Non-invasive Genetic Sampling Research Articles

Spatial proximity moderates genotype uncertainty in genetic tagging studies

Accelerating declines of an increasing number of animal populations worldwide necessitate methods to reliably and efficiently estimate demographic parameters such as population density and trajectory. Standard methods for estimating demographic parameters from noninvasive genetic samples are inefficient because lower-quality samples cannot be used, and they assume individuals are identified without error. We introduce the genotype spatial partial identity model (gSPIM), which integrates a genetic classification model with a spatial population model to combine both spatial and genetic information, thus reducing genotype uncertainty and increasing the precision of demographic parameter estimates. We apply this model to data from a study of fishers (Pekania pennanti) in which 37% of hair samples were originally discarded because of uncertainty in individual identity. The gSPIM density estimate using all collected samples was 25% more precise than the original density estimate, and the model identified and corrected three errors in the original individual identity assignments. A simulation study demonstrated that our model increased the accuracy and precision of density estimates 63 and 42%, respectively, using three replicated assignments (e.g., PCRs for microsatellites) per genetic sample. Further, the simulations showed that the gSPIM model parameters are identifiable with only one replicated assignment per sample and that accuracy and precision are relatively insensitive to the number of replicated assignments for high-quality samples. Current genotyping protocols devote the majority of resources to replicating and confirming high-quality samples, but when using the gSPIM, genotyping protocols could be more efficient by devoting more resources to low-quality samples.

Estimating Abundance of Siberian Roe Deer Using Fecal-DNA Capture-Mark-Recapture in Northeast China.

Simple SummaryEstimation of population abundance or density is necessary for managing deer populations. However, there is no estimation of Siberian roe deer (Capreolus pygargus) in the Lesser Xing’an Mountains, northeast China where the density of roe deer is much lower than that of European or North American populations. We used fecal-DNA capture-mark-recapture to estimate the abundance and density in Liangshui National Nature Reserve. We collected 422 fecal pellet groups during two sampling periods in 2016, identified them to be 77 individuals by the DNA microsatellite technique and estimated the abundance of roe deer to be 87 deer (80–112, 95% CI) using the Program CAPTURE. Density was estimated to be 2.9 deer/km2 (2.7–3.7, 95% CI). Our study estimated the roe deer population abundance by a feces-based capture-mark-recapture approach in northeast China, successfully demonstrating the applicability of this feces sampling method in monitoring deer populations in this area. It also contributes to the development of low-density deer population ecology and management.It is necessary to estimate the population abundance of deer for managing their populations. However, most estimates are from high-density populations inhabiting the forests of North America or Europe; there is currently a lack of necessary knowledge regarding low-density deer populations in different forest habitats. In this article, we used fecal DNA based on the capture-mark-recapture method to estimate the population abundance of Siberian roe deer (Capreolus pygargus) in Liangshui National Nature Reserve in the Lesser Xing’an Mountains, northeast China, where the deer population was found to be of a low density by limited studies. We used a robust survey design to collect 422 fecal pellet groups in 2016 and extracted DNA from those samples, generating 265 different genotypes; we thus identified 77 deer individuals based on six microsatellite markers (Roe1, Roe8, Roe9, BM757, MB25 and OarFCB304). With capture and recapture records of these 77 individuals, the abundance of roe deer was estimated to be 87 deer (80–112, 95% CI) using the Program CAPTURE. Using an effective sampling area which resulted from the mean maximum recapture distance (MMRD), we converted the population abundance to a density of 2.9 deer/km2 (2.7–3.7, 95% CI). Our study estimated the roe deer population abundance by a feces-based capture-mark-recapture approach in northeast China, successfully demonstrating the applicability of non-invasive genetic sampling in monitoring populations of deer in this area, which contributes to the development of low-density deer population ecology and management.

Scent marking in wolves Canis lupus inhabiting managed lowland forests in Poland

In wolves Canis lupus, scent marking plays an important role in territory defence. In Europe, studies on patterns of scent marking in wolves have mostly been conducted in mountains or primeval forests, but since these areas are characterised by low human activity, the impact of people on this behaviour has been neglected. We conducted a study that combined genetic methods with an analysis of the spatial distribution of wolf territory markings in lowland managed forests with high human activity. We found that scent markings are deposited by all members of wolf family groups. Wolves most intensively marked crossroads and their vicinity, especially on roads only accessible for four-wheel drive cars. Our study provides further evidence that crossroads of forest roads play a crucial role in wolf scent marking. The results of our study may be useful during inventories of wolf populations based on collecting indirect signs of their presence or non-invasive genetic sampling.

