Multi-seasonal eDNA metabarcoding highlights a resurgence in fish diversity across a severely impacted estuarine ecosystem

Aquatic ecosystems have been in an alarming state of decline for decades. In particular, estuarine ecosystems have experienced long-term declines in fish diversity due to factors such as habitat degradation, pollution, and altered hydrology. Monitoring these systems is often limited by the difficulty and cost of conventional survey methods. In this study, we applied environmental DNA (eDNA) metabarcoding to assess fish diversity in the Mersey Estuary (UK), a historically severely impacted system. Monthly water samples were collected over a year (2023–2024) across saline, brackish, and freshwater zones. Overall, 69 species were detected, surpassing both historical (46 species) and recent (39 species) records. Richness was highest in the upper freshwater zones, and several species were recorded returning to the estuary for the first time since pre-industrial times (~1850s). Peak species richness occurred during winter (December–February). Species composition varied monthly and spatially, though not consistently by season. Approximately 15% of detected species were diadromous, with the endangered Atlantic salmon (Salmo salar) being frequently detected during its key spawning period (October-December), for example. The results presented here indicate a resurgence in estuarine fish diversity in the Mersey and highlight eDNA metabarcoding as a rapid, sensitive tool for monitoring both contemporary and historically absent species, supporting conservation and restoration efforts.