SPECIES DETECTION: AMPHIBIANS
FROGS AND TOADS
Stevenson B. C., Borchers D. L., Altwegg R., Swift R. J., Gillespie D. M., Measey G. J. (2015): A general framework for animal density estimation from acoustic detections across a fixed microphone array. Methods in Ecology and Evolution 6: 38-48.
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Acoustic monitoring can be an efficient, cheap, non-invasive alternative to physical trapping of individuals. Spatially explicit capture–recapture (SECR) methods have been proposed to estimate calling animal abundance and density from data collected by a fixed array of microphones. However, these methods make some assumptions that are unlikely to hold in many situations, and the consequences of violating these are yet to be investigated. We generalize existing acoustic SECR methodology, enabling these methods to be used in a much wider variety of situations. We incorporate time-of-arrival (TOA) data collected by the microphone array, increasing the precision of calling animal density estimates. We use our method to estimate calling male density of the Cape Peninsula Moss Frog Arthroleptella lightfooti. Our method gives rise to an estimator of calling animal density that has negligible bias, and 95% confidence intervals with appropriate coverage. We show that using TOA information can substantially improve estimate precision. Our analysis of the A. lightfooti data provides the first statistically rigorous estimate of calling male density for an anuran population using a microphone array. This method fills a methodological gap in the monitoring of frog populations and is applicable to acoustic monitoring of other species that call or vocalize.
Secondi J., Dejean T., Valentini A., Audebaud B., Miaud C. (2016): Detection of a global aquatic invasive amphibian, Xenopus laevis, using environmental DNA. Amphibia-Reptilia 37: 131-136.
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Detection is crucial in the study and control of invasive species but it may be limited by methodological issues. In amphibians, classical survey techniques exhibit variable detection probability depending on species and are often constrained by climatic conditions often requiring several site visits. Furthermore, detection may be reduced at low density because probability capture (passive traps), or activity (acoustic surveys) drop. Such limits may impair the study of invasive species because low density is typical of the onset of colonisation on a site. In the last few years, environmental DNA (eDNA) methods have proved their ability to detect the presence of aquatic species. We developed here an eDNA method to detect Xenopus laevis in ponds. This austral African species is now present worldwide because of its use in biology and as a pet. Populations have settled and expanded on several continents so that it is now considered as one of the major invasive amphibians in the World. We detected the presence of X. laevis at density as low as 1 ind/100 m2 and found a positive relationship between density in ponds and rate of DNA amplification. Results show that eDNA can be successfully applied to survey invasive populations of X. laevis even at low density in order to confirm suspected cases of introduction, delimit the expansion of a colonized range, or monitor the efficiency of a control program.
Measey G. J., Stevenson B. C., Scott T., Altwegg R., Borchers D. L. (2017). Counting chirps: acoustic monitoring of cryptic frogs. Journal of Applied Ecology 54: 894-902.
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Global amphibian declines have resulted in a vital need for monitoring programmes that follow population trends. Monitoring using advertisement calls is ideal as choruses are undisturbed during data collection. However, methods currently employed by managers frequently rely on trained observers and/or do not provide density data on which to base trends. This study explores the utility of monitoring using acoustic spatially explicit capture–recapture (aSCR) with time of arrival (ToA) and signal strength (SS) as a quantitative monitoring technique to measure call density of a threatened but visually cryptic anuran, the Cape peninsula moss frog Arthroleptella lightfooti. The relationships between temporal and climatic variables (date, rainfall, temperature) and A. lightfooti call density at three study sites on the Cape peninsula, South Africa, were examined. Acoustic data, collected from an array of six microphones over 4 months during the winter breeding season, provided a time series of call density estimates. Model selection indicated that call density was primarily associated with seasonality fitted as a quadratic function. Call density peaked mid-breeding season. At the main study site, the lowest recorded mean call density (0·160 calls m−2 min−1) occurred in May and reached its peak mid-July (1·259 calls m−2 min−1). The sites differed in call density, but also the effective sampling area. The monitoring technique, acoustic spatially explicit capture–recapture (aSCR), quantitatively estimates call density of calling animals without disturbing them or their environment. In addition, time of arrival (ToA) and signal strength (SS) data significantly add to the accuracy of call localization, which in turn increases precision of call density estimates without the need for specialist field staff. This technique appears ideally suited to aid the monitoring of visually cryptic, acoustically active species.
Bailey L. L., Jones P., Thompson K. G., Foutz H. P., Logan J. M., Wright F. B., Crockett H. J. (2019): Determining presence of rare amphibian species: Testing and combining novel survey methods. Journal of Herpetology 53: 115-124.
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Data deficiencies exist for ∼20–25% of amphibian and reptile species worldwide, typically excluding them from conservation consideration and funding opportunities. Current species status is often unknown at historic locations or previously unsurveyed areas. We present an iterative study to assess and optimize novel survey methods for a declining amphibian species (Boreal Toad, Anaxyrus boreas boreas) using a combination of methods. We found that if toads are present in a drainage, searching riparian areas multiple times during the active season is an efficient way of detecting the occurrence of previously unknown breeding populations. Once a breeding population has been located, traditional visual encounter surveys yield high probabilities of species detection for monitoring efforts (p̂vis ≈ 0.80). Supplementing streamside surveys when toads are not detected with other survey methods (e.g., environmental DNA [eDNA] samples) at suitable breeding locations can help confirm the species is absent, provided ≥10 eDNA samples are collected. Moreover, employing both visual surveys and eDNA samples can simultaneously yield distributional information on amphibian species and target pathogens, if pathogen presence is evaluated for all captured amphibian species and environmental samples are tested for both amphibian and pathogen DNA. Our iterative process of designing, testing, optimizing, and combining sampling methods to determine current species distribution should serve as a model for other rare amphibian and reptile species and provide managers better information with which to plan mitigation and conservation efforts.
Lin M., Zhang S., Yao M. (2019): Effective detection of environmental DNA from the invasive American bullfrog. Biological Invasions 21: 2255-2268.
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The American bullfrog (Rana catesbeiana) is one of the most damaging invasive species worldwide. Recently developed environmental DNA (eDNA)-based methods have proven to be promising tools to detect aquatic invasive species at low abundance. To develop an eDNA-based assay for bullfrog monitoring, we designed a primer and probe set that targeted the bullfrog DNA and compared its species specificity in in silico PCR and in vitro quantitative PCR. We demonstrated that our primers successfully amplified the bullfrog DNA, but not DNA of native anuran species in the Beijing area, in silico and in vitro. Additionally, in silico PCR identified only a small number of potentially amplifiable species occurring in North America. In contrast, a previously reported primer pair that specifically targeted the bullfrog in France amplified the DNA of a wide range of teleost fish and amphibians in silico and a common native species in vitro. We also compared three commonly used eDNA extraction methods—the phenol–chloroform–isoamyl alcohol (PCI) method, cetyltrimethylammonium bromide (CTAB)-based extraction, and Qiagen’s DNeasy blood and tissue kit—for their efficiency in recovering bullfrog eDNA from laboratory aquariums. The CTAB method showed a significantly higher extraction efficacy than the PCI method or the DNeasy kit and considerably outperformed the other methods in terms of time efficiency and costs. Our study provides a useful primer set and extraction protocol for eDNA-based active surveillance of bullfrog invasions over a potentially wide geographical range, which has important implications for the management of this invasive species.
