Non-invasive Genetic Sampling Research Articles

Rapid species identification of pygmy rabbits (Brachylagus idahoensis) from faecal pellet DNA

The pygmy rabbit (Brachylagus idahoensis) is a small lagomorph of the western United States that specializes in sagebrush (Artemisia spp.) habitat. Intensive habitat loss and modification have increased the vulnerability of pygmy rabbit populations, but the current geographic distribution and population status remain unclear. To aid in detection and population monitoring, we developed a species identification test that uses mitochondrial DNA species-specific primers to distinguish among six sympatric lagomorph species using DNA isolated from faecal pellets. Applying this test, we successfully identified the species of origin for all pellet samples that produced a positive PCR result (77% of 283 pellets collected). Pellets collected during the winter (December-February) had higher PCR success rate (93%) than pellets collected at other times of the year (72%). This test, using non-invasive genetic sampling of faecal pellets, provides an efficient method for assessing site occupancy and distribution of pygmy rabbits and other lagomorphs across large geographic areas.

Evaluation of noninvasive genetic sampling methods for cougars in Yellowstone National Park

AbstractConventional methods for monitoring cougar, Puma concolor, populations involve capture, tagging, and radio‐collaring, but these methods are time‐consuming, expensive, and logistically challenging. For difficult‐to‐study species such as cougars, noninvasive genetic sampling (NGS) may be a useful alternative. The ability to identify individuals from samples collected through NGS methods provides many opportunities for developing population‐monitoring tools, but the utility of these survey methods is dependent upon collection of samples and accurate genotyping of those samples. In January 2003, we initiated a 3‐yr evaluation of NGS methods for cougars using a radio‐collared population in Yellowstone National Park (YNP), USA. Our goals were to: 1) determine which DNA collection method, hair snares or snow tracking, provided a better method for obtaining samples for genetic analysis, 2) evaluate reliability of the genetic data derived from hair samples collected in the field, and 3) evaluate the potential of NGS for demographic monitoring of cougar populations. Snow tracking yielded more hair samples and was more cost effective than snagging hair with rub pads. Samples collected from bed sites and natural hair snags (e.g., branch tips, thorn bushes) while snow tracking accurately identified and sexed 22 individuals (9 F, 13 M). The ratio of the count from snow tracking to the count from radio‐telemetry was 15:24 in winter 2004, 13:12 in 2005, and 22:29 for both years combined. Annual capture probabilities for obtaining DNA from snow tracking varied considerably between years for females (0.42 in 2004 and 0.88 in 2005) but were more consistent for males (0.77 in 2004 and 0.88 in 2005). Our results indicate that snow tracking can be an efficient, reliable NGS method for cougars in YNP and has potential for estimating demographic and genetic parameters of other carnivore populations in similar climates. © 2011 The Wildlife Society.

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas’ habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas’ habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

THE PARASITES OF GIANT PANDAS: INDIVIDUAL-BASED MEASUREMENT IN WILD ANIMALS

There is a growing recognition of parasites as a significant factor in the successful conservation of endangered species. Determining parasite infection and load in free-ranging populations traditionally is done via necropsy or coproscopy. For studies of wild animals, fecal sample collection can result in bias because the individual identity of animals is unknown and multiple samples may be collected from the same individual, yet treated as unrelated samples. We studied parasite load in wild giant pandas (Ailuropoda melanoleuca) across six mountain ranges in China. Genetic identification was used to determine the exact number of individuals sampled. The parasite fauna consisted of five species, dominated by Baylisascaris shroederi. The pattern of statistical difference between mountains was artificially inflated when animal identity was not included in the model. Our results suggest that caution should be exercised in inferring patterns from comparative parasitologic studies when samples cannot be attributed to specific individuals. Using noninvasive genetic sampling to avoid such bias should form a standard tool in the management of endangered species and their parasites.

A Multi-Regional Assessment of the American Pika (Ochotona Princeps) in National Parks

American pikas are conspicuous and charismatic inhabitants of mountainous regions of the western United States. Due to their sensitivity to high temperatures, they are considered an important early warning indicator species for detecting the ecological effects of climate change. This study addresses the potential threat of climate change to American pikas by assessing current occupancy patterns, modeling current gene flow patterns, and combining these two components to project habitat-specific models of occupancy and connectivity into the future under various climate change scenarios. The preliminary results presented here reflect field work conducted in Grand Teton National Park during the summer of 2010 and 2011. We conducted occupancy surveys, collected non-invasive genetic samples, and conducted preliminary genetic analyses.

