Development and validation of four environmental DNA assays for species of conservation concern in the South-Central United States

We describe the development and testing of qPCR assays to detect four species of amphibians and reptiles of conservation concern in the South Central United States through environmental DNA (eDNA) samples. The target species include the Ringed Salamander (Ambystoma annulatum), Three-toed Amphiuma (Amphiuma tridactylum), Crawfish Frog (Rana areolata), and Chicken Turtle (Deirochelys reticularia). A set of primers and probes amplifying a 64-72 bp target regions were designed for each species from DNA sequence data for either the mitochondrial Cytochrome Oxidase I or Cytochrome B gene. All assays were assessed for target specificity, with no evidence of amplification in closely related or sympatrically-distributed non-target species. In vitro tests indicate that all assays consistently detect focal species down to concentrations of 2 x 10(- 9)pg/mu L. We evaluated the utility of qPCR assays on the eDNA samples collected during field surveys across Eastern Oklahoma, focusing on counties with vouchered historical records for the target species. Although detection rates were low for field applications of the assays, positive detection ofAmbystoma annulatum,Rana areolata, andDeirochelys reticularia, but notAmphiuma tridactylum, were recorded. These assays can provide a practical tool for a non-invasive, genetic monitoring program that allows for both rapid detection and tracking of native aquatic and semi-aquatic amphibian and reptile species of conservation concern.

Automated summary

This study describes the development and testing of qPCR assays for detecting four species of amphibians and reptiles in the South Central United States using environmental DNA samples. The assays demonstrated high specificity and sensitivity, with positive detections of some target species in field surveys, providing a practical tool for genetic monitoring of conservation concern species.