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      Repurposing environmental DNA samples—detecting the western pearlshell ( Margaritifera falcata) as a proof of concept

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          Abstract

          Information on the distribution of multiple species in a common landscape is fundamental to effective conservation and management. However, distribution data are expensive to obtain and often limited to high‐profile species in a system. A recently developed technique, environmental DNA ( eDNA) sampling, has been shown to be more sensitive than traditional detection methods for many aquatic species. A second and perhaps underappreciated benefit of eDNA sampling is that a sample originally collected to determine the presence of one species can be re‐analyzed to detect additional taxa without additional field effort. We developed an eDNA assay for the western pearlshell mussel ( Margaritifera falcata) and evaluated its effectiveness by analyzing previously collected eDNA samples that were annotated with information including sample location and deposited in a central repository. The eDNA samples were initially collected to determine habitat occupancy by nonbenthic fish species at sites that were in the vicinity of locations recently occupied by western pearlshell. These repurposed eDNA samples produced results congruent with historical western pearlshell surveys and permitted a more precise delineation of the extent of local populations. That a sampling protocol designed to detect fish was also successful for detecting a freshwater mussel suggests that rapidly accumulating collections of eDNA samples can be repurposed to enhance the efficiency and cost‐effectiveness of aquatic biodiversity monitoring.

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          Next-generation monitoring of aquatic biodiversity using environmental DNA metabarcoding.

          Global biodiversity in freshwater and the oceans is declining at high rates. Reliable tools for assessing and monitoring aquatic biodiversity, especially for rare and secretive species, are important for efficient and timely management. Recent advances in DNA sequencing have provided a new tool for species detection from DNA present in the environment. In this study, we tested whether an environmental DNA (eDNA) metabarcoding approach, using water samples, can be used for addressing significant questions in ecology and conservation. Two key aquatic vertebrate groups were targeted: amphibians and bony fish. The reliability of this method was cautiously validated in silico, in vitro and in situ. When compared with traditional surveys or historical data, eDNA metabarcoding showed a much better detection probability overall. For amphibians, the detection probability with eDNA metabarcoding was 0.97 (CI = 0.90-0.99) vs. 0.58 (CI = 0.50-0.63) for traditional surveys. For fish, in 89% of the studied sites, the number of taxa detected using the eDNA metabarcoding approach was higher or identical to the number detected using traditional methods. We argue that the proposed DNA-based approach has the potential to become the next-generation tool for ecological studies and standardized biodiversity monitoring in a wide range of aquatic ecosystems.
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            Molecular Detection of Vertebrates in Stream Water: A Demonstration Using Rocky Mountain Tailed Frogs and Idaho Giant Salamanders

            Stream ecosystems harbor many secretive and imperiled species, and studies of vertebrates in these systems face the challenges of relatively low detection rates and high costs. Environmental DNA (eDNA) has recently been confirmed as a sensitive and efficient tool for documenting aquatic vertebrates in wetlands and in a large river and canal system. However, it was unclear whether this tool could be used to detect low-density vertebrates in fast-moving streams where shed cells may travel rapidly away from their source. To evaluate the potential utility of eDNA techniques in stream systems, we designed targeted primers to amplify a short, species-specific DNA fragment for two secretive stream amphibian species in the northwestern region of the United States (Rocky Mountain tailed frogs, Ascaphus montanus, and Idaho giant salamanders, Dicamptodon aterrimus). We tested three DNA extraction and five PCR protocols to determine whether we could detect eDNA of these species in filtered water samples from five streams with varying densities of these species in central Idaho, USA. We successfully amplified and sequenced the targeted DNA regions for both species from stream water filter samples. We detected Idaho giant salamanders in all samples and Rocky Mountain tailed frogs in four of five streams and found some indication that these species are more difficult to detect using eDNA in early spring than in early fall. While the sensitivity of this method across taxa remains to be determined, the use of eDNA could revolutionize surveys for rare and invasive stream species. With this study, the utility of eDNA techniques for detecting aquatic vertebrates has been demonstrated across the majority of freshwater systems, setting the stage for an innovative transformation in approaches for aquatic research.
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              Robust Detection of Rare Species Using Environmental DNA: The Importance of Primer Specificity

              Environmental DNA (eDNA) is being rapidly adopted as a tool to detect rare animals. Quantitative PCR (qPCR) using probe-based chemistries may represent a particularly powerful tool because of the method’s sensitivity, specificity, and potential to quantify target DNA. However, there has been little work understanding the performance of these assays in the presence of closely related, sympatric taxa. If related species cause any cross-amplification or interference, false positives and negatives may be generated. These errors can be disastrous if false positives lead to overestimate the abundance of an endangered species or if false negatives prevent detection of an invasive species. In this study we test factors that influence the specificity and sensitivity of TaqMan MGB assays using co-occurring, closely related brook trout (Salvelinus fontinalis) and bull trout (S. confluentus) as a case study. We found qPCR to be substantially more sensitive than traditional PCR, with a high probability of detection at concentrations as low as 0.5 target copies/µl. We also found that number and placement of base pair mismatches between the Taqman MGB assay and non-target templates was important to target specificity, and that specificity was most influenced by base pair mismatches in the primers, rather than in the probe. We found that insufficient specificity can result in both false positive and false negative results, particularly in the presence of abundant related species. Our results highlight the utility of qPCR as a highly sensitive eDNA tool, and underscore the importance of careful assay design.
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                Author and article information

                Contributors
                jdysthe@fs.fed.us
                Journal
                Ecol Evol
                Ecol Evol
                10.1002/(ISSN)2045-7758
                ECE3
                Ecology and Evolution
                John Wiley and Sons Inc. (Hoboken )
                2045-7758
                05 February 2018
                March 2018
                : 8
                : 5 ( doiID: 10.1002/ece3.2018.8.issue-5 )
                : 2659-2670
                Affiliations
                [ 1 ] U.S. Forest Service National Genomics Center for Wildlife and Fish Conservation Rocky Mountain Research Station Missoula MT USA
                [ 2 ] Ecology Center and Wildland Resources Department Utah State University Logan UT USA
                Author notes
                [*] [* ] Correspondence

                Joseph C. Dysthe, U.S. Forest Service, National Genomics Center for Wildlife and Fish Conservation, Rocky Mountain Research Station, Missoula, MT, USA.

                Email: jdysthe@ 123456fs.fed.us

                Author information
                http://orcid.org/0000-0002-6790-7841
                http://orcid.org/0000-0003-2904-005X
                Article
                ECE33898
                10.1002/ece3.3898
                5838043
                29531684
                0ee653ee-de4f-4b74-b922-bff7a9023132
                © 2018 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.

                This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 19 October 2017
                : 20 December 2017
                : 11 January 2018
                Page count
                Figures: 1, Tables: 5, Pages: 12, Words: 8483
                Funding
                Funded by: National Genomics Center for Wildlife and Fish Conservation – USDA Forest Service Rocky Mountain Research Station
                Funded by: USDA Forest Service Beaverhead‐Deer Lodge National Forest
                Categories
                Original Research
                Original Research
                Custom metadata
                2.0
                ece33898
                March 2018
                Converter:WILEY_ML3GV2_TO_NLMPMC version:version=5.3.2.2 mode:remove_FC converted:05.03.2018

                Evolutionary Biology
                noninvasive sampling,quantitative pcr,sample archive
                Evolutionary Biology
                noninvasive sampling, quantitative pcr, sample archive

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