Next-Generation Sequencing of Ancient and Recent Fungarium Specimens

Fungaria are an unmatched resource for providing genetic data from authoritative, taxonomically-correct fungal species, especially type specimens. These specimens serve to anchor species hypotheses by enabling the correct taxonomic placement of taxa in systematic studies. The DNA from ancient specimens older than 30 years is commonly fragmented, and sometimes highly contaminated by exogenous, non-target fungal DNA, making conventional PCR amplification and Sanger sequencing difficult or impossible. Here, we present the results of DNA extraction, PCR amplification of the ITS2 region, and Illumina MiSeq Nano sequencing of nine recent and 11 ancient specimens, including seven type specimens. The taxa sampled included a range of large and fleshy, to small and tough, or small, melanized specimens of Discina, Gyromitra, Propolis, Stictis, and Xerotrema, with a culture of Lasiosphaeria serving as a positive control. DNA was highly fragmented and in very low quantity for most samples, resulting in inconclusive or incorrect results for all but five samples. Taxonomically-correct sequences were generated from the holotype specimens of G. arctica, G. korshinskii, and G. leucoxantha, from the neotype of G. ussuriensis, and from the positive control. Taxonomic assignments were confirmed through morphology, top BLASTn hits, and maximum likelihood phylogenetic analyses. Though this study was not cost-effective due to the small number of samples submitted and few generating correct sequences, it did produce short DNA barcode fragments for four type specimens that are essential for their correct taxonomic placement in our ongoing systematic studies.