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Anna M. McKee

Publications and source records attributed to Anna M. McKee.

14 recordsLinked to original sources
USGS2024

Phytoplankton, taste-and-odor compounds, and cyanotoxin occurrence in four water-supply reservoirs in the Triangle area of North Carolina, April–October 2014

Prior to 2014, local utilities and State agencies monitored for cyanotoxins and taste-and-odor (T&O) compounds and reported occasional detections in three water-supply reservoirs in Wake County, North Carolina. Comparable data for cyanotoxins and T&O compounds were lacking for other water-supply reservoirs in the Triangle area of North Carolina. This report assesses whether cyanotoxins and T&O compounds occurred in four previously unmonitored North Carolina Triangle area water-supply reservoirs at levels that exceed existing North Carolina and U.S. Environmental Protection Agency recreational and drinking water health advisory, guidance, and criterion levels based on data collected during the peak phytoplankton growth period in 2014. Samples were collected from five sites across the study reservoirs (Cane Creek Reservoir, West Fork Eno River Reservoir, B. Everett Jordan Lake, and University Lake) between April and October 2014 and analyzed for physical characteristics, chemical constituents, phytoplankton communities, cyanotoxins, and T&O compounds. Lake stratification during the sampling period in 2014 could indicate that the deep zones of the water column, during stratified anoxic conditions, may serve as possible sources of nutrients and metals for algal growth and other biogeochemical processes. Differences in phytoplankton communities were attributed to variability in environmental conditions across the sites and sampling events. Differences generally were greater among sites than among sampling events for phytoplankton communities and environmental conditions. Phytoplankton community assemblages, within reservoirs, often were dominated by cyanobacteria that contained genera capable of producing T&O compounds and cyanotoxins during summer and fall months. The occurrence and associated biovolumes of potential producers of cyanotoxins and T&O compounds varied across the sites and sampling events. Of 20 samples collected during the study, the T&O compound geosmin and the cyanotoxin microcystin were present in 19 and 18 samples, respectively. While not harmful, the aesthetically displeasing geosmin concentrations periodically exceeded the human detection threshold of 15 nanograms per liter at most sites. The T&O compound 2-methylisoborneol (MIB) was detected in 11 of 20 samples, with concentrations below the human detection threshold of 15 nanograms per liter in all but one sample. The cyanotoxin anatoxin-a was detected in two of the samples. No other cyanotoxins were detected during the study. In general, results did not indicate the biovolume of any given phytoplankton genera in the study was correlated with increased concentrations of MIB, geosmin, or microcystin. Results from this study indicated that microcystin concentrations in the water-supply reservoirs in the Triangle area were below EPA-recommended recreational level of 8 micrograms per liter, but periodically exceeded the EPA finished-water 10-day health advisory level of 0.3 microgram per liter for bottle-fed infants and preschool-age children. This suggests longer term data collection may be necessary to better understand the magnitude and frequency of cyanotoxin concentrations in these four water-supply reservoirs, particularly those with an elevated risk of exceeding the EPA 10-day health advisory levels in the finished drinking water or those with a higher frequency of T&O compound occurrence.

North Carolina↗
USGS2023

Dead-end hollow fiber ultrafiltration capture of environmental DNA for freshwater mussel (Unionidae) species detection with metabarcoding

