Aligments used in phylogenetic analyzes

The diversity of microscopic hyphomycetes is still an ongoing effort and many species remain to be described. While studying lichen organismal composition in western Canada, metatranscriptome data revealed the presence of an unknown species of <i>Retiarius</i> (<i>Orbiliaceae</i>, Ascomycota), a pollen-parasitic fungus with no previous records in the region. We developed genus-specific primers to amplify <i>Retiarius</i> DNA in lichen and adjacent substrate extractions and detected multiple lineages of <i>Retiarius</i> across a wide geographic range. We proceeded to screen accumulations of pollen on the undersurface of lichen thalli to isolate any pollen-associated fungi. Using dilution series and direct-PCR for identification, we isolated two strains of <i>Retiarius</i> with morphology unlike any described member of the genus. Inclusion of DNA from these strains in a multilocus phylogeny using ITS1-5.8S-ITS2 (ITS), partial nuc 28S rDNA (28S) and nuc small subunit (18S) confirmed their evolutionarily distinct position in the genus. We describe these two species here as <i>Retiarius canadensis</i> and <i>R</i>. <i>crescentus</i>, respectively. The former possesses trinacrium-shaped conidia, similar to <i>R</i>. <i>bovicornutus</i> and <i>R</i>. <i>revayae</i>, but morphometrically different; and the latter is distinguished by its canoe-shaped conidia, a morphological character heretofore unknown from <i>Retiarius</i>.