Multi-taxon environmental DNA analysis reveals distributions and pathways of Pelophylax nigromaculatus invasion in Iwamizawa, Hokkaido, Japan

Abstract This study explores the recent expansion of the translocated invasive Black-spotted pond frog Pelophylax nigromaculatus in Iwamizawa, Hokkaido, Japan, focusing on dispersal routes and habitat uses at landscape and field scales. Environmental DNA (eDNA) surveys across rivers and agricultural canals confirmed the occurrence of P. nigromaculatus in Iwamizawa for the first time, and revealed that P. nigromaculatus primarily dispersed via agricultural canals connected to paddy fields rather than larger natural rivers. Smaller canals facilitated species movement, whereas rivers posed potential barriers. Detailed eDNA analysis and visual observations in two adjacent paddy lots—one with an open-ditch system and the other without—demonstrated that frogs likely entered from the irrigation canals. The open-ditch lot supported a population due to continuous inundation even during midseason drainage, whereas reduced water levels in the conventional paddy lot restricted long-term microhabitat use. In contrast to the invasive species, the native Japanese tree frog Dryophytes japonicus was not detected by eDNA in the open-ditch lot, reflecting a habitat segregation of the species. These findings support the important role of agricultural canal networks and connectivity between rivers, canals, and paddy fields in driving amphibian invasions, offering critical insights into the conservation and control of invasive species.