Weak population spatial genetic structure and low infraspecific specificity for fungal partners in the rare mycoheterotrophic orchid Epipogium aphyllum.

Some plants abandoned photosynthesis and developed full dependency on fungi for nutrition. Most of the so-called mycoheterotrophic plants exhibit high specificity towards their fungal partners. We tested whether natural rarity of mycoheterotrophic plants and usual small and fluctuating population size make their populations more prone to genetic differentiation caused by restricted gene flow and/or genetic drift. We also tested whether these genetic characteristics might in turn shape divergent fungal preferences. We studied the mycoheterotrophic orchid Epipogium aphyllum, addressing the joint issues of genetic structure of its populations over Europe and possible consequences for mycorrhizal specificity within the associated fungal taxa. Out of 27 sampled E. aphyllum populations, nine were included for genetic diversity assessment using nine nuclear microsatellites and plastid DNA. Population genetic structure was inferred based on the total number of populations. Individuals from 17 locations were included into analysis of genetic identity of mycorrhizal fungi of E. aphyllum based on barcoding by nuclear ribosomal DNA. Epipogium aphyllum populations revealed high genetic diversity (uHe = 0.562) and low genetic differentiation over vast distances (FST = 0.106 for nuclear microsatellites and FST = 0.156 for plastid DNA). Bayesian clustering analyses identified only two genetic clusters, with a high degree of admixture. Epipogium aphyllum genets arise from panmixia and display locally variable, but relatively high production of ramets, as shown by a low value of rarefied genotypic richness (Rr = 0.265). Epipogium aphyllum genotype control over partner selection was negligible as (1) we found ramets from a single genetic individual associated with up to 68% of the known Inocybe spp. associating with the plant species, (2) and partner identity did not show any geographic structure. The absence of mosaicism in the mycorrhizal specificity over Europe may be linked to preferential allogamous habit of E. aphyllum and significant gene flow, which tend to promote host generalism.

Adapting through glacial cycles: insights from a long-lived tree (Taxus baccata).

Despite the large body of research devoted to understanding the role of Quaternary glacial cycles in the genetic divergence of European trees, the differential contribution of geographic isolation and/or environmental adaptation in creating population genetic divergence remains unexplored. In this study, we used a long-lived tree (Taxus baccata) as a model species to investigate the impact of Quaternary climatic changes on genetic diversity via neutral (isolation-by-distance) and selective (isolation-by-adaptation) processes. We applied approximate Bayesian computation to genetic data to infer its demographic history, and combined this information with past and present climatic data to assess the role of environment and geography in the observed patterns of genetic structure. We found evidence that yew colonized Europe from the East, and that European samples diverged into two groups (Western, Eastern) at the beginning of the Quaternary glaciations, c. 2.2 Myr before present. Apart from the expected effects of geographical isolation during glacials, we discovered a significant role of environmental adaptation during interglacials at the origin of genetic divergence between both groups. This process may be common in other organisms, providing new research lines to explore the effect of Quaternary climatic factors on present-day patterns of genetic diversity.

Molinia caerulea alters forest Quercus petraea seedling growth through reduced mycorrhization.

Oak regeneration is jeopardized by purple moor grass, a well-known competitive perennial grass in the temperate forests of Western Europe. Below-ground interactions regarding resource acquisition and interference have been demonstrated and have led to new questions about the negative impact of purple moor grass on ectomycorrhizal colonization. The objective was to examine the effects of moor grass on root system size and ectomycorrhization rate of oak seedlings as well as consequences on nitrogen (N) content in oak and soil. Oak seedlings and moor grass tufts were planted together or separately in pots under semi-controlled conditions (irrigated and natural light) and harvested 1 year after planting. Biomass, N content in shoot and root in oak and moor grass as well as number of lateral roots and ectomycorrhizal rate in oak were measured. Biomass in both oak shoot and root was reduced when planting with moor grass. Concurrently, oak lateral roots number and ectomycorrhization rate decreased, along with a reduction in N content in mixed-grown oak. An interference mechanism of moor grass is affecting oak seedlings performance through reduction in oak lateral roots number and its ectomycorrhization, observed in conjunction with a lower growth and N content in oak. By altering both oak roots and mycorrhizas, moor grass appears to be a species with a high allelopathic potential. More broadly, these results show the complexity of interspecific interactions that involve various ecological processes involving the soil microbial community and need to be explored in situ.

