Death caps (Amanita phalloides) frequently establish from sexual spores, but individuals can grow large and live for more than a decade in invaded forests.

Global change is reshaping Earth's biodiversity, but the changing distributions of nonpathogenic fungi remain largely undocumented, as do mechanisms enabling invasions. The ectomycorrhizal Amanita phalloides is native to Europe and invasive in North America. Using population genetics and genomics, we sought to describe the life history traits of this successfully invading symbiotic fungus. To test whether death caps spread underground using hyphae, or aboveground using sexual spores, we mapped and genotyped mushrooms from European and US sites. Larger genetic individuals (genets) would suggest spread mediated by vegetative growth, while many small genets would suggest dispersal mediated by spores. To test whether genets are ephemeral or persistent, we also sampled from populations over time. At nearly every site and across all time points, mushrooms resolve into small genets. Individuals frequently establish from sexual spores. But at one Californian site, a single individual measuring nearly 10 m across dominated. At two Californian sites, the same genetic individuals were discovered in 2004, 2014, and 2015, suggesting single individuals (both large and small) can reproduce repeatedly over relatively long timescales. A flexible life history strategy combining both mycelial growth and spore dispersal appears to underpin the invasion of this deadly perennial ectomycorrhizal fungus.

Biogeographic history of a large clade of ectomycorrhizal fungi, the Russulaceae, in the Neotropics and adjacent regions.

The biogeography of neotropical fungi remains poorly understood. Here, we reconstruct the origins and diversification of neotropical lineages in one of the largest clades of ectomycorrhizal fungi in the globally widespread family Russulaceae. We inferred a supertree of 3285 operational taxonomic units, representing worldwide internal transcribed spacer sequences. We reconstructed biogeographic history and diversification and identified lineages in the Neotropics and adjacent Patagonia. The ectomycorrhizal Russulaceae have a tropical African origin. The oldest lineages in tropical South America, most with African sister groups, date to the mid-Eocene, possibly coinciding with a boreotropical migration corridor. There were several transatlantic dispersal events from Africa more recently. Andean and Central American lineages mostly have north-temperate origins and are associated with North Andean uplift and the general north-south biotic interchange across the Panama isthmus, respectively. Patagonian lineages have Australasian affinities. Diversification rates in tropical South America and other tropical areas are lower than in temperate areas. Neotropical Russulaceae have multiple biogeographic origins since the mid-Eocene involving dispersal and co-migration. Discontinuous distributions of host plants may explain low diversification rates of tropical lowland ectomycorrhizal fungi. Deeply diverging neotropical fungal lineages need to be better documented.

Facilitation of Balsam Fir by Trembling Aspen in the Boreal Forest: Do Ectomycorrhizal Communities Matter?

Succession is generally well described above-ground in the boreal forest, and several studies have demonstrated the role of interspecific facilitation in tree species establishment. However, the role of mycorrhizal communities for tree establishment and interspecific facilitation, has been little explored. At the ecotone between the mixed boreal forest, dominated by balsam fir and hardwood species, and the boreal forest, dominated by black spruce, several stands of trembling aspen can be found, surrounded by black spruce forest. Regeneration of balsam fir seems to have increased in the recent decades within the boreal forest, and it seems better adapted to grow in trembling aspen stands than in black spruce stands, even when located in similar abiotic conditions. As black spruce stands are also covered by ericaceous shrubs, we investigated if differences in soil fungal communities and ericaceous shrubs abundance could explain the differences observed in balsam fir growth and nutrition. We conducted a study centered on individual saplings to link growth and foliar nutrient concentrations to local vegetation cover, mycorrhization rate, and mycorrhizal communities associated with balsam fir roots. We found that foliar nutrient concentrations and ramification indices (colonization by mycorrhiza per length of root) were greater in trembling aspen stands and were positively correlated to apical and lateral growth of balsam fir saplings. In black spruce stands, the presence of ericaceous shrubs near balsam fir saplings affected ectomycorrhizal communities associated with tree roots which in turn negatively correlated with N foliar concentrations. Our results reveal that fungal communities observed under aspen are drivers of balsam fir early growth and nutrition in boreal forest stands and may facilitate ecotone migration in a context of climate change.

Hidden fungal diversity from the Neotropics: Geastrum hirsutum, G. schweinitzii (Basidiomycota, Geastrales) and their allies.

