A Preliminary Study of the Mitochondrial Genome of Leptobotia rotundilobus: Structural Characteristics and Insights into the Phylogeny of Leptobotinae

Leptobotia rotundilobus is a newly described species in the subfamily Leptobotinae (Teleostei: Cypriniformes), which is endemic to China. Research on this recently discovered species is preliminary, characterized by limited baseline data and the absence of a fully sequenced mitochondrial genome. To elucidate the structural features of the mitochondrial genome of L. rotundilobus, we performed whole-genome sequencing using next-generation sequencing technology and analyzed its genomic composition, gene content, and structural variation through genome assembly and bioinformatics. The complete circular sequence, spanning 16,593 bp, comprises 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and a typical control region (D-loop), all arranged in the canonical order. The overall base composition of the genome was determined to be 30.8% adenine (A), 24.4% thymine (T), 28.6% cytosine (C), and 16.2% guanine (G). This A+T bias (55.2%) is consistent with the mitochondrial genomes of other Leptobotia, which may affect secondary structure. The ratio of non-synonymous (Ka) to synonymous substitutions (Ks) of 13 PCGs of 16 Leptobotinae species is far less than 1 (0.012–0.063), indicating strong negative or purifying selection on the mitogenome in these species. Moreover, to investigate the phylogenetic relationships within the subfamily Leptobotinae, particularly within the genus Leptobotia, we constructed multiple phylogenetic trees of the mitogenome and concatenated 13 PCGs of 39 sequences with Sinibotia superciliaris as an outgroup. The phylogentic trees using the maximum likelihood (ML) and Bayesian inference (BI) methods consistently indicate that: (1) after correcting the species identification error, L. rotundilobus is closely related to L. micra; and (2) the species of Leptobotia and Parabotia each form a monophyletic group. This study provides new insights into the taxonomy and phylogenetic relationships of Leptobotinae, with a particular focus on the genus Leptobotia, thereby contributing to the clarification of the systematics, origin, and evolution of Botiidae.