Extensive gene flow over Europe and possible speciation over Eurasia in the ectomycorrhizal basidiomycete Laccaria amethystina complex.

Biogeographical patterns and large-scale genetic structure have been little studied in ectomycorrhizal (EM) fungi, despite the ecological and economic importance of EM symbioses. We coupled population genetics and phylogenetic approaches to understand spatial structure in fungal populations on a continental scale. Using nine microsatellite markers, we characterized gene flow among 16 populations of the widespread EM basidiomycete Laccaria amethystina over Europe (i.e. over 2900 km). We also widened our scope to two additional populations from Japan (10(4) km away) and compared them with European populations through microsatellite markers and multilocus phylogenies, using three nuclear genes (NAR, G6PD and ribosomal DNA) and two mitochondrial ribosomal genes. European L. amethystina populations displayed limited differentiation (average F(ST) = 0.041) and very weak isolation by distance (IBD). This panmictic European pattern may result from effective aerial dispersal of spores, high genetic diversity in populations and mutualistic interactions with multiple hosts that all facilitate migration. The multilocus phylogeny based on nuclear genes confirmed that Japanese and European specimens were closely related but clustered on a geographical basis. By using microsatellite markers, we found that Japanese populations were strongly differentiated from the European populations (F(ST) = 0.416), more than expected by extrapolating the European pattern of IBD. Population structure analyses clearly separated the populations into two clusters, i.e. European and Japanese clusters. We discuss the possibility of IBD in a continuous population (considering some evidence for a ring species over the Northern Hemisphere) vs. an allopatric speciation over Eurasia, making L. amethystina a promising model of intercontinental species for future studies.

A multiscale approach to detect selection in nonmodel tree species: Widespread adaptation despite population decline in Taxus baccata L.

Detecting the molecular basis of local adaptation and identifying selective drivers is still challenging in nonmodel species. The use of purely population genetic approaches is limited by some characteristics of genetic systems, such as pleiotropy and polygenic control, and parallel evidence from phenotypic-based experimental comparisons is required. In long-lived organisms, the detection of selective pressures might also be precluded by evolutionary lag times in response to the environment. Here, we used the English yew to showcase an example of a multiscale integrative approach in a nonmodel species with limited plant and genomic resources. We combined information from two independent sources, phenotypes in a common environment and genomic data in natural populations, to investigate the signature of selection. Growth differences among populations in a common environment, and phenological patterns of both shoot elongation and male strobili maturation, were associated with climate clines, providing evidence for local adaptation and guiding us in the selection of populations for genomic analyses. We used information on over 25,000 SNPs from c. 1,200 genes to infer the demographic history and to test for molecular signatures of selection at different levels: SNP, gene, and biological pathway. Our results confirmed an overall demographic history of population decline, but we also found evidence for putative local adaptation at the molecular level. We identified or confirmed several candidate genes for positive and negative selection in forest trees, including the pseudo-response regulator 7 (PRR7), an essential component of the circadian clock in plants. In addition, we successfully tested an approach to detect polygenic adaptation in biological pathways, allowing us to identify the flavonoid biosynthesis pathway as a candidate stress-response pathway that deserves further attention in other plants. Finally, our study contributes to the emerging view that explaining contemporary standing genetic variation requires considering adaptation to past climates, especially for long-lived trees.

Evidence from population genetics that the ectomycorrhizal basidiomycete Laccaria amethystina is an actual multihost symbiont.

