Nearly 90% of fungal diversity, one of the most speciose branches in the tree of life, remains undescribed. Lichenized fungi as symbiotic associations are still a challenge for species delimitation, and current species diversity is vastly underestimated. The ongoing democratization of Next-Generation Sequencing is turning the tables. Particularly, reference-based RADseq allows for metagenomic filtering of the symbiont sequence and yields robust phylogenomic trees of closely related species. We implemented reference-based RADseq to disentangle the evolution of neuropogonoid lichens, which inhabit harsh environments and belong to Usnea (Parmeliaceae, Ascomycota), one of the most taxonomically intriguing genera within lichenized fungi. Full taxon coverage of neuropogonoid lichens was sampled for the first time, coupled with phenotype characterizations. More than 20,000 loci of 126 specimens were analyzed through concatenated and coalescent-based methods, including time calibrations. Our analysis addressed the major taxonomic discussions over recent decades. Subsequently, two species are newly described, namely U. aymondiana and U. fibriloides, and three species names are resurrected. The late Miocene and Pliocene-Pleistocene boundary is inferred as the timeframe for neuropogonoid lichen diversification. Ultimately, this study helped fill the gap of fungal diversity by setting a solid backbone phylogeny which raises new questions about which factors may trigger complex evolutionary scenarios.
Keywords: lichen-forming fungi; next-generation sequencing; phylogenomics; species complex; species delimitation; systematics.