Species richness and distribution of Sphaeriidae surveyed with Environmental DNA metabarcoding

Freshwater bivalves of the family Sphaeriidae (fingernail, pea, and pill clams) are difficult to survey and identify due to their small size and overlapping morphological traits. Environmental DNA (eDNA) metabarcoding offers a cost-effective method for assessing species richness and distributional patterns at large scales. We evaluated sphaeriid species richness and distribution at 15 sites in the Maumee River, Ohio, USA, based on two eDNA metabarcoding assays (broad and targeted), and we compared our results with those from a traditional benthic macroinvertebrate survey. We detected seven molecular operational taxonomic units (MOTUs) in the Maumee River, including Sphaerium transversum, five MOTUs representing Euglesa spp., and one MOTU representing Odhneripisidum sp. Sphaerium transversum was widely distributed, occurring at 10 sites, but Euglesa and Odhneripisidum were restricted to one to four sites in the upper river. Distributional patterns were broadly similar between both metabarcoding assays and benthic surveys. However, eDNA metabarcoding provided species-level identifications, resulting in higher species richness. Environmental DNA sampling augments and enhances traditional benthic surveys, but greater eDNA sample replication is needed to improve detection, and additional sphaeriid reference sequences are needed to improve species-level identification.