Insufficient water sample volumes can be a limiting factor for detecting species with environmental DNA (eDNA) from aquatic habitats. We compared detections of freshwater mussel (Unionidae) communities using large water sample volumes and dead-end hollow fiber ultrafiltration (D-HFUF or DEUF) with traditional eDNA filtration methods that use relatively small water sample volumes. Unionid species were detected in approximately 50-L D-HFUF eDNA samples with two mitochondrial DNA metabarcoding markers (COI and ND1) and compared to species detection results from eDNA captured from commonly used 1-L samples filtered with polyethersulfone (PES) filters at three lotic sites in Georgia and Missouri. Of the 431,560 COI and 1,035,472 ND1 reads from all environmental samples of both filter types that passed quality control, 95% (410,755 reads) of COI reads and 85% (883,472 reads) of ND1 reads were assigned to a unionid species. Nineteen different freshwater mussel species were detected across all D-HFUF samples, and 11 species were detected across all PES samples. Reads assigned to the genus Elliptio could not be resolved beyond the genus level with either marker. From D-HFUF samples, 15 and 16 mussel species were detected with the COI and ND1 markers, respectively. From PES samples, nine and seven species were detected with the COI and ND1 markers, respectively. More mussel species were detected at each site in D-HFUF samples than in PES samples regardless of whether results from both markers were combined or evaluated separately. Our results demonstrate the merit of further exploration and optimization of D-HFUF for capturing eDNA from high-volume water samples to facilitate detection of unionids and likely other aquatic organisms.

Environmental DNA↗
USGS2021

Occurrence and distribution of mercury in streams and reservoirs in the Triangle Area of North Carolina, July 2007–June 2009

During the time period 2001–2006, the U.S. Geological Survey reported mercury-concentration measurements that exceeded the North Carolina water-quality criterion (NCWQC) of 0.012 microgram per liter for total recoverable mercury in streams and reservoirs across the Triangle Area of North Carolina. Mercury data were sparse, however, generally consisting of only one or two water samples per year. Additional monitoring and data analysis were needed to better determine the occurrence and distribution of mercury in the Triangle Area for all seasons and waterbody types as well as associations between mercury concentrations and water-quality and land-use parameters. Water at fifteen reservoir and 14 stream sites across the Triangle Area was sampled at various times between August 2007 and June 2009, with water samples collected from both the surfaces and bottoms of the water columns in reservoirs and from the surfaces of streams. A bed sediment sample was also collected at all reservoir sites and at all but one stream site. A total of 301 water samples was collected at reservoir sites. Filtered and total recoverable mercury were detected in at least one water sample collected from each reservoir site. A total of 77 water samples was collected from stream sites with filtered mercury detected in samples from one-half of these sites, and total recoverable mercury detected in at least one water sample from all but two sites. Total recoverable and filtered mercury concentrations exceeded the NCWQC for mercury more frequently in reservoir than in stream samples. Differences in sampling frequencies among seasons and between streams and reservoirs, however, may have negatively biased overall estimates of mercury concentrations in streams relative to reservoirs. Filtered mercury concentrations in surface-water samples from reservoirs and total recoverable mercury concentrations in bottom samples from reservoirs were highest in the fall, whereas no seasonal trends in filtered or total recoverable mercury were detected from stream samples. Total mercury concentrations were calculated for the bulk sample on the basis of the percentage of the grains in the bulk sample whose diameters that were smaller than 0.0625 millimeters. Total mercury concentrations in bed sediment were generally higher for samples from reservoir sites compared to streams sites, although the highest total mercury concentration in bed sediment was from a stream site. Concentrations of total recoverable mercury in water samples from stream sites all fell within the general range for streams and lakes without on-site significant anthropogenic sources (for example, mercury mines or industrial pollution), whereas samples collected from eight reservoir sites had total mercury concentrations in a range characteristic of sites affected by mercury mines or industrial pollution. Results suggested that litterfall may be a source of mercury in streams, whereas atmospheric deposition is likely a dominant source for reservoirs; however, high concentrations of filtered and total recoverable mercury concentrations in the fall season in some reservoir-water samples may warrant further analysis of potential hydrologic factors. Mercury concentrations in all water and bed sediment samples were below levels expected to cause adverse effects to humans and aquatic biota, indicating that mercury levels at the study sites in the Triangle Area were unlikely to cause an immediate health risk to humans or aquatic organisms. The high variability among several sample replicates for total recoverable mercury, however, indicated that inferences from total recoverable mercury concentrations can be tenuous.

