Monique Gardes

A sequence database for the identification of ectomycorrhizal basidiomycetes by phylogenetic analysis

We have assembled a sequence database for 80 genera of Basidiomycota from the Hymenomycete lineage (sensu Swann & Taylor 1993) for a small region of the mitochondrial large subunit rRNA gene. Our taxonomic sample is highly biased toward known ectomycorrhizal (EM) taxa, but also includes some related saprobic species. This gene fragment can be amplified directly from mycorrhizae, sequenced, and used to determine the family or subfamily of many unknown mycorrhizal basidiomycetes. The method is robust to minor sequencing errors, minor misalignments, and method of phylogenetic analysis. Evolutionary inferences are limited by the small size and conservative nature of the gene fragment. Nevertheless two interesting patterns emerge: (i) the switch between ectomycorrhizae and saprobic lifestyles appears to have happened convergently several and perhaps many times; and (ii) at least five independent lineages of ectomycorrhizal fungi are characterized by very short branch lengths. We estimate that two of these groups radiated in the mid-Tertiary, and we speculate that these radiations may have been caused by the expanding geographical range of their host trees during this period. The aligned database, which will continue to be updated, can be obtained from the following site on the WorldWide Web: http://mendel.berkeley.edu/boletus.html.

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.

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

BackgroundMycorrhizal 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.ResultsSporocarps 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.ConclusionsPatterns 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. scalpturatums 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.

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.

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.

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.

Spatial Heterogeneity in Mycorrhizal Populations and Communities: Scales and Mechanisms

The importance of a spatial context in understanding the ecology and evolution of organisms has become increasingly clear. Although there is a growing awareness of the importance of mycorrhizal fungi in many communities and ecosystems, much of this understanding is based on a spatially homogenized view of these soil fungi. This homogenized approach may limit our understanding of how these organisms interact with plants and other biota in the field. As an attempt to advance a spatial framework for understanding mycorrhizal ecology, we review our current understanding of the spatial structure of communities and populations of ectomycorrhizal and arbuscular mycorrhizal fungi at the scale of landscapes, communities, and individual host root systems. A variety of potential mechanisms such as disturbance, abiotic and biotic dispersal of mycorrhizal propagules, and biotic interactions may be responsible for generating and maintaining this spatial variation of populations and communities, but the links between observed spatial patterns and mechanisms have yet to be formed. Future work assessing the potential functional significance of spatial variation of mycorrhizal fungi for plant communities and ecosystem function, as well as measuring spatial variation in mycorrhizal function, will continue to advance our understanding of the spatial template for mycorrhizal–plant interactions in the field.