SPECIES DETECTION: REPTILES
CROCODILES
Shirley M. H., Dorazio R. M., Abassery E., Elhady A. A., Mekki M. S., Asran H. H. (2012): A sampling design and model for estimating abundance of Nile crocodiles while accounting for heterogeneity of detectability of multiple observers. The Journal of Wildlife Management 76: 966-975.
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As part of the development of a management program for Nile crocodiles in Lake Nasser, Egypt, we used a dependent double-observer sampling protocol with multiple observers to compute estimates of population size. To analyze the data, we developed a hierarchical model that allowed us to assess variation in detection probabilities among observers and survey dates, as well as account for variation in crocodile abundance among sites and habitats. We conducted surveys from July 2008–June 2009 in 15 areas of Lake Nasser that were representative of 3 main habitat categories. During these surveys, we sampled 1,086 km of lake shore wherein we detected 386 crocodiles. Analysis of the data revealed significant variability in both inter- and intra-observer detection probabilities. Our raw encounter rate was 0.355 crocodiles/km. When we accounted for observer effects and habitat, we estimated a surface population abundance of 2,581 (2,239–2,987, 95% credible intervals) crocodiles in Lake Nasser. Our results underscore the importance of well-trained, experienced monitoring personnel in order to decrease heterogeneity in intra-observer detection probability and to better detect changes in the population based on survey indices. This study will assist the Egyptian government establish a monitoring program as an integral part of future crocodile harvest activities in Lake Nasser.
Ezat M. A., Fritsch C. J., Downs C. T. (2018): Use of an unmanned aerial vehicle (drone) to survey Nile crocodile populations: A case study at Lake Nyamithi, Ndumo game reserve, South Africa. Biological Conservation 223: 76-81.
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Observer bias and inexperience are challenging aspects of crocodile survey methods for determining population numbers and structure. Aerial surveys with either a helicopter or a fixed winged aircraft are generally preferred methods to ground surveys; however, the high cost of the former is a limiting factor. Recently unmanned aerial vehicles (UAVs) or drones have been proposed for surveys because of their potential of improving over traditional techniques of wildlife monitoring and as they have relatively lower costs. We investigated of the suitability of a UAV to determine numbers and structure of the Nile crocodile, Crocodylus niloticus, population during winter at Lake Nyamithi, Ndumo Game Reserve in South Africa. We used the UAV for eight flights covering ~132 ha. We also conducted a diurnal ground survey of crocodiles for comparison. Using the UAV, 287 crocodiles were identified and body length measured accurately for size class allocation whereas only 211 crocodiles were counted in the diurnal ground survey. Consequently, the UAV aerial survey recorded 26% more crocodiles. The potential of using UAVs to estimate crocodile population size and measure the total length (TL) of individuals accurately and precisely at a relatively low cost should improve management actions, enable monitoring of the crocodile populations annually and importantly avoid observer bias. Implications of this may facilitate improved crocodilian survey techniques.
Naveda-Rodríguez A., Utreras V., Zapata-Ríos G. (2020): A standardised monitoring protocol for the black caiman (Melanosuchus niger). Wildlife Research 47: 317-325.
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Estimating population abundance can be plagued by the violation of methodological assumptions, which can be overcome with standardised protocols. The black caiman (BC) is considered a conservation-dependent species, and previous abundance estimates are surrounded by uncertainty and flaws in the survey (e.g. different survey design and efforts) and analytical approach used (e.g. relative abundance index, which ignores imperfect detection). Its conservation status assessment demands the implementation of a standardised monitoring protocol. The protocol provides guidelines to collect and analyse data in a consistent manner to survey BC. Besides accounting for imperfect detection to produce reliable abundance estimates, the protocol aimed to be easily implemented by park rangers, and to fit field observations into a hierarchical modelling approach to assess how environmental variables affects detectability and abundance. The protocol subdivides a 20-km transect into 10 2-km segments; each transect is surveyed four consecutive nights, starting at 1900 hours and finishing when the 20 km are completed. For each caiman detected, the observers estimated head size to classify the individual by age. We tested the protocol in Ecuador during January and December 2017, and field data were analysed using N-mixture models. We compared abundance estimates derived with this protocol with commonly used relative abundance indexes. We surveyed 460 km that resulted in 177 detections. Percentage of moonlight and distance from human settlement best explained detectability and abundance respectively. Mean detection probability was 0.14 (95% BCI: 0.1–0.18), whereas absolute abundance was 196 (95% BCI: 147–370). The overall adult to immature ratio was 1 : 1.3. This is the first estimate of detectability and absolute abundance for BC by using a standardised survey with a clearly defined and repeatable survey and analysis methods. Relative abundance indexes did not reflect absolute abundance estimates. We recommend the use of this protocol in future surveys across the Amazon region to effectively evaluate BC conservation status. Population size cannot be estimated from relative abundance indexes; they lead to bias estimates for ignoring imperfect detection. We discourage the use of relative abundance indexes to evaluate the conservation status of this species.
Rose A., Fukuda Y., Campbell H. A. (2020): Using environmental DNA to detect estuarine crocodiles, a cryptic-ambush predator of humans. Human–Wildlife Interactions 14: 64-72.