Tingley R., Greenlees M., Oertel S., van Rooyen A. R., Weeks A. R. (2019): Environmental DNA sampling as a surveillance tool for cane toad Rhinella marina introductions on offshore islands. Biological Invasions 21: 1-6.
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Containing the spread of established invasive species is critical for minimizing their ecological impact. Effective containment requires sensitive sampling methods capable of detecting new introductions when invaders are at low density. Here we explore whether environmental DNA (eDNA) sampling could be used as a surveillance tool to detect new incursions of aquatic invasive species on offshore islands. We develop an eDNA molecular assay for invasive cane toads (Rhinella marina) in Australia, validate our assay on the mainland, and apply it to an offshore island (Moreton Island) that is a target of ongoing cane toad surveillance. Our eDNA assay correctly identified four mainland sites at which cane toads were observed, as well as a fifth site within 1 km of known populations. Five additional sites outside the cane toad’s current distribution tested negative for cane toad eDNA. Site occupancy detection models indicated that two water samples and three qPCR replicates were sufficient to achieve a cumulate detection probability > 0.95. Applying our eDNA assay to samples from 19 sites on an offshore island over a 2-year period revealed the absence of cane toad eDNA, in line with our current understanding of cane toad distribution. Our results suggest that eDNA sampling could be strategically applied to meet the Australian Commonwealth’s objective of maintaining cane toad-free offshore islands.
Hobbs J., Adams I. T., Round J. M., Goldberg C. S., Allison M. J., Bergman L. C., Mirabzadeh A., Allen H., Helbing C. C. (2020): Revising the range of Rocky Mountain tailed frog, Ascaphus montanus, in British Columbia, Canada, using environmental DNA methods. Environmental DNA 2: 350-361.
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The Rocky Mountain tailed frog, Ascaphus montanus, is a species at-risk in Canada. Based upon time- and area-constrained physical search surveys completed between 1996 and 2004, its Canadian distribution was defined as occurring in 19 tributaries and reaches within the Yahk and west side Flathead River Basins of British Columbia. We undertook a five-year (2014–2018 inclusive) environmental DNA (eDNA) survey to reassess the distribution of Rocky Mountain tailed frog, focusing on tributaries proximal to known extant occurrence records. Seventeen days of field sampling were performed over the five-year period. Targeted qPCR-based eDNA approaches proved more effective than conventional physical search methods for detecting tailed frogs due to relatively rapid field collection, low cost of filter materials, elimination of observer bias, and higher detection probabilities compared to conventional time-constrained survey methods. One hundred and forty sites were examined (138 for eDNA plus two visual only). Thirty-two of the 138 sites (23%) tested positive for Rocky Mountain tailed frog DNA, including from the four extant populations sampled, whereas visual observations occurred at only seven of the sites (5%) during the survey. During the study, we evaluated two tailed frog tests and the mitigation of false negatives through testing for qPCR inhibition and sample degradation, and we demonstrate their utility in evaluating eDNA data quality. These results expand the extant range of Rocky Mountain tailed frog in the Flathead, Wigwam, and Yahk watersheds and add two new watersheds (Moyie and Tepee) by identifying five newly recorded occupied drainages in Canada: Elder Creek, Upper Wigwam River, Tepee Creek, Gilnockie Creek, and Elmer Creek. These data are important to refine and augment wildlife habitat conservation areas for Rocky Mountain tailed frog.
Bedwell M. E., Hopkins K. V., Dillingham C., Goldberg C. S. (2021): Evaluating Sierra Nevada yellow‐legged frog distribution using environmental DNA. The Journal of Wildlife Management 85: 945-952.
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Genetic tools that identify species from trace DNA samples could supplement traditional survey methods to clarify distributional limits of rare species. For species with legal habitat protection, elevational limits of distributions are used to determine where management actions may affect endangered species. The endangered Sierra Nevada yellow-legged frog (Rana sierrae) generally is found down to 1,370 m, but in the Plumas National Forest, California, USA, there are a number of historical records below this elevation, resulting in protections extending to 1,067 m. This species is phenotypically similar to the foothill yellow-legged frog (R. boylii), with which it occasionally hybridizes. We used a combination of genetic methods to investigate the fine-scale distribution of the Sierra Nevada yellow-legged frog in the Plumas National Forest. We collected and analyzed environmental DNA (eDNA) samples from all accessible lower elevation sites with records of Sierra Nevada yellow-legged frog (n = 17) and swabbed 220 individuals for genetic identification from 2016–2018 to clarify the distribution of this endangered species. We created a climatic suitability model using the validated Sierra Nevada yellow-legged frog records and current (1970–2000) climate models to assess additional highly suitable localities for Sierra Nevada yellow-legged frog presence using eDNA capture. We did not confirm detection of Sierra Nevada yellow-legged frog eDNA at any historical sites and identified all swabbed individuals from below 1,370 m (n = 144) as foothill yellow-legged frogs. We located a new Sierra Nevada yellow-legged frog site (at 1,919 m) during surveys guided by the climatic suitability model. It does not appear after extensive eDNA and genetic sampling that the Sierra Nevada yellow-legged frog occurs below 1,370 m in this portion of their range at present. Our results show that eDNA sampling can be used as an effective management tool to evaluate historical locations and previously unknown suitable localities for current presence of a species of interest.
Barata I. M., Razafindraibe J. H., Ravelojaona R. N., Ralovarisoa E., Mullin K. E., Hudson M. A., Dawson J. (2022): First population estimates of two Critically Endangered frogs from an isolated forest plateau in Madagascar. Oryx 56: 897-903.