Genetic evidence of recent population contraction in the southernmost population of giant pandas

Anthropogenic habitat loss and fragmentation have been implicated in the endangerment and extinction of many species. Here we assess genetic variation and demographic history in the southernmost population of giant pandas (Ailuropoda melanoleuca) that continues to be threatened by habitat degradation and fragmentation, using noninvasive genetic sampling, mitochondrial control region sequence and 12 microsatellite loci. Compared to other giant panda populations, this population has medium-level genetic diversity based on the measure of both mitochondrial and nuclear markers. Mitochondrial DNA-based demographic analyses revealed that no historical population expansion or contraction has occurred, indicating a relatively stable population size. However, a Bayesian-coalescent method based on the observed allele distribution and allele frequencies of microsatellite clearly did detect, quantify and date a recent decrease in population size. Overall, the results indicate that a population contraction in the order of 95-96% has taken place over the last 910-999 years and is most likely due to anthropogenic habitat loss. These findings highlight the need for a greater focus on habitat protection and restoration for the long-term survival of this giant panda population.

Deforestation and cattle ranching drive rapid range expansion of capybara in the Gran Chaco ecosystem

Anthropogenic habitat alteration has the capacity to alter the distribution of species. Capybara (Hydrochoerus hydrochaeris) are a widely distributed rodent throughout most of South America, but are restricted to areas of standing water. As the Gran Chaco ecosystem of Paraguay is converted from dry tropical forest to pastureland, we hypothesize that this alteration creates potential for invasion by capybara into newly fragmented areas. We surveyed throughout the Chaco to estimate the distribution of capybara, and we collected noninvasive genetic samples. We used ecological niche modeling based on six environmental or climatic variables, and we modeled both the current distribution of capybara and the distribution of capybara 80 years ago. We then verified the hypothesized demographic signal generated with our model using phylogeographic analyses of 386 bp of the mtDNA control region. Comparison of present and past models suggested that populations expanded into the Gran Chaco after forest was converted to pastureland. Analyses of the mitochondrial D-loop supported the rapid range expansion scenario. We also found evidence of secondary contact of two distinct phylogroups which had previously been disjunct. Anthropogenic land transformation appeared to be a major factor influencing the distribution, as predicted by the niche model and confirmed by genetic data. Habitat modification altered connectivity of populations across the landscape. In addition, long separated clades of capybara are now admixed throughout the Paraguayan Chaco. The invasion of a large bodied herbivore into the High Chaco region may exacerbate the degradation of forest and prevent forest regeneration. As the reservoir host of several zoonotic diseases, the expansion and contact of two previously disjunct capybara populations has implications for disease emergence.

Noninvasive genome sampling in chimpanzees

The inevitable has happened: genomic technologies have been added to our noninvasive genetic sampling repertoire. In this issue of Molecular Ecology, Perry et al. (2010) demonstrate how DNA extraction from chimpanzee faeces, followed by a series of steps to enrich for target loci, can be coupled with next-generation sequencing. These authors collected sequence and single-nucleotide polymorphism (SNP) data at more than 600 genomic loci (chromosome 21 and the X) and the complete mitochondrial DNA. By design, each locus was 'deep sequenced' to enable SNP identification. To demonstrate the reliability of their data, the work included samples from six captive chimps, which allowed for a comparison between presumably genuine SNPs obtained from blood and potentially flawed SNPs deduced from faeces. Thus, with this method, anyone with the resources, skills and ambition to do genome sequencing of wild, elusive, or protected mammals can enjoy all of the benefits of noninvasive sampling.

A new PCR assay for reliable molecular sexing of endangered Tasmanian devils (Sarcophilus harrisii) from non-invasive genetic samples

Sex identification is a critical component of threatened species monitoring programs. For cryptic, rare and endangered species that have low detection probabilities, efficient monitoring can be achieved by analysing non-invasively collected DNA with molecular genetic techniques. In this study, we report a multiplex PCR-based sexing assay for the endangered Tasmanian devil (Sarcophilus harrisii). The assay uses a new species-specific primer set that amplifies a fragment of the SRY gene and an autosomal microsatellite marker as an internal positive control. We show that this assay provides highly accurate and robust sex identification (99% accuracy [95%CI 96.5–0.99.8]) where sex could be assigned of devil DNA from tissue, hair and faeces. This simple, yet reliable sexing assay is an important step towards effective monitoring and management of Tasmanian devils.

