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

Environmental (e)DNA assays are valuable tools for monitoring presence and distribution of cryptic species. Like many freshwater mussels, the dwarf wedgemussel, Alasmidonta heterodon numbers have dwindled and its range has diminished. As of its listing in 1993, only 10 to 20 locations were known to persist of the 70 Atlantic slope locations known historically. A 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 oxidase I (COI) gene. This SNP defines northern and southern major phylogenetic lineages. The primers match exactly the previously determined cytochrome oxidase I sequences of twenty 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 and/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, virus, and dissolved DNA). In addition to the specific application described here, the methodological approaches used in this study are widely applicable to the study of conservation issues including, but not limited to general aquatic biodiversity, phylogenetic studies, and detection of pathogenic microbes.