II. Laboratory methods
Genomic DNA was extracted from tissue samples using the DNeasy Blood Tissue Kit (Qiagen, Venlo, Netherlands) following the manufacturer’s protocols. Seven molecular markers were amplified and sequenced in this study: two mtDNA (Cytochrome b [cytb ], ND2 ) and five nuDNA (Rhodopsin intron 3 [Rho-3 ], Sex determining region Y box containing gene 9 intron 2 [Sox9-2 ], Cyclin B2 intron 3 [CCNB2-3 ], Diazepam binding inhibitor intron 2 [DBI-2 ], Ubiquitin carboxyl-terminal hydrolase intron 2 [UCH-2 ]). Polymerase chain reaction (PCR) was performed in 30 μL reactions with 3 μL 10X Buffer (1X/μL), 2.4 μL dNTP (0.2 pm/μL), 1.5 μL each primer (0.5 pm/μL), 0.2 μL Taq polymerase (1U/μL), 20.4 μL distilled water, and 1 μL template DNA (10 ng/μL). The 10X Buffer, dNTP, and Taq polymerase were from the i-star TaqTM DNA polymerase kit (iNtRON Biotechnology, Seongnam, Gyeonggi, Korea). Detailed PCR conditions for each marker are in Table S3 , and primer information is in Table S4 .
The size of PCR products was confirmed with electrophoresis on a 1% agarose gel. When multiple bands were found on the gel, PCR products were run again on a 2% agarose gel and the correct-sized band was excised. The PCR products and gel fragments were purified using DNA purification columns (Zymo Research, Irvine, CA, USA). Sequencing was performed in the both directions using the PCR primers on an Applied Biosystems 3730XL machine and Big Dye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) at the National Instrumentation Center for Environmental Management of Seoul National University (Seoul, Korea). Due to the large size of cytb(>1000 bp), an additional primer (Bufo3-Inner1) was used for sequencing (Fong et al., 2020; Table S4 ).