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In the largely deforested areas of Madagascar, small forest fragments remain as last refuges for amphibian diversity. Isolated populations of the Critically Endangered Anodonthyla vallani and Anilany helenae persist in the fragmented forest of Ambohitantely but little information is available to inform their management and any conservation interventions. We generated estimates of population size and occupancy for both species in the largest fragment of Ambohitantely Special Reserve using acoustic survey data collected from 84 sites along 12 transects in December 2018. We used a single-season occupancy model to estimate detection and occupancy and a Royal–Nichols model to estimate abundance and population size. Anilany helenae and A. vallani had high occupancy rates (80 and 93%, respectively) whereas their detection rates differed (34 and 55%, respectively). Abundance and occupancy were best explained by vegetation structure whereas detection was influenced by time of survey and rainfall. For our sampled sites the estimated population sizes of males were 855 for A. vallani, with an estimated density of 52 individuals/ha, and 388 for A. helenae, with an estimated population density of 23 individuals/ha. Given their relatively low densities, small population sizes and restricted ranges, any further habitat loss could have drastic consequences for these populations. Our results provide guidance for future species-focused studies, and can inform conservation management at the local scale. Our work will help to improve species monitoring in Madagascar and elsewhere, especially for range-restricted non-charismatic amphibians.
Breton B. A. A., Beaty L., Bennett A. M., Kyle C. J., Lesbarrères D., Vilaça S. T., Wikston M. J., Wilson C. C., Murray D. L. (2022): Testing the effectiveness of environmental DNA (eDNA) to quantify larval amphibian abundance. Environmental DNA 4: 1229-1240.
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Environmental DNA (eDNA) monitoring is rapidly becoming an established approach for detecting the presence of aquatic organisms and may also be useful for indexing or estimating species abundance. However, the link between eDNA concentration and abundance of individuals (i.e., density or biomass) remains tenuous and may vary widely across species and environmental conditions. We investigated the relationship between eDNA concentration and abundance in two common and closely related amphibians in eastern North America, the wood frog (Rana sylvatica), and northern leopard frog (R. pipiens). We manipulated tadpole density in 80-L mesocosms and documented the relationship between tadpole density and biomass and eDNA concentration through time. The two species differed in the amount of detectible genetic material produced, despite having comparable biomass. Concentration of eDNA increased with tadpole numbers and was primarily correlated with tadpole density in wood frogs and biomass in leopard frogs. eDNA degradation rates were rapid and comparable between species, with tadpoles becoming indetectable within 5 days post-removal from the mesocosm, irrespective of tadpole density. Overall, our findings support that eDNA concentration has potential for tracking amphibian abundance in wetlands, but that indices of abundance are likely to be coarse and species-specific calibration will be required. Future research should address how biotic and abiotic factors influence eDNA production, degradation, and recovery across species and through time before relying on eDNA for monitoring amphibian abundance in nature.
Dubois‐Gagnon M. P., Bernatchez L., Bélisle M., Dubois Y., Mazerolle M. J. (2022): Distribution of the boreal chorus frog (Pseudacris maculata) in an urban environment using environmental DNA. Environmental DNA 4: 326-338.
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The boreal chorus frog (Pseudacris maculata) is at risk of extinction in parts of its range in Canada. Our objectives were to quantify the influence of local and landscape characteristics on the occurrence of the species in wetlands in southern Québec. We hypothesized that site occupancy depends on local characteristics and landscape characteristics contributing to site connectivity. We developed an environmental DNA (eDNA) method to detect the species and compared the detection probability of this method to traditional call surveys. We collected water samples from a total of 180 sites (90 in 2017, 110 in 2018), whereas we surveyed a subset of 63 sites using both eDNA and call surveys in 2018. Site occupancy varied across years, but was higher in sites where the species had been previously detected during the last 12 years by other studies. Site occupancy did not vary with other local and landscape characteristics, in part due to an apparent decrease in the number of sites occupied by the species since the last 12 years. Detection probability via eDNA (0.81; 95% CI: [0.31; 0.98]) did not differ from that of call surveys (0.62; 95% CI: [0.25; 0.89]). To identify the optimal sampling period for the boreal chorus frog, future studies should estimate the detection probability of eDNA during the breeding season and the larval development period of the species.
Everts T., Van Driessche C., Neyrinck S., De Regge N., Descamps S., De Vocht A., Jacquemyn H., Brys R. (2022): Using quantitative eDNA analyses to accurately estimate American bullfrog abundance and to evaluate management efficacy. Environmental DNA 4: 1052-1064.
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Biological invasions contribute now more than ever to the global homogenization of fauna and flora. Large-scale monitoring programs are, therefore, needed to detect incipient invasions and to evaluate management interventions. As conventional monitoring methods are constrained by large costs, environmental DNA (eDNA)-based methods are increasingly recognized as valuable monitoring tools. However, accurately estimating species abundance from eDNA concentrations in natural systems remains challenging and consequently hinders their integration in management applications. Here, we used droplet digital PCR (ddPCR) in eDNA surveys to estimate the abundance of invasive American bullfrogs (Lithobates catesbeianus). We first introduced bullfrog tadpoles in natural ponds to assess the relationship between abundances and eDNA concentrations under field conditions. Next, we combined eDNA sampling with fyke netting in naturally colonized ponds to investigate whether bullfrog eDNA concentrations can estimate bullfrog capture success and conventional abundance measures obtained via depletion sampling. Finally, we evaluated eradication measures by comparing bullfrog eDNA concentrations before and after fyke netting. We found a strong linear relationship between the numbers of introduced tadpoles and eDNA concentrations (r2 = 0.988). Bullfrog eDNA concentrations were not only linearly related to the catch-per-unit-effort (r2 = 0.739), but also to conventional abundance estimates (r2 = 0.716), particularly when eDNA concentrations were standardized for pond area (r2 = 0.834) and volume (r2 = 0.888). Bullfrog tadpoles were only captured when eDNA concentrations exceeded 1.5 copies µl−1, indicating that quantitative eDNA analyses enable the localization of breeding ponds. We found a significant reduction in eDNA concentrations after fyke netting proportional to the number of captured bullfrogs. These results demonstrate that eDNA quantification is a reliable tool that accurately estimates bullfrog abundance in natural lentic systems. We show that quantitative eDNA analyses can complement the toolbox of natural resource managers and facilitate the coordination of eradication campaigns targeting alien invasive species.
Falaschi M., Gibertini C., Lo Parrino E., Muraro M., Barzaghi B., Manenti R., Ficetola G. F. (2022): Assessing population trends of species with imperfect detection: double count analyses and simulations confirm reliable estimates in brown frogs. Animals 12: 2085.
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Most animal species are detected imperfectly and overlooking individuals can result in a biased inference of the abundance patterns and underlying processes. Several techniques can incorporate the imperfect detection process for a more accurate estimation of abundance, but most of them require repeated surveys, i.e., more sampling effort compared to single counts. In this study, we used the dependent double-observer approach to estimate the detection probability of the egg clutches of two brown frog species, Rana dalmatina and R. latastei. We then simulated the data of a declining population at different levels of detection probability in order to assess under which conditions the double counts provided better estimates of population trends compared to naïve egg counts, given the detectability of frog clutches. Both species showed a very high detection probability, with average values of 93% for Rana dalmatina and 97% for R. latastei. Simulations showed that not considering imperfect detection reduces the power of detecting population trends if detection probability is low. However, at high detection probability (>80%), ignoring the imperfect detection does not bias the estimates of population trends. This suggests that, for species laying large and easily identifiable egg clutches, a single count can provide useful estimates if surveys are correctly timed.
