Evaluation of eDNA for groundwater invertebrate detection and monitoring: a case study with endangered Stygobromus (Amphipoda: Crangonyctidae)

Effective conservation and management of biodiversity is limited by a lack of critical knowledge on species’ distributions and abundances. This problem is particularly exacerbated for species living in habitats that are exceptionally difficult to access or survey, such as groundwater habitats. Environmental DNA (eDNA) represents a rapid, noninvasive, and potentially cost-effective new tool for detection and monitoring of biodiversity that occur in such habitats. In this study, we investigated the utility of eDNA in detecting the federally endangered Hay’s Spring Amphipod Stygobromus hayi and a co-occurring common congener S. tenuis potomacus from unique groundwater-associated habitats—hypotelminorheic seepage springs—in the Washington, DC metro area. We developed taxon-specific primers and probes for each species to amplify Stygobromus DNA using qPCR. In silico and in vitro validation demonstrated specificity of each designed assay. Assays were then used to screen water samples collected from ten seepage springs. Stygobromus hayi was detected at four seepage springs, including one potential new locality, while S. t. potomacus was detected at four springs, two of which were new localities. This study is the first to our knowledge to successfully employ an eDNA approach to detect rare or threatened invertebrates from subterranean ecosystems. Our study highlights challenges of employing an eDNA approach for the detection and monitoring of invertebrates in groundwater habitats that are difficult to study, including accounting for PCR inhibition and the potential for cryptic genetic diversity.