Non-invasive genetic sampling of deer: a method for DNA extraction and genetic analysis from antlers

We describe a non-invasive technique to isolate genomic DNA from connective tissue present in the antlers of deer of the genus Hippocamelus. This method is simpler and more effective than conventional more-destructive procedures which damage the collection material. This method is applicable to all cervids that annually regenerate their antlers.

Low wolverine (Gulo gulo) density in a national park complex of the Canadian Rocky Mountains

Large carnivores are sensitive to human-caused extirpation due to large home ranges, low population densities, and low reproductive rates. Protected areas help maintain populations by acting as sources, but human-caused mortality, habitat displacement, and edge effects occurring at protected area boundaries may reduce that function. The national parks Banff, Yoho, and Kootenay in the Canadian Rocky Mountains are refugia for large carnivores, including wolverines (Gulo gulo (Linnaeus, 1758)). Despite growing conservation concern, empirical baseline population data for wolverines remain scarce throughout their range, including most of Canada. We hypothesized (i) that in these national parks, wolverine density matched values expected for high-quality habitat, and (ii) that edge effects decreased density towards park boundaries. We conducted systematic non-invasive genetic sampling surveys covering >7000 km2 (2011 and 2013). Using spatial capture–recapture models, we estimated mean (±SE) female (1.5 ± 0.3 and 1.4 ± 0.3 wolverine/1000 km2), male (1.8 ± 0.4 and 1.5 ± 0.3 wolverine/1000 km2), and combined (3.3 ± 0.5 and 3.0 ± 0.4 wolverine/1000 km2) densities for 2011 and 2013, respectively. These estimates were lower than predictions based on density extrapolation from nearby high-quality habitat, and density decreased towards park boundaries. To benefit the population, we recommend creating buffer zones around parks that protect female habitat and prohibit harvest.

The curse of observer experience: Error in noninvasive genetic sampling.

Noninvasive genetic sampling (NGS) is commonly used to study elusive or rare species where direct observation or capture is difficult. Little attention has been paid to the potential effects of observer bias while collecting noninvasive genetic samples in the field, however. Over a period of 7 years, we examined whether different observers (n = 58) and observer experience influenced detection, amplification rates, and correct species identification of 4,836 gray wolf (Canis lupus) fecal samples collected in Idaho and Yellowstone National Park, USA and southwestern Alberta, Canada (2008–2014). We compared new observers (n = 33) to experienced observers (n = 25) and hypothesized experience level would increase the overall success of using NGS techniques in the wild. In contrast to our hypothesis, we found that new individuals were better than experienced observers at detecting and collecting wolf scats and correctly identifying wolf scats from other sympatric carnivores present in the study areas. While adequate training of new observers is crucial for the successful use of NGS techniques, attention should also be directed to experienced observers. Observer experience could be a curse because of their potential effects on NGS data quality arising from fatigue, boredom or other factors. The ultimate benefit of an observer to a project is a combination of factors (i.e., field savvy, local knowledge), but project investigators should be aware of the potential negative effects of experience on NGS sampling.

Investigating a simplified method for noninvasive genetic sampling in East African mammals using silica dried scat swabs.

Swabbing scat has proved to be an effective noninvasive method to collect DNA from mammals in the field. Previously, this method has relied on preservative liquids or freezing to preserve the DNA collected on swabs. In this study, we determine the effectiveness of using silica to simply dry the swab in field as an alternative way to prevent DNA degredation. Four species were included in the study; reticulated giraffe, impala, fringe‐eared oryx, and lion. Swabs were taken at multiple time points for giraffe and impala scat samples, with the lion and oryx sampled opportunistically. Mitochondrial DNA was successfully amplified and sequenced from scat swabs from all species; however, effectiveness varied between species, with 81.8% amplification success rate from swabs taken from impala scat compared to 25% amplification success rate in giraffe. This variation in success rate was overcome by taking multiple swabs, thus increasing the probability of a successful amplification. The true merit of this method is in its simplicity and cheapness; no preservative liquids were required to be brought into the field, at no stage in the 2 weeks of field sampling were samples frozen, and no commercial kits were used for DNA extraction.

Marking behaviour and census of Eurasian otters (Lutra lutra) in riverine habitats: what can scat abundances and non-invasive genetic sampling tell us about otter numbers?