Moss W. E., Harper L. R., Davis M. A., Goldberg C. S., Smith M. M., Johnson P. T. (2022): Navigating the trade‐offs between environmental DNA and conventional field surveys for improved amphibian monitoring. Ecosphere 13: e3941.
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The need for efficient, accurate biodiversity monitoring is growing, especially for globally imperiled taxa, such as amphibians. Environmental DNA (eDNA) analysis holds enormous potential for enhancing monitoring programs, but as this tool is increasingly adopted, it is imperative for users to understand its potential benefits and shortcomings. We conducted a comparative study to evaluate the efficacy of two eDNA methodologies (quantitative (q)PCR and metabarcoding) and conventional field sampling approaches (seining, dipnetting, and visual encounter surveys) in a system of 20 ponds containing six different amphibian species. Using an occupancy modeling framework, we estimated differences in detection sensitivity across methods, with a focus on how eDNA survey design could be further optimized. Overall, both metabarcoding and qPCR were competitive with or improved upon conventional methods. Specifically, qPCR (species-specific approach) was the most effective technique for detecting two rare species, the California tiger salamander (Ambystoma californiense) and California red-legged frog (Rana draytonii), with a detection probability of >0.80 per survey. Metabarcoding (community approach) estimated amphibian diversity with comparable rates to field techniques on average, and detected an additional 41 vertebrate taxa. However, for two abundant species (western toads, Anaxyrus boreas, and Pacific chorus frogs, Pseudacris regilla), field techniques outperformed metabarcoding, especially as individuals metamorphosed. Our results indicate that eDNA approaches would be most effective when paired with visual encounter surveys to detect terrestrial life stages, and that more optimization, specifically primer choice and validation, is needed. By comparing methods across a diverse set of ponds and species, we provide guidance for future studies integrating eDNA approaches into amphibian monitoring.
Mullin K. E., Barata I. M., Dawson J., Orozco-terWengel P. (2022): First extraction of eDNA from tree hole water to detect tree frogs: a simple field method piloted in Madagascar. Conservation Genetics Resources 14: 99-107.
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Environmental DNA (eDNA) is becoming an increasingly used tool for monitoring cryptic species within terrestrial and aquatic systems. We present the first method for extracting water from tree holes for eDNA studies of tree-dwelling frogs, and the first use of eDNA for amphibian monitoring in Madagascar. This pilot study expands on a previously developed method and aims to provide a simple field protocol for DNA extraction from very small water samples, using a relatively inexpensive kit compared to other collection methods. We collected 20 ml of water from tree holes in Ambohitantely Special Reserve in Madagascar, with the aim to survey for the Critically Endangered tree frog Anodonthyla vallani, and we developed species specific cytochrome c oxidase 1 primers for this species. While our two samples did not detect A. vallani, we successfully extracted up to 16.6 ng/µl of eDNA from the samples and using 16S rRNA primers barcoded the tree frog Plethodontohyla mihanika in one of the samples. Despite just two samples being collected, we highlight the future potential of eDNA from tree holes for investigating cryptic habitat specialist amphibians given we extracted frog eDNA from just 20 ml of water. The method provides a rapid, simple, and cost-effective method which can assist cryptic species monitoring in challenging and time-consuming field conditions and should be developed further for frog surveying in Madagascar and beyond. The newly developed primers can be used for further work using this eDNA method to survey threatened Anodonthyla frog species.
Perl R. G. B., Avidor E., Roll U., Malka Y., Geffen E., Gafny S. (2022): Using eDNA presence/non‐detection data to characterize the abiotic and biotic habitat requirements of a rare, elusive amphibian. Environmental DNA 4: 642-653.
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The environmental DNA (eDNA) approach has already been established as a valuable tool for the detection and monitoring of rare and elusive species. However, its application is not limited to assessing whether or not a species is present in a given area. In this study, we collected environmental data from 48 aquatic locations that had previously been investigated in an eDNA-based study. We sought to determine the abiotic and biotic factors that could explain the presence or non-detection of Hula painted frog DNA at those locations, in order to characterize this rare species’ little studied habitat requirements. We found that the detection probability of this species decreased substantially with increasing phosphorus loads as well as in the presence of the wetland plant Lythrum salicaria. By contrast, the detection probability increased markedly when Phragmites australis or Ludwigia stolonifera constituted part of the dominant aquatic vegetation. Our results expand the knowledge on this elusive frog species and contribute valuable information for future habitat restoration plans. They further show that eDNA data can also be used to characterize the putative habitats of species where such data are scarce or even totally lacking.
Saeed M., Rais M., Akram A., Williams M. R., Kellner K. F., Hashsham S. A., Davis D. R. (2022): Development and validation of an eDNA protocol for monitoring endemic Asian spiny frogs in the Himalayan region of Pakistan. Scientific Reports 12: 5624.
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Wildlife monitoring programs are instrumental for the assessment of species, habitat status, and for the management of factors affecting them. This is particularly important for species found in freshwater ecosystems, such as amphibians, as they have higher estimated extinction rates than terrestrial species. We developed and validated two species-specific environmental DNA (eDNA) protocols and applied them in the field to detect the Hazara Torrent Frog (Allopaa hazarensis) and Murree Hills Frog (Nanorana vicina). Additionally, we compared eDNA surveys with visual encounter surveys and estimated site occupancy. eDNA surveys resulted in higher occurrence probabilities for both A. hazarensis and N. vicina than for visual encounter surveys. Detection probability using eDNA was greater for both species, particularly for A. hazarensis. The top-ranked detection model for visual encounter surveys included effects of both year and temperature on both species, and the top-ranked occupancy model included effects of elevation and year. The top-ranked detection model for eDNA data was the null model, and the top-ranked occupancy model included effects of elevation, year, and wetland type. To our knowledge, this is the first time an eDNA survey has been used to monitor amphibian species in the Himalayan region.
Chen Y., Tournayre O., Tian H., Lougheed S. C. (2023): Assessing the breeding phenology of a threatened frog species using eDNA and automatic acoustic monitoring. PeerJ 11: e14679.
