Development of eDNA markers for the Rio Grande Silvery Minnow

Abstract

We designed a pair of environmental DNA (eDNA) quantitative real-time polymerase chain reaction (qPCR) assays for the detection of the Rio Grande Silvery Minnow (Hybognathus amarus), validating each assay with eDNA samples collected from tanks holding H. amarus as well as from locations the species is known to occupy within the Middle Rio Grande River in New Mexico, United States. Both assays demonstrated high specificity to H. amarus in cross-amplification tests of nontarget species. We recommend the assays be used in tandem (multiplex qPCR), as most eDNA studies have demonstrated that multi-assay surveys increase detection power, which is key when monitoring for a rare species for which the target eDNA is a minor component of the overall eDNA pool and likely degraded. The multiplex approach is also helpful when the monitored locale contains compounds that, when co-extracted with eDNA, may inhibit qPCR. The two assays appear to be an effective eDNA tool for H. amarus, and could contribute to the Middle Rio Grande Endangered Species Collaborative Program’s genetic monitoring efforts by providing a rapid, non-invasive sampling method for the detection of this endangered species.