Growing human-wildlife conflicts and legal conservation obligations increased the need for precise information on Eurasian otter (Lutra lutra) population parameters for species status assessment and wildlife management measures. Scat surveys have become the method of choice to monitor species distribution range, abundance and habitat use. Although methodological concerns exist, scat abundance is often used as main indicator for otter population estimates and trends. To evaluate whether scat counts serve as good proxy for otter densities in linear stream habitats, we modelled the relationship between old/fresh scat abundance and otter numbers on two spatial scales, including also seasonal effects. Actual otter densities and marking behaviour were ascertained by genotyping of otter faeces collected at 218 marking sites along 132.6 km of four salmonid streams. Otter densities in our study areas ranged from 0.16 to 0.28 otters per stream kilometre. The age of scats and the level of spatial scale were determining factors when evaluating the suitability of scat abundance as index for otter densities. Covering large parts of an otter’s territory, fresh scat abundance increased with otter density, irrespectively of season. On the spatial scale of single marking sites, the relationship was not well supported, and no relationship could be found when using old scats in the models. Otter marking behaviour and differences in marking site use intensity, as well as seasonal differences in traceability and accumulation of old scats were discussed to bias the relationship between scat abundance and otter numbers.

Monitoring a New England Cottontail Reintroduction with Noninvasive Genetic Sampling

ABSTRACTCareful monitoring of reintroduced threatened species is essential for informing conservation strategies and evaluating reintroduction efforts in an adaptive management context. We used noninvasive genetic sampling to monitor a reintroduction of a threatened shrubland specialist, the New England cottontail (Sylvilagus transitionalis), in southeastern New Hampshire, USA. We monitored the apparent survival and breeding success of founder individuals and tracked changes in population size and genetic diversity for 5 years following an initial reintroduction in 2013. We released 42 rabbits, documented 29 unique offspring in years following releases through noninvasive surveys, and identified 6 founder individuals and 9 recruited offspring that bred. Apparent survival of founders was variable and greatest in the first year of the reintroduction. Predation was the primary cause of mortality and greatest in the first month after release and after heavy snowfall. Population size remained small but relatively stable until a stochastic decline in the fourth year following reintroduction, followed by a slight rebound after population augmentation and offspring production by wild‐born rabbits. Genetic diversity increased after the initial founders with diverse genetic backgrounds were released and then they and their subsequent offspring bred. We documented successful dispersal 700 m from the release site to a high‐quality patch of habitat, which remained occupied throughout the study. For New England cottontail reintroductions to be successful in the long term, releases will be needed at multiple patches within dispersal distance, and habitat corridors need to be restored among patches to create a functioning metapopulation. For small or isolated reintroduced populations, continued intensive monitoring is needed to detect stochastic declines in population size or changes in sex ratios and guide subsequent management reactions via additional reintroductions or population augmentations. Noninvasive genetic sampling is a valuable tool to monitor reintroductions of the New England cottontail and other threatened species to provide managers with detailed information to inform decision‐making in an adaptive management framework. © 2020 The Wildlife Society.

Genetic Diversity and Prediction Analysis of Small Isolated Giant Panda Populations After Release of Individuals.

Release of individuals is an effective conservation approach to protect endangered species. To save this small isolated giant panda population in Liziping Nature Reserve, a few giant pandas have been released to this population. Here we assess genetic diversity and future changes in the population using noninvasive genetic sampling after releasing giant pandas. In this study, a total of 28 giant pandas (including 4 released individuals) were identified in the Liziping, China. Compared with other giant panda populations, this population has medium-level genetic diversity; however, a Bayesian-coalescent method clearly detected, quantified, and dated a recent decrease in population size. The predictions for genetic diversity and survival of the population in the next 100 years indicate that this population has a high risk of extinction. We show that released giant pandas can preserve genetic diversity and improve the probability of survival in this small isolated giant panda population. To promote the recovery of this population, we suggest that panda release should be continued and this population will need to release 10 males and 20 females in the future.

A DNA toolbox for non-invasive genetic studies of sambar deer (Rusa unicolor)

Invasive sambar deer (Rusa unicolor) are having significant detrimental impacts on natural environments in south-eastern Australia. Little, however, is known about their ecology, limiting evidence-based management strategies directed at reducing deer impacts. Genetic data, generated from DNA isolated from deer scats, can be used to fill ecological knowledge gaps. This study outlines a non-invasive genetic sampling strategy by which good-quality DNA from a single deer scat can be used to determine (1) species of origin, (2) sex and (3) a unique DNA profile. DNA from deer tissue and sambar deer scat samples were used to develop and optimise molecular methods to collect reliable genetic information. A DNA toolbox is presented that describes how to find, collect and store scat samples, isolate DNA and use molecular markers to generate informative genetic data. Generating genetic data using this approach will support studies aimed at acquiring ecological knowledge about sambar deer. Such knowledge will be critical for developing evidence-based recommendations to improve on-ground management decisions for sambar deer.

