The use of molecular markers for identifying species diversity and functional variation of ectomycorrhizal fungi

Ectomycorrhiza (EM) is a symbiosis between soil living fungi and the roots of woody plants. EM fungal communities are important in forest nutrient cycling, they are species rich and their structures vary between habitats and over time. An EM community in a phosphorus-poor forest was exposed to apatite and EM mycelia actively colonizing ingrowth bags were analyzed. Internal transcribed spacer (ITS) amplification; cloning, sequencing and identification against databases were used to identify EM species. The fungal colonization of bags was mainly EM. The biomass tended to be higher in apatite bags but the difference was not statistically significant. In average there were 13 species per bag and 80% of the EM sequences belonged to Tylospora fibrillosa, Amphinema sp., Tomentellopsis submollis and Xerocomus badius. Phylogenetic species (PS) within the EM Paxillus involutus species group were recognized using multilocus sequencing (MLS). Concordance of five gene genealogies showed that this group comprised at least four PS. The ITS phylogenetic tree corresponded well to the MLS trees, allowing linking of ITS sequences to species. Three of the PS corresponded to described morphospecies; PS I to P. obscurosporus, PS II to P. involutus s. str. and PS III to P. validus. Differences in gene expression levels during mycorrhizal contact between birch and three P. involutus s.l. strains, ATCC 200175, MAJ and NAU were compared with microarray technology. Different compatibility with birch resulted in quantitative differences in gene expression as well as divergence in nucleotide sequences of symbiosis-regulated genes. The transcriptomes were more similar for compatible isolates than between incompatible isolates, a similarity not reflected in the genomic distances seen by comparative genomic hybridization (CGH). The differences are probably coupled to changes in promoter elements and transcription factors. P. involutus s.l. isolates were examined in microcosms for their ability to form mycorrhiza with birch and spruce. Compatible and incompatible isolates were found within PS I, II and III. The gene contents of three incompatible isolates were compared with compatible isolates within the same PS using microarray-based CGH. The proportions of genes that varied in copy numbers between the compatible/incompatible isolates within PS I, PS II and PS III were 3.5, 6.3 and 1.3%, respectively. Only three genes showed similar patterns of variation between all three pairs. Our data suggests that host incompatibility has arisen repeatedly in several lineages within the P. involutus species group. The genomic mechanisms that could account for these phenotypic differences appear to differ between lineages