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Climate change has driven shifts in breeding phenology of many amphibians, causing phenological mismatches (e.g., predator-prey interactions), and potentially population declines. Collecting data with high spatiotemporal sensitivity on hibernation emergence and breeding times can inform conservation best practices. However, monitoring the phenology of amphibians can be challenging because of their cryptic nature over much of their life cycle. Moreover, most salamanders and caecilians do not produce conspicuous breeding calls like frogs and toads do, presenting additional monitoring challenges. In this study, we designed and evaluated the performance of an environmental DNA (eDNA) droplet digital PCR (ddPCR) assay as a non-invasive tool to assess the breeding phenology of a Western Chorus Frog population (Pseudacris maculata mitotype) in Eastern Ontario and compared eDNA detection patterns to hourly automatic acoustic monitoring. For two eDNA samples with strong PCR inhibition, we tested three methods to diminish the effect of inhibitors: diluting eDNA samples, adding bovine serum albumin to PCR reactions, and purifying eDNA using a commercial clean-up kit. We recorded the first male calling when the focal marsh was still largely frozen. Chorus frog eDNA was detected on April 6th, 6 days after acoustic monitoring revealed this first calling male, but only 2 days after males attained higher chorus activity. eDNA signals were detected at more sampling locales within the marsh and eDNA concentrations increased as more males participated in the chorus, suggesting that eDNA may be a reasonable proxy for calling assemblage size. Internal positive control revealed strong inhibition in some samples, limiting detection probability and quantification accuracy in ddPCR. We found diluting samples was the most effective in reducing inhibition and improving eDNA quantification. Altogether, our results showed that eDNA ddPCR signals lagged behind male chorusing by a few days; thus, acoustic monitoring is preferable if the desire is to document the onset of male chorusing. However, eDNA may be an effective, non-invasive monitoring tool for amphibians that do not call and may provide a useful complement to automated acoustic recording. We found inhibition patterns were heterogeneous across time and space and we demonstrate that an internal positive control should always be included to assess inhibition for eDNA ddPCR signal interpretations.
SALAMANDERS AND NEWTS
Olson Z. H., Briggler J. T., Williams R. N. (2012): An eDNA approach to detect eastern hellbenders (Cryptobranchus a. alleganiensis) using samples of water. Wildlife Research 39: 629-636.
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Environmental DNA, or eDNA, methods are a novel application of non-invasive genetic sampling in which DNA from organisms is detected via sampling of water or soil, typically for the purposes of determining the presence or absence of an organism. eDNA methods have the potential to revolutionise the study of rare or endangered taxa. We evaluated the efficacy of eDNA sampling to detect populations of an amphibian of conservation concern, the eastern hellbender (Cryptobranchus a. alleganiensis), indirectly from their aquatic environments. We developed species-specific primers, validated their specificity and sensitivity, and assessed the utility of our methods in silico and in laboratory trials. In the field, we collected water samples from three sites with known densities of hellbenders, and from one site where hellbenders do not occur. We filtered water samples, extracted DNA from filters, and assayed the extraction products for hellbender DNA by using polymerase chain reaction (PCR) and gel electrophoresis. Our methods detected hellbenders at densities approaching the lowest of reported natural densities. The low-density site (0.16 hellbenders per 100 m2) yielded two positive amplifications, the medium-density site (0.38 hellbenders per 100 m2) yielded eight positive amplifications, and the high-density site (0.88 hellbenders per 100 m2) yielded 10 positive amplifications. The apparent relationship between density and detection was obfuscated when river discharge was considered. There was no amplification in any negative control. eDNA methods may represent a cost-effective means by which to establish broad-scale patterns of occupancy for hellbenders. eDNA can be considered a valuable tool for detecting many species that are otherwise difficult to study.
Rees H. C., Bishop K., Middleditch D. J., Patmore J. R., Maddison B. C., Gough K. C. (2014): The application of eDNA for monitoring of the Great Crested Newt in the UK. Ecology and Evolution 4: 4023-4032.
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Current ecological surveys for great crested newts are time-consuming and expensive and can only be carried out within a short survey window. Additional survey methods which would facilitate the detection of rare or protected species such as the great crested newt (Triturus cristatus) would be extremely advantageous. Environmental DNA (eDNA) analysis has been utilized for the detection of great crested newts in Denmark. Here, the same methodology has been applied to water samples taken from UK ponds concurrently with conventional field surveying techniques. Our eDNA analysis exhibited an 84% success rate with a kappa coefficient of agreement between field and eDNA surveys of 0.86. One pond determined to be negative for great crested newt by field survey was positive by eDNA analysis, revealing the potential for improved detection rates using this methodology. Analysis of water samples collected in late summer indicates that eDNA analysis could be used to detect great crested newt after the optimal survey window for current field techniques had passed. Consequently, eDNA analysis could augment currently stipulated techniques for great crested newt surveying as a relatively quick and inexpensive tool for collecting great crested newt presence and distribution data within the UK instead of or prior to full field surveys.
Biggs J., Ewald N., Valentini A., Gaboriaud C., Dejean T., Griffiths R. A., Foster J., Wilkinson J. W., Arnell A., Brotherton P., Williams P., Dunn F. (2015): Using eDNA to develop a national citizen science-based monitoring programme for the great crested newt (Triturus cristatus). Biological Conservation 183: 19-28.
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The use of environmental DNA (eDNA) is rapidly emerging as a potentially valuable survey technique for rare or hard to survey freshwater organisms. For the great crested newt (Triturus cristatus) in the UK, the substantial cost and manpower requirements of traditional survey methods have hampered attempts to assess the status of the species. We tested whether eDNA could provide the basis for a national citizen science-based monitoring programme for great crested newts by (i) comparing the effectiveness of eDNA monitoring with torch counts, bottle trapping and egg searches and (ii) assessing the ability of volunteers to collect eDNA samples throughout the newt’s UK range. In 35 ponds visited four times through the breeding season, eDNA detected newts on 139 out of 140 visits, a 99.3% detection rate. Bottle traps, torch counts and egg searches were significantly less effective, detecting newts 76%, 75% and 44% of the time. eDNA was less successful at predicting newt abundance being positively, but weakly, correlated with counts of the number of newts. Volunteers successfully collected eDNA samples across the UK with 219 of 239 sites (91.3%) correctly identified as supporting newts. 8.7% of sites generated false negatives, either because of very small newt populations or practical difficulties in sample collection. There were no false positives. Overall, we conclude that eDNA is a highly effective survey method and could be used as the basis for a national great crested newt monitoring programme.
Pierson T. W., McKee A. M., Spear S. F., Maerz J. C., Camp C. D., Glenn T. C. (2016): Detection of an enigmatic Plethodontid salamander using environmental DNA. Copeia 104: 78-82.
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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.
Katano I., Harada K., Doi H., Souma R., Minamoto T. (2017): Environmental DNA method for estimating salamander distribution in headwater streams, and a comparison of water sampling methods. Plos One 12: e0176541.