It is commonly assumed that ectomycorrhizal (ECM) fungi associated with temperate forest tree roots are not host-specific. Because this assumption relies on species delineations based on fruitbodies morphology or ribosomal DNA sequences, host-specific, cryptic biological species cannot be ruled out. To demonstrate that Laccaria amethystina has true generalist abilities, we sampled 510 fruitbodies on three French sites situated 150-450 km away from each other. At each site, populations from monospecific stands (Abies alba, Castanea europea and Fagus sylvatica) or mixed stands (F. sylvatica + Quercus robur or Q. robur +Carpinus betulus) were sampled. Three different sets of markers were used for genotyping: (i) five microsatellite loci plus the ribosomal DNA intergenic spacer, (ii) the mitochondrial large ribosomal DNA subunit, and (iii) direct amplification of length polymorphism (DALP), a new method for fungi providing dominant markers. Evidence for allogamous populations (with possible inbreeding at local scale) and possibly for biparental mitochondrial inheritance was found. All markers congruently demonstrated that L. amethystina populations show little structure at this geographical scale, indicating high gene flow (as many as 50% of founding spores in all populations being of external origin). Our results also showed that host species contributed even less to population differentiation, and there was no evidence for cryptic biological species. This first in situ demonstration of a true multihost ability in an ECM species is discussed in terms of ecology and evolutionary biology.

A pantropically introduced tree is followed by specific ectomycorrhizal symbionts due to pseudo-vertical transmission.

Global trade increases plant introductions, but joint introduction of associated microbes is overlooked. We analyzed the ectomycorrhizal fungi of a Caribbean beach tree, seagrape (Coccoloba uvifera, Polygonacaeae), introduced pantropically to stabilize coastal soils and produce edible fruits. Seagrape displays a limited symbiont diversity in the Caribbean. In five regions of introduction (Brazil, Japan, Malaysia, Réunion and Senegal), molecular barcoding showed that seagrape mostly or exclusively associates with Scleroderma species (Basidiomycota) that were hitherto only known from Caribbean seagrape stands. An unknown Scleroderma species dominates in Brazil, Japan and Malaysia, while Scleroderma bermudense exclusively occurs in Réunion and Senegal. Population genetics analysis of S. bermudense did not detect any demographic bottleneck associated with a possible founder effect, but fungal populations from regions where seagrape is introduced are little differentiated from the Caribbean ones, separated by thousands of kilometers, consistently with relatively recent introduction. Moreover, dry seagrape fruits carry Scleroderma spores, probably because, when drying on beach sand, they aggregate spores from the spore bank accumulated by semi-hypogeous Scleroderma sporocarps. Aggregated spores inoculate seedlings, and their abundance may limit the founder effect after seagrape introduction. This rare pseudo-vertical transmission of mycorrhizal fungi likely contributed to efficient and repeated seagrape/Scleroderma co-introductions.

Out of Asia: Biogeography of fungal populations reveals Asian origin of diversification of the Laccaria amethystina complex, and two new species of violet Laccaria.

Purple Laccaria are ectomycorrhizal basidiomycetes associated with temperate forests all over the Northern Hemisphere in at least two taxa: Laccaria amethysteo-occidentalis in North America, and L. amethystina complex in Eurasia, as shown by Vincenot et al. (2012). Here, we combine a further study of the genetic structure of L. amethystina populations from Europe to southwestern China and Japan, using neutral Single Sequence Repeat (SSR; microsatellite) markers; and a systematic description of two novel Asian species, namely Laccaria moshuijun and Laccaria japonica, based on ecological, morphological, and molecular criteria (rDNA sequences). Population genetics provides evidence of the ancient isolation of three regional groups, with strong signal for speciation, and suggests a centre of origin of modern populations closest to present-day Chinese populations. Phylogenetic analyses confirm speciation at the molecular level, reflected in morphological features: L. moshuijun samples (from Yunnan, China) display strongly variable cheilocystidia, while L. japonica samples (from Japan) present distinctive globose to subglobose spores and clavate cheilocystidia. This study of a species complex primarily described with an extremely wide ecological and geographical range sheds new light on the biodiversity and biogeography of ectomycorrhizal fungi.

Population genetics of ectomycorrhizal fungi: from current knowledge to emerging directions.