Increased DNA amplification success of non-invasive genetic samples by successful removal of inhibitors from faecal samples collected in the field

The use of non-invasive genetic sampling (NGS) is becoming increasingly important in the study of wild animal populations. Obtaining DNA from faecal samples is of particular interest because faeces can be collected without deploying sample capture devices. However, PCR amplification of DNA extracted from faeces is problematic because of high concentrations of inhibitors. Here we present a method for increasing the successful application of donor DNA extracted from faecal samples through inhibitor reduction. After standard extraction with a DNA stool kit we used a ‘Concentrated Chelex Treatment’ (CCT) that increased the amplification success from 31.7 to 61.4% of loci. Our results suggest that darker supernatant and samples with more precipitate contain more inhibitors than lighter samples and samples with little or no precipitate. We expect the use of this technique to have wide applicability within conservation biology for research and management that relies on NGS of wild animal populations.

Addressing challenges in non invasive capture-recapture based estimates of small populations: a pilot study on the Apennine brown bear

It is often difficult to determine optimal sampling design for non-invasive genetic sampling, especially when dealing with rare or elusive species depleted of genetic diversity. To address this problem, we ran a hair-snag pilot study on the remnant Apennine brown bear population. We used occupancy models to estimate the performance of an improved field protocol, a meta-analysis approach to indirectly model capture probability, and simulations to evaluate the effect of genotyping errors on the accuracy of capture-recapture population estimates. In spring 2007 we collected 70 bear hair samples in 15 5 × 5 km cells, using 5 10-day trapping sessions. Bear detectability was higher in 2007 than in a previous attempt on the same population in 2004, reflecting improved field protocols and sampling design. However, individual capture probability was 0.136 (95% CI = 0.120–0.152), still below the minimum requirements of capture-mark-recapture closed population models. We genotyped hair samples (n = 63) at 9 microsatellite loci, obtaining 94% Polymerase Chain Reaction success, and 13 bear genotypes. Estimated PIDsib was 0.00594, and per-genotype error rate was 0.13, corresponding to a 99% probability of correct individual identification. Simulation studies showed that the effect of non-corrected or filtered genetic errors on the accuracy of population estimates was negligible only when individual capture probability was >0.2. Our results underline how the interaction among field protocols, sampling strategies and genotyping errors may affect the accuracy of DNA-based estimates of small and genetically depleted populations, and warned us about the feasibility of a survey using only traditional hair-snag sampling. In this and similar cases, indications from pilot studies can provide cost-effective means to evaluate the efficiency of designed sampling and modelling procedures.

Noninvasive Estimation of Minimum Population Sizes and Variability of the Major Histocompatibility Complex in the Andean Condor

Abstract. Estimating indices of abundance of threatened species is crucial to preserving biodiversity. Over the last few decades, noninvasive genetic sampling has proven to be a more straightforward and less expensive approach than capture—mark—recapture analyses. In particular, molted feathers have become extremely popular for the monitoring of bird populations. Diagnostic molecular markers such as microsatellites, however, are still not available for many avian species of conservation concern. Highly polymorphic genes of the major histocompatibility complex (MHC), on the other hand, have become reasonably accessible during the last few years. We tested the suitability of MHC profiles as DNA fingerprints to assist the identification of individuals of a scavenger difficult to monitor through traditional approaches, the Andean Condor (Vultur gryphus). To achieve this aim, we isolated polymorphic and putatively functional genes of MHC class I (exon 3, 6 alleles) and MHC class IIB (exon 2, 11 alleles). Singl...

Efficient, Noninvasive Genetic Sampling for Monitoring Reintroduced Wolves

ABSTRACT Traditional methods of monitoring gray wolves (Canis lupus) are expensive and invasive and require extensive efforts to capture individual animals. Noninvasive genetic sampling (NGS) is an alternative method that can provide data to answer management questions and complement already‐existing methods. In a 2‐year study, we tested this approach for Idaho gray wolves in areas of known high and low wolf density. To focus sampling efforts across a large study area and increase our chances of detecting reproductive packs, we visited 964 areas with landscape characteristics similar to known wolf rendezvous sites. We collected scat or hair samples from 20% of sites and identified 122 wolves, using 8–9 microsatellite loci. We used the minimum count of wolves to accurately detect known differences in wolf density. Maximum likelihood and Bayesian single‐session population estimators performed similarly and accurately estimated the population size, compared with a radiotelemetry population estimate, in both years, and an average of 1.7 captures per individual were necessary for achieving accurate population estimates. Subsampling scenarios revealed that both scat and hair samples were important for achieving accurate population estimates, but visiting 75% and 50% of the sites still gave reasonable estimates and reduced costs. Our research provides managers with an efficient and accurate method for monitoring high‐density and low‐density wolf populations in remote areas.