Taxonomy of Geastrum species in the neotropics has been subject to divergent opinions among specialists. In our study, type collections were reassessed and compared with recent collections in order to delimit species in Geastrum, sect. Myceliostroma, subsect. Epigaea. A thorough review of morphologic features combined with barcode and phylogenetic analyses (ITS and LSU nrDNA) revealed six new species (G. neoamericanum, G. rubellum, G. brunneocapillatum, G. baculicrystallum, G. rubropusillum and G. courtecuissei). In additon, the presence of hairs on the exoperidium, a commonly used feature to diagnose Geastrum species, proved to be ineffective because it is a derived character within subsect. Epigaea.

Erratum: Author Correction: Evolutionary history of plant hosts and fungal symbionts predicts the strength of mycorrhizal mutualism.

[This corrects the article DOI: 10.1038/s42003-018-0120-9.].

Evolutionary history of plant hosts and fungal symbionts predicts the strength of mycorrhizal mutualism.

Most plants engage in symbioses with mycorrhizal fungi in soils and net consequences for plants vary widely from mutualism to parasitism. However, we lack a synthetic understanding of the evolutionary and ecological forces driving such variation for this or any other nutritional symbiosis. We used meta-analysis across 646 combinations of plants and fungi to show that evolutionary history explains substantially more variation in plant responses to mycorrhizal fungi than the ecological factors included in this study, such as nutrient fertilization and additional microbes. Evolutionary history also has a different influence on outcomes of ectomycorrhizal versus arbuscular mycorrhizal symbioses; the former are best explained by the multiple evolutionary origins of ectomycorrhizal lifestyle in plants, while the latter are best explained by recent diversification in plants; both are also explained by evolution of specificity between plants and fungi. These results provide the foundation for a synthetic framework to predict the outcomes of nutritional mutualisms.

Patterns of diversity, endemism and specialization in the root symbiont communities of alder species on the island of Corsica.

We investigated whether the diversity, endemicity and specificity of alder symbionts could be changed by isolation in a Mediterranean glacial refugium. We studied both ectomycorrhizal (EM) fungi and nitrogen-fixing actinobacteria associated with alders, and compared their communities in Corsica and on the European continent. Nodules and root tips were sampled on the three alder species present in Corsica and continental France and Italy. Phylogenies based on internal transcribed spacer (ITS) and a multilocus sequence analysis approach were used to characterize fungal and Frankia species, respectively. Patterns of diversity, endemism and specialization were compared between hosts and regions for each symbiont community. In Corsica, communities were not generally richer than on the mainland. The species richness per site depended mainly on host identity: Alnus glutinosa and Alnus cordata hosted richer Frankia and EM communities, respectively. Half of the Frankia species were endemic to Corsica against only 4% of EM species. Corsica is not a hotspot of diversity for all alder symbionts but sustains an increased frequency of poor-dispersers such as hypogeous fungi. Generalist EM fungi and host-dependent profusely sporulating (Sp+) Frankia were abundantly associated with Corsican A. cordata, a pattern related to a more thermophilic and xerophylic climate and to the co-occurrence with other host trees.

Alder and the Golden Fleece: high diversity of Frankia and ectomycorrhizal fungi revealed from Alnus glutinosa subsp. barbata roots close to a Tertiary and glacial refugium.

BACKGROUND: Recent climatic history has strongly impacted plant populations, but little is known about its effect on microbes. Alders, which host few and specific symbionts, have high genetic diversity in glacial refugia. Here, we tested the prediction that communities of root symbionts survived in refugia with their host populations. We expected to detect endemic symbionts and a higher species richness in refugia as compared to recolonized areas. METHODS: We sampled ectomycorrhizal (EM) root tips and the nitrogen-fixing actinomycete Frankia communities in eight sites colonized by Alnus glutinosa subsp. barbata close to the Caucasus in Georgia. Three sites were located in the Colchis, one major Eurasian climatic refugia for Arcto-Tertiary flora and alders, and five sites were located in the recolonized zone. Endemic symbionts and plant ITS variants were detected by comparing sequences to published data from Europe and another Tertiary refugium, the Hyrcanian forest. Species richness and community structure were compared between sites from refugia and recolonized areas for each symbionts. RESULTS: For both symbionts, most MOTUs present in Georgia had been found previously elsewhere in Europe. Three endemic Frankia strains were detected in the Colchis vs two in the recolonized zone, and the five endemic EM fungi were detected only in the recolonized zone. Frankia species richness was higher in the Colchis while the contrary was observed for EM fungi. Moreover, the genetic diversity of one alder specialist Alnicola xanthophylla was particularly high in the recolonized zone. The EM communities occurring in the Colchis and the Hyrcanian forests shared closely related endemic species. DISCUSSION: The Colchis did not have the highest alpha diversity and more endemic species, suggesting that our hypothesis based on alder biogeography may not apply to alder's symbionts. Our study in the Caucasus brings new clues to understand symbioses biogeography and their survival in Tertiary and ice-age refugia, and reveals that isolated host populations could be of interest for symbiont diversity conservation.

