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Natural history collections, including name-bearing type specimens, are an important source of genetic information. These data can be critical for appropriate taxonomic revisions in cases where the phylogenetic position of name-bearing type specimens needs to be identified, including morphologically cryptic lichen-forming fungal species. Here, we use high-throughput metagenomic shotgun sequencing to generate genome-scale data from decades-old (i.e., more than 30 years old) isotype specimens representing three vagrant taxa in the lichen-forming fungal genus Rhizoplaca, including one species and two subspecies. We also use data from high-throughput metagenomic shotgun sequencing to infer the phylogenetic position of an enigmatic collection, originally identified as R. haydenii, that failed to yield genetic data via Sanger sequencing. We were able to construct a 1.64 Mb alignment from over 1200 single-copy nuclear gene regions for the Rhizoplaca specimens. Phylogenomic reconstructions recovered an isotype representing Rhizoplaca haydenii subsp. arbuscula within a clade comprising other specimens identified as Rhizoplaca haydenii subsp. arbuscula, while an isotype of R. idahoensis was recovered within a clade with substantial phylogenetic substructure comprising Rhizoplaca haydenii subsp. haydenii and other specimens. Based on these data and morphological differences, Rhizoplaca haydenii subsp. arbuscula is elevated to specific rank as Rhizoplaca arbuscula. For the enigmatic collection, we were able to assemble the nearly complete nrDNA cistron and over 50 Mb of the mitochondrial genome. Using these data, we identified this specimen as a morphologically deviant form representing Xanthoparmelia aff. subcumberlandia. This study highlights the power of high-throughput metagenomic shotgun sequencing in generating larger and more comprehensive genetic data from taxonomically important herbarium specimens.

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