Comparative assessment of SSR and SNP markers for inferring the population genetic structure of the common fungus Armillaria cepistipes.

During the last years, simple sequence repeats (SSRs, also known as microsatellites) and single-nucleotide polymorphisms (SNPs) have become the most popular molecular markers for describing neutral genetic variation in populations of a wide range of organisms. However, only a limited number of studies has focused on comparing the performance of these two types of markers for describing the underlying genetic structure of wild populations. Moreover, none of these studies targeted fungi, the group of organisms with one of the most complex reproductive strategies. We evaluated the utility of SSRs and SNPs for inferring the neutral genetic structure of Armillaria cepistipes (basidiomycetes) at different spatial scales. For that, 407 samples were collected across a small (150 km2) area in the Ukrainian Carpathians and a large (41 000 km2) area in the Swiss Alps. All isolates were analyzed at 17 SSR loci distributed throughout the whole genome and at 24 SNP loci located in different single-copy conserved genes. The two markers showed different patterns of structure within the two spatial scales studied. The multi-allelic SSR markers seemed to be best suited for detecting genetic structure in indigenous fungal populations at a rather small spatial scale (radius of ~50-100 km). The pattern observed at SNP markers rather reflected ancient divergence of distant (~1000 km) populations that in addition are separated by mountain ranges. Despite these differences, both marker types were suitable for detecting the weak genetic structure of the two A. cepistipes populations investigated.

Genetic signatures of variation in population size in a native fungal pathogen after the recent massive plantation of its host tree.

Historical fluctuations in forests' distribution driven by past climate changes and anthropogenic activities can have large impacts on the demographic history of pathogens that have a long co-evolution history with these host trees. Using a population genetic approach, we investigated that hypothesis by reconstructing the demographic history of Armillaria ostoyae, one of the major pathogens of the maritime pine (Pinus pinaster), in the largest monospecific pine planted forest in Europe (south-western France). Genetic structure analyses and approximate Bayesian computation approaches revealed that a single pathogen population underwent a severe reduction in effective size (12 times lower) 1080-2080 generations ago, followed by an expansion (4 times higher) during the last 4 generations. These results are consistent with the history of the maritime pine forest in the region characterized by a strong recession during the last glaciation (~19 000 years ago) and massive plantations during the second half of the nineteenth century. Results suggest that recent and intensive plantations of a host tree population have offered the opportunity for a rapid spread and adaptation of their pathogens.

The population biology of fungal invasions.

Fungal invasions are increasingly recognized as a significant component of global changes, threatening ecosystem health and damaging food production. Invasive fungi also provide excellent models to evaluate the generality of results based on other eukaryotes. We first consider here the reasons why fungal invasions have long been overlooked: they tend to be inconspicuous, and inappropriate methods have been used for species recognition. We then review the information available on the patterns and mechanisms of fungal invasions. We examine the biological features underlying invasion success of certain fungal species. We review population structure analyses, revealing native source populations and strengths of bottlenecks. We highlight the documented ecological and evolutionary changes in invaded regions, including adaptation to temperature, increased virulence, hybridization, shifts to clonality and association with novel hosts. We discuss how the huge census size of most fungi allows adaptation even in bottlenecked, clonal invaders. We also present new analyses of the invasion of the anther-smut pathogen on white campion in North America, as a case study illustrating how an accurate knowledge of species limits and phylogeography of fungal populations can be used to decipher the origin of invasions. This case study shows that successful invasions can occur even when life history traits are particularly unfavourable to long-distance dispersal and even with a strong bottleneck. We conclude that fungal invasions are valuable models to contribute to our view of biological invasions, in particular by providing insights into the traits as well as ecological and evolutionary processes allowing successful introductions.

The chestnut blight fungus world tour: successive introduction events from diverse origins in an invasive plant fungal pathogen.