North Carolina↗
USGS2021

Executive summary and annotated bibliography of selected references from “Microbial and viral indicators of pathogens and human health risks from recreational exposure to waters impaired by fecal contamination” with related project ideas for Gwinnett County, Georgia

This document was prepared in cooperation with Gwinnett County, Georgia, to supplement the journal article “Microbial and Viral Indicators of Pathogens and Human Health Risks from Recreational Exposure to Waters Impaired by Fecal Contamination” (published in Journal of Sustainable Water in the Built Environment ). The document includes an executive summary of the article, project ideas for Gwinnett County to enhance its bacterial monitoring program, and an annotated bibliography of selected references from the article. Although tailored to Gwinnett County, the project ideas are based on the state of the science of monitoring for fecal-associated pathogens and pathogen indicators in impaired surface waters and may be of interest to water resources divisions of other municipalities.

Georgia↗
USGS2021

Feral swine as sources of fecal contamination in recreational waters

Recreational waters are primary attractions at many national and state parks where feral swine populations are established, and thus are possible hotspots for visitor exposure to feral swine contaminants. Microbial source tracking (MST) was used to determine spatial and temporal patterns of fecal contamination in Congaree National Park (CONG) in South Carolina, U.S.A., which has an established population of feral swine and is a popular destination for water-based recreation. Water samples were collected between December 2017 and June 2019 from 18 surface water sites distributed throughout CONG. Host specific MST markers included human (HF183), swine (Pig2Bac), ruminant (Rum2Bac), cow (CowM3), chicken (CL), and a marker for shiga toxin producing Escherichia coli (STEC; stx2 ). Water samples were also screened for culturable Escherichia coli ( E. coli ) as part of a citizen science program. Neither the cow nor chicken MST markers were detected during the study. The human marker was predominantly detected at boundary sites or could be attributed to upstream sources. However, several detections within CONG without concurrent detections at upstream external sites suggested occasional internal contamination from humans. The swine marker was the most frequently detected of all MST markers, and was present at sites located both internal and external to the Park. Swine MST marker concentrations ≥ 43 gene copies/mL were associated with culturable E. coli concentrations greater than the U.S. Environmental Protection Agency beach action value for recreational waters. None of the MST markers showed a strong association with detection of the pathogenic marker ( stx2 ). Limited information about the health risk from exposure to fecal contamination from non-human sources hampers interpretation of the human health implications.

South Carolina↗
USGS2021

Microbial and viral indicators of pathogens and human health risks from recreational exposure to waters impaired by fecal contamination

Fecal indicator bacteria (FIB) (e.g., fecal coliforms, Escherichia coli , and enterococci) have been used for decades to monitor for and protect the public from waterborne pathogens from fecal contamination. However, FIB may not perform well at predicting the presence of waterborne pathogens or human health outcomes from recreational exposure to fecal-contaminated surface waters. Numerous factors can influence the relationship between FIB and pathogens or human health outcomes, including the source(s) of contamination, the type of pathogen(s) present, differences in the survival and behavior of FIB and pathogens in the wastewater conveyance and treatment process, and varying environmental conditions. As a result, different indicators, such as source-specific microbial source tracking (MST) markers and viral fecal indicators, have been used as possible surrogates to better approximate pathogen abundance and human health risks in recreational waters. The performance of these alternative indicators has been mixed, with some promise of viral indicators better approximating viral pathogens than bacterial fecal indicators, and FIB generally more closely associated with bacterial and protozoal pathogen presence than human MST markers. Many of the assays to detect and quantify fecal indicators and pathogens are polymerase chain reaction-based assays, which detect and quantify nucleic acid [deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)] sequences specific to a target of interest. Recent advances in DNA and RNA sequencing technologies may push the field toward metabarcoding approaches, where multiple targets can be detected and quantified simultaneously. Metabarcoding is currently more applicable to bacterial and protozoal assessments than viral assessments based on a lack of universal metabarcoding markers for viruses. Innovative technologies, such as biosensors and nanotechnologies, may provide more sensitive and accurate tools to detect and quantify pathogens. When a specific pathogen is of concern for a recreational water body, a practical approach in estimating the likelihood of human health outcomes is the application of quantitative microbial risk assessments (QMRAs). Quantitative microbial risk assessments can be used to model the likelihood of pathogen-specific human health outcomes from recreational exposure as a function of a surrogate indicator. Inputs for QMRAs include the ratio between the indicator to be monitored and the pathogen of interest, the concentration of the indicator, the amount of water ingested, and the likelihood of the health outcome based on the estimated amount of pathogen consumed. There are numerous unknowns about the behavior and survival of fecal indicators and pathogens in environmental waters. Developing accurate models to predict pathogen concentrations from fecal indicators in recreational waters will require a better understanding of these unknowns. Current methods and technologies for detecting and quantifying fecal indicators and pathogens are limited due to the rare and patchy nature of pathogens. Technological advances may help improve sensitivity for detecting and quantifying pathogens.