MycoDB, a global database of plant response to mycorrhizal fungi.

Plants form belowground associations with mycorrhizal fungi in one of the most common symbioses on Earth. However, few large-scale generalizations exist for the structure and function of mycorrhizal symbioses, as the nature of this relationship varies from mutualistic to parasitic and is largely context-dependent. We announce the public release of MycoDB, a database of 4,010 studies (from 438 unique publications) to aid in multi-factor meta-analyses elucidating the ecological and evolutionary context in which mycorrhizal fungi alter plant productivity. Over 10 years with nearly 80 collaborators, we compiled data on the response of plant biomass to mycorrhizal fungal inoculation, including meta-analysis metrics and 24 additional explanatory variables that describe the biotic and abiotic context of each study. We also include phylogenetic trees for all plants and fungi in the database. To our knowledge, MycoDB is the largest ecological meta-analysis database. We aim to share these data to highlight significant gaps in mycorrhizal research and encourage synthesis to explore the ecological and evolutionary generalities that govern mycorrhizal functioning in ecosystems.

Paxillus rubicundulus (Boletales, Paxillaceae) and two new alder-specific ectomycorrhizal species, Paxillus olivellus and Paxillus adelphus, from Europe and North Africa.

Paxillus rubicundulus P.D. Orton has been shown to be a complex of at least three ectomycorrhizal taxa strictly associated with alders (Alnus) in Europe, P. rubicundulus s. str., and two undescribed clades. To assess the taxonomic status of these three clades and their phylogenetic relationships, phylogenetic analyses of two independent gene regions (ITS and gpd), combined with macro- and micromorphological comparisons of genetically identified specimens, were carried out. A total of 85 sequences were successfully obtained from basidiomata and alder mycorrhizae collected in France and Algeria and combined with GenBank and UNITE sequences. The phylogenetic results and estimates of genetic diversity confirmed that the three clades are distinct species, often found in sympatry. As a result, P. rubicundulus s. str. was redefined based on the revision of type material, and Paxillus adelphus and Paxillus olivellus are introduced as new Linnaean names. The often used name Paxillus filamentosus is rejected since it could not be applied to any of the new species. The three species are distinguished micromorphologically by spore size and shape. They are widely distributed in Europe, North Africa and western Asia; P. rubicundulus is rare, and all species have a limited host range.

What determines Alnus-associated ectomycorrhizal community diversity and specificity? A comparison of host and habitat effects at a regional scale.

· Global-scale analyses of ectomycorrhizal (ECM) fungi communities emphasize host plant families as the main drivers of diversity. This study aims to test, on Alnus-ECM communities, which fungi are said to be 'host-specific', to what extent host species, habitat and distance explain their alpha and beta diversity variations, and their specificity. · In France, ECM communities associated with two subgenera and five species of Alnus, were sampled on 165 trees from 39 lowland to subalpine sites. In all, 1178 internal transcribed spacer (ITS) sequences of ECM fungi clustered in 86 molecular operational taxonomic units (MOTUs). · The species richness was low but still variable, and the evenness of communities was lower on organic soils and in Corsica. Similarity between communities was influenced both by host, soil parameters, altitude and longitude, but not by climate and distance. A large majority of 'specific' fungi were shared between host species within a subgenus, and showed habitat preferences within the subgenus distribution range. · Our study confirms that Alnus ECM communities are low in diversity, highly conserved at a regional scale, and partly shared between congeneric host species. A large part of alpha and beta diversity variations remained unexplained, and other processes may shape these communities.

Comparative phylogenies and host specialization in the alder ectomycorrhizal fungi Alnicola, Alpova and Lactarius (Basidiomycota) in Europe.

