In Golden Gate Park, a large proportion of MLGs (61 of the 90 in total) were recovered from only a single tree. This contrasts with the presence of one to a few diploid individuals, spanning the roots of many adjacent trees, as documented in past studies of A. mellea populations in California (Rizzo et al., 1998; Baumgartner & Rizzo, 2002). At such study sites, the hosts (grapevines and pear trees) were planted on land that was cleared of infected forest stands, and thus the colonization of the successive crops was primarily a consequence of root infection by resident mycelia. Assuming that resident mycelia were initially rare to absent from much of Golden Gate Park, the planted trees constituted a potential site for the establishment of infections by spores; colonization of the park was mainly by new infection foci. A similar colonization strategy was revealed in the root pathogen Onnia tomentosa, which was shown to initially colonize conifer stands by basidiospores, and then to expand to colonize adjacent trees through mycelial growth (Germain et al., 2009). Similarly, the presence of many small genotypes of the ECM fungus Suillus pungens in a post-fire stand of Pinus muricata suggested that the recolonization of the new seedlings occurred mainly by spores, and not by the resident mycelium that dominated the mature stand before the fire (Bruns et al., 2002). This is in contrast with other tree pathogens, such as Phellinus tremulae and, to a lesser extent, Heterobasidion annosum, whose populations are typically characterized by small genotypes, independent of the forest age, suggesting that spores are the primary dispersal mode for these two pathogens (Holmer et al., 1994; Garbelotto et al., 1999). Because A. mellea can occur on the roots of asymptomatic trees (Baumgartner & Rizzo, 2001), and we sampled only symptomatic trees, our collection may have been biased towards the detection of small, unique MLGs. However, the intensive sampling carried out over the course of 2 yr (309 total samples from mycelial fans and sporocarps, exhaustive examination of symptomatic trees) was the most feasible approach to mapping the A. mellea population of the 409-ha park.
A strong spatial genetic structure was detected when all isolates were maintained in the spatial autocorrelation analyses. These results reflect the spatial grouping of most repeated MLGs up to c. 130 m, reflecting the contribution of mycelial growth to the clustering of repeated MLGs at such distances. However, we detected two MLGs with greater spatial dimensions (322.1 and 342.8 m in length) in the arboretum of Golden Gate Park. These largest two MLGs may represent resident mycelia that pre-dated the planting of the park, although the exact locations of the original forested areas are unknown. A thick layer of wood chips (c. 30 cm) and frequent irrigation in the arboretum (P. Ehrlich, Presidio of San Francisco, Golden Gate National Recreation Area, pers. comm.) lead to excessive soil moisture, which is known to increase mycelial growth of A. mellea within susceptible root tissue (Redfern & Filip, 1991). Alternatively, it is possible that humans have moved infected plant material within the arboretum (e.g. grinding down an infected stump and then using the wood chips as mulch 300 m away), thereby creating the appearance of a large MLG that is actually made up of discontinuous fragments of the same MLG on distant trees. Further evidence in support of human-mediated fragmentation of these two large MLGs is the unexpected increase in the probability of genotypic identity at large distances (Arnaud-Haond et al., 2007).
The examination of the convergence point of the two spatial autocorrelograms that either included or excluded repeated MLGs provided an estimate of the average clonal subrange of A. mellea. The clonal subrange has previously been calculated primarily for plant species, for example, Cymodocea nodosa, a rhizomatous seagrass that spreads both by vegetative and sexual propagules (Alberto et al., 2005). We estimated the clonal subrange for a fungal species with analogous modes of dispersal. Spatial autocorrelograms including and excluding repeated MLGs merged at an average distance of 131.8 m, indicating that this is the characteristic maximum size (clonal subrange) of an A. mellea MLG in Golden Gate Park. Indeed, all MLGs were < 100 m in length, with the exception of two (322.1 and 342.8 m), the latter of which probably resulted from human-mediated fragmentation in the heavily managed arboretum of the park (see previous paragraph). After excluding these two largest MLGs from the analysis, a clonal subrange of 97.2 m was calculated, reflecting the spatial patterns of vegetative growth of A. mellea in a human-managed, ornamental landscape, where the host population probably pre-dates much of the A. mellea population. This clonal subrange can theoretically be used as a minimum sampling distance between trees, when one seeks to maximize the genotypic richness of an A. mellea population. Nonetheless, our measurements are specific to the population studied here, and confirmation in other environments (e.g. natural forests, plantations) is required. In addition, the comparisons of clonal subrange values in different habitats could allow an assessment of the effects of habitat characteristics (e.g. age, host density, levels of disturbance) on the reproductive mode of A. mellea (clonality vs sexual reproduction).