Journal of Sustainable Water in the Built Environm↗
USGS2020

Monitoring and real-time modeling of Escherichia coli bacteria for the Chattahoochee River, Chattahoochee River National Recreation Area, Georgia, 2000–2019

The Chattahoochee River National Recreation Area (CRNRA) is a National Park Service unit/park with 48 miles of urban waterway in the Atlanta metropolitan area. The Chattahoochee River within the CRNRA is a popular place for water-based recreation but is known to periodically experience elevated levels of fecal-coliform bacteria associated with warm-blooded animals that can result in a variety of pathogen-related human illnesses. In 2000, the National Park Service entered into a public-private partnership with the U.S. Geological Survey (USGS) and the Chattahoochee Riverkeeper, called the Chattahoochee River BacteriALERT program, to monitor Escherichia coli ( E. coli ), which is a fecal indicator bacteria and a proxy for human health risk from waterborne pathogens. The BacteriALERT network monitors E. coli densities at three stations on the Chattahoochee River within the CRNRA, at Norcross (USGS station 02335000), Powers Ferry (USGS station 02335880), and Atlanta (USGS station 02336000). E. coli densities determined from water samples were compared to the U.S. Environmental Protection Agency’s Beach Action Value (BAV) of 235 colony forming units per 100 milliliters to assess whether conditions were considered safe for freshwater, primary contact recreational use. Sample E. coli densities exceeded the BAV for 15.5 percent of the samples collected at Norcross (n = 1,969) and 30.3 percent of the samples at Atlanta (n = 1,938) for the study period October 23, 2000, to May 23, 2019, and 33.6 percent of the samples from Powers Ferry (n = 134) for the study period May 5, 2016, to May 23, 2019. Models to predict E. coli densities in near real-time were developed for the three BacteriALERT stations. Models were developed using forward-stepwise multiple linear regression with the Bayesian Information Criteria and were calibrated with samples collected between October 4, 2007, and May 23, 2019. Explanatory variables included season, turbidity, water temperature, streamflow, upstream tributary streamflows, and temporal trend. The most statistically significant explanatory variables in the models were turbidity, upstream tributary streamflows, and season. The Norcross model had an increasing trend in E. coli densities of 2.3 percent per year. A significant trend was not detected for the Atlanta station, while trends were not assessed for Powers Ferry models due to the short (3-year) calibration period. Model adjusted R 2 s ranged from 0.686 (Atlanta) to 0.795 (Norcross with time trend) indicating that the models explained a substantial portion of the variations in E. coli densities. Evaluation of model predictions and residuals indicated that models were well posed and exhibited little bias. The models performed well in accurately determining compliance and exceedance of the BAV with low misidentification rates ranging from 3.5 percent (Norcross) to 11.3 percent (Powers Ferry). Misidentification was most common for densities near the BAV, and misidentification rates in the study were low despite fairly low model precisions because E. coli densities were infrequently near the BAV. The precisions of the models developed herein were comparable to the more complex models developed by Lawrence (2012) that were never implemented in the BacteriALERT program due to their computational complexity. The predictive E. coli models developed herein will improve the ability to assess the health risks of water-based recreational activities in the CRNRA in near real-time.

