High-resolution insights into the geographic differentiation and hybridisation of Odontobutis gobies through integrated eDNA and SNP analyses

Understanding fine-scale population genetic structure is essential for biodiversity conservation and evolutionary research, but conventional phylogeographic studies often face labour and financial cost constraints. This study proposes a two-step survey strategy that integrates environmental DNA (eDNA) analysis and PCR-based genome-wide SNP genotyping, aiming to evaluate its effectiveness by comprehensively characterising the population structure of widely distributed species. As a model system, we selected the odontobutid gobies, Odontobutis obscurus and O. hikimius , which occur in western Japan. Initially, water samples were collected from 335 sites across western Japan. Subsequently, tissue sampling for SNP analysis was conducted at 49 sites representing the regional groups and species. The eDNA analysis revealed two major mitochondrial clades within O. obscurus , each comprising multiple geographically distinct groups. Subsequently, tissue sampling and SNP analysis were conducted at representative sites of each regional group and species. Nuclear genomic SNP data (661 loci) corroborated the deep divergence between the two clades of O. obscurus and, unexpectedly, indicated that O. hikimius , whose range lies at their boundary, originated through hybridisation between them. Geographic patterns of the regional groups inferred from both mitochondrial and nuclear data were largely explained by historical geological events such as mountain uplift and ancient river system dynamics, and provide unprecedentedly detailed insight into the population structuring of the focal species. This study demonstrates that the integration of eDNA and SNP analyses provides a cost-effective and scalable approach for high-resolution phylogeographic surveys.