When repeated MLGs were removed from the spatial autocorrelation analysis, a steady decrease in kinship coefficient was observed for the first three distance classes (up to an average spatial distance of 119 m), with a significant and positive spatial autocorrelation detected at the first distance class (average spatial distance of 30 m). This IBD pattern is weak overall and only detectable at the shortest spatial distance class. One of the assumptions of the IBD model is that the population is at equilibrium between genetic drift and gene dispersal (Rousset, 1997). This assumption can be violated, for instance, in a founder population. In our case, we presume that the A. mellea population of Golden Gate Park is probably at drift dispersal equilibrium, in part because over 100 yr have passed since the establishment of the park (McLaren, 1872). Moreover, the fungus reproduces sexually each year, based on our observations of sporocarps. Consequently, the large number of sexual generations that have occurred in the A. mellea population of Golden Gate Park have probably brought the population to drift dispersal equilibrium, and thus its structure could be shaped by IBD (Hardy & Vekemans, 1999).
Theoretically, a nonrandom spatial distribution of genotypes, which is indicative of spatial genetic structure, can be caused by various factors, such as limited gene dispersal in space (i.e. IBD), local adaptation (e.g. host specialization) or the presence of physical barriers to gene flow (e.g. a mountain range) among genetically differentiated clusters of genotypes (Guillot et al., 2009). Among these factors, limited gene dispersal has the most support as the contributing factor to spatial genetic structure. Indeed, there was no evidence of genetically differentiated clusters of MLGs within the park (e.g. no clustering of genotypes infecting pine trees). Furthermore, in several instances, a single MLG infected multiple tree species, and this is consistent with the broad host range of A. mellea (Raabe, 1962) and the lack of reports of host specialization from the literature. Our finding of significant spatial genetic structure is, rather, in agreement with direct estimates of basidiospore dispersal in A. novae-zelandiae: most basidiospores are deposited within a few meters from the sporocarps, whereas deposition up to 150 m is much less frequent (Power et al., 2008). The IBD pattern suggests that spore dispersal mainly occurs over a few meters. This very localized dispersal may provide ecological benefits. All spores from a given sporocarp of A. mellea can undergo plasmogamy with their diploid parent (Anderson & Ullrich, 1982). The diploid nucleus often displaces the haploid nucleus within the haploid mycelium (Rizzo & May, 1994). In this way, a diploid–haploid mating between parent and progeny expands the parent’s mycelium, and thus its substrate base. The parental genotype persists, and thus we may have underestimated local spore dispersal, because such haploid–diploid matings following local dispersal of haploid spores are undetectable by our genotyping methods.
Our findings suggest that spore dispersal is spatially restricted in A. mellea, but rare instances of long-distance dispersal may promote genetic homogeneity between spatially separate subpopulations. Indeed, gene flow through spore dispersal has been suggested as a means of preventing genetic differentiation among subpopulations of A. ostoyae and A. mellea at spatial scales beyond hundreds of kilometers (Prospero et al., 2008; Baumgartner et al., 2010). Long-distance spore dispersal is thus plausible and is probably important for the colonization of new habitat by A. mellea. Similarly, low levels of genetic differentiation have been found among distant subpopulations of wood decay fungi (Stenlid & Gustafsson, 2001). For example, distant subpopulations of the forest pathogen Heterobasidion annosum are not genetically differentiated (Stenlid et al., 1994), in spite of evidence that most spores are deposited within a few meters of a sporocarp, and only 0.1% of the total released spores are estimated to be trapped 100 m away (Kallio, 1970). Like H. annosum, large population sizes, high spore production per sporocarp and rare, long-distance spore dispersal for A. mellea probably counteract genetic divergence at a large spatial scale. Alternatively, although individual-based methods, such as spatial autocorrelations, characterize contemporary gene dispersal, population-based methods estimate evolutionary averages of dispersal. The latter approach depends on the establishment of an a priori definition of a subpopulation that may not accurately reflect the population structure. The spatial distribution of genetic diversity is also affected by historical events (e.g. establishment by multiple founder events vs a single, massive invasion by a large panmictic group). Moreover, gene flow that causes genetic homogeneity between subpopulations is the product of the migration rate (i.e. the fraction of dispersers entering the subpopulation each generation) and the effective population size (Slatkin, 1987). Pathogen populations are typically characterized by very large population sizes (e.g. millions of spores are produced per mushroom), and therefore subpopulations may be genetically homogeneous in the presence of very low migration rates (Rieux et al., 2011; Travadon et al., 2011). Our study illustrates that, when the distribution of genotypes is examined at a local spatial scale, an indirect estimate of the scale at which fungal spore dispersal occurs can be obtained from genetic and spatial data, and thus provides an alternative to the labor-intensive, direct trapping methods.