Non-invasive Genetic Sampling Research Articles

Evaluating the efficacy of non-invasive genetic sampling of the Northern Pacific rattlesnake with implications for other venomous squamates

A common challenge for conservation genetic studies is finding minimally invasive sampling methods that maximize the quantity and quality of data produced. Conventional approaches rely on tissue or blood sampling, which typically require lengthy handling times, and can be hazardous for high-risk species, such as venomous snakes. Finding alternative, less invasive techniques is imperative in such circumstances. We compared DNA quantity and genotyping success of blood samples to those from buccal swabs, cloacal swabs, and scale clippings in the Northern Pacific rattlesnake (Crotalus oreganus oreganus). Buccal swabs and scales yielded significantly less DNA compared to blood, whereas cloacal samples were not significantly different. Cloacal swabs produced the highest PCR success and lowest genotyping errors commensurate to the blood samples, but differences were not significant between sample types. Our findings suggest cloacal swabbing as an efficient, less invasive alternative for providing high quality genotypic data for squamate reptiles (venomous or nonvenomous).

Identifying gray wolf packs and dispersers using noninvasive genetic samples

Many animals, including gray wolves (Canis lupus), live in social groups. Genetic techniques can help reveal the structure and composition of social groups, providing valuable information about group and population dynamics. We evaluated the effectiveness of using noninvasive genetic sampling (NGS) of fecal and hair samples at wolf rendezvous sites combined with spatial and genetic assignment criteria for assigning individuals to packs, detecting dispersers and lone wolves, determining the number of packs in an area, and obtaining group metrics. We applied this approach in 4 study areas covering 13,182 km2 in Idaho, USA while concurrently monitoring wolves using telemetry techniques. We assigned pack affiliation to 78–97% of individuals across study areas and identified 12 potential dispersers. We detected a successful gene flow event by reconstructing a breeding male's genotype and tracing it back to a pack of origin using genetic assignment techniques. Average pack size was consistent between our NGS- and telemetry-based counts (x¯ = 10 for both), and both methods detected similar age composition within groups (31% pups and 69% adults for NGS and 33% pups and 67% adults for telemetry). Our NGS approach has the advantage of providing pack metrics including sex ratio, inferred breeders, and intra-pack relatedness that telemetry and observational techniques alone cannot. This NGS field sampling strategy combined with our pack assignment method was successful and provides an approach for characterizing functional social groups in the absence of previously acquired NGS, telemetry, or other observational data that may not be available when sampling new areas. © 2016 The Wildlife Society.

Describing a developing hybrid zone between red wolves and coyotes in eastern North Carolina, USA.

When hybridizing species come into contact, understanding the processes that regulate their interactions can help predict the future outcome of the system. This is especially relevant in conservation situations where human activities can influence hybridization dynamics. We investigated a developing hybrid zone between red wolves and coyotes in North Carolina, USA to elucidate patterns of hybridization in a system heavily managed for preservation of the red wolf genome. Using noninvasive genetic sampling of scat, we surveyed a 2880 km2 region adjacent to the Red Wolf Experimental Population Area (RWEPA). We combined microsatellite genotypes collected from this survey with those from companion studies conducted both within and outside the RWEPA to describe the gradient of red wolf ancestry. A total of 311 individuals were genotyped at 17 loci and red wolf ancestry decreased along an east–west gradient across the RWEPA. No red wolves were found outside the RWEPA, yet half of individuals found within this area were coyotes. Hybrids composed only 4% of individuals within this landscape despite co‐occurrence of the two species throughout the RWEPA. The low proportion of hybrids suggests that a combination of active management and natural isolating mechanisms may be limiting intermixing within this hybrid system.

Estimating population density and connectivity of American mink using spatial capture-recapture.

Estimating the abundance or density of populations is fundamental to the conservation and management of species, and as landscapes become more fragmented, maintaining landscape connectivity has become one of the most important challenges for biodiversity conservation. Yet these two issues have never been formally integrated together in a model that simultaneously models abundance while accounting for connectivity of a landscape. We demonstrate an application of using capture-recapture to develop a model of animal density using a least-cost path model for individual encounter probability that accounts for non-Euclidean connectivity in a highly structured network. We utilized scat detection dogs (Canis lupus familiaris) as a means of collecting non-invasive genetic samples of American mink (Neovison vison) individuals and used spatial capture-recapture models (SCR) to gain inferences about mink population density and connectivity. Density of mink was not constant across the landscape, but rather increased with increasing distance from city, town, or village centers, and mink activity was associated with water. The SCR model allowed us to estimate the density and spatial distribution of individuals across a 388 km² area. The model was used to investigate patterns of space usage and to evaluate covariate effects on encounter probabilities, including differences between sexes. This study provides an application of capture-recapture models based on ecological distance, allowing us to directly estimate landscape connectivity. This approach should be widely applicable to provide simultaneous direct estimates of density, space usage, and landscape connectivity for many species.

