High‐Resolution Insights Into the Geographic Differentiation and Hybridisation of Odontobutis Gobies Through Integrated eDNA and Satsuki Tsuji, Yugo Miuchi, Naoki Shibata, Katsutoshi Watanabe

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 the effectiveness of this strategy by comprehensively characterising the population structure of widely distributed species.

As a model system, we investigated two closely related freshwater gobies, Odontobutis obscurus and O. hikimius , endemic to western Japan. Water samples were collected from 335 sites to characterise mitochondrial diversity via eDNA metabarcoding. Based on these results, tissue samples were subsequently collected from 49 representative sites and used for PCR‐based genome‐wide SNP analysis.

eDNA analysis revealed two major mitochondrial clades within O. obscurus , each comprising several geographically structured groups. Nuclear SNP data (661 loci) confirmed the deep divergence between these clades and further revealed that O. hikimius , whose range lies at the boundary between the two clades, likely originated through historical hybridisation.

Both mitochondrial and nuclear data revealed concordant geographic structuring, which could be explained by past geological events such as mountain uplift and ancient river system dynamics. These findings provide unprecedented resolution of the population structure in these species.

This study demonstrates that integrating eDNA and SNP data offers a cost‐effective and scalable framework for high‐resolution phylogeographic surveys. The approach is particularly valuable for non‐model species and can inform biodiversity monitoring and conservation strategies at a global scale.