Spatial genetic structure and dispersal of giant pandas on a mountain-range scale

Understanding the spatial genetic structure of populations can provide insight into the ecological or evolutionary processes of the species, and enable wise conservation decisions. We examined the spatial genetic structure of a giant panda (Ailuropoda melanoleuca) population in a heterogeneous mountainous landscape using noninvasive genetic sampling and 12 microsatellite loci. Nonrandom genetic structure was detected through spatial autocorrelation analysis, demonstrating a significantly positive autocorrelation over closer distances. Additional spatial analyses showed significantly positive genetic correlation among spatially-proximate males, and no correlation among females and among male–female pairs. These findings suggest a female-biased dispersal pattern and cryptic family grouping among giant pandas on a large mountain-range scale. The spatial extent of genetic structure occurred within 12.5 km, measured by a least-cost path distance model integrating information of habitat quality and habitat preferences of this species. Using the bearing analysis of PASSAGE, we found that directional genetic autocorrelations were in agreement with habitat structure, and habitat heterogeneity may affect the direction of giant panda dispersal. The characterization of spatial genetic structure can provide potentially valuable information for the conservation and management of giant pandas and their habitat.

Multiplex pre‐amplification for noninvasive genetic sampling: is the extra effort worth it?

Microsatellite genotyping of hair and faeces using standard polymerase chain reaction (PCR) resulted in low success rates and high error rates in a 2003-2004 pilot study using noninvasive genetic sampling for the brown bear (Ursus arctos) in the Italian Alps. Thus, we evaluated the performance of multiplex pre-amplification for improving microsatellite genotyping results. Brown bear faecal DNA extracts of varying quality (n = 33) and hair DNA extracts of poor (n = 32) and good (n = 34) quality were used to compare standard PCR and pre-amplification. In contrast to previous studies, there was no significant difference between methods for individual locus amplification success, genotyping error and genotyping success rates for scat and hair samples. The use of pre-amplification requires an additional investment of time and resources, and our results raise questions about the universal value of pre-amplification approaches. We suggest that researchers carefully evaluate the performance of pre-amplification compared to standard PCR using field-collected samples from the study area of interest before engaging in large-scale noninvasive genetic analyses.

Evaluation of Bear Rub Surveys to Monitor Grizzly Bear Population Trends

Abstract: Wildlife managers need reliable estimates of population size, trend, and distribution to make informed decisions about how to recover at‐risk populations, yet obtaining these estimates is costly and often imprecise. The grizzly bear (Ursus arctos) population in northwestern Montana, USA, has been managed for recovery since being listed under the United States Endangered Species Act in 1975, yet no rigorous data were available to evaluate the program's success. We used encounter data from 379 grizzly bears identified through bear rub surveys to parameterize a series of Pradel model simulations in Program MARK to assess the ability of noninvasive genetic sampling to estimate population growth rates. We evaluated model performance in terms of 1) power to detect gender‐specific and population‐wide declines in population abundance, 2) precision and relative bias of growth rate estimates, and 3) sampling effort required to achieve 80% power to detect a decline within 10 years. Simulations indicated that ecosystem‐wide, annual bear rub surveys would exceed 80% power to detect a 3% annual decline within 6 years. Robust‐design models with 2 simulated surveys per year provided precise and unbiased annual estimates of trend, abundance, and apparent survival. Designs incorporating one survey per year require less sampling effort but only yield trend and apparent survival estimates. Our results suggest that systematic, annual bear rub surveys may provide a viable complement or alternative to telemetry‐based methods for monitoring trends in grizzly bear populations.

