Morpho-Molecular Identification of Common Freshwater Loaches Collected From Different Ecosystems of Bangladesh

The biodiversity of hill stream fishes, especially loaches, in Bangladesh is declining, despite limited exploration. Conservation strategies can flourish through the effective identification of species. Ambiguous loaches were collected to resolve taxonomic confusion. Morphology was analyzed through morphometric and meristic traits, with mitochondrial COI and nuclear RAG1 genes as molecular markers. Sixteen morphotypes are found in 60 specimens, and the molecular study confirmed the existence of seven species. Furthermore, the species delimitation method ASAP suggests seven species according to the best partition. A low level of intraspecific diversity is found in the RAG1 gene analysis compared with the COI gene. The lowest interspecific genetic divergence (3.7%) was observed between Botia lohachata and Botia rostrata , whereas Canthophrys gongota showed the highest interspecific genetic divergence (24.45%) with B. lohachata. A potential barcoding gap of 2.52% in the COI gene is typically the threshold for distinguishing intra-species from inter-species comparisons. In the maximum likelihood phylogenetic tree of both COI and RAG1 gene sequences, the species are divided into distinct clades with high bootstrap support, and specimens within the same species do not converge. Morphology, along with the molecular data of this study, will strengthen conservation efforts for loaches.