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Negative human–wildlife interactions can be better managed by early detection of the wildlife species involved. However, many animals that pose a threat to humans are highly cryptic, and detecting their presence before the interaction occurs can be challenging. We describe a method whereby the presence of the estuarine crocodile (Crocodylus porosus), a cryptic and potentially dangerous predator of humans, was detected using traces of DNA shed into the water, known as environmental DNA (eDNA). The estuarine crocodile is present in waterways throughout Southeast Asia and Oceania and has been responsible for >1,000 attacks upon humans in the past decade. A critical factor in the crocodile’s capability to attack humans is their ability to remain hidden in turbid waters for extended periods, ambushing humans that enter the water or undertake activities around the waterline. In northern Australia, we sampled water from aquariums where crocodiles were present or absent, and we were able to discriminate the presence of estuarine crocodile from the freshwater crocodile (C. johnstoni), a closely related sympatric species that does not pose a threat to humans. Further, we could detect the presence of estuarine crocodiles within an hour of its entry and up to 72 hours after the crocodiles were removed from aquariums. We conclude that eDNA could be a valuable tool for reducing human–wildlife conflict through early detection of the species.
Ahizi M. N., Kouman C. Y., Ouattara A., Kouamé N. D. P., Dede A., Fairet E., Shirley M. H. (2021): Detectability and impact of repetitive surveys on threatened West African crocodylians. Ecology and Evolution 11: 15062-15076.
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West African crocodylians are among the most threatened and least studied crocodylian species globally. Assessing population status and establishing a basis for population monitoring is the highest priority action for this region. Monitoring of crocodiles is influenced by many factors that affect detectability, including environmental variables and individual- or population-level wariness. We investigated how these factors affect detectability and counts of the critically endangered Mecistops cataphractus and the newly recognized Crocodylus suchus. We implemented 195 repetitive surveys at 38 sites across Côte d’Ivoire between 2014 and 2019. We used an occupancy-based approach and a count-based GLMM analysis to determine the effect of environmental and anthropogenic variables on detection and modeled crocodile wariness over repetitive surveys. Despite their rarity and level of threat, detection probability of both species was relatively high (0.75 for M. cataphractus and 0.81 for C. suchus), but a minimum of two surveys were required to infer absence of either species with 90% confidence. We found that detection of M. cataphractus was significantly negatively influenced by fishing net encounter rate, while high temperature for the previous 48 h of the day of the survey increased C. suchus detection. Precipitation and aquatic vegetation had significant negative and positive influence, respectively, on M. cataphractus counts and showed the opposite effect for C. suchus counts. We also found that fishing encounter rate had a significant negative effect on C. suchus counts. Interestingly, survey repetition did not generally affect wariness for either species, though there was some indication that at least M. cataphractus was more wary by the fourth replicate. These results are informative for designing future survey and monitoring protocols for these threatened crocodylians in West Africa and for other endangered crocodylians globally.
LIZARDS
Ariefiandy A., Purwandana D., Seno A., Ciofi C., Jessop T. S. (2013): Can camera traps monitor Komodo dragons a large ectothermic predator?. Plos One 8: e58800.
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Camera trapping has greatly enhanced population monitoring of often cryptic and low abundance apex carnivores. Effectiveness of passive infrared camera trapping, and ultimately population monitoring, relies on temperature mediated differences between the animal and its ambient environment to ensure good camera detection. In ectothermic predators such as large varanid lizards, this criterion is presumed less certain. Here we evaluated the effectiveness of camera trapping to potentially monitor the population status of the Komodo dragon (Varanus komodoensis), an apex predator, using site occupancy approaches. We compared site-specific estimates of site occupancy and detection derived using camera traps and cage traps at 181 trapping locations established across six sites on four islands within Komodo National Park, Eastern Indonesia. Detection and site occupancy at each site were estimated using eight competing models that considered site-specific variation in occupancy (ψ)and varied detection probabilities (p) according to detection method, site and survey number using a single season site occupancy modelling approach. The most parsimonious model [ψ (site), p (site*survey); ω = 0.74] suggested that site occupancy estimates differed among sites. Detection probability varied as an interaction between site and survey number. Our results indicate that overall camera traps produced similar estimates of detection and site occupancy to cage traps, irrespective of being paired, or unpaired, with cage traps. Whilst one site showed some evidence detection was affected by trapping method detection was too low to produce an accurate occupancy estimate. Overall, as camera trapping is logistically more feasible it may provide, with further validation, an alternative method for evaluating long-term site occupancy patterns in Komodo dragons, and potentially other large reptiles, aiding conservation of this species.
Reinhardt T., van Schingen M., Windisch H. S., Nguyen T. Q., Ziegler T., Fink P. (2019): Monitoring a loss: Detection of the semi‐aquatic crocodile lizard (Shinisaurus crocodilurus) in inaccessible habitats via environmental DNA. Aquatic Conservation: Marine and Freshwater Ecosystems 29: 353-360.
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Assessing the conservation status of a species is strongly dependent upon data on species distribution and abundance. With the emergence of novel methods for species monitoring – such as the use of environmental DNA (eDNA) – monitoring success can be improved at reduced expenditure in the field, particularly in remote regions and terrains where access is difficult or dangerous. The highly endangered crocodile lizard (Shinisaurus crocodilurus Ahl, 1930) inhabits fragmented sites of the remaining evergreen forest with running water systems in a narrow distribution range in southern China and north‐east Vietnam. Crocodile lizards spend most of the day within or above water bodies, which are commonly remote and inaccessible. To monitor recent spatial occurrences, and to confirm the persistence or extinction of previously reported populations (especially in heavily altered habitats), the suitability of using eDNA and quantitative polymerase chain reaction (qPCR) was tested as an alternative method for monitoring this semiaquatic lizard. To assess the accuracy and limitations of this method, eDNA results from the field were compared with eDNA data from mesocosms and census data on the actual abundance of this species in the field. Environmental DNA of the crocodile lizard was detected in all of the positive controls, and in four of six natural sites; thus, all data collected using traditional field surveys were confirmed with eDNA results. eDNA monitoring was found to be a reliable method for assessing the viability of populations; we suggest that it should be developed as a tool for efficient wildlife management, particularly under difficult field and funding conditions.
