Genomic DNA was extracted using the phenol-chloroform method as described in Sambrook et al. (1989 ). Partial fragments of mitochondrial NADH dehydrogenase subunit 2 (ND2) and cytochrome b (cyt b) were sequenced for all specimens. In addition, 39 ND2 sequences and 35 cyt b sequences were downloaded from GenBank. Data from the closely related, monotypic Laotriton were also downloaded. For the nuclear genes, fragments of the genes encoding the brain-derived neurotrophic factor (BDNF) and proopiomelanocortin (POMC) were sequenced from a subset of samples consisting of 83 individuals (Supplementary Table S1). These samples were selected to represent all major mitochondrial matrilines. PCR conditions of these gene fragments were performed as in Vieites et al. (2007 (link)) and Wu et al. (2010b (link)).
Purifications were performed using the Gel Extraction Mini Kit (Watson BioTechnologies, China) and sequencing was performed in both directions using the BigDye Terminator Cycle Sequencing Kit (v.2.0; Applied Biosystems, USA) by an ABI PRISM 3730 automated DNA sequencer (Applied Biosystems, USA). Nucleotide sequences were aligned using Clustal X v.1.81 (Thompson et al., 1997 (link)) with default parameters and manually checked in mega v.6.0.6 (Kumar et al., 2018 ).
Purifications were performed using the Gel Extraction Mini Kit (Watson BioTechnologies, China) and sequencing was performed in both directions using the BigDye Terminator Cycle Sequencing Kit (v.2.0; Applied Biosystems, USA) by an ABI PRISM 3730 automated DNA sequencer (Applied Biosystems, USA). Nucleotide sequences were aligned using Clustal X v.1.81 (Thompson et al., 1997 (link)) with default parameters and manually checked in mega v.6.0.6 (Kumar et al., 2018 ).