DNA extraction, PCR amplification, and sequencing

A total of 40 collected specimens were sequenced to obtain a molecular barcode. Genomic DNA was extracted from ethanol-preserved samples using the PowerSoil DNA extraction kit (MoBio, Carlsbad, CA, USA), following the manufacturer’s protocol. The oligonucleotide primers SP635F and SP1411R were used to amplify a portion of the 28S ribosomal subunit (Thacker et al., 2013), yielding an approximately 650bp fragment. PCR reaction products were gel-purified and cleaned using the Wizard PCR Preps DNA Purification System (Promega, Madison, WI, USA). Forward and reverse sequencing reactions were performed at the University of Alabama at Birmingham (UAB) Center for AIDS Research (CFAR) DNA Sequencing Core Facility. Forward and reverse sequences were then compared to ensure the accuracy of sequencing reactions in CodonCode Aligner software (CodonCode, Dedham, MA, USA) and Geneious (version 6.1.8; Biomatters Limited, Auckland, New Zealand), yielding a final consensus sequence for each voucher specimen. A representative sequence for each species is archived in GenBank under accession numbers {"type":"entrez-nucleotide","attrs":{"text":"KU746954","term_id":"1005053578","term_text":"KU746954"}}KU746954, {"type":"entrez-nucleotide","attrs":{"text":"KU746955","term_id":"1005053579","term_text":"KU746955"}}KU746955, {"type":"entrez-nucleotide","attrs":{"text":"KU746956","term_id":"1005053580","term_text":"KU746956"}}KU746956, {"type":"entrez-nucleotide","attrs":{"text":"KU746957","term_id":"1005053581","term_text":"KU746957"}}KU746957, {"type":"entrez-nucleotide","attrs":{"text":"KU746958","term_id":"1005053582","term_text":"KU746958"}}KU746958, {"type":"entrez-nucleotide","attrs":{"text":"KU746959","term_id":"1005053583","term_text":"KU746959"}}KU746959, {"type":"entrez-nucleotide","attrs":{"text":"KU746960","term_id":"1005053584","term_text":"KU746960"}}KU746960 and {"type":"entrez-nucleotide","attrs":{"text":"KU746961","term_id":"1005053585","term_text":"KU746961"}}KU746961.