Welbourne D. J., Claridge A. W., Paull D. J., Ford F. (2020): Camera-traps are a cost-effective method for surveying terrestrial squamates: A comparison with artificial refuges and pitfall traps. Plos One 15: e0226913.
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Fundamental data on the distributions, diversity, and threat status of terrestrial snakes and lizards (hereafter squamates) is limited. This is due to the cryptic nature of species in this faunal group, and to limitations in the effectiveness of the survey methods used to detect these species. Camera-traps are a useful tool for detecting numerous vertebrate species, yet their use for detecting squamates has been limited. Here, we apply recent methodological advancements in camera-trapping and assessed the utility of camera-traps for inventorying a squamate assemblage by comparing camera-trapping survey results with two widely used labour-intensive methods: artificial refuges and pitfall traps. We conducted a 74-day survey using camera-traps and, concurrently, four by four-day surveys using labour-intensive methods. Given the duration and three detection methods, we compared seven variants of survey protocol, including using each method alone or all methods simultaneously. We compared both the effectiveness and cost-effectiveness of each survey protocol by estimating the number of species detected at the transect level, and by calculating the costs of conducting those surveys. We found the camera-trapping survey was most cost-effective, costing 687 AUD (CI 534–912) per squamate species detected, compared with the 2975 AUD (CI 2103–4486) per squamate species detected with the labour-intensive methods. Using all methods together was less cost-effective than using camera-traps alone. Additionally, there was a 99% probability that camera-traps would detect more species per transect than the labour-intensive methods examined. By focusing the analysis at the level of the survey, rather than the level of the device, camera-traps are both a more effective and cost-effective technique for surveying terrestrial squamates. Where circumstances are appropriate, those wildlife researchers and managers currently using camera-traps for non-squamate surveys, can adopt the methods presented to incorporate squamate surveys with little upfront cost. Additionally, researchers currently using traditional techniques can be confident that switching to camera-traps will likely yield improved results. Still, camera-traps are not a panacea and careful consideration into the benefits and usefulness of these techniques in individual circumstances is required.
Purwandana D., Ariefiandy A., Azmi M., Nasu S. A., Dos A. A., Jessop T. S. (2021): Turning ghosts into dragons: improving camera monitoring outcomes for a cryptic low-density Komodo dragon population in eastern Indonesia. Wildlife Research 49: 295-302.
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Detection probability is a key attribute influencing population-level wildlife estimates necessary for conservation inference. Increasingly, camera traps are used to monitor threatened reptile populations and communities. Komodo dragon (Varanus komodoensis) populations have been previously monitored using camera traps; however, considerations for improving detection probability estimates for very low-density populations have not been well investigated. Here we compare the effects of baited versus non-baited camera monitoring protocols to influence Komodo dragon detection and occupancy estimates alongside monitoring survey design and cost considerations for ongoing population monitoring within the Wae Wuul Nature Reserve on Flores Island, Indonesia. Twenty-six camera monitoring stations (CMS) were deployed throughout the study area with a minimum of 400 m among CMS to achieve independent sampling units. Each CMS was randomly assigned as a baited or non-baited camera monitoring station and deployed for 6 or 30 daily sampling events. Baited camera monitoring produced higher site occupancy estimates with reduced variance. Komodo dragon detection probability estimates were 0.15 ± 0.092–0.22 (95% CI), 0.01 ± 0.001–0.03, and 0.03 ± 0.01–0.04 for baited (6 daily survey sampling events), unbaited (6 daily survey sampling events) and long-unbaited (30 daily survey sampling events) sampling durations respectively. Additionally, the provision of baited lures at cameras had additional benefits for Komodo detection, survey design and sampling effort costs. Our study indicated that baited cameras provide the most effective monitoring method to survey low-density Komodo dragon populations in protected areas on Flores. We believe our monitoring approach now lends itself to evaluating population responses to ecological and anthropogenic factors, hence informing conservation efforts in this nature reserve.
Kyle K. E., Allen M. C., Dragon J., Bunnell J. F., Reinert H. K., Zappalorti R., Lockwood J. L. (2022): Combining surface and soil environmental DNA with artificial cover objects to improve terrestrial reptile survey detection. Conservation Biology 36: e13939.
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Reptiles are increasingly of conservation concern due to their susceptibility to habitat loss, emerging disease, and harvest in the wildlife trade. However, reptile populations are often difficult to monitor given the frequency of crypsis in their life history. This difficulty has left uncertain the conservation status of many species and the efficacy of conservation actions unknown. Environmental DNA (eDNA) surveys consistently elevate the detection rate of species they are designed to monitor, and while their use is promising for terrestrial reptile conservation, successes in developing such surveys have been sparse. We tested the degree to which inclusion of surface and soil eDNA sampling into conventional artificial-cover methods elevates the detection probability of a small, cryptic terrestrial lizard, Scincella lateralis. The eDNA sampling of cover object surfaces with paint rollers elevated per sample detection probabilities for this species 4–16 times compared with visual surveys alone. We readily detected S. lateralis eDNA under cover objects up to 2 weeks after the last visual detection, and at some cover objects where no S. lateralis were visually observed in prior months. With sufficient sampling intensity, eDNA testing of soil under cover objects produced comparable per sample detection probabilities as roller surface methods. Our results suggest that combining eDNA and cover object methods can considerably increase the detection power of reptile monitoring programs, allowing more accurate estimates of population size, detection of temporal and spatial changes in habitat use, and tracking success of restoration efforts. Further research into the deposition and decay rates of reptile eDNA under cover objects, as well as tailored protocols for different species and habitats, is needed to bring the technique into widespread use.
