Molecular identification, species delimitation, and phylogenetic analysis of Bornean leaffish (Pristolepis spp.) using cytochrome oxidase I (COI) gene

Abstract

The genus Pristolepis comprises a group of freshwater leaffishes widely distributed across South and Southeast Asia, yet their taxonomy and evolutionary relationships remain insufficiently resolved. In this study, we employed mitochondrial DNA barcoding of the cytochrome c oxidase subunit I (COI) gene to investigate molecular identification, phylogenetic relationships, and species boundaries of Pristolepis with particular emphasis on Bornean populations. A total of newly generated and publicly available sequences was analyzed to reconstruct phylogenetic relationships, evaluate genetic divergence using the Kimura 2-Parameter (K2P) distance model, assess barcode gap patterns, and infer species limits using multiple delimitation approaches. Phylogenetic reconstruction revealed well-supported lineages corresponding to recognized species, including Pristolepis grootii , Pristolepis fas-ciata , Pristolepis rubripinnis , and Pristolepis marginata . The Bornean specimens of P. grootii formed a distinct and cohesive clade, clearly separated from other congeners. In contrast, P. fasciata exhibited substantial genetic structuring across its distribution, with multiple geographically associated lineages detected in the phylogeny and consistently recovered by species delimitation analyses. Genetic distance analyses further revealed relatively high intraspecific divergence within P. fasciata compared to other species, while barcode gap analysis indicated overlapping intra-and interspecific divergence values. These results suggest that