Stepwise diversification and recent decline revealed by mitochondrial and genome-wide SNP Data: Phylogeography and conservation insights into Opsariichthys rubriventris in Southern China

Opsariichthys rubriventris (Cyprinidae), a recently described cyprinid endemic in Guangdong’s streams, faces conservation risks due to its restricted range. Here we analyzed population genetic structure using mitochondrial cytochrome b (cyt b ) and genome-wide single nucleotide polymorphisms (SNPs) across seven populations in the Pearl River basin and adjacent river systems. Our results revealed extremely low within-population diversity and positive inbreeding coefficients ( F IS ) across all populations, indicating small effective population sizes. Strong genetic differentiation ( F ST up to 0.743) among rivers suggested limited connectivity. Phylogenetic analyses of cyt b and SNPs resolved four geographically discrete lineages corresponding to the Moyang, Xi, Dong, and Mingxi Rivers, with the Moyang lineage being basal. Divergence time estimates place the earliest split at ~1.74 million years ago (Ma), with further mid-Pleistocene divergences. Biogeographic reconstructions support a stepwise expansion from the Moyang to the Xi, then to the Dong, and finally to the Mingxi River. Gene flow is highly asymmetric and largely confined within the Dong River. Demographic reconstructions indicate recent population declines without postglacial expansion, highlighting vulnerability. Based on our findings, we further proposed six evolutionarily significant units (ESUs) and four management units (MUs), all of which should be regarded as equally important conservation units, while the relatively higher genetic diversity observed in the Dong River lineage provides additional information for management planning. These findings underscore the urgent need for habitat restoration and genetic monitoring to conserve this endemic species in South China’s threatened freshwater ecosystems.