Madani G., Pietsch R., Beranek C. T. (2023): Where are my dragons? Replicating refugia to enhance the detection probability of an endangered cryptic reptile. Acta Oecologica 119: 103910.
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Rare and cryptic species require effective monitoring methods in order to track populations over time. Monitoring surveys utilizing artificial refugia are an increasingly applied tool for detecting herpetofauna; yet for many species, limited information is available on the factors that influence optimal detectability. Most monitoring programs utilize only one type of artificial refuge. This approach has limitations as it may overlook the dynamic habitat needs of some species, with different refugia required under different conditions. Here we investigate whether using two different types of artificial refugia could maximize overall detection of the endangered Monaro grassland earless dragon Tympanocryptis osbornei by providing refugia that match habitat use. Tympanocryptis osbornei is a grassland specialist endemic to the Monaro Plains of southern NSW which occurs within a fragmented and altered agricultural landscape. Artificial refuge habitat in the form of customized replica ‘spider’ tubes and concrete roof tiles were utilized to infer the highest detection probability when compared to traditional rock rolling techniques. We found tubes to be more successful than tiles (mean: 0.021, 0.010–0.037 95% Bayesian credible interval (BCI) vs mean: 0.0096, 0.0045–0.018 95% BCI). However, this was dependent on air temperature, as tiles became more effective between 25 and 30 °C when dragons were more likely to be using surface shelters. Artificial refugia on a transect level (12 tubes and 12 tiles) had the greatest detection probability (mean: 0.11, 0.04–0.23 95% BCI) compared to individually checked objects. Our results identify the best current methods and conditions to detect T. osbornei and provide insights into additional considerations for optimizing survey efforts, timing, and detectability for other small reptiles in temperate climates. We recommend a combination of artificial refugia be considered in future surveys for cryptic reptiles.
SNAKES
Piaggio A. J., Engeman R. M., Hopken M. W., Humphrey J. S., Keacher K. L., Bruce W. E., Avery M. L. (2014): Detecting an elusive invasive species: a diagnostic PCR to detect Burmese python in Florida waters and an assessment of persistence of environmental DNA. Molecular Ecology Resources 14: 374-380.
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Recent studies have demonstrated that detection of environmental DNA (eDNA) from aquatic vertebrates in water bodies is possible. The Burmese python, Python bivittatus, is a semi‐aquatic, invasive species in Florida where its elusive nature and cryptic coloration make its detection difficult. Our goal was to develop a diagnostic PCR to detect P. bivittatus from water‐borne eDNA, which could assist managers in monitoring this invasive species. First, we used captive P. bivittatus to determine whether reptilian DNA could be isolated and amplified from water samples. We also evaluated the efficacy of two DNA isolation methods and two DNA extraction kits commonly used in eDNA preparation. A fragment of the mitochondrial cytochrome b gene from P. bivittatus was detected in all water samples isolated with the sodium acetate precipitate and the QIA amp DNA Micro Kit. Next, we designed P. bivittatus ‐specific primers and assessed the degradation rate of eDNA in water. Our primers did not amplify DNA from closely related species, and we found that P. bivittatus DNA was consistently detectable up to 96 h. Finally, we sampled water from six field sites in south Florida. Samples from five sites, where P. bivittatus has been observed, tested positive for eDNA. The final site was negative and had no prior documented evidence of P. bivittatus. This study shows P. bivittatus eDNA can be isolated from water samples; thus, this method is a new and promising technique for the management of invasive reptiles.
Hunter M. E., Oyler-McCance S. J., Dorazio R. M., Fike J. A., Smith B. J., Hunter C. T., Reed R. N., Hart K. M. (2015): Environmental DNA (eDNA) sampling improves occurrence and detection estimates of invasive Burmese pythons. Plos One 10: e0121655.
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Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors. Generic sampling design and terminology are proposed to standardize and clarify interpretations of eDNA-based occupancy models.
Kucherenko A., Herman J.., III E. M. E., Urakawa H. (2018): Terrestrial snake environmental DNA accumulation and degradation dynamics and its environmental application. Herpetologica 74: 38-49.
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There is an increasing need for effective biomonitoring tools that quantify patterns of habitat occupancy by reptile species. Environmental DNA (eDNA) has been regarded as an emerging tool to detect specific target species; however, the dynamics of accumulation and degradation of eDNA in terrestrial environments are poorly understood. This study determines the time required for terrestrial snakes to leave enough eDNA behind to become detectable (accumulation time) as well as its persistence (degradation time). By targeting mitochondrial cytochrome oxidase subunit I and 12S rRNA genes of Red Cornsnakes (Pantherophis guttatus) in a controlled laboratory setting, we found that eDNA can be detected 3.5 h after the snakes had contact with soil and for up to 6 d after their removal. Estimated accumulation rate of Pantherophis guttatus eDNA per gram of snake biomass per hour was 12.6 μg. We also evaluated the applicability of eDNA detection under field conditions by targeting the mitochondrial cytochrome b gene of a cryptic invasive species in South Florida, Burmese Pythons (Python bivittatus). Soil samples were derived from two groups of field sites: telemetry-monitored refugia (i.e., radiotelemetry evidence of python presence) and telemetry-absent refugia (i.e., no telemetry evidence, but monitored with a burrow camera at time of sample collection). We were able to detect the presence of python eDNA in 66.7% of the telemetry-monitored sites that fit within our laboratory-defined residence and degradation time window. Additionally, at the telemetry-absent sites, no eDNA from Burmese Pythons was detected and burrow cameras did not detect their presence. We concluded that eDNA technology using soil can be an effective detection tool for terrestrial snakes, particularly when used with other traditional tracking and sampling methods.