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Environmental DNA (eDNA) has recently been used for detecting the distribution of macroorganisms in various aquatic habitats. In this study, we applied an eDNA method to estimate the distribution of the Japanese clawed salamander, Onychodactylus japonicus, in headwater streams. Additionally, we compared the detection of eDNA and hand-capturing methods used for determining the distribution of O. japonicus. For eDNA detection, we designed a qPCR primer/probe set for O. japonicus using the 12S rRNA region. We detected the eDNA of O. japonicus at all sites (with the exception of one), where we also observed them by hand-capturing. Additionally, we detected eDNA at two sites where we were unable to observe individuals using the hand-capturing method. Moreover, we found that eDNA concentrations and detection rates of the two water sampling areas (stream surface and under stones) were not significantly different, although the eDNA concentration in the water under stones was more varied than that on the surface. We, therefore, conclude that eDNA methods could be used to determine the distribution of macroorganisms inhabiting headwater systems by using samples collected from the surface of the water.
Rees H. C., Baker C. A., Gardner D. S., Maddison B. C., Gough K. C. (2017): The detection of great crested newts year round via environmental DNA analysis. BMC Research Notes 10: 327.
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Analysis of environmental DNA (eDNA) is a method that has been used for the detection of various species within water bodies. The great crested newt (Triturus cristatus) has a short eDNA survey season (mid-April to June). Here we investigate whether this season could be extended into other months using the current methodology as stipulated by Natural England. Here we present data to show that in monthly water samples taken from two ponds (March 2014–February 2015) we were able to detect great crested newt DNA in all months in at least one of the ponds. Similar levels of great crested newt eDNA (i.e. highly positive identification) were detected through the months of March–August, suggesting it may be possible to extend the current survey window. In order to determine how applicable these observations are for ponds throughout the rest of the UK, further work in multiple other ponds over multiple seasons is suggested. Nevertheless, the current work clearly demonstrates, in two ponds, the efficacy and reproducibility of eDNA detection for determining the presence of great crested newts.
Walker D. M., Leys J. E., Dunham K. E., Oliver J. C., Schiller E. E., Stephenson K. S., Kimrey J. T., Wooten J., Rogers M. W. (2017): Methodological considerations for detection of terrestrial small-body salamander eDNA and implications for biodiversity conservation. Molecular Ecology Resources 17: 1223-1230.
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Environmental DNA (eDNA) can be used as an assessment tool to detect populations of threatened species and provide fine-scale data required to make management decisions. The objectives of this project were to use quantitative PCR (qPCR) to: (i) detect spiked salamander DNA in soil, (ii) quantify eDNA degradation over time, (iii) determine detectability of salamander eDNA in a terrestrial environment using soil, faeces, and skin swabs, (iv) detect salamander eDNA in a mesocosm experiment. Salamander eDNA was positively detected in 100% of skin swabs and 66% of faecal samples and concentrations did not differ between the two sources. However, eDNA was not detected in soil samples collected from directly underneath wild-caught living salamanders. Salamander genomic DNA (gDNA) was detected in all qPCR reactions when spiked into soil at 10.0, 5.0, and 1.0 ng/g soil and spike concentration had a significant effect on detected concentrations. Only 33% of samples showed recoverable eDNA when spiked with 0.25 ng/g soil, which was the low end of eDNA detection. To determine the rate of eDNA degradation, gDNA (1 ng/g soil) was spiked into soil and quantified over seven days. Salamander eDNA concentrations decreased across days, but eDNA was still amplifiable at day 7. Salamander eDNA was detected in two of 182 mesocosm soil samples over 12 weeks (n = 52 control samples; n = 65 presence samples; n = 65 eviction samples). The discrepancy in detection success between experiments indicates the potential challenges for this method to be used as a monitoring technique for small-bodied wild terrestrial salamander populations.
Harper L. R., Lawson Handley L., Hahn C., Boonham N., Rees H. C., Gough K. C., Lewis E., Adams I. P., Brotherton P., Phillips S., Hänfling B. (2018): Needle in a haystack? A comparison of eDNA metabarcoding and targeted qPCR for detection of the great crested newt (Triturus cristatus). Ecology and Evolution 8: 6330-6341.
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Environmental DNA (eDNA) analysis is a rapid, cost-effective, non-invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species-specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and “metabarcoding” have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real-time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using high-throughput sequencing technology. With qPCR and a detection threshold of 1 of 12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4 of 12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species-specific surveys.
Sakai Y., Kusakabe A., Tsuchida K., Tsuzuku Y., Okada S., Kitamura T., Tomita S., Mukai T., Tagami M., Takagi M., Yaoi Y. (2019): Discovery of an unrecorded population of Yamato salamander (Hynobius vandenburghi) by GIS and eDNA analysis. Environmental DNA 1: 281-289.
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Biodiversity loss is a serious environmental problem, and human activities might be primarily responsible for the marked decline in animal populations globally. Amphibians, in particular, have significantly decreased in number in recent decades. One example is the endangered Yamato salamander (Hynobius vandenburghi), which is distributed in Central Japan and has a very restricted distribution in Gifu Prefecture. We aimed to discover new populations of H. vandenburghi using a combination of GIS and environmental DNA (eDNA) analysis. Firstly, we designed two primer sets for amplifying Hynobius species targeting mitochondrial 12S rRNA and cytochrome b genes. Next, we performed aquarium experiments to detect H. vandenburghi DNA in aquarium water. We also conducted sequential eDNA detection surveys in five known habitats in Gifu City, Japan, 17 times from January to August 2016. Following these basic eDNA studies, we used GIS to characterize the vegetation and topography of known habitats of H. vandenburghi. We collected water samples in the potential habitats identified by GIS and analyzed eDNA for the presence of H. vandenburghi using the designed primers. Finally, we conducted physical collection surveys in these potential habitats. We successfully developed two Hynobius-specific primer sets and detected H. vandenburghi eDNA in aquarium water. The eDNA of the target species was detected in almost all (94%–100%) samples from four habitats, whereas only 29% of samples were positive for one habitat. We identified five potential habitats by GIS analysis. The DNA of H. vandenburghi was detected in three of five potential habitats. Finally, we discovered a new population in one of the potential sites. Our approach combining GIS and eDNA enabled the detection of novel population of an endangered amphibian species. This study was conducted by high school students under the supervision of teachers with the help of university researchers, suggesting the applicability of eDNA studies as a tool of citizen science. The combination of GIS and eDNA will allow to detect cryptic populations on which conservation efforts may be focused and to alert people to the need for conservation action.