Clonal expansion has been observed in several invasive fungal plant pathogens colonizing new areas, raising the question of the origin of clonal lineages. Using microsatellite markers, we retraced the evolutionary history of introduction of the chestnut blight fungus, Cryphonectria parasitica, in North America and western Europe. Combining discriminant analysis of principal components and approximate Bayesian computation analysis, we showed that several introduction events from genetically differentiated source populations have occurred in both invaded areas. In addition, a low signal of genetic recombination among different source populations was suggested in North America. Finally, two genetic lineages were present in both invaded areas as well as in the native areas, suggesting the existence of genetic lineages with a high capacity to establish in diverse environments and host species. This study confirmed the importance of multiple introductions, but questioned the role of genetic admixture in the success of introduction of a fungal plant pathogen.

Multiple introductions of divergent genetic lineages in an invasive fungal pathogen, Cryphonectria parasitica, in France.

The occurrence of multiple introductions may be a crucial factor in the successful establishment of invasive species, but few studies focus on the introduction of fungal pathogens, despite their significant effect on invaded habitats. Although Cryphonectria parasitica, the chestnut blight fungus introduced in North America and Europe from Asia during the 20th century, caused dramatic changes in its new range, the history of its introduction is not well retraced in Europe. Using 10 microsatellite loci, we investigated the genetic diversity of 583 isolates in France, where several introductions have been hypothesized. Our analyses showed that the seven most frequent multilocus genotypes belonged to three genetic lineages, which had a different and geographically limited distribution. These results suggest that different introduction events occurred in France. Genetic recombination was low among these lineages, despite the presence of the two mating types in each chestnut stand analysed. The spatial distribution of lineages suggests that the history of introductions in France associated with the slow expansion of the disease has contributed to the low observed rate of recombination among the divergent lineages. However, we discuss the possibility that environmental conditions or viral interactions could locally reduce recombination among genotypes.

Genetic structure of an expanding Armillaria root rot fungus (Armillaria ostoyae) population in a managed pine forest in southwestern France.

The Landes de Gascogne forest (southwestern France) is the largest maritime pine (Pinus pinaster) plantation in Europe. Armillaria root disease (Armillaria ostoyae) has been reported since the early 1920s in the coastal area (western sector), but its incidence over the last 20 years has increased in the eastern sector. We investigated the genetic structure of the A. ostoyae population in this forest, focusing particularly on geographical differentiation potentially indicative of disease expansion in this area. In total, 531 isolates obtained from mycelial fans on symptomatic trees or undecayed stumps in 31 different disease foci were genotyped at five microsatellite loci. In 20 of these disease foci, a single genotype dominated, reflecting a predominantly clonal local spread of A. ostoyae. By contrast, at the regional scale, A. ostoyae probably spreads mostly via basidiospores (sexual spores), as no genotype common to several disease foci was identified. The absence of a clear pattern of isolation by distance may indicate either substantial gene flow or stochastic colonisation independent of spatial distance. The gradient of genetic diversity from the coast inwards and the greater genetic divergence of the eastern disease foci are consistent with the expansion of the A. ostoyae population from the coast eastwards.

Geostatistical genetic analysis for inferring the dispersal pattern of a partially clonal species: example of the chestnut blight fungus.

Spatial genetic analyses can be used to infer dispersal processes in natural populations. For partially clonal species with alternating sexual and asexual reproduction, the repetition of genotypes must be taken into account in analyses. The methods currently employed to evaluate the relevance of the spatial scale used for the estimation of gene flow are not suitable for these species. We investigated recently developed methods for taking into account repeated genotypes and for determining whether the sampling scale is large enough to capture all the spatial genetic structure existing within a population. We applied these methods to a fungal plant pathogen species, Cryphonectria parasitica, which has caused the death of many American and European chestnut trees since its introduction from Asia at the beginning of the 20th century. These methods were found to be useful for unravelling the effects of clonality and historical gene flow on the spatial genetic structure, and indicated that dispersal processes have probably occurred over a larger spatial scale than previously assumed.

Population genetics of fungal diseases of plants.

Although parasitism is one of the most common lifestyles among eukaryotes, population genetics on parasites lag for behind those on free-living organisms. Yet, the advent of molecular markers offers great tools for studying important processes, such as dispersal, mating systems, adaptation to host and speciation. Here we highlight some studies that used molecular markers to address questions about the population genetics of fungal (including oomycetes) plant pathogens. We conclude that population genetics approaches have provided tremendous insights into the biology of a few fungal parasites and warrant more wide use in phytopathology. However, theoretical advances are badly needed to best apply the existing methods. Fungi are of prime interest not only because they are major parasites of plants and animals, but they also constitute tractable and highly useful models for understanding evolutionary processes. We hope that the emerging field of fungal evolution will attract more evolutionary biologists in the near future.