Population genetic structure and habitat connectivity for jaguar (Panthera onca) conservation in Central Belize

BackgroundConnectivity among jaguar (Panthera onca) populations will ensure natural gene flow and the long-term survival of the species throughout its range. Jaguar conservation efforts have focused primarily on connecting suitable habitat in a broad-scale. Accelerated habitat reduction, human-wildlife conflict, limited funding, and the complexity of jaguar behaviour have proven challenging to maintain connectivity between populations effectively. Here, we used non-invasive genetic sampling and individual-based conservation genetic analyses to assess genetic diversity and levels of genetic connectivity between individuals in the Cockscomb Basin Wildlife Sanctuary and the Maya Forest Corridor. We used expert knowledge and scientific literature to develop models of landscape permeability based on circuit theory with fine-scale landscape features as ecosystem types, distance to human settlements and roads to predict the most probable jaguar movement across central Belize.ResultsWe used 12 highly polymorphic microsatellite loci to identify 50 individual jaguars. We detected high levels of genetic diversity across loci (HE = 0.61, HO = 0.55, and NA = 9.33). Using Bayesian clustering and multivariate models to assess gene flow and genetic structure, we identified one single group of jaguars (K = 1). We identified critical areas for jaguar movement that fall outside the boundaries of current protected areas in central Belize. We detected two main areas of high landscape permeability in a stretch of approximately 18 km between Sittee River Forest Reserve and Manatee Forest Reserve that may increase functional connectivity and facilitate jaguar dispersal from and to Cockscomb Basin Wildlife Sanctuary. Our analysis provides important insights on fine-scale genetic and landscape connectivity of jaguars in central Belize, an area of conservation concern.ConclusionsThe results of our study demonstrate high levels of relatively recent gene flow for jaguars between two study sites in central Belize. Our landscape analysis detected corridors of expected jaguar movement between the Cockscomb Basin Wildlife Sanctuary and the Maya Forest Corridor. We highlight the importance of maintaining already established corridors and consolidating new areas that further promote jaguar movement across suitable habitat beyond the boundaries of currently protected areas. Continued conservation efforts within identified corridors will further maintain and increase genetic connectivity in central Belize.

Gut microbiota and their putative metabolic functions in fragmented Bengal tiger population of Nepal.

Bengal tigers (Panthera tigris tigris) serve a pivotal role as an apex predator in forest ecosystems. To increase our knowledge on factors impacting the viability and health of this endangered species, we studied the gut microbiota in 32 individual Bengal tigers from three geographically separated areas (Chitwan National Park (CNP), Bardia National Park (BNP) and Suklaphanta Wildlife Reserve (SWR)) in Nepal, using noninvasive genetic sampling methods. Gut microbiota influence the immune system, impact various physiological functions, and modulates metabolic reactions, that ultimately impact the host health, behavior and development. Across the tiger populations in Nepal, we found significant differences in the composition of microbial communities based on their geographic locations. Specifically, we detected significant differences between CNP and the other two protected areas (CNP vs BNP: pseudo t = 1.944, P = 0.006; CNP vs SWR: pseudo t = 1.9942, P = 0.0071), but no differences between BNP and SWR. This mirrors what has been found for tiger gene flow in the same populations, suggesting gut microbiota composition and host gene flow may be linked. Furthermore, predictive metagenome functional content analysis (PICRUSt) revealed a higher functional enrichment and diversity for significant gut microbiota in the Chitwan tiger population and the lowest enrichment and diversity in Suklaphanta. The CNP tiger population contained higher proportions of microbiota that are associated with predicted functions relevant for metabolism of amino acid, lipid, xenobiotics biodegradation, terpenoides and polyketides than the SWR population. We conclude the tiger population structure, gut microbiota profile and associated functional metabolic categories are correlated, with geographically most separated CNP and SWR tiger population having the most distinct and different host genotype and microbiota profiles. Our work dramatically expands the understanding of tiger microbiota in wild populations and provides a valuable case study on how to investigate genetic diversity at different hierarchical levels, including hosts as well as their microbial communities.

