DNA isolation, PCR amplification, DNA sequencing

DNA was extracted from dried specimens using either the E.Z.N.A. Forensic DNA Kit (Omega Bio-tek) or the DNeasy Plant Pro Kit (Qiagen). Partial nuclear large subunit rDNA (nrLSU) genes were PCR amplified with primers LROR and LR5 under the following cycling conditions: (1) 95 °C for 15 min, (2) then 30 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min, (3) then 72 °C for 5 min. Partial translation elongation factor 1 alpha (tef1) and second largest subunit of RNA polymerase II (rpb2) genes (both nuclear protein-coding genes) were PCR amplified based on primers and protocols outlined in Davoodian et al. (2021) and Khmelnitsky et al. (2019), respectively. In some cases, it was necessary to dilute the gDNA extractions with 10 parts distilled, deionized water prior to PCR to achieve successful amplification. Sanger sequencing was conducted in forward and reverse directions at the Australian Genome Research Facility in Melbourne, Australia and Macrogen in the USA (formerly https://www.macrogenusa.com).