Applying environmental DNA metabarcoding to calculate an index of biotic integrity for freshwater fish

Multi-metric indices like the Index of Biotic Integrity (IBI) are important biomonitoring tools for Clean Water Act compliance in the United States (US) but have also been implemented worldwide across many taxonomic groups. Environmental DNA (eDNA) metabarcoding could complement IBIs by increasing detection sensitivity for rare taxa while also reducing monitoring costs. To date, no studies have examined the efficacy of using eDNA metabarcoding to calculate IBIs in the US. Here, we used eDNA metabarcoding to calculate a fish-based IBI for streams and rivers of the Tennessee River Basin in northern Alabama, US. We collected water samples from 50 stream and river sites across a gradient of land use intensity, extracted eDNA from these samples, and sequenced the eDNA using vertebrate-specific primers. We compared our eDNA-IBI to a previous fish-IBI implemented by the state of Alabama using conventional sampling, as well as predicted biological condition of these streams from the US Environmental Protection Agency (EPA) based on a benthic macroinvertebrate multi-metric index (BMMI). We found a highly significant, positive relationship between the eDNA-IBI and fish-IBI for the same streams but a weaker, non-significant positive relationship between the eDNA-IBI and BMMI. Notably, the former was recovered despite eDNA-IBI sampling being conducted nine years after the conventional fish sampling at slightly different locations, the fish-IBI having used greater sampling effort throughout the year (including spring and autumn rather than only summer sampling), and a lack of reference DNA sequences that prevented eDNA detection for some species detected by conventional fish sampling. Accordingly, our study provides a baseline for how an eDNA-IBI may work relative to multi-metric indices calculated from conventional sampling, which can be improved through future directions identified and discussed in our paper. ### Competing Interest Statement The authors have declared no competing interest. US Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) Hatch Project, ILLU-875-976 University of Illinois Urbana-Champaign Graduate College Dissertation Travel Grant Harley J. van Cleave Research Award University of Illinois Urbana-Champaign Graduate College Dissertation Completion Fellowship Program in Ecology, Evolution, and Conservation Biology summer research stipend supported by USDA NIFA Hatch Project, ILLU-875-984