Witzel N. A., Taheri A., Miller B. T., Hardman R. H., Withers D. I., Spear S. F., Sutton W. B. (2020): Validation of an environmental DNA protocol to detect a stream‐breeding amphibian, the Streamside Salamander (Ambystoma barbouri). Environmental DNA 2: 554-564.
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Environmental DNA (eDNA), or DNA that is shed into the environment by an organism, can be used to detect the presence of cryptic species. However, eDNA methodology requires validation of an assay in both laboratory and field environments. Here, we describe the development of a quantitative PCR (qPCR) assay and field protocol for detecting a secretive amphibian, the Streamside Salamander (Ambystoma barbouri). This fossorial species is rarely encountered because adults are only active for several months during the winter when they breed and deposit eggs underneath rocks within intermittent streams. We designed and validated a qPCR assay for A. barbouri against five ambystomatid congeners and the Southern Two-lined Salamander (Eurycea cirrigera), which occur within the range of A. barbouri in the focal study area. We detected DNA of A. barbouri in the laboratory to 0.0004 ng/µl, while all other congeners were rarely detected below 40 ng/µl. We collected 1-L water samples from 45 streams from December 2016 to May 2017 and during April 2018 to validate our methods in the field. Our assay was effective at detecting A. barbouri in 24 of 45 streams. We confirmed physical presence of A. barbouri (adults, larvae, and/or eggs) at 21 out of 24 of these positive sites. The detection probability was 0.85 ± 0.05; CI: 0.75, 0.95 within a single sampling event that incorporated 5 water samples from each of 17 repeat-visit sites. The per water sample sensitivity was 68% (163/238) and specificity was 100% (22/22). The per site visit sensitivity was 85% (46/54) and specificity was 100% (6/6). Lastly, the per site sensitivity was 95% (21/22) and specificity was 100% (3/3). Collectively, this protocol outlines an efficient and cost-effective method to detect A. barbouri and provides a technique to rapidly identify sites where breeding is occurring and to determine the distribution of this species.
Osathanunkul M., Minamoto T. (2021): eDNA‐based detection of a vulnerable crocodile newt (Tylototriton uyenoi) to influence government policy and raise public awareness. Diversity and Distributions 27: 1958-1965.
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Although at least five Tylototriton species were recorded in Thailand, only Tylototriton verrucosus is registered as a protected species under the Wildlife Preservation and Protection Act in Thailand. Populations of T. uyenoi are now severely declining, caused by anthropogenic activities. As intense human pressure is having profound effects on the diminishment in T. uyenoi populations, a conservation plan is needed. Information such as the abundance and distribution of a species is necessary. Yet, current established survey methods are either time-consuming or labour-intensive. Here, eDNA-based detection was developed for tracking the presence of the T. uyenoi. We target the surveillance of T. uyenoi using eDNA. Primers and a probe specific to T. uyenoi were designed and tested for their specificity and sensitivity. Water samples were collected once a month from August to January at three sites in Doi Suthep and at three extra sites within the range of the species. Three hundred ml samples of water were collected and filtered. Environmental DNA was extracted and then subjected to qPCR assay in an attempt to detect T. uyenoi. The qPCR assay was found to be species-specific to T. uyenoi. Both PCR and qPCR did not result in any positive detection of three congeners or other non-target species. The LOD and the LOQ of the assay were determined by an analysis of the standard curve, and it was found that the LOD and the LOQ were 7.99 and 9.0 copies per reaction, respectively. Environmental DNA was detected in water samples from all sites where T. uyenoi has been known to occur, but detection varied among sites and sampling times. In addition, low amounts of eDNA were detected in three sites with unknown occupancy of newts, but within the species’ range. Our findings suggest that eDNA survey is a powerful tool for tracking T. uyenoi throughout the year regardless of the sampling site conditions. Similar to other amphibians, T. uyenoi is severely declining due to anthropogenic factors. In order to have an effective conservation plan, knowledge of a species’ distribution is needed. To our knowledge, this was the first study that used eDNA to track the crocodile newt in Thailand.
Plante F., Bourgault P., Dubois Y., Bernatchez L. (2021): Environmental DNA as a detection and quantitative tool for stream‐dwelling salamanders: A comparison with the traditional active search method. Environmental DNA 3: 1128-1141.
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As amphibians are showing significant signs of decline, adequate information and understanding of target species are essential for taking appropriate conservation measures. In recent years, environmental DNA has seen notable growth as a monitoring tool and testing this emergent method with various species has become an important step toward a better understanding of its benefits and limits for studying specific taxa. This study focused on using species-specific qPCR assays developed in our research group to test the eDNA method for three stream-dwelling salamander species of the Plethodontidae family: the Spring salamander (Gyrinophilus porphyriticus), the Northern dusky salamander (Desmognathus fuscus), and the Northern two-lined salamander (Eurycea bislineata). The traditional active search method and the eDNA method were compared for both their ability to detect species as well as to provide a quantitative assessment of populations in 24 headwater streams in Québec, eastern Canada. For all three species, eDNA was detected in every stream where the target species was observed during the active search method. Moreover, eDNA was detected in nine streams where the target species was not identified with the active search. A marginally significant association was found between eDNA concentration and salamander density for D. fuscus only. All species showed high variability for eDNA concentration between qPCR technical replicates and between samples of a given stream. The results of this study lead us to conclude that eDNA can be an excellent method for detection of stream-dwelling salamanders. Given the inconsistent quantitative aspect of eDNA with the studied species, the future of these stream-dwelling salamander monitoring most likely lies in the combined use of both eDNA and active search methods. Hence, active search could continue to offer useful small-scale detection and reliable quantitative data while eDNA could be implemented as an efficient and promising tool for large-scale detection.
Bell F. G., Flores A. F., Sena K. L., Maigret T. A., Leow C. J., Sams R. B., Peyton D. K., Brammell B. F. (2022): Development and validation of qPCR assays for use in eDNA detection of Southern Two-lined (Eurycea cirrigera) and Norher Dusky (Desmognathus fuscus) salamanders. Herpetological Conservation and Biology 17: 398-412.
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Environmental DNA (eDNA) uses DNA shed from organisms into the environment to detect their presence and provides an effective, non-invasive method to survey organisms in an efficient manner. Recent works have emphasized the need for careful development and both laboratory and field validation of eDNA assays. We developed species specific qPCR assays for Southern Two-lined (Eurycea cirrigera) and Northern Dusky (Desmognathus fuscus) salamanders, two salamanders found throughout much of the eastern USA. We designed primers and probes based on sequences obtained from locally collected specimens and screened for specificity against 19 salamander species that occur sympatrically with these species in various parts of their range. We collected 38 water samples from streams in a minimally disturbed forest in eastern Kentucky, USA, and we analysed samples for both E. cirrigera and D. fuscus DNA. There were 16 samples that were positive for E. cirrigera and 21 for D. fuscus DNA. We cloned and sequenced four E. cirrigera and five D. fuscus amplicons from filtered water to verify identity. These data add to the growing pool of knowledge concerning eDNA monitoring of salamander species and provide useful reference data as well as valuable molecular tools for future monitoring and range delineation studies.
