Performance of eDNA Filtration Methods for Monitoring Fish Diversity in a Hyper‐Tidal Estuary

Abstract

Environmental DNA (eDNA)-based monitoring has become an established and efficient method for surveying biodiversity inaquatic systems. However, there is a need to compare and standardize sampling methods across different ecosystem types, par-ticularly complex ecosystems such as estuaries, where unique challenges exist for monitoring fish populations due to fluctuatingenvironmental factors. Here, we compare species richness obtained from eDNA metabarcoding data using four different eDNAfiltration methods: three manual filtration methods with different pore sizes (0.45, 1.2, and 5 μm) and a newly established pas-sive method, the metaprobe. The study was applied across a salinity gradient in a hyper-tidal estuarine ecosystem. Overall, 44fish species were detected across the four methods used. The 0.45 μm filter recovered the highest richness (39 species), then themetaprobe method (35), followed by the 1.2 μm (34) and 5 μm (33) filters. Filter performance between salinity gradients revealedthat the 0.45 μm and the 1.2 μm methods recovered the highest species richness across all sampled zones. The 0.45 μm also hadthe most consistent detection probabilities using representative species from each zone. While the 0.45 μm method appeared tobe the optimal method, each of the methods can be considered a viable and comparable option for biomonitoring in dynamicecosystems such as estuaries and rivers. In particular, the passive metaprobe (used in a freshwater system for the first time here)performed well in comparison to the manual filtering methods despite a short deployment time. This study provides criticalinsights for optimizing fish diversity assessments using eDNA metabarcoding in estuarine ecosystems, providing a valuableframework for future monitoring efforts in similar systems worldwide.