BACKGROUND: Mycorrhizal fungi form intimate associations with their host plants that constitute their carbon resource and habitat. Alnus spp. (Betulaceae) are known to host an exceptional species-poor and specialized ectomycorrhizal (ECM) fungal community compared to other tree species, but the host-specificity pattern and its significance in terms of fungal diversification and speciation remain poorly documented. The degree of parallel speciation, host switching, and patterns of biogeography were explored in the historical associations between alders and three ECM taxa of Basidiomycetes: Alnicola (Agaricales), Alpova (Boletales), and Lactarius (Russulales). The aim was to develop an evolutionary framework on host specificity and diversification of Basidiomycetes in this highly specialized plant-fungus symbiosis. RESULTS: Sporocarps of Alnicola (220), Lactarius (61) and Alpova (29) were collected from stands of the four European alder species (A. alnobetula including the endemic subsp. suaveolens in Corsica, A. cordata, A. glutinosa, A. incana) in Western Europe (mainly in France and Austria), from 1995 to 2009. Specimens were morphologically identified to the species level. From these, 402 sequences of four DNA regions (ITS, rpb2, gpd, and the V9 domain of the mit-SSU rDNA) were successfully obtained and analyzed in addition with 89 sequences available in GenBank and UNITE databases. Phylogenetic analyses were conducted on all sequence data sets (individual and combined) using maximum likelihood reconstruction and Bayesian inference. Fungal phylogenies are compared and discussed in relation to the host, with a focus on species boundaries by associating taxonomic, systematic and molecular information. CONCLUSIONS: Patterns of host specificity and phylogenies of Alnicola and Lactarius suggest coevolution as a basal factor of speciation in relation with the subgeneric diversification of Alnus, possibly due to the very selective pressure of the host. A second element of the historical associations between Alnus and its fungal symbionts is a host-dependent speciation (radiation without host change), here observed in Alnicola and Alpova in relation with Alnus subgen. Alnus. Finally host shifts from Alnus subgen. Alnus to A. alnobetula are found in most lineages of Alnicola (at least four times), Alpova (twice) and Lactarius (once), but they do not represent such a common event as could be expected by geographic proximity of trees from the two subgenera. However, active or very recent host extensions clearly occurred in Corsica, where some fungi usually associated with Alnus glutinosa on mainland Europe locally extend there to A. alnobetula subsp. suaveolens without significant genetic or morphological deviation.

Phylogenetic species delimitation in ectomycorrhizal fungi and implications for barcoding: the case of the Tricholoma scalpturatum complex (Basidiomycota).

Population studies have revealed that the fungal ectomycorrhizal morphospecies Tricholoma scalpturatum consists of at least two genetically distinct groups that occur sympatrically in several geographical areas. This discovery prompted us to examine species boundaries and relationships between members formerly assigned to T. scalpturatum and allied taxa using phylogenetic analyses. Sequence data were obtained from three nuclear DNA regions [internal transcribed spacer (ITS), gpd and tef], from 101 carpophores collected over a large geographical range in Western Europe, and some reference sequences from public databases. The ITS was also tested for its applicability as DNA barcode for species delimitation. Four highly supported phylogenetic clades were detected. The two previously detected genetic groups of T. scalpturatum were assigned to the phylospecies Tricholoma argyraceum and T. scalpturatum. The two remaining clades were referred to as Tricholoma cingulatum and Tricholoma inocybeoides. Unexpectedly, T. cingulatum showed an accelerated rate of evolution that we attributed to narrow host specialization. This study also reveals recombinant ITS sequences in T. inocybeoides, suggesting a hybrid origin. The ITS was a useful tool for the determination of species boundaries: the mean value of intraspecific genetic distances in the entire ITS region (including 5.8S rDNA) was <0.2%, whereas interspecific divergence estimates ranged from 1.78% to 4.22%. Apart from giving insights into the evolution of the T. scalpturatum complex, this study contributes to the establishment of a library of taxonomically verified voucher specimens, an a posteriori correlation between phenotype and genotype, and DNA barcoding of ectomycorrhizal fungi.

Facilitated establishment of Quercus ilex in shrub-dominated communities within a Mediterranean ecosystem: do mycorrhizal partners matter?

Positive plant-plant interaction is a widespread phenomenon, especially in harsh environments, which can contribute to secondary successions. Here, we investigated whether Arbutus unedo positively influences Quercus ilex establishment in shrub communities by abiotic and/or biotic interactions in a Mediterranean forest ecosystem, where we previously showed that A. unedo and Q. ilex share numerous species of mycorrhizal fungi. In a first field experiment, patterns of Q. ilex survivorship were documented. During the summer following germination, a majority of seedlings survived in A. unedo chaparral (AU), whereas most of them died in previous succession stages dominated by Erica arborea (EA). These results showed that survival of the Q. ilex seedling is succession stage dependent, probably due to the differential effects of the summer drought. In a second experiment, Q. ilex seedlings were used as bait plants to investigate the mycorrhizal inoculum in EA and AU. Morphotyping and molecular typing revealed 2.5 times higher colonization in AU than in EA, with more diverse fungi. Our results demonstrate that A. unedo facilitates mycorrhization of Q. ilex by hosting compatible ectomycorrhizal symbionts and positively influences seedling survival by buffering abiotic conditions. A comprehensive understanding of facilitation should thus include investigations of the soil biological patterns.

