Validating environmental DNA metabarcoding for marine fishes in diverse ecosystems using a public aquarium

Environmental DNA metabarcoding has been widely touted as a powerful tool for monitoring biodiversity in marine ecosystems. However, this method still requires thorough validation and standardization before it can be widely applied for ecological monitoring. The potential utility of environmental DNA metabarcoding is greatest in systems with high levels of diversity, yet environmental DNA metabarcoding has typically been validated in closed systems with relatively low levels of diversity. Additionally, the use of a multiple marker approach has been minimally explored under controlled, closed systems in the literature. Using a pilot study, we assess the ability of eDNA metabarcoding to capture biodiversity in a highly diverse closed system at the Ripley's Aquarium of Canada in Toronto, Ontario. Our pilot study highlights several key knowledge gaps that must be addressed before metabarcoding can be employed for widespread use in ecological monitoring. We found that environmental DNA metabarcoding recovered just over 50% of target species and 80% of target genera within a closed marine system containing 107 species and 44 genera using previously published markers for COI, 12S, and 16S. Additionally, COI and 12S were found to identify fewer target species than 16S, with COI generating the most off‐target identifications, but maximum detection success was achievable by combining all three markers. We discuss numerous key limitations which currently present barriers to the application of eDNA metabarcoding for studying highly diverse marine systems. These include marker selection, data validation and confidence, and the complexity of abundant and diverse novel systems. This study highlights important, yet incompletely resolved, challenges of environmental DNA metabarcoding for the detection of marine fishes in a diverse closed system environment using a multiple marker approach.