Complete Mitochondrial Genome and Phylogenetic Analysis of the Chungtien Schizothoracin (Ptychobarbus chungtienensis)

Background: The Chungtien schizothoracin (Ptychobarbus chungtienensis) is a threatened freshwater fish endemic to the Qinghai–Tibet Plateau and adjacent high-altitude regions of northwestern Yunnan, China. Although a complete mitochondrial genome of P. chungtienensis has been previously reported, direct comparison with a mitogenome generated using high-accuracy long-read sequencing can provide additional information on mitochondrial genome structure and sequence variation. This study aimed to assemble and annotate a complete mitogenome of P. chungtienensis using PacBio HiFi sequencing and to compare its mitogenomic characteristics with previously published Ptychobarbus mitogenomes. Methods: High-molecular-weight genomic DNA from a single specimen was sequenced using PacBio HiFi long-read technology. The mitochondrial genome was assembled using MitoHiFi, annotated using MitoFinder followed by manual curation, and compared with previously published Ptychobarbus mitogenomes. Phylogenetic relationships were evaluated using maximum-likelihood analysis with expanded taxon sampling, and selection pressure on the 13 mitochondrial protein-coding genes was assessed using dN/dS-based branch and branch-site models. Results: The assembled mitogenome is 16,583 bp in length and contains the typical 37 mitochondrial genes, including 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes, together with a control region and the origin of light-strand replication (OL). The overall A + T content was 54.97%. Direct comparison with the previously reported 16,970 bp mitogenome showed that the 387 bp length difference was concentrated in non-coding regions, particularly the control region and the tRNA-Thr–tRNA-Pro intergenic region. Phylogenetic analysis based on 22 complete mitogenomes placed the newly assembled P. chungtienensis sequence in a strongly supported mitochondrial clade with Schizothorax macropogon (bootstrap = 100%), whereas the previously reported P. chungtienensis sequence clustered with P. kaznakovi (bootstrap = 100%), indicating that the two P. chungtienensis records represent distinct mitochondrial lineages. The dN/dS values of all 13 mitochondrial protein-coding genes were below 1, and neither branch nor branch–site analyses detected significant evidence of lineage-specific positive selection. Conclusions: This long-read-based mitogenome provides a high-quality genomic resource for P. chungtienensis and reveals substantial mitochondrial sequence and lineage variation among available records. These results provide a basis for comparative mitogenomic and conservation genetic studies while also indicating that species-level phylogenetic relationships and high-altitude adaptation should not be inferred from mitochondrial data alone.