Ratsch R., Kingsbury B. A., Jordan M. A. (2020): Exploration of environmental DNA (eDNA) to detect Kirtland’s Snake (Clonophis kirtlandii). Animals 10: 1057.
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Environmental DNA (eDNA) surveys utilize DNA shed by organisms into their environment in order to detect their presence. This technique has proven effective in many systems for detecting rare or cryptic species that require high survey effort. One potential candidate for eDNA surveying is Kirtland’s Snake (Clonophis kirtlandii), a small natricine endemic to the midwestern USA and threatened throughout its range. Due to its cryptic and fossorial lifestyle, it is also a notoriously difficult snake to survey, which has limited efforts to understand its ecology. Our goal was to utilize eDNA surveys for this species to increase detection probability and improve survey efficiency to assist future conservation efforts. We conducted coverboard surveys and habitat analyses to determine the spatial and temporal activity of snakes, and used this information to collect environmental samples in areas of high and low snake activity. In addition, we spiked artificial crayfish burrows with Kirtland’s Snake feces to assess the persistence of eDNA under semi-natural conditions. A quantitative PCR (qPCR) assay using a hydrolysis probe was developed to screen the environmental samples for Kirtland’s Snake eDNA that excluded closely related and co-occurring species. Our field surveys showed that snakes were found in the spring during the first of two seasons, and in areas with abundant grass, herbaceous vegetation, and shrubs. We found that eDNA declines within a week under field conditions in artificial crayfish burrows. In environmental samples of crayfish burrow water and sediment, soil, and open water, a single detection was found out of 380 samples. While there may be physicochemical and biological explanations for the low detection observed, characteristics of assay performance and sampling methodology may have also increased the potential for false negatives. We explored these outcomes in an effort to refine and advance the successful application of eDNA surveying in snakes and groundwater microhabitats.
Katz A. D., Harper L. R., Sternhagen E. C., Pearce S. E., Melder C. A., Sperry J. H., Davis M. A. (2021): Environmental DNA is effective in detecting the federally threatened Louisiana Pinesnake (Pituophis ruthveni). Environmental DNA 3: 409-425.
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Successful conservation of rare, threatened, or endangered (RTE) species is dependent upon rapid and accurate assessment of their distribution and abundance. However, assessments are challenging as RTE species typically exist as numerically small populations in often fragmented habitats and can possess complex natural histories. Environmental DNA (eDNA) analysis may provide a rapid, cost-effective means of assessing RTE species presence/absence in viable habitat patches. We evaluated the efficacy of eDNA surveillance for the Louisiana Pinesnake (Pituophis ruthveni), an elusive, semi-fossorial, nonvenomous colubroid snake endemic to Louisiana and Texas, USA, that has dramatically declined in both distribution and abundance. We developed two quantitative polymerase chain reaction (qPCR) assays that target the mitochondrial cytochrome c oxidase subunit I (COI) and mitochondrially encoded ATP synthase membrane subunit 6 (ATP6) genes. We validated each assay in silico, in vitro, and in situ, and investigated the influence of eDNA extraction method and genetic marker on assay performance. Both assays were highly sensitive and successfully detected the Louisiana Pinesnake under artificial and field conditions, including bedding samples collected from captive snake enclosures (100%), soil samples from Louisiana Pinesnake release sites (100%), and soil samples from sites where Louisiana Pinesnakes were documented via radio telemetry (45%). Although differences between genetic markers were negligible, assay performance was strongly influenced by eDNA extraction method. Informed by our results, we discuss methodological and environmental factors influencing Louisiana Pinesnake eDNA detection and quantification, broader implications for management and conservation of the Louisiana Pinesnake and other terrestrial reptiles and provide recommendations for future research. We suggest that eDNA surveys can more effectively assess Louisiana Pinesnake occupancy than conventional sampling, highlighting the need for comprehensive eDNA monitoring initiatives to better identify suitable habitat that will promote persistence of this imperiled species going forward.
Matthias L., Allison M. J., Maslovat C. Y., Hobbs J., Helbing C. C. (2021): Improving ecological surveys for the detection of cryptic, fossorial snakes using eDNA on and under artificial cover objects. Ecological Indicators 131: 108187.
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Performing ecological surveys for secretive, fossorial snakes is challenging. Traditional survey methods involve visual observation under artificial cover objects (ACOs); this is labor-intensive and requires multiple consistent surveys of suitable habitats. Detection of snake DNA deposited under ACOs represents an innovative method for species detection. However, for terrestrial species, common issues with soil-based methods include the challenges of adequately removing enzyme inhibitors that reduce environmental DNA (eDNA) detection and potential photodegradation of DNA taken from surface samples. These issues may be circumvented by obtaining swabs and soil samples directly from the underside of ACOs for eDNA analysis. We demonstrate the application of this method in surveys of sharp-tailed snake (Contia tenuis), an endangered species under the Canadian Species at Risk Act. We describe the design and validation of a new quantitative real-time polymerase chain reaction (qPCR)-based eDNA eCOTE3 assay with high specificity and sensitivity for sharp-tailed snake. We developed a practical and robust protocol for obtaining eDNA samples by swabbing the underside of ACOs and collecting soil samples under ACOs. Traditional surveys were conducted over two successive years (2018–19) on 220 paired ACOs at 110 sites monitored between 12 and 30 times each. Of the 6,060 ACO visits, only 24 resulted in sharp-tailed snake observations (0.4% success rate) illustrating the considerable difficulty in detecting these snakes. During this same time, 109 swabs were taken directly from the undersides of ACOs and 78 soil samples were collected from a subset of these ACOs. Of the 24 occurrences where sharp-tailed snakes were visually observed, 13 of 23 ACO swabs (57%) and nine of 20 soil samples (45%) tested positive for DNA. eDNA deposition is likely low because of the small size and behavior of this cryptic species, yet DNA was detected from soil exposed to captured snakes for only 10 min. Nevertheless, sharp-tailed snake eDNA was detected at eight sites (9%) from ACO swabs (n = 86) and seven sites (13%) from soil samples (n = 56) where snakes were not observed. This is an overall detection rate of 25% (14/56) for swab and soil samples testing positive in sites where both were tested, representing a substantial reduction in the effort required for detection of this species. Given the time-consuming nature of traditional surveys, eDNA holds great promise as a complementary survey tool for this terrestrial species. While further work is needed to delineate DNA deposition rates, this work represents a significant advance in monitoring a challenging species.