Does faecal matter reflect location? An initial assessment of isotopic variability between consumed prey remains and faecal matter for wild jaguars

ABSTRACTFaecal isotopic analysis may complement other non-invasive wildlife survey tools for monitoring landscape use by carnivores, such as motion-detecting cameras and non-invasive genetic sampling. We analysed carbon, nitrogen, and strontium isotopes in faecal matter produced by jaguars (Panthera onca) as well as bones from consumed prey at the Mountain Pine Ridge Forest Reserve (MPR) in Belize, Central America. The MPR is ideally suited for a spatial isotope study as vegetation and geology both vary considerably. The isotopic composition of faecal matter should reflect the habitat and geology where consumed prey lived. We used bone from consumed prey recovered from jaguar scats as a proxy for diet. Faecal matter and bone showed comparable spatial isotopic trends, suggesting that the isotopic composition of jaguar faeces can be used to detect foraging in different habitats (pine forest versus broadleaf forest) or on different geologies (Mesozoic carbonates; Palaeozoic granite, contact metamorphics, and metasediments). This result is reassuring as bones are not always present in carnivore scats. Studying landscape use by cryptic and wide-ranging carnivore species like jaguars remains challenging. Isotopic analysis of faecal matter complements the existing array of non-invasive spatial monitoring tools.

Long-term inference of population size and habitat use in a socially dynamic population of wild western lowland gorillas

Genetic estimation of population sizes has been critical for monitoring cryptic and rare species; however, population estimates do not inherently reveal the permanence or stability of the population under study. Thus, it is important to monitor not only the number of individuals in a population, but also how they are associated in groups and how those groups are distributed across the landscape. Adding to the challenge of obtaining such information with high precision for endangered and elusive species is the need for long-term collection of such data. In this study we compare sampling approaches and genotype non-invasive genetic samples to estimate the number and distribution of wild western lowland gorillas occupying a ~ 100 km2 area in Loango National Park, Gabon, for the periods 2005–2007 and 2014–2017. Based on the number of genotyped individuals we inferred a minimum of 83 gorillas during the first and 81 gorillas during the second study period. We also obtained similar capture–recapture population size estimates for the two periods despite variance in social dynamics like group formations, group dissolutions and individual dispersal. We furthermore found area fidelity for two groups that were sampled for 10–12 years, despite variation in group membership. Our results revealed how individual movements link groups in a ‘network’ and show that western lowland gorilla populations can show a high degree of temporal and geographic stability concurrent with substantial social dynamics.

Demographic fragmentation of a protected wolverine population bisected by a major transportation corridor

Roads fragment ecosystems around the globe, but the effects of this fragmentation on biodiversity remain poorly understood. Wolverines (Gulo gulo) are snow-dependent carnivores that occur at low densities and they exhibit low genetic diversity at the southern extent of their range where they are snow-limited and fragmented by human development. Therefore, understanding the effect of roads and transportation infrastructure on population connectivity in protected strongholds such as national parks is crucial to effective wolverine management in a changing climate. We assessed whether the Trans-Canada Highway, Canada's largest east-west transportation corridor, affects wolverine gene flow in the Rocky Mountains. We used noninvasive genetic sampling methods (i.e., hair traps, backtracking) to collect DNA samples (i.e. hair, scat) from an 8000 km2 area of Banff, Kootenay, and Yoho National Parks and provincial lands and then used population and individual-based genetic analyses (e.g., assignment tests, principal coordinates analysis) to examine genetic structure across the highway in the national parks complex. We collected 2586 DNA samples between 2010 and 2013 from which we identified 49 unique individuals (29 males, 20 females). We detected weak population structure in males and relatively strong genetic differentiation in females spanning the highway with complementary nuclear and mitochondrial DNA analyses. Our results demonstrate that sex-biased dispersal across a major highway can lead to genetic isolation and demographic fragmentation in a protected carnivore population, highlighting the urgent need to maintain connectivity for wildlife over an expanding global road network in the face of climate change, landscape degradation, and loss of biodiversity.

