Zong, S.; Bauknecht, R.; Seybold, H.; Albouy, C.; Pellissier, L.

Environmental DNA (eDNA) provides a powerful tool for biodiversity monitoring in large river ecosystems. However, current studies often rely on subjective site selection and lack systematic sampling designs. This can limit the ability to capture the full spectrum of environmental conditions that species depend on, thereby compromising sampling efficiency. To address this challenge, we propose utilizing remote sensing-based clustering for environmental stratification of sampling designs, thereby enhancing detection capabilities and increasing the objectivity of eDNA sampling. Using GBIF-based fish species distribution models and simulated distributions along the Danube, we demonstrate that this approach enhances detection efficiency compared to conventional random and regular sampling methods. To facilitate practical implementation, we developed a tool to help fieldwork planners of river sampling campaigns automatically apply this method and select stratified sampling sites without the need for extensive data processing. Finally, we demonstrate that eDNA detection occurred most frequently within the range of 0-20 km downstream of the expected modeled distribution of species, suggesting that the diffusion of the signal should be further considered in the sampling design process. Our findings highlight the potential of remote sensing-based stratification to create more efficient and objective sampling designs but suggest that sampling design should be further combined with hydrological information to optimize cost-efficient sampling. The development of standard and robust sampling protocols will help advance more cost-effective eDNA-based biodiversity monitoring in riverine ecosystems.