Total genomic DNA was extracted from a clipped toe using the Ezup Column Animal Genomic DNA purification Kit (Sangon Biotech Company, Shanghai, China) in compliance with the manufacturer’s standard protocols for tissue. We amplified 10 overlapping genes via normal PCR and long-accurate PCR methods slightly modified from Zhang et al. [45 (link)]. Additionally, the ND5 fragments were successfully obtained using primers designed by Cai et al. [46 (link)]. After obtaining the ND5 gene fragment, four sets of primers were designed to sequence adjacent gene fragments, including the region from ND5(1) to ND5(2) genes, the region from Cytb to ND5(1) genes, and the region from ND5(2) to tRNAPro genes (Figure 1 ). An overview of all fragments is given in Table 1 and Table S2 . Unexpectedly, the amplicons generated from Cytb to ND5(1), ND5(1) to ND5(2), and ND5(2) to tRNAPro fragments were found to have lengths of approximately 3400 bp, 3300 bp, and 4100 bp, respectively. These PCR products underwent bidirectional sequencing utilizing the primer-walking method from Sangon Biotech Company (Shanghai, China).
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