Georgia↗
USGS2020

Microbial source tracking (MST) in Chattahoochee River National Recreation Area: Seasonal and precipitation trends in MST marker concentrations, and associations with E. coli levels, pathogenic marker presence, and land use

Escherichia coli levels in recreational waters are often used to predict when fecal-associated pathogen levels are a human health risk. The reach of the Chattahoochee River that flows through the Chattahoochee River National Recreation Area (CRNRA), located in the Atlanta-metropolitan area, is a popular recreation area that frequently exceeds the U.S. Environmental Protection Agency beach action value (BAV) for E. coli . A BacteriALERT program has been implemented to provide real-time E. coli estimates in the reach and notify the public of potentially harmful levels of fecal-associated pathogens as indicated by surrogate models based on real-time turbidity measurements from continuous water quality monitoring stations. However, E. coli does not provide information about the sources of fecal contamination and its accuracy as a human health indicator is questionable when sources of contamination are non-human. The objectives of our study were to investigate, within the Park and surrounding watersheds, seasonal and precipitation-related patterns in microbial source tracking marker concentrations of possible sources (human, dog, and ruminant), assess correlations between source contamination levels and culturable E. coli levels, determine which sources best explained model-based E. coli estimates above the BAV and detection of esp2 (a marker for the esp gene associated with pathogenic strains of Enterococcus faecium and Enterococcus faecalis) , and investigate associations between source contamination levels and land use features. Three BacteriALERT sites on the Chattahoochee River were sampled six times per season in the winter and summer from December 2015 through September 2017, and 11 additional stream sites (synoptic sites) from the CRNRA watershed were sampled once per season. Samples were screened with microbial source tracking (MST) quantitative PCR (qPCR) markers for humans (HF183 Taqman), dogs (DogBact), and ruminants (Rum2Bac), the esp2 qPCR marker, and culturable E. coli. At the BacteriALERT sites, HF183 Taqman concentrations were higher under wet conditions DogBact concentrations were greater in the winter and under wet conditions, and Rum2Bac concentrations were comparatively low throughout the study with no difference across seasons or precipitation conditions. Concentrations of HF183 Taqman, DogBact, and Rum2Bac were positively correlated with culturable E. coli concentrations; however, DogBact had the largest R 2 value among the three markers, and the forward stepwise regression indicated it was the best predictor of culturable E. coli concentrations at the BacteriALERT sites. Recursive partitioning indicated that BAV exceedances of model-based E. coli estimates were best explained by DogBact concentrations ≥3 gene copies per mL (CN/mL). Detections of esp2 at BacteriALERT sites were best explained by DogBact concentrations ≥11 CN/mL, while detections of esp2 at synoptic sites were best explained by HF183 Taqman ≥29 CN/mL. At the synoptic sites, HF183 Taqman levels were associated with wastewater treatment plant density. However, this relationship was driven primarily by a single site, suggesting possible conveyance issues in that catchment. esp2 detections at synoptic sites were positively associated with development within a 2-km radius and negatively associated with development within the catchment, suggesting multiple sources of esp2 in the watershed. DogBact and Rum2Bac were not associated with the land use features included in our analyses. Implications for Park management include: 1) fecal contamination levels were highest during wet conditions and in the off season when fewer visitors are expected to be participating in water-based recreation, 2) dogs are likely contributors to fecal contamination in the CRNRA and may be sources of pathogenic bacteria indicating further investigation of the origins of this contamination may be warranted as would be research to understand the human health risks from exposure to dog fecal contamination, and 3) high levels of the human marker at one site in the CRNRA watershed suggests more extensive monitoring in that catchment may locate the origin of human fecal contamination detected during this study.