Optimization of Scat Detection Methods for a Social Ungulate, the Wild Pig, and Experimental Evaluation of Factors Affecting Detection of Scat.

Collection of scat samples is common in wildlife research, particularly for genetic capture-mark-recapture applications. Due to high degradation rates of genetic material in scat, large numbers of samples must be collected to generate robust estimates. Optimization of sampling approaches to account for taxa-specific patterns of scat deposition is, therefore, necessary to ensure sufficient sample collection. While scat collection methods have been widely studied in carnivores, research to maximize scat collection and noninvasive sampling efficiency for social ungulates is lacking. Further, environmental factors or scat morphology may influence detection of scat by observers. We contrasted performance of novel radial search protocols with existing adaptive cluster sampling protocols to quantify differences in observed amounts of wild pig (Sus scrofa) scat. We also evaluated the effects of environmental (percentage of vegetative ground cover and occurrence of rain immediately prior to sampling) and scat characteristics (fecal pellet size and number) on the detectability of scat by observers. We found that 15- and 20-m radial search protocols resulted in greater numbers of scats encountered than the previously used adaptive cluster sampling approach across habitat types, and that fecal pellet size, number of fecal pellets, percent vegetative ground cover, and recent rain events were significant predictors of scat detection. Our results suggest that use of a fixed-width radial search protocol may increase the number of scats detected for wild pigs, or other social ungulates, allowing more robust estimation of population metrics using noninvasive genetic sampling methods. Further, as fecal pellet size affected scat detection, juvenile or smaller-sized animals may be less detectable than adult or large animals, which could introduce bias into abundance estimates. Knowledge of relationships between environmental variables and scat detection may allow researchers to optimize sampling protocols to maximize utility of noninvasive sampling for wild pigs and other social ungulates.

Fur and faeces: an experimental assessment of non-invasive DNA sampling for the European pine marten

Non-invasive genetic sampling using materials such as faeces or hair can be used to monitor wildlife populations, although DNA quality is often poor. Improving sampling efficiency and minimising factors that reduce DNA quality are therefore critical. After a severe decline, the European pine marten, Martes martes, has reclaimed much of its former range in Scotland, UK. Recording this rapid range expansion requires developing techniques for accurate monitoring, but this is hampered by the species’ elusive behaviour. We tested two sampling methods, hair collected from hair tubes and faeces (scat) collected along tracks, to assess the effects of key environmental and sampling variables on DNA quality and sampling efficiency. For hair, we tested the influence of hair tube location (distance from forest tracks) on collection rate and sex ratio of animals successfully sampled. For scats, we assessed the effect of time since defecation (1 to 16 days) on genotyping error rates and success under two contrasting environmental conditions (exposed to rainfall or sheltered). We found no bias in the collection rate or sex ratio of animals detected by hair samples with differing proximity to forest tracks. DNA amplification failure for scats exposed to rainfall increased from 28 to 65 % over the 16-day experimental period. During periods of low rainfall, the length of collection sessions could therefore be extended to increase sample number without risk of DNA degradation. Lack of bias in hair collection rates with proximity to forest tracks provides justification for tube placement close to tracks, as this reduces survey effort. These findings provide guidance for the development of efficient and cost-effective non-invasive sampling of Scottish pine martens.

Fine-Scale Habitat Segregation between Two Ecologically Similar Top Predators.

