LAUSR

Comparative phylogeography provides crucial insights into evolutionary processes shaping biodiversity patterns by analysing spatial genetic variations across multiple species. However, conventional capture‐based methods are often labour‐intensive, particularly for multi‐species analyses. Environmental DNA (eDNA) analysis has significant advantages in comparative phylogeography, including simplified field surveys requiring only water collection and the potential to simultaneously analyse multiple species from a single sample. To further expand the eDNA application and demonstrate its utility in comparative phylogeographic studies, this study employed eDNA analysis to simultaneously analyse the phylogeographic patterns in a species‐rich freshwater goby group (Rhinogobius) in the Japanese Archipelago. DNA amplification was performed on eDNA samples collected from 573 sites across the archipelago using newly designed group‐specific primers targeting the mitochondrial cytochrome b region of Rhinogobius. High‐throughput sequencing detected haplotypes of all nine known species (or species groups) occurring in this region, followed by phylogenetic and network analyses. The eDNA analysis successfully revealed the genetic population structures across multiple species. A landlocked species, R. flumineus, exhibited fine‐scale population differentiation shaped by geomorphological barriers, while amphidromous species showed broader genetic patterns likely influenced by ocean currents and their ecological traits. The phylogenetic and phylogeographic patterns reconstructed by the eDNA analysis were almost completely concordant with previously identified patterns of limited groups based on conventional methods, demonstrating the reliability of eDNA‐based comparative phylogeography. This study highlights the potential of eDNA to complement and partially replace conventional methods, facilitating large‐scale comparative phylogeographic research to gain new insights into spatial patterns and evolutionary processes of biodiversity.