Despite the ecological and societal importance of large rivers, fish sampling remains costly and limited to specific habitats (e.g., river banks). Using an eDNA metabarcoding approach, we regularly sampled 500 km of a large river (Rhône River). Comparisons with long-term electrofishing surveys demonstrated the ability of eDNA metabarcoding to qualitatively and quantitatively reveal fish assemblage structures (relative species abundance) but eDNA integrated a larger space than the classical sampling location. Combination of a literature review and field data showed that eDNA behaves in the water column like fine particulate organic matter. Its detection distance varied from a few km in a small stream to more than 100 km in a large river. To our knowledge, our results are the first demonstration of the capacity of eDNA metabarcoding to describe longitudinal fish assemblage patterns in a large river, and metabarcoding appears to be a reliable, cost-effective method for future monitoring.

Summary: The need for sensitive biological measures of aquatic ecosystem integrity applicable at large spatial scales has been highlighted by the implementation of the European Water Framework Directive. Using fish communities as indicators of habitat quality in rivers, we developed a multi‐metric index to test our capacity to (i) correctly model a variety of metrics based on assemblage structure and functions, and (ii) discriminate between the effects of natural vs. human‐induced environmental variability at a continental scale. Information was collected for 5252 sites distributed among 1843 European rivers. Data included variables on fish assemblage structure, local environmental variables, sampling strategy and a river basin classification based on native fish fauna similarities accounting for regional effects on local assemblage structure. Fifty‐eight metrics reflecting different aspects of fish assemblage structure and function were selected from the available literature and tested for their potential to indicate habitat degradation. To quantify possible deviation from a ‘reference condition’ for any given site, we first established and validated statistical models describing metric responses to natural environmental variability in the absence of any significant human disturbance. We considered that the residual distributions of these models described the response range of each metric, whatever the natural environmental variability. After testing the sensitivity of these residuals to a gradient of human disturbance, we finally selected 10 metrics that were combined to obtain a European fish assemblage index. We demonstrated that (i) when considering only minimally disturbed sites the index remains invariant, regardless of environmental variability, and (ii) the index shows a significant negative linear response to a gradient of human disturbance. Synthesis and applications. In this reference condition modelling approach, by including a more complete description of environmental variability at both local and regional scales it was possible to develop a novel fish biotic index transferable between catchments at the European scale. The use of functional metrics based on biological attributes of species instead of metrics based on species themselves reduced the index sensitivity to the variability of fish fauna across different biogeographical areas.