Similar, coexisting species often segregate along the spatial ecological axis. Here, we examine if two top predators (jaguars and pumas) present different fine-scale habitat use in areas of coexistence, and discuss if the observed pattern can be explained by the risk of interference competition between them. Interference competition theory predicts that pumas should avoid habitats or areas used by jaguars (the dominant species), and as a consequence should present more variability of niche parameters across study areas. We used non-invasive genetic sampling of faeces in 12 different areas and sensor satellite fine-scale habitat indices to answer these questions. Meta-analysis confirmed differences in fine-scale habitat use between jaguars and pumas. Furthermore, average marginality of the realized niches of pumas was more variable than those of jaguars, and tolerance (a measure of niche breadth) was on average 2.2 times higher in pumas than in jaguars, as expected under the interference competition risk hypothesis. The use of sensor satellite fine-scale habitat indices allowed the detection of subtle differences in the environmental characteristics of the habitats used by these two similar top predators, which, as a rule, until now were recorded using the same general habitat types. The detection of fine spatial segregation between these two top predators was scale-dependent.

Gene flow and genetic structure of the puma and jaguar in Mexico

Gene flow among populations and subpopulations homogenizes allele frequencies. This mechanism is strongly influenced by species dispersal ability, frequently correlating genetic variation with distance among individuals, which is also known as an isolation-by-distance pattern. Species with high dispersal abilities are expected to show a limited isolation-by-distance pattern compared to those with reduced dispersal. Here, we use non-invasive genetic sampling of faeces to evaluate how isolation-by-distance is differentially structured in jaguar and puma populations in Mexico. We have optimized and validated a reliable and standardized non-invasive genetic sampling protocol to monitor pumas based on 12 microsatellite markers, as well as applied a previously published and consistent protocol for jaguars. We found that jaguars and pumas were not uniform and panmictic populations. Spatial trends in allele frequencies for both species generated clinal patterns. However, pumas showed a stronger isolation-by-distance pattern than jaguars, which was expected since pumas seem to have a lower dispersal ability than jaguars. The genetic structures of both species differed at the level of subpopulations. These results reinforce the differences in intensity of isolation-by-distance between two generalist species with high dispersal ability.

Non-invasive genetic sampling reveals diet shifts, but little difference in endoparasite richness and faecal glucocorticoids, in Belizean felids inside and outside protected areas

Abstract:Many Neotropical felids are threatened with extinction due to direct effects of habitat destruction and/or human persecution. However, indirect and synergistic effects of human-felid conflict remain under-studied and potentially include increased stress and diet shifts that may negatively impact felid health. We hypothesized that faecal glucocorticoid metabolites (FGM) and endoparasite species richness (ESR) would be higher, and diet would shift, for felids outside protected areas where conflict occurs. In north-western Belize, a scat-detector dog located 336 faecal samples, identified to species and individual using DNA analyses. DNA amplification success was substantially higher within protected areas than outside. We detected jaguar, puma, ocelot, jaguarundi and domestic cat. FGMs were higher in puma and jaguarundi than in other felids, while ESR was similar across felids with domestic cats exhibiting the highest number of genera. Diet partitioning occurred among felids, but domestic cats may compete with ocelot and jaguarundi for small prey. Outside of protected areas, large cats shifted their diet to smaller prey and livestock remains were not found. Contrary to our hypotheses, FGM and ESR did not differ inside versus outside protected areas, but sample sizes were low in human-modified areas. We provide a baseline on wild felid adrenal activity, endoparasites and diet and suggest improvements to increase sample sizes outside protected areas. Our research provides a template for expanding non-invasive sampling approaches more widely across the range of Neotropical felids.

Monitoring coyote population dynamics with fecal DNA and spatial capture-recapture

Estimating coyote (Canis latrans) density and other demographic parameters is difficult, particularly for populations that exist at low density. This is the situation for recently established coyote populations in the eastern United States where populations may be below carrying capacity and growth unregulated. We used non-invasive fecal DNA collected from 5 scat sampling sessions over 2.5 years to estimate population parameters (i.e., density, apparent survival, recruitment, and population growth) for coyotes at 2 different sites in the Ridge and Valley region of the central Appalachians in Virginia, USA. We identified individuals using microsatellite genotypes and estimated apparent survival for the local population at both sites across the 5 sessions in a single Cormack–Jolly–Seber model. We estimated density for each site and session separately using single session spatial replicates of 0.5-km transect segments as traps in a spatial capture–recapture model. Finally, we derived estimates of recruitment and population growth using an ad hoc robust design approach. We were able to estimate population parameters, even though coyote densities at both sites were low. Generally, derived recruitment and apparent survival were inversely related across sites, however, precision in estimates was poor. Thus, although there appeared to be some differences in demographic estimates for local coyote populations, uncertainty in parameters was too great to detect changes in demographic rates over short