Phylogenetic, structural and functional diversification of nitrate transporters in three ecologically diverse clades of mushroom-forming fungi

Nrt2 encodes a transporter (NRT2) that facilitates nitrate assimilation in most lineages of Dikarya. We used degenerate PCR to examine nrt2 sequence diversity and phylogeny in three clades of mushroom-forming basidiomycetes (Hebeloma, Hydnangiaceae and Psathyrellaceae) and tested for positive selection on nrt2 in each lineage. We investigated the relative expression of nrt2 paralogs in Hebeloma helodes and compared the patterns of expression between three Hebeloma and one Gymnopilus species, with quantitative PCR. Four lines of evidence of functional diversification in the nitrate assimilation system in mushroom-forming fungi emerged: (1) paralogs of nrt2 were recovered in Hebeloma and Hydnangiaceae, but distribution was not complete; (2) two paralogs in H. helodes appear to be differently expressed; (3) structural differences in NRT2 are apparent between paralogs and lineages; and (4) nrt2 is under significant positive selection in each lineage, and at least 6 sites are identified as under positive selection in the ectomycorrhizal family, Hydnangiaceae.