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 sympatrically-distributed 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.
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Acknowledgements
This research was supported by grants from the Research Council of the University of Oklahoma (OU) Norman Campus to CDS, an ODWC (F16AF01213 [T-91-R-1]) grant to CDS, JLW, TY, and LS. Additional financial support for assay development was provided by an OU Graduate Student Senate award to ESF. No live animals were impacted as part of this research; all tissue samples or DNA extracts were obtained from freezer stocks of previously captured live animals, collected on necessary Institutional Animal Care and Use Committee (IACUC) approvals and state collecting permits at time of capture. The authors thank the members of the Siler Lab and Souza Lab for fieldwork and DNA extraction assistance. We thank the various land managers of Army Corps of Engineers lakes, National Wildlife Refuges, Oklahoma State Parks, The Nature Conservancy, and ODWC Wildlife Management Areas for allowing access to researchers for water sample collection. We thank the following entities and people for loaning us tissue samples: Florida Museum of Natural History (FLMNH) Genetic Resources Repository (GRR), Louisiana State University Museum of Zoology (LSUMZ) Genetic Resources Collection, Sam Noble Museum (OMNH) Oklahoma Collection of Genomic Resources (OCGR), and Dr. Day Ligon of Missouri State University. Lastly, we thank anonymous reviewers for their critical evaluations of this manuscript.
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CDS, JLW, TY, and LS contributed to study conception and design. CDS, JLW, TY, and LS secured funding for assay development, testing, and field survey work, with additional funding secured by ESF for assay development. All authors contributed to field surveys. Assay development was undertaken by ESF and TY. DNA extractions and screening were overseen by JLW, TY, ESF, and CDS. Data analysis and summary of the eDNA sample screening were performed by JLW, with contributions from CDS. The first draft of the manuscript was written by CDS and JLW, with contributions and reviews by ESF, TY, and LS. All authors read and approved the final manuscript.
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Siler, C.D., Freitas, E.S., Yuri, T. et al. Development and validation of four environmental DNA assays for species of conservation concern in the South-Central United States. Conservation Genet Resour 13, 35–40 (2021). https://doi.org/10.1007/s12686-020-01167-3
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DOI: https://doi.org/10.1007/s12686-020-01167-3