Mitogenome Assembly and Annotation

The total DNA was isolated from a single specimen and used as the PCR template. Primers were designed to produce amplicons overlapping by approximately 100 bp. PCR products were sequenced bidirectionally by the Bio-Transduction Lab in Wuhan using the Sanger method and the same set of primers that were used for the amplification (Additional file 1: Table S1). Sequenced data were quality-inspected using sequencing chromatograms; 20–30 bases at both ends were deleted from each amplicon. The mitogenome was assembled manually using DNAstar v7.1 [55 ], ensuring that overlaps between fragments were identical. PCGs and rRNAs were approximately located using DNAstar and MITOS [56 (link)] and then manually fine-tuned according to the orthologous sequences using BLAST and BLASTx [57 (link)] and PhyloSuite [58 , 59 (link)]. tRNAs were annotated using MITOS [56 (link)] and ARWEN [60 (link)] tools, but some tRNAs had to be identified manually via comparisons with related species. The secondary structure of tRNAs was further studied using ARWEN. Tools such as tRNAscan, which searches for a complete cloverleaf structure, are not suitable for the identification of tRNAs that exhibit nonstandard secondary structures. ARWEN was designed especially for this purpose: it first identifies only the most conserved domain, the anticodon stem, and subsequently searches for the presence of D-stem and T-stem structures, and the search for an acceptor stem then provides specificity. Due to this high sensitivity, ARWEN is also prone to producing a substantial false discovery rate [47 (link)]. The assembled circular mitogenome was visualised using OGDRAW [61 (link)].