Comparing opportunistic and systematic sampling methods for non‐invasive genetic monitoring of a small translocated brown bear population

Summary 1. Non‐invasive genetic sampling (NGS) of hair and faeces has become an important tool for monitoring wildlife populations, but many managers question the feasibility and cost‐effectiveness of these methods for long‐term monitoring. To address this question, more studies are needed that simultaneously evaluate the effectiveness and efficiency of multiple NGS designs in the same study area. 2. In 2003–2004, we carried out an experimental study of NGS for a small brown bear Ursus arctos population established by translocation in the Italian Alps. We evaluated and compared the effectiveness and efficiency of three NGS approaches including two systematic designs, baited hair traps and transect sampling of hair and faeces, and opportunistic collection of faecal and hair samples. Effectiveness was evaluated in terms of the number of samples collected, bears identified, genotyping success and error rate, detection frequencies, individual movement and spatial distribution of the species. We also evaluated the suitability of the data collected for population size estimation using single‐ and multi‐session approaches. Efficiency was assessed by calculating total cost/genotyped sample, cost/unique bear identified and cost/bear sample. 3. During 2 years of sampling, 1164 samples and 15 unique genotypes were obtained. From these genotypes, we documented reproduction, an increase in the minimum population size of bears in the study area and important information on specific bears causing damages to property. 4. The optimal sampling strategy combined systematic hair trapping and opportunistic sampling, as the pooled data set efficiently provided large sample quantities, the highest number of identified bears, multiple individual detections, information on bear distribution and suitable data for population size estimation. 5. We provide an example of how the efficiency of NGS monitoring can be improved by integrating sampling into routine duties of existing field personnel. 6. Synthesis and applications. This study provides baseline data for monitoring brown bears in the Italian Alps and has important implications for NGS of other small populations in human‐dominated landscapes. Conservation of small populations in such habitats will benefit from multiple strategies that obtain critical demographic, spatial and genetic information in a cost‐effective manner.

Piloting a Non-Invasive Genetic Sampling Method for Evaluating Population-Level Benefits of Wildlife Crossing Structures

Intuitively, wildlife crossing structures should enhance the viability of wildlife populations. Previous research has demonstrated that a broad range of species will use crossing structures, however, questions remain as to whether these measures actually provide benefits to populations. To assess this, studies will need to determine the number of individuals using crossings, their sex, and their genetic relationships. Obtaining empirical data demonstrating population-level benefits for some species can be problematic and challenging at best. Molecular techniques now make it possible to identify species, individuals, their sex, and their genetic relatedness from hair samples collected through non-invasive genetic sampling (NGS). The authors describe efforts to pilot a method to assess potential population-level benefits of wildlife crossing structures. They tested the feasibility of a prototype NGS system designed to sample hair from black bears (Ursus americanus) and grizzly bears (U. arctos) at two wildlife underpasses. The piloted hair-sampling method did not deter animal use of the trial underpasses and was effective at sampling hair from more than 90% of the bear crossing events at the underpasses. Hair samples were also obtained from non-target carnivore species, including three out of five (60%) cougar (Puma concolor) crossing events. Individual identification analysis revealed that three female and two male grizzly bears used one wildlife underpass, whereas two female and three male black bears were identified as using the other underpass. Of the 36 hair samples from bears analyzed, five failed, resulting in an 87% extraction success rate, and six more were only identified to species. Overall, 70% of the hair samples from bears collected in the field had sufficient DNA for extraction purposes. Preliminary data from our NGS suggest the technique can be a reliable method to assess the population-level benefits of Banff wildlife crossings. Furthermore, NGS can be an important tool for the conservation value of wildlife crossings for other taxa, and we urge others to carry out evaluations of this emerging methodology.

Non-invasive genetic sampling of faecal material and hair from the grey-headed flying-fox (Pteropus poliocephalus)

Remote-sampling DNA from animals offers obvious benefits for species that are difficult to sample directly and is less disruptive for species of conservation concern. Here we report the results of a pilot study investigating non-invasive DNA sampling of the grey-headed flying-fox (Pteropus poliocephalus), a threatened species that is restricted to the east coast of Australia. We successfully extracted DNA from fresh scats and hair, each of which was of sufficient quality for amplifying mitochondrial DNA markers and microsatellites. A single-locus multitube approach was used to investigate amplification success and genotyping reliability. Faecal samples yielded a higher proportion of successful amplifications and consensus genotype assignments than hair samples. We outline measures that may be utilised to minimise microsatellite genotyping error for future studies. These indirect approaches to obtaining genetic data show much promise given the difficult nature of directly sampling flying-foxes and related species.