Walkup D. K., Ryberg W. A., Pierce J. B., Smith E., Childress J., East F., Pierce B. L., Brown P., Fielder C. M., Hibbitts T. J. (2023): Testing the detection of large, secretive snakes using camera traps. Wildlife Society Bulletin 47: e1408.
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Novel technologies, such as camera traps, have expanded the opportunities for species detection, especially for rare species. Corresponding changes in data processing must occur to handle the large volume of data gathered from technology like camera traps. Automated image data processing, usually by running images through different types of computer algorithms, is an overarching goal to reduce the number of images that researchers must manually review. However, differences in camera trap setups and species characteristics can make automatic processing a challenge. Here, we evaluated the detection accuracy and efficiency of a time-lapse triggered camera trapping technique combined with a pixel change detection algorithm as part of a monitoring program for a translocated population of the rare and federally threatened Louisiana Pinesnake (Pituophis ruthveni). We paired 5 cameras with automated pit tag readers to collect observations of P. ruthveni. We evaluated an image dataset of 1,500,187 images, collected over 7 months, both manually (i.e., researchers looking at each individual picture to determine snake presence) and automatically using a change detection algorithm. There were 18 P. ruthveni observations recorded by the tag readers, 7 of which occurred while a paired camera was not operational. Ten of the tag reader P. ruthveni observations were captured by the paired camera trap, with an additional P. ruthveni observation from a paired camera trap not recorded by the tag reader. There were 132 snake observations of 13 additional species and 18 observations of unknown snakes from the camera traps. The algorithm reduced the number of images reviewers evaluated by an average of 78.5% per camera (range = 37.3–98.7%) but had a 54.5% success rate at detecting observations of P. ruthveni (47.1% for individual images), and a slightly lower 48.9% success rate detecting other large snakes. Large snakes were 4 times more likely to be flagged by the algorithm than small snakes. Our time-lapse triggered camera trapping technique performed well with respect to P. ruthveni detection accuracy, compared to the tag readers. However, further research is needed to improve quality assurances of camera trap image filtering and object recognition algorithms across different sites or environments.
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Ballouard J. M., Bonnet X., Gravier C., Ausanneau M., Caron S. (2016): Artificial water ponds and camera trapping of tortoises, and other vertebrates, in a dry Mediterranean landscape. Wildlife Research 43: 533-543.
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Mediterranean areas offer a mosaic of favourable microhabitats to reptiles (e.g. open zones, thorny bushes) and are considered as biodiversity hotspots for these organisms. However, in these dry and hot environments, reptiles remain sheltered most of the time. They generally escape observation, posing difficulties to perform inventories. Trap sampling or rock-turning surveys commonly used to detect reptiles entail important logistical constraints, may perturb fragile microhabitats, and are not appropriate for chelonians. Alternative simple and cost-effective methods are desired. We tested the efficiency of camera trapping in a dry Mediterranean landscape, notably to detect threatened Hermann’s tortoises. We tested whether small artificial freshwater ponds could attract animals in the field of view of the cameras to increase detectability. We also tested whether sand tracks survey around ponds could improve the method. We used a small number of cameras with ponds (5 in 2011, 7 in 2012), thereby maintaining low logistical costs. We randomly filled three ponds and emptied three ponds every 7 days. We set the time-lapse function of each camera with an interval of 5 min and inspected the sand tracks every 2 or 3 days. We used information from 39 radio-tracked tortoises to better estimate the detectability performances of the camera–pond system. This technique was effective to detect tortoises (n = 348 observations) and five other reptiles (among the 11 species present in the study area). Large numbers of birds and mammals were observed (n = 4232, n = 43 species at least), thereby increasing the biodiversity list of the surveyed area. We detected 28% of the radio-tracked tortoises present in the monitored area. Filled ponds were more attractive and sand track survey completed camera monitoring. Camera trapping associated with small ponds represent a useful tool to perform rapid inventories of the fauna in Mediterranean habitats, especially to detect the emblematic Hermann’s tortoise and other cryptic reptiles (e.g. snakes). The low cost–efficiency ratio of this method allows performing multiple counting surveys, and thus may help collect robust data necessary to justify the protection of key habitats that are coveted by property developers.
Lacoursière-Roussel A., Dubois Y., Normandeau E., Bernatchez L. (2017): Improving herpetological surveys in eastern North America using the environmental DNA method. Genome 59: 991-1007.
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Among vertebrates, herpetofauna has the highest proportion of declining species. Detection of environmental DNA (eDNA) is a promising method towards significantly increasing large-scale herpetological conservation efforts. However, the integration of eDNA results within a management framework requires an evaluation of the efficiency of the method in large natural environments and the calibration of eDNA surveys with the quantitative monitoring tools currently used by conservation biologists. Towards this end, we first developed species-specific primers to detect the wood turtle (Glyptemys insculpta) a species at risk in Canada, by quantitative PCR (qPCR). The rate of eDNA detection obtained by qPCR was also compared to the relative abundance of this species in nine rivers obtained by standardized visual surveys in the Province of Québec (Canada). Second, we developed multi-species primers to detect North American amphibian and reptile species using eDNA metabarcoding analysis. An occurrence index based on the distribution range and habitat type was compared with the eDNA metabarcoding dataset from samples collected in seven lakes and five rivers. Our results empirically support the effectiveness of eDNA metabarcoding to characterize herpetological species distributions. Moreover, detection rates provided similar results to standardized visual surveys currently used to develop conservation strategies for the wood turtle. We conclude that eDNA detection rates may provide an effective semiquantitative survey tool, provided that assay calibration and standardization is performed.
