Detecting the undetectable: Characterization, optimization, and validation of an eDNA detection assay for the federally endangered dwarf wedgemussel, Alasmidonta heterodon (Bivalvia: Unionoida)

Environmental DNA (eDNA) assays are valuable tools for monitoring the presence and distribution of cryptic species. Like many freshwater mussels, the numbers of dwarf wedgemussel, Alasmidonta heterodon, have dwindled and its range has diminished. As of its listing in 1993, only 10-20 locations were known to persist out of the 70 Atlantic slope locations known historically. A quantitative polymerase chain reaction (qPCR) assay to detect the presence of A. heterodon was developed that uses two probes to accommodate a single-nucleotide polymorphism (SNP) in the probe-binding site within the cytochrome c oxidase subunit I (COI) gene. This SNP defines northern and southern major phylogenetic lineages. The primers match exactly the previously determined COI sequences of 20 dwarf wedgemussel individuals representing Atlantic slope populations from North Carolina, Virginia, Maryland, New York, and New Hampshire. Other than for the qPCR assay described here, these primers can be used for sequencing or metabarcoding to further delineate dwarf wedgemussel populations phylogenetically. A simple eDNA preparation method is introduced using flocculation to concentrate free DNA in solution as well as cellular material (including shed animal cells, bacteria, viruses, and dissolved DNA). In addition to the specific application described here, the methodological approaches used in this study are widely applicable to conservation, including, but not limited to, general aquatic biodiversity, phylogenetic studies, and the detection of pathogenic microbes.