High sexual reproduction and limited contemporary dispersal in the ectomycorrhizal fungus Tricholoma scalpturatum: new insights from population genetics and spatial autocorrelation analysis.

Dispersal and establishment are fundamental processes influencing the response of species to environmental changes, and the long-term persistence of populations. A previous study on the symbiotic ectomycorrhizal fungus Tricholoma scalpturatum revealed strong genetic differentiations between populations in Western Europe, suggesting restricted dispersal for this wind-dispersed cosmopolitan fungus. Two distinct genetic groups (genetic groups 1 and 2), co-occurring in some locations, were also identified and could correspond to cryptic species. In the present work, we examine the reproductive strategy and dispersal biology of the two T. scalpturatum's genetic groups. Variable molecular markers (intersimple sequence repeats and intergenic spacer 2-restriction fragment length polymorphisms) and spatial autocorrelation analyses were used to examine fine-scale patterns (< 140 m) of genetic structure, in an effort to determine the physical scale at which genetic structure exists. A total of 473 fruit bodies were mapped and collected over 3 years from two plots located in the south of France, including 219 and 254 samples from group 1 and group 2, respectively. High genetic diversity and the presence of numerous small genets were observed in both groups. Autocorrelation analyses revealed significant positive spatial genetic structures of genets at close distances (up to few metres for both groups). Mantel tests confirmed this isolation-by-distance pattern. These results clearly demonstrate high sexual reproduction and spatial structuring of genets at very small geographical scales in this wind-dispersed ectomycorrhizal fungal species, a pattern consistent with restricted contemporary dispersal of spores.

Molecular evolution of mitochondrial ribosomal DNA in the fungal genus Tricholoma: barcoding implications.

The molecular evolution of the V6 and V9 domains of the mitochondrial SSU-rDNA was investigated to evaluate the use of these sequences for DNA barcodes in the Basidiomycota division. The PCR products from 27 isolates belonging to 11 Tricholoma species were sequenced. Both domains in the isolates belonging to the same species had identical sequences. All the species possess distinctive V9 sequences due to point mutations and insertion/deletion events. Secondary structures revealed that the insertion-deletion events occurred in regions not directly involved in the maintenance of the standard SSU-rRNA structure. The inserted sequences possess conserved motifs that enable their alignment among phylogenetically distant species. Hence, the V9 domain by displaying identical sequences within species, an adequate divergence level, easy amplification, and alignment represents an alternative molecular marker for the Basidiomycota division and opens the way for this sequence to be used as specific molecular markers of the fungal kingdom.

Population evidence of cryptic species and geographical structure in the cosmopolitan ectomycorrhizal fungus, Tricholoma scalpturatum.

Tricholoma scalpturatum is an ectomycorrhizal fungus that forms symbioses with roots of diverse trees and shrubs. It is commonly encountered in a wide range of habitats, across temperate ecosystems. A previous study has revealed a high genetic diversity at a local scale, and ruderal abilities. To examine genetic structure at a large geographical scale, a total of 164 basidiocarps were collected from 30 populations located in Western Europe, from Spain to Scandinavia. These samples were analyzed by three molecular methods with different levels of resolution: inter-simple sequence repeats (ISSRs), restriction fragment length polymorphisms (RFLPs) in the rDNA internal transcribed spacer (ITS), and ITS sequence analysis. Considerable genetic variation was found, and the morphospecies was separated into two genetic groups that were distinct from each other. The ISSR data and the relatively low percentage value (96%) of shared sequence polymorphisms in the ITS between isolates from the two groups, strongly suggest cryptic species and long-lasting separation. No geographical exclusion was detected for these two widely distributed taxa. However, high estimates of population differentiation were observed in each group, including between populations less than a few kilometers apart. This result provides evidence for limited gene flow and/or founding effects. It also indicates that T. scalpturatum does not constitute a random mating population, and the hypothesis of endemism cannot be excluded for this cosmopolitan wind-dispersed fungus.

Population dynamics of the ectomycorrhizal fungal species Tricholoma populinum and Tricholoma scalpturatum associated with black poplar under differing environmental conditions.