Georgia↗
USGS2019

Adapterama II: Universal amplicon sequencing on Illumina platforms (TaggiMatrix)

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5’ end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (eight forward primers + 12 reverse primers yield 8 x 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

PeerJ↗
USGS2019

South Atlantic Water Science Center Strategic Science Plan: 2019–23

Executive Summary The South Atlantic Water Science Center Strategic Science Planning Team has developed a unified strategic science plan to guide the science vision of the South Atlantic Water Science Center (SAWSC) in response to the merging of the Georgia, North Carolina, and South Carolina Water Science Centers. This plan proposes a path forward to keep SAWSC science activities relevant to the many diverse needs of stakeholders in the South Atlantic region (Georgia, North Carolina, and South Carolina) and considers the hydrologic setting and issues of the region. This plan advises the creation of five working groups to address five priority science topics for the period 2019–23 and beyond. The five priority science topics are (1) Foundational Data, (2) Effects of Land-Use Change, (3) Coastal Plain Science, (4) Water Availability, and (5) Hazards. From the goals laid forth in this plan for each priority science topic, the working groups plan to devise a set of strategic actions and milestones to be achieved by the SAWSC to provide valuable and relevant data, research, and assessments in the South Atlantic region. In this report, the “South Atlantic region” is used to describe the area encompassed by the States of North Carolina, South Carolina, and Georgia.

Open-File Report↗
USGS2017

New distributional records of the stygobitic crayfish Cambarus cryptodytes (Decapoda: Cambaridae) in the Floridan Aquifer System of southwestern Georgia

Cambarus cryptodytes (Dougherty Plain Cave Crayfish) is an obligate inhabitant of groundwater habitats (i.e., a stygobiont) with troglomorphic adaptations in the Floridan aquifer system of southwestern Georgia and adjacent Florida panhandle, particularly in the Dougherty Plain and Marianna Lowlands. Documented occurrences of Dougherty Plain Cave Crayfish are spatially distributed as 2 primary clusters separated by a region where few caves and springs have been documented; however, the paucity of humanly accessible karst features in this intermediate region has inhibited investigation of the species' distribution. To work around this constraint, we employed bottle traps to sample for Dougherty Plain Cave Crayfish and other groundwater fauna in 18 groundwater-monitoring wells that access the Floridan aquifer system in 10 counties in southwestern Georgia. We captured 32 Dougherty Plain Cave Crayfish in 9 wells in 8 counties between September 2014 and August 2015. We detected crayfish at depths ranging from 17.9 m to 40.6 m, and established new county records for Early, Miller, Mitchell, and Seminole counties in Georgia, increasing the number of occurrences in Georgia from 8 to 17 sites. In addition, a new US Geological Survey (USGS) Hydrologic Unit Code 8 (HUC8) watershed record was established for the Spring Creek watershed. These new records fill in the distribution gap between the 2 previously known clusters in Georgia and Jackson County, FL. Furthermore, this study demonstrates that deployment of bottle traps in groundwater-monitoring wells can be an effective approach to presence—absence surveys of stygobionts, especially in areas where surface access to groundwater is limited.

Geogia↗
USGS2016

Detection of an enigmatic plethodontid Salamander using Environmental DNA

The isolation and identification of environmental DNA (eDNA) offers a non-invasive and efficient method for the detection of rare and secretive aquatic wildlife, and it is being widely integrated into inventory and monitoring efforts. The Patch-Nosed Salamander (Urspelerpes brucei) is a tiny, recently discovered species of plethodontid salamander known only from headwater streams in a small region of Georgia and South Carolina. Here, we present results of a quantitative PCR-based eDNA assay capable of detecting Urspelerpes in more than 75% of 33 samples from five confirmed streams. We deployed the method at 31 additional streams and located three previously undocumented populations of Urspelerpes. We compare the results of our eDNA assay with our attempt to use aquatic leaf litterbags for the rapid detection of Urspelerpes and demonstrate the relative efficacy of the eDNA assay. We suggest that eDNA offers great potential for use in detecting other aquatic and semi-aquatic plethodontid salamanders.

Copeia↗
USGS2015

Assessment of environmental DNA for detecting presence of imperiled aquatic amphibian species in isolated wetlands

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.

Alabama, Florida, Georgia, South Carolina↗