Estimating anisotropic pollen dispersal: a case study in Quercus lobata.

The pollen dispersal distribution is an important element of the neighbourhood size of plant populations. Most methods aimed at estimating the dispersal curve assume that pollen dispersal is isotropic, but evidence indicates that this assumption does not hold for many plant species, particularly wind-pollinated species subject to prevailing winds during the pollination season. We propose here a method of detecting anisotropy of pollen dispersal and of gauging its intensity, based on the estimation of the differentiation of maternal pollen clouds (TWOGENER extraction), assuming that pollen dispersal is bivariate and normally distributed. We applied the new method to a case study in Quercus lobata, detecting only a modest level of anisotropy in pollen dispersal in a direction roughly similar to the prevailing wind direction. Finally, we conducted a simulation to explore the conditions under which anisotropy can be detected with this method, and we show that while anisotropy is detectable, in principle, it requires a large volume of data.

Gene flow, historical population dynamics and genetic diversity within French Guianan populations of a rainforest tree species, Vouacapoua americana.

Both gene flow and historical events influence the genetic diversity of natural populations. One way to understand their respective impact is to analyze population genetic structure at large spatial scales. We studied the distribution of genetic diversity of 17 populations of Vouacapoua americana (Caesalpiniaceae) in French Guiana, using nine microsatellite loci. Low genetic diversity was observed within populations, with a mean allelic richness and gene diversity of 4.1 and 0.506, respectively, which could be due to low effective population size and/or past bottlenecks. Using the regression between Fst/(1-Fst), estimated between pairs of populations, and the logarithm of the geographical distance, the spatial genetic structure can partly be explained by isolation-by-distance and limited gene flow among populations. This result is in agreement with the species' biology, including seed and pollen dispersal by rodents and insects, respectively. In contrast, no clear genetic signal of historical events was found when examining genetic differentiation among populations in relation to biogeographical hypotheses or by testing for bottlenecks within populations. Our conclusion is that nuclear spatial genetic structure of V. americana, at the geographic scale of French Guiana, is better explained by gene flow rather than by historical events.

Chloroplast diversity in Vouacapoua americana (Caesalpiniaceae), a neotropical forest tree.

The chloroplast genome has been widely used to describe genetic diversity in plant species. Its maternal inheritance in numerous angiosperm species and low mutation rate are suitable characters when inferring historical events such as possible recolonization routes. Here we have studied chloroplast DNA variation using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) with seven pairs of primers and four restriction enzymes in 14 populations of Vouacapoua americana (Caesalpiniaceae) a neotropical tree sampled throughout French Guiana. Population diversity (Hs), total gene diversity (Ht) and differentiation among populations (GST) were estimated using Nei's method as 0.09, 0.87 and 0.89, respectively. This is consistent with the limited gene flow associated with synzoochory in this species. The genetic structure observed in the north of French Guiana suggests that historical events such as contractions and recent recolonizations have had a large impact on the distribution of genetic diversity in this species.

Evidence of low gene flow in a neotropical clustered tree species in two rainforest stands of French Guiana.

The spatial genetic structure of the neotropical, clustered tree species Vouacapoua americana (Aublet) was studied in two natural forest stands (Paracou and Nouragues) in French Guiana. Using eight microsatellite loci, V. americana is characterized by a marked genetic structure at small spatial distances (under 30-60 m), in agreement with the limited seed dispersal by rodent species. Gene flow through pollen is also shown to be mainly restricted to less than 100 m. This result suggests that most pollination events (mediated through small insects) are probably limited to within-patches of individuals, which might explain the high genetic differentiation among patches (F(ST) = 0.11) separated by less than 2 km. We also assume that stronger genetic structure in Paracou is likely to be due to lower seed dispersal by rodents, large spatial distances separating patches, or a recent recolonization event.