Adams C. I., Hoekstra L. A., Muell M. R., Janzen F. J. (2019): A brief review of non-avian reptile environmental DNA (eDNA), with a case study of painted turtle (Chrysemys picta) eDNA under field conditions. Diversity 11: 50.
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Environmental DNA (eDNA) is an increasingly used non-invasive molecular tool for detecting species presence and monitoring populations. In this article, we review the current state of non-avian reptile eDNA work in aquatic systems, and present a field experiment on detecting the presence of painted turtle (Chrysemys picta) eDNA. Thus far, turtle and snake eDNA studies have shown mixed results in detecting the presence of these animals under field conditions. However, some instances of low detection rates and non-detection occur for these non-avian reptiles, especially for squamates. We explored non-avian reptile eDNA quantification by sampling four lentic ponds with different densities (0 kg/ha, 6 kg/ha, 9 kg/ha, and 13 kg/ha) of painted turtles over three months to detect differences in eDNA using a qPCR assay amplifying the COI gene of the mtDNA genome. Only one sample of the highest-density pond amplified eDNA for a positive detection. Yet, estimates of eDNA concentration from pond eDNA were rank-order correlated with turtle density. We present the “shedding hypothesis”—the possibility that animals with hard, keratinized integument do not shed as much DNA as mucus-covered organisms—as a potential challenge for eDNA studies. Despite challenges with eDNA inhibition and availability in water samples, we remain hopeful that eDNA can be used to detect freshwater turtles in the field. We provide key recommendations for biologists wishing to use eDNA methods for detecting non-avian reptiles.
Akre T. S., Parker L. D., Ruther E., Maldonado J. E., Lemmon L., McInerney N. R. (2019): Concurrent visual encounter sampling validates eDNA selectivity and sensitivity for the endangered wood turtle (Glyptemys insculpta). Plos One 14: e0215586.
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Environmental DNA (eDNA) has been used to record the presence of many different organisms in several different aquatic and terrestrial environments. Although eDNA has been demonstrated as a useful tool for the detection of invasive and/or cryptic and declining species, this approach is subject to the same considerations that limit the interpretation of results from traditional survey techniques (e.g. imperfect detection). The wood turtle is a cryptic semi-aquatic species that is declining across its range and, like so many chelonian species, is in-need of a rapid and effective method for monitoring distribution and abundance. To meet this need, we used an eDNA approach to sample for wood turtle presence in northern Virginia streams. At the same time, we used repeat visual encounter surveys in an occupancy-modelling framework to validate our eDNA results and reveal the relationship of detection and occupancy for both methods. We sampled 37 stream reaches of varying size within and beyond the known distribution of the wood turtle across northern Virginia. Wood turtle occupancy probability was 0.54 (0.31, 0.76) and while detection probability for wood turtle occupancy was high (0.88; 0.58, 0.98), our detection of turtle abundance was markedly lower (0.28; 0.21, 0.37). We detected eDNA at 76% of sites confirmed occupied by VES and at an additional three sites where turtles were not detected but were known to occur. Environmental DNA occupancy probability was 0.55 (0.29, 0.78); directly comparable to the VES occupancy estimate. Higher probabilities of detecting wood turtle eDNA were associated with higher turtle densities, an increasing number of days since the last rainfall, lower water temperatures, and lower relative discharges. Our results suggest that eDNA technology holds promise for sampling aquatic chelonians in some systems, even when discharge is high and biomass is relatively low, when the approach is validated and sampling error is quantified.
Feng W., Bulté G., Lougheed S. C. (2020): Environmental DNA surveys help to identify winter hibernacula of a temperate freshwater turtle. Environmental DNA 2: 200-209.
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Overwintering is a critical part of the annual cycle of animals living at high latitudes, and selection of overwintering sites (hibernacula) is important to population persistence. Identifying the overwintering sites of aquatic species is challenging in areas where water bodies are frozen for significant parts of the year. We tested whether environmental DNA (eDNA) approaches could help to locate them. We conducted environmental DNA surveys of underwater overwintering sites of the northern map turtle (Graptemys geographica), a species of conservation concern in Canada. We collected water samples under the ice in winter across a mid-sized temperate lake and used quantitative PCR with a species-specific probe to quantify concentrations of map turtle eDNA. We found localized eDNA signals consistent with known overwintering sites and one previously suspected site. The latter was further confirmed using underwater remote operated vehicle (ROV) visual surveys. Our study confirms that eDNA can offer insights on a critical part of the annual cycle of aquatic species, for which we know very little.
Harper K. J., Goodwin K. D., Harper L. R., LaCasella E. L., Frey A., Dutton P. H. (2020): Finding Crush: environmental DNA analysis as a tool for tracking the green sea turtle Chelonia mydas in a marine estuary. Frontiers in Marine Science 6: 810.