Poor implementation of non-invasive sampling in wildlife genetics studies

The Earth’s biodiversity is currently experiencing immense pressure from habitat loss, overexploitation, global climate change, and invasive species, which escalate the global extinction crisis. Comprehensive knowledge of the extent and impact of biodiversity loss is therefore critical for determining species vulnerability and prioritizing conservation goals. An integral part of wildlife conservation research and management is nowadays genetic sampling. Animal DNA has been traditionally obtained invasively, from blood or other tissues, however public concerns over animal welfare require that animals are affected as little as possible during research. One of the ways to minimize the impact on wildlife animal welfare is to use non-invasive genetic sampling. Even though non-invasive genetic sampling techniques have been developed for many animal species, it is not clear how often they are being implemented. Here, I present an overview of recently published articles on genetics in amphibians, birds, carnivores, molluscs and rodents, for which I examined whether they used a lethal, invasive or non-invasive DNA sampling technique. Disappointingly, only 22% of the identified relevant studies implemented the available non-invasive genetic sampling method. I conclude highlighting the need for better implementation of non-invasive DNA collection methods in wildlife research through raising awareness, increasing financial support, and introducing more stringent criteria for obtaining research permits.

Empirical comparisons of abundance estimators for two sympatric carnivores using noninvasive genetic sampling

Abundance estimators are often evaluated with simulations, or by comparing estimates to populations of known size. Advances in noninvasive genetic sampling have fueled an increase in the use of noninvasive genetic sampling-based capture–recapture. However, when working with free-ranging populations of unknown size, managers often lack data necessary to select the appropriate estimator. This leads to uncertainty regarding how choice of estimator or sampling design influence estimates, and managers may select estimators based on funding or logistical constraints. Alternatively, comparing estimates from multiple estimators can provide managers with greater confidence in estimates, or highlight potential differences. We used noninvasive genetic sampling to estimate the abundance of free-ranging kit foxes Vulpes macrotis and coyotes Canis latrans. We generated estimates of abundance with two non-spatial likelihood-based estimators: 1) robust design Huggins capture–recapture models and 2) single-occasion capture with replacement (CAPWIRE) models. We compared these with recently published estimates derived from spatially explicit capture–recapture (SECR) models. For both species, estimates from Huggins models were generally lower than those from SECR. Abundance estimates from CAPWIRE, which was developed specifically for noninvasive genetic sampling and generates estimates from a single sampling occasion, tended to be biased low with high precision. Our results suggest that choice of estimator and sampling design can significantly influence estimates, and that the relationship between estimators varied between species. Our results further suggest that single-occasion sampling often employed with CAPWIRE abundance estimation may produce biased results and be inappropriate for species requiring dispersed sampling strategies.

A DNA toolbox for non-invasive genetic studies of sambar deer (Rusa unicolor)

Invasive sambar deer (Rusa unicolor) are having significant detrimental impacts on natural environments in south-eastern Australia. Little, however, is known about their ecology, limiting evidence-based management strategies directed at reducing deer impacts. Genetic data, generated from DNA isolated from deer scats, can be used to fill ecological knowledge gaps. This study outlines a non-invasive genetic sampling strategy by which good-quality DNA from a single deer scat can be used to determine (1) species of origin, (2) sex and (3) a unique DNA profile. DNA from deer tissue and sambar deer scat samples were used to develop and optimise molecular methods to collect reliable genetic information. A DNA toolbox is presented that describes how to find, collect and store scat samples, isolate DNA and use molecular markers to generate informative genetic data. Generating genetic data using this approach will support studies aimed at acquiring ecological knowledge about sambar deer. Such knowledge will be critical for developing evidence-based recommendations to improve on-ground management decisions for sambar deer.

Less invasive monitoring of cougars in Colorado's front range

ABSTRACTFrom 2014 to 2016, in the Front Range of Colorado, USA, we assessed noninvasive approaches to sampling cougar (Puma concolor) populations in an attempt to provide a new method that would be less field intensive, less expensive, and could be applied over large spatial extents compared with current methods. We assessed the use of predator calls to lure cougars to a site with remote camera traps for detection and also evaluated hair snags at sites to noninvasively identify individual animals. Predator calls effectively attracted cougars to specific sites with an average of 82 unique photographic detections of cougars per survey year (0.03 detections/trap‐night). However, obtaining hair samples from these animals was less effective because animals did not always pass through hair snags and ability to uniquely identify individuals by genotype was poor. We evaluated different approaches to estimating cougar density and found mark–resight to be a viable option in our study system. Mark–resight density estimate after correcting for partial use of the sampling area by cougars was 4.1 cougars/100 km2 (95% CI = 2.4, 5.8). Our results indicate that combining methods of noninvasive genetic sampling and auditory calls to monitor cougar populations can provide reliable density estimates over large geographic areas and areas with significant amounts of inaccessible private lands. © 2019 The Wildlife Society.