Applying Environmental DNA Metabarcoding to Calculate an Index of Biotic Integrity for Freshwater Fish

ABSTRACT Multi‐metric indices like the Index of Biotic Integrity (IBI) are important biomonitoring tools for Clean Water Act compliance in the USA. Environmental DNA (eDNA) metabarcoding could complement IBIs by increasing detection sensitivity for rare taxa while also reducing monitoring costs. To date, there is a lack of studies that have examined the efficacy of using eDNA metabarcoding to calculate IBIs in the USA. Here, we used eDNA metabarcoding to calculate a fish‐based IBI for streams and rivers of the Tennessee River Basin in northern Alabama, USA. 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 benthic macroinvertebrates. We found a significant, positive relationship between the eDNA‐IBI and fish‐IBI, as well as shared relationships to a measure of in‐stream habitat quality, but a weaker, non‐significant positive relationship to predicted biological condition from benthic macroinvertebrates. Notably, the former was recovered despite eDNA‐IBI sampling being conducted 9 years after the conventional fish sampling at slightly different locations (although distance did not explain variation), 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. 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.