Characterization of Three Complete Mitochondrial Genomes of Hemiculter and Comparative Mitogenomic Analysis of Cultrinae Fishes

The genus Hemiculter includes closely related freshwater fishes widely distributed across East Asia, and several species are difficult to distinguish using external morphology alone. Mitochondrial markers have been widely used for species identification and phylogenetic inference in fishes, but single-gene approaches may be insufficient when nominal species show recent divergence, mitochondrial introgression, or taxonomic inconsistency. In this study, we newly assembled and characterized three complete mitogenomes of Hemiculter: H. leucisculus from Korea, H. leucisculus from Mongolia, and H. eigenmanni from Korea. The three mitogenomes contained the typical 37 mitochondrial genes and showed conserved genome organization, base composition, codon usage, and tRNA structures. Comparative analysis of concatenated 13 protein-coding genes revealed uneven sequence variability, with relatively high nucleotide diversity in ND2, ND1, ND6, and ND5. Pairwise K2P distances and phylogenetic analyses showed that the Korean and Mongolian H. leucisculus mitogenomes formed a distinct clade, evidencing that sequences assigned to H. leucisculus did not form a single consensus mitochondrial lineage. Barcode-gap overlap appeared to reflect discordance between current species assignments and mitogenomic relationships rather than the absence of informative barcode genes. These results indicate that Hemiculter mitogenomes are structurally conserved but informative for detecting lineage-level divergence and taxonomic inconsistency. Our findings suggest that the H. leucisculus–H. eigenmanni complex requires further integrative reassessment using broader sampling, voucher-based morphology, and nuclear genomic evidence.