Fungi combine sexual reproduction and clonal propagation. The balance between these two reproductive modes affects establishment dynamics, and ultimately the evolutionary potential of populations. The pattern of colonization was studied in two species of ectomycorrhizal fungi: Tricholoma populinum and Tricholoma scalpturatum. The former is considered to be a host specialist whereas T. scalpturatum is a generalist taxon. Fruit bodies of both basidiomycete species were mapped and collected over several years from a black poplar (Populus nigra) stand, at two different sites. Multilocus genotypes (= genets) were identified based on the analysis of random amplified polymorphic DNA (RAPD) patterns, inter-simple sequence repeat (ISSR) patterns and restriction fragment length polymorphisms (RFLPs) in the ribosomal DNA intergenic spacer (rDNA IGS). The genetic analyses revealed differences in local population dynamics between the two species. Tricholoma scalpturatum tended to capture new space through sexual spores whereas T. populinum did this by clonal growth, suggesting trade-offs in allocation of resources at the genet level. Genet numbers and sizes strongly differ between the two study sites, perhaps as a result of abiotic disturbance on mycelial establishment and genet behaviour.

Phylogeny of the ectomycorrhizal mushroom genus Alnicola (Basidiomycota, Cortinariaceae) based on rDNA sequences with special emphasis on host specificity and morphological characters.

Alnicola (=Naucoria, pro parte) is a mushroom genus of strictly temperate, obligately ectomycorrhizal species, traditionally included in the family Cortinariaceae. Most Alnicola spp. are primarily host specific on Alnus, although a few are mycobionts of Salix or other hosts. The different species of Alnicola exhibit unique morphological (cystidia, pileipellis) and cytological (dikaryotic or monokaryotic hyphae) characters. This makes the genus Alnicola of particular interest for studying the evolution of host specificity and morphological characters in ectomycorrhizal basidiomycetes. We used a combination of classical morphological and phylogenetic methods (rDNA ITS and LSU sequences) to address the following questions: (i) Is Alnicola monophyletic? And (ii) Are characters like host specificity or microscopical structures synapomorphic for certain clades? The study included nearly all currently known European Alnicola sp. Our results demonstrated that, on one hand, the genus Alnicola is polyphyletic, with sistergroup relationships to Hebeloma, Anamika or the clades /Hymenogaster I and /Hymenogaster II. On the other hand, Alnicola splits into three well-supported clades corresponding to the sections Alnicola, Submelinoides, and Salicicolae. The strict host-specificity to Alnus is a derived character and has occurred at least twice. The following morphological characters are synapomorphic for defined clades: the spindle-shaped hymenial cystidia for sect. Alnicola, the hymeniform pileipellis for sect. Submelinoides, and monocaryotic/clampless hyphae for sect. Salicicolae and its sistergroup /Hymenogaster II. As a taxonomical consequence, polyphyly of Alnicola implies that the sects. Submelinoides and Salicicolae need to be segregated from Alnicola.

Genetic diversity in Tetrachaetum elegans, a mitosporic aquatic fungus.

Tetrachaetum elegans Ingold is a saprobic aquatic hyphomycete for which no sexual stage has yet been described. It occurs most commonly during the initial decay of tree leaves in temperate freshwater habitats and typically sporulates under water. Dispersal of the aquatic fungus takes place primarily in the water column and has a large passive component. Differences in substrate composition (e.g. quality of leaf litter) may also play a role in the distribution of different species or genotypes. The population genetic structure of T. elegans was studied using amplified fragment length polymorphism (AFLP) multilocus fingerprints. The populations were isolated from the leaf litter of three different tree genera, sampled in nine streams distributed throughout a mixed deciduous forest. Molecular markers were developed for 97 monosporic isolates using four selective primer pairs. A total of 247 fragments were scored, of which only 32 were polymorphic. Significant stream differentiation was detected for the isolates considered in this study. Analysis of molecular variance revealed that 20% of the genetic variation observed was the result of differences between streams. No correlation between genetic and geographical distances was found but a few multilocus genotypes were observed in different locations. Altogether these results suggest that environmental barriers play a role in the population structure of this aquatic fungus. No clear-cut effect of leaf litter composition on genetic variation could be demonstrated. Finally, tests of linkage disequilibrium between the 32 polymorphic AFLP loci as well as simulations did not provide a final answer regarding clonality in T. elegans. Indeed, it was possible to reject linkage equilibrium at different sampling levels and show that full linkage was unlikely.