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Environmental DNA (eDNA) analysis is a rapid, non-invasive method for species detection and distribution assessment using DNA released into the surrounding environment by an organism. eDNA analysis is recognised as a powerful tool for detecting endangered or rare species in a range of ecosystems. Although the number of studies using eDNA analysis in marine systems is continually increasing, there appears to be no published studies investigating the use of eDNA analysis to detect sea turtles in natural conditions. We tested the efficacy of two primer pairs known to amplify DNA fragments of differing lengths (488 and 253 bp) from Chelonia mydas tissues for detecting C. mydas eDNA in water samples. The capture, extraction, and amplification of C. mydas eDNA from aquaria (Sea World, San Diego, CA, United States) and natural water (San Diego Bay, CA, United States) were successful using either primer set. The primer pair providing the shorter amplicon, LCMint2/H950g, demonstrated the ability to distinguish cross-reactive species by melt curve analysis and provided superior amplification metrics compared to the other primer set (LTCM2/HDCM2); although primer dimer was observed, warranting future design refinement. Results indicated that water samples taken from deeper depths might improve detection frequency, consistent with C. mydas behaviour. Overall, this pilot study suggests that with refinement of sampling methodology and further assay optimisation, eDNA analysis represents a promising tool to monitor C. mydas. Potential applications include rapid assessment across broad geographical areas to pinpoint promising locations for further evaluation with traditional methods.
Barreto J., Cajaíba L., Teixeira J. B., Nascimento L., Giacomo A., Barcelos N., Fettermann T., Martins A. (2021): Drone-monitoring: Improving the detectability of threatened marine megafauna. Drones 5: 14.
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Unmanned aerial vehicles (UAVs; or drones) are an emerging tool to provide a safer, cheaper, and quieter alternative to traditional methods of studying marine megafauna in a natural environment. The UFES Nectology Laboratory team developed a drone-monitoring to assess the impacts on megafauna related to the Fundão dam mining tailings disaster in the Southeast Brazilian coast. We have developed a systematic pattern to optimize the available resources by covering the largest possible area. The fauna observer can monitor the environment from a privileged angle with virtual reality and subsequently analyzes each video captured in 4k, allowing to deepening behavioral ecology knowledge. Applying the drone-monitoring method, we have observed an increasing detectability by adjusting the camera angle, height, orientation, and speed of the UAV; which saved time and resources for monitoring turtles, sea birds, large fish, and especially small cetaceans efficiently and comparably.
Farrell J. A., Whitmore L., Mashkour N., Rollinson Ramia D. R., Thomas R. S., Eastman C. B., Burkhalter B., Yetsko K., Mott C., Wood L., Zirkelbach B., Meers L., Kleinsasser P., Stock S., Libert E., Herren R., Eastman S., Crowder W., Bovery C., Anderson D., Godfrey D., Condron N., Duffy D. J. (2022): Detection and population genomics of sea turtle species via noninvasive environmental DNA analysis of nesting beach sand tracks and oceanic water. Molecular Ecology Resources 22: 2471-2493.
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Elusive aquatic wildlife, such as endangered sea turtles, are difficult to monitor and conserve. As novel molecular and genetic technologies develop, it is possible to adapt and optimize them for wildlife conservation. One such technology is environmental (e)DNA – the detection of DNA shed from organisms into their surrounding environments. We developed species-specific green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtle probe-based qPCR assays, which can detect and quantify sea turtle eDNA in controlled (captive tank water and sand samples) and free ranging (oceanic water samples and nesting beach sand) settings. eDNA detection complemented traditional in-water sea turtle monitoring by enabling detection even when turtles were not visually observed. Furthermore, we report that high throughput shotgun sequencing of eDNA sand samples enabled sea turtle population genetic studies and pathogen monitoring, demonstrating that noninvasive eDNA techniques are viable and efficient alternatives to biological sampling (e.g., biopsies and blood draws). Genetic information was obtained from sand many hours after nesting events, without having to observe or interact with the target individual. This greatly reduces the sampling stress experienced by nesting mothers and emerging hatchlings, and avoids sacrificing viable eggs for genetic analysis. The detection of pathogens from sand indicates significant potential for increased wildlife disease monitoring capacity and viral variant surveillance. Together, these results demonstrate the potential of eDNA approaches to ultimately help understand and conserve threatened species such as sea turtles.
Sellés-Ríos B., Flatt E., Ortiz-García J., García-Colomé J., Latour O., Whitworth A. (2022): Warm beach, warmer turtles: using drone-mounted thermal infrared sensors to monitor sea turtle nesting activity. Frontiers in Conservation Science 3: 954791.
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For decades sea turtle projects around the world have monitored nesting females using labor-intensive human patrolling techniques. Here we describe the first empirical testing of a drone-mounted thermal infrared sensor for nocturnal sea turtle monitoring; on the Osa peninsula in Costa Rica. Preliminary flights verified that the drone could detect similar sea turtle activities as identified by on-the-ground human patrollers – such as turtles, nests and tracks. Drone observers could even differentiate tracks of different sea turtle species, detect sea turtle hatchlings, other wildlife, and potential poachers. We carried out pilot flights to determine optimal parameters for detection by testing different thermal visualization modes, drone heights, and gimbal angles. Then, over seven nights, we set up a trial to compare the thermal drone and operators’ detections with those observed by traditional patrollers. Our trials showed that thermal drones can record more information than traditional sea turtle monitoring methods. The drone and observer detected 20% more sea turtles or tracks than traditional ground-based patrolling (flights and patrols carried out across the same nights at the same time and beach). In addition, the drone operator detected 39 other animals/predators and three potential poachers that patrollers failed to detect. Although the technology holds great promise in being able to enhance detection rates of nesting turtles and other beach activity, and in helping to keep observers safer, we detail challenges and limiting factors; in drone imagery, current cost barriers, and technological advances that need to be assessed and developed before standardized methodologies can be adopted. We suggest potential ways to overcome these challenges and recommend how further studies can help to optimize thermal drones to enhance sea turtle monitoring efforts worldwide.