Grimm-Seyfarth A. (2022): Environmental and training factors affect canine detection probabilities for terrestrial newt surveys. Journal of Veterinary Behavior 57: 6-15.
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Amphibians are of great interest to scientific research, but many populations are highly threatened and declining worldwide. Although varieties of aquatic survey methods exist, traditional methods for terrestrial surveys are very time-consuming but often not very effective. A novel method to detect terrestrial amphibians is the use of wildlife detection dogs. While their use for and factors affecting detection rates of mammals and reptiles are well documented, scientific literature on amphibian detection dogs is just emerging. The purpose of this study was to investigate the effects of environmental (habitat, weather) and training factors on detection probabilities for a newt detection dog. An experienced wildlife detection dog was trained and tested on smooth (Lissotriton vulgaris) and great crested newts (Triturus cristatus). Environmental and training parameters were recorded for 101 test trials and used as explanatory parameters in a binomial GLM. I found that detection probability strongly depended on temperature, whereby optimal temperatures varied by habitat. Detection probabilities were lowest in short grass, but there was no strong difference among forest habitats. They were higher for males than females and for great crested than smooth newts. For this dog, detection probabilities were also higher if the dog was working off the leash than on it, and when the dog was cooperative than fatigued. Dog performance increased over time with a strong increase at the beginning and a plateau at 92% detection probability. However, detection rates of this specific dog slightly decreased when the dog was working for more than two hours. The findings of this study provide a valuable basis for future deployments of this and other amphibian detection dogs. Dogs can certainly work in a variety of different habitats, although directed off-leash searching with enough time in complex habitats and specific training for small species with low detection distances may enhance their performance. The study design might also consider the temperature and humidity at which the dog will be deployed. A regular assessment of the detection dog using blind tests will give an indication of its reliability. Assessments similar to this study may further be used to estimate detection probability for a particular dog under given field conditions. Regular blind tests will show when the detection rates reached a plateau, which may then give an indication of on its reliability. Altogether, results suggest that newt detection dogs may provide a highly promising survey method, which can certainly be transferred for detecting other amphibians in terrestrial habitats.
Kaganer A. W., Stapleton G. S., Bunting E. M., Hare M. P. (2022): Aquatic eDNA can advance monitoring of a small‐bodied terrestrial salamander and amphibian pathogen. Environmental DNA 4: 1164-1175.
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Successful conservation efforts for threatened species depend on accurate characterization of their distribution, habitat use, and threats. However, surveillance can be challenging for species with small size, cryptic coloring, or elusive behavior. Environmental DNA (eDNA) monitoring can provide a sensitive and noninvasive alternative to traditional surveillance techniques by detecting trace DNA shed by a target species into their environment. We evaluated the efficacy of eDNA monitoring for the four-toed salamander (Hemidactylium scutatum), a terrestrial salamander threatened throughout much of its range. Additionally, we integrated eDNA monitoring for ranavirus, a widespread pathogen of ectothermic vertebrates, to efficiently evaluate a potential disease threat to H. scutatum. We designed a novel species-specific quantitative PCR (qPCR) assay for H. scutatum, multiplexed the assay with a previously developed ranavirus qPCR assay, and validated the multiplexed tests in silico, in vitro, and in situ. We collected aquatic eDNA weekly for 8 weeks from pools with historical reports of H. scutatum (n = 6) and contemporary reports of H. scutatum on associated uplands (n = 4). We identified H. scutatum eDNA at low quantities (< 12 copies/reaction) on multiple sampling days in two pools with historic species presence. Ranavirus eDNA was widespread; nine pools tested positive including the pools where H. scutatum eDNA was detected. Ranavirus eDNA quantities increased, peaked, then decreased (i.e., were not sporadic in time) in several pools but never rose in abundance to levels associated with ranavirosis in other studies. Our results suggest that H. scutatum eDNA is distributed sporadically at low quantities in pools; we recommend that future surveillance efforts prioritize increased frequency and quantity of sample collection per site visit and include repeat surveys of candidate sites. Further work to characterize Ranavirus eDNA movement, temporal trends, relationship to infectious virion, and decay is needed to help advance this tool.
Hoy M. S., Ostberg C. O. (2023): Development of a quantitative PCR assay for detecting northwest salamander (Ambystoma gracile) in environmental DNA samples. Conservation Genetics Resources 15: 109-112.
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We developed a primer and probe based quantitative PCR assay for use with environmental DNA to detect Northwest salamander (Ambystoma gracile), a species endemic to the temperate Pacific coastal region of North America. The assay targets a region in the mitochondrial DNA (mtDNA) D-loop gene. Tests of the assay were performed in silico (using the NCBI BLAST tool), in vitro (using DNA extracted from A. gracile and related species) and in situ (water samples collected from a lake with a known population of A. gracile). This assay will be useful in efforts to monitor the distribution and occupancy of this species.
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Knudsen S. W., Hesselsøe M., Rytter M., Lillemark M. R., Tøttrup A. P., Rahbek C., Sheard J. K., Thomsen P. F., Agersnap S., Mortensen P. B., Møller P. R. (2023): Detection of environmental DNA from amphibians in Northern Europe applied in citizen science. Environmental DNA.
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Many species of amphibians in Northern Europe are threatened and the local distributions are rarely described in detail. Application of modern molecular methods provides an important supplementary tool for monitoring the distribution and diversity of amphibians. For this purpose, we designed, tested, validated, and optimized 14 species-specific assays on genomic DNA extracted from tissue samples to use for quantitative polymerase chain reaction (qPCR) setups targeting mitochondrial DNA from amphibians in freshwater samples. The tests confirmed species specificity for all assays. Considering a systematic definition of the limit of detection for each of the assays, the presented qPCR assays are unlikely to return false positive detection from any co-occurring species in northern Europe. For field validation, the qPCR assays were applied in a large-scale nationwide citizen science project in which sampling and qPCR analysis was carried out by high school students. Data from the citizen science project returned the expected results when compared to the known regional distribution of the target species and confirmed the presence of nine out of 14 Danish species of amphibians in the collected freshwater samples. Four out of 2550 qPCR test sets carried out by the high school students required a professional reanalysis in multiple replicates due to initial unexpected results. This emphasizes that efforts from citizen science may generate large amounts of valuable data, as long as the results are carefully scrutinized by experts.