Development and validation of four environmental DNA assays for species of conservation concern in the South-Central Uni
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METHODS AND RESOURCES ARTICLE
Development and validation of four environmental DNA assays for species of conservation concern in the South-Central United States Cameron D. Siler1,2 · Elyse S. Freitas1,2 · Tamaki Yuri1,3 · Lara Souza4 · Jessa L. Watters1 Received: 6 December 2019 / Accepted: 18 August 2020 © Springer Nature B.V. 2020
Abstract 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 sympatricallydistributed non-target species. In vitro tests indicate that all assays consistently detect focal species down to concentrations of 2 × 10− 9 pg/µL. 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 of Ambystoma annulatum, Rana areolata, and Deirochelys reticularia, but not Amphiuma 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. Keywords Ambystoma annulatum · Amphiuma tridactylum · Cytochrome B · Cytochrome Oxidase I · Deirochelys reticularia · eDNA · Oklahoma · Rana areolata As organisms interact with aquatic or semi-aquatic environments, they leave behind genetic material (e.g., urine, feces, skin, reproductive output) termed environmental DNA (or Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12686-020-01167-3) contains supplementary material, which is available to authorized users. * Cameron D. Siler [email protected] 1
Sam Noble Oklahoma Museum of Natural History, 2401 Chautauqua Ave., Norman, OK 73072‑7029, USA
2
Department of Biology, University of Oklahoma, 730 Van Vleet Oval, Norman, OK 73019, USA
3
Present Address: Museum of Biological Diversity, Department of Evolution, Ecology and Organismal Biology, The Ohio State University, 1315 Kinnear Rd, Columbus, OH 43212, USA
4
Oklahoma Biological Survey & Department of Microbiology and Plant Biology, University of Oklahoma, 770 Van Vleet Oval, Norman, OK 73019, USA
eDNA). Techniques have recently been developed to detect eDNA, which has applications to monitoring and conserving wild populations (Bohmann et al. 201
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