Ectomycorrhizal (EM) fungi are major microbial components of boreal, temperate and Mediterranean forests, as well as some tropical forest ecosystems. Nearly two decades of studies have clarified many aspects of their population biology, based on several model species from diverse lineages of fungi where the EM symbiosis evolved, i.e. among Hymenomycetes and, to a lesser extent, among Ascomycetes. In this review, we show how tools for individual recognition have changed, shifting from the use of somatic incompatibility reactions to dominant and non-specific markers (such as random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP)) and, more recently, to co-dominant and specific markers (such as microsatellites and single nucleotide polymorphisms (SNPs)). At the same time, the theoretical focus has also changed. In earlier studies, a major aim was the description of genet size and popul/ation strategy. For example, we show how some studies supported or challenged the simple, classical model of colonization of new forest stands by ruderal (R) species, propagating by spores and forming small genets, progressively replaced in older forests by more competitive (C) species, propagating by mycelial growth and forming larger genets. By contrast, more recent studies give insights into some genetic traits, such as partners' assortment (allo- versus autogamy), genetic structure of populations and gene flow that turn out to depend both on distance and on whether spores are animal- or wind-dispersed. We discuss the rising awareness that (i) many morphospecies contain cryptic biological species (often sympatric) and (ii) trans- and inter-continental species may often contain several biological species isolated by distance. Finally, we show the emergence of biogeographic approaches and call for some aspects to be developed, such as fine-scale and long-term population monitoring, analyses of subterranean populations of extra-radical mycelia, or more model species from the tropics, as well as from the Ascomycetes (whose genetic idiosyncrasies are discussed). With the rise of the '-omics' sciences, analysis of population structure for non-neutral genes is expected to develop, and forest management and conservation biology will probably profit from published and expected work.

Fungal associates of Pyrola rotundifolia, a mixotrophic Ericaceae, from two Estonian boreal forests.

Pyrola rotundifolia (Ericaceae, Pyroleae tribe) is an understorey subshrub that was recently demonstrated to receive considerable amount of carbon from its fungal mycorrhizal associates. So far, little is known of the identity of these fungi and the mycorrhizal anatomy in the Pyroleae. Using 140 mycorrhizal root fragments collected from two Estonian boreal forests already studied in the context of mixotrophic Ericaceae in sequence analysis of the ribosomal DNA internal transcribed spacer region, we recovered 71 sequences that corresponded to 45 putative species in 19 fungal genera. The identified fungi were mainly ectomycorrhizal basidiomycetes, including Tomentella, Cortinarius, Russula, Hebeloma, as well as some ectomycorrhizal and/or endophytic ascomycetes. The P. rotundifolia fungal communities of the two forests did not differ significantly in terms of species richness, diversity and nutritional mode. The relatively high diversity retrieved suggests that P. rotundifolia does not have a strict preference for any fungal taxa. Anatomical analyses showed typical arbutoid mycorrhizae, with variable mantle structures, uniseriate Hartig nets and intracellular hyphal coils in the large epidermal cells. Whenever compared, fungal ultrastructure was congruent with the molecular identification. Similarly to other mixotrophic and autotrophic pyroloids in the same forests, P. rotundifolia shares its mycorrhizal fungal associates with surrounding trees that are likely a carbon source for pyroloids.

Genome structure impacts molecular evolution at the AvrLm1 avirulence locus of the plant pathogen Leptosphaeria maculans.

Leptosphaeria maculans, a dothideomycete fungus causing stem canker on oilseed rape, develops gene-for-gene interactions with its host plants. It has the ability to rapidly adapt to selection pressure exerted by cultivars harbouring novel resistance genes as exemplified recently by the 3-year evolution towards virulence at the AvrLm1 locus in French populations. The AvrLm1 avirulence gene was recently cloned and shown to be a solo gene within a 269 kb non-coding, heterochromatin-like region. Here we describe the sequencing of the AvrLm1 genomic region in one avirulent and two virulent isolates to investigate the molecular basis of evolution towards virulence at the AvrLm1 locus. For these virulent isolates, the gain of virulence was linked to a 260 kb deletion of a chromosomal segment spanning AvrLm1 and deletion breakpoints were identical or similar. Among the 460 isolates analysed from France, Australia and Mexico, a similar large deletion was apparent in > 90% of the virulent isolates. Deletion breakpoints were also strongly conserved in most of the virulent isolates, which led to the hypothesis that a unique deletion event leading to the avrLm1 virulence has diffused in pathogen populations. These data finally suggest that retrotransposons are key drivers in genome evolution and adaptation to novel selection pressure in L. maculans.