Hidden diversity – DNA metabarcoding reveals hyper-diverse benthic invertebrate communities

Freshwater ecosystems, such as streams, are facing increasing pressures from agricultural land use. Aquatic insects and other macroinvertebrates have historically been used as indicators of ecological condition and water quality in freshwater biomonitoring programs; however, many of these protocols use coarse taxonomic resolution (e.g., family) when identifying macroinvertebrates. The use of family-level identification can mask species-level diversity, as well as patterns in community composition in response to environmental variables. Recent literature stresses the importance of robust biomonitoring to detect trends in insect decline globally, though most of these studies are carried out in terrestrial habitats. Here, we incorporate molecular identification (DNA metabarcoding) into a stream biomonitoring sampling design to explore the diversity and variability of aquatic macroinvertebrate communities at small spatial scales. We sampled twenty southern Ontario streams in an agricultural landscape for aquatic macroinvertebrates and, using DNA metabarcoding, revealed incredibly rich benthic communities which were largely comprised of rare taxa detected only once per stream despite multiple biological replicates. In addition to numerous rare taxa, our species pool estimates indicated that after 240 samples from twenty streams, there was a large proportion of taxa present which remained undetected by our sampling regime. When comparing different levels of taxonomic resolution, we observed that using OTUs revealed over ten times more taxa than family-level identification. A single insect family, the Chironomidae, contained over one third of the total number of OTUs detected in our study. Within-stream dissimilarity estimates were consistently high for all taxonomic groups (invertebrate families, invertebrate OTUs, chironomid OTUs), indicating stream communities are very dissimilar at small spatial scales. While we predicted that increased land use would homogenize benthic communities, this was not supported as within-stream dissimilarity was unrelated to land use. ### Competing Interest Statement The authors have declared no competing interest.