Molecular species delimitation in the genus Acrossocheilus based on mitochondrial sequences

Accurate species delimitation is essential for phylogeny, phylogeography, ecology, conservation, and biogeography. In China, the cyprinid genus Acrossocheilus is of economic and ecological importance, but species boundaries remain controversial because several nominal species exhibit overlapping diagnostic characters and uncertain phylogenetic affinities. Here, we analyzed 31 publicly available complete mitochondrial genomes representing 22 cyprinid taxa, including 16 nominal Acrossocheilus taxa, three Onychostoma taxa, and three Spinibarbus outgroups. We extracted and concatenated the 13 mitochondrial protein-coding genes (ATP6, ATP8, COX1, COX2, COX3, CYTB, ND1, ND2, ND3, ND4, ND4L, ND5, and ND6) and reconstructed phylogenetic relationships using Bayesian inference and maximum-likelihood methods. Species boundaries were evaluated using four complementary approaches: Generalized Mixed Yule Coalescent (GMYC), Bayesian Poisson Tree Processes (bPTP), Automatic Barcode Gap Discovery (ABGD), and Bayesian Phylogenetics and Phylogeography (BPP). The combined analyses supported 15 molecular operational taxonomic units (MOTUs), with bPTP and ABGD showing high congruence when ABGD prior intraspecific divergence was set between 0.001 and 0.0144. The results highlighted several taxonomically important patterns, including possible conspecificity among A. longipinnis , A. iridescens , and A. barbodon ; divergent placements of two A. paradoxus accessions; and unresolved generic boundaries between Acrossocheilus and Onychostoma . Because the present inference is based only on mitochondrial loci and uneven taxon sampling, these MOTUs should be interpreted as testable molecular hypotheses for future integrative taxonomy using nuclear markers, broader sampling, and morphology-based validation.