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. Therefore, there is an urgent need for more accessible and rapid monitoring methods that will ultimately allow for restoration programmes to be implemented. In this study, we deployed environmental DNA (eDNA) metabarcoding as a fish monitoring tool in the Mersey estuarine system (UK), where ecosystem productivity has been severely impacted by water quality degradation since the Industrial Revolution. Monthly water samples were collected over a year (2023-2024) throughout the estuary, covering saline, brackish and freshwater zones. Overall, 69 species of fish were detected, increasing the number of known species within the estuary in comparison to both historical and contemporary records (46 and 39, respectively), with a generally higher observed richness within the upper estuary sampled zones where the water chemistry is predominantly freshwater. Notably, we identified several species returning to the system for the first time since pre-industrial times. Peak species richness was recorded during the winter season (December-February). Species compositions varied significantly by month and spatially by zone within months, but not when grouped seasonally. Furthermore, around [~]15% of the species detected were diadromous, with the endangered Atlantic salmon Salmo salar, for example, being frequently detected during its key migratory period. This study highlights the resurgence in fish diversity in a once biologically depleted estuarine ecosystem and demonstrates how eDNA metabarcoding can be implemented for detecting historically absent species, thereby enhancing restoration monitoring efforts globally.