Evidence for extensive diversity in the pollen-parasitic genus Retiarius in western North America, including two new species associated with lichens

Understanding the diversity of microscopic hyphomycetes is an ongoing effort, and many species remain undescribed. While studying lichen organismal composition in western Canada, metagenomic data revealed the presence of an unknown species of Retiarius (Orbiliaceae, Ascomycota), a genus of pollen-parasitic fungus with no previous records in the region. We developed genus-specific primers to amplify Retiarius DNA in lichen and adjacent substrate extractions, successfully detecting multiple lineages of Retiarius across a wide geographic range within North America. We proceeded to screen accumulations of pollen on the undersurface of lichen thalli to isolate any pollen-associated fungi. Using dilution series and PCR for identification, we isolated two specifically distinct strains of Retiarius with morphology unlike any described member of the genus. Inclusion of DNA from these strains in a multilocus phylogeny using the internal transcribed spacer regions 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 Retiarius canadensis and R. crescentus. The former possesses trinacrium-shaped conidia, similar to those of R. bovicornutus and R. revayae but morphometrically different, and the latter is distinguished by its canoe-shaped conidia, a morphological character heretofore unknown from Retiarius.