Assessment of Environmental DNA for Detecting Presence of Imperiled Aquatic Amphibian Species in Isolated Wetlands

Abstract

Environmental DNA (eDNA) is an emerging tool that allows low-impact sampling for aquatic species by isolating DNA from water samples and screening for DNA sequences specific to species of interest. However, researchers have not tested this method in naturally acidic wetlands that provide breeding habitat for a number of imperiled species, including the frosted salamander (Ambystoma cingulatum), reticulated flatwoods salamanders (Ambystoma bishopi), striped newt (Notophthalmus perstriatus), and gopher frog (Lithobates capito). Our objectives for this study were to develop and optimize eDNA survey protocols and assays to complement and enhance capture-based survey methods for these amphibian species. We collected three or more water samples, dipnetted or trapped larval and adult amphibians, and conducted visual encounter surveys for egg masses for target species at 40 sites on 12 different longleaf pine (Pinus palustris) tracts. We used quantitative PCRs to screen eDNA from each site for target species presence. We detected flatwoods salamanders at three sites with eDNA but did not detect them during physical surveys. Based on the sample location we assumed these eDNA detections to indicate the presence of frosted flatwoods salamanders. We did not detect reticulated flatwoods salamanders. We detected striped newts with physical and eDNA surveys at two wetlands. We detected gopher frogs at 12 sites total, three with eDNA alone, two with physical surveys alone, and seven with physical and eDNA surveys. We detected our target species with eDNA at 9 of 11 sites where they were present as indicated from traditional surveys and at six sites where they were not detected with traditional surveys. It was, however, critical to use at least three water samples per site for eDNA. Our results demonstrate eDNA surveys can be a useful complement to traditional survey methods for detecting imperiled pond-breeding amphibians. Environmental DNA may be particularly useful in situations where detection probability using traditional survey methods is low or access by trained personnel is limited.

Comparison of capture and storage methods for aqueous macrobial eDNA using an optimized extraction protocol: advantage of enclosed filter

“…; McKee et al . ). Aqueous eDNA monitoring provides possibilities to upscale species distribution surveys considerably, because much less effort in time and resources are required compared to conventional methods (Dejean et al .…”

“…Smart et al 2015;Simmons et al 2016) and monitoring rare and/or threatened species for conservation (e.g. Zhan et al 2013;McKee et al 2015). Aqueous eDNA monitoring provides possibilities to upscale species distribution surveys considerably, because much less effort in time and resources are required compared to conventional methods (Dejean et al 2012;Davy, Kidd & Wilson 2015).…”

Environmental DNA (eDNA) Assays for Invasive Populations of Black Carp in North America

“…; McKee et al. ; Lance and Guan, in press). However, PCR inhibition seemed unlikely to have substantially impacted our results, given that we utilized Environmental Master Mix 2.0 (Thermo Fisher Scientific, Waltham, Massachusetts) for our assays, and Environmental Master Mix 2.0 has been demonstrated to confer considerable robustness to qPCR in the presence of inhibitors (Jane et al.…”

“…Because eDNA is often found in very low concentrations, it is not uncommon for eDNA qPCR protocols to incorporate such large numbers of cycles (McKee et al. ; Sigsgaard et al. ; Smart et al.…”

Development, validation, and evaluation of an assay for the detection of wood frogs (Rana sylvatica) in environmental DNA

“…Further, the use of eDNA techniques for the detection of semi-aquatic species in ephemeral wetlands (e.g. wood frogs) has not been extensively assessed (McKee et al 2015a). Conditions in ephemeral wetlands may be unfavorable for preservation and detection of DNA due to elevated temperatures, high sediment load, and high acidity contrasted with lakes and streams (Dejean et al 2011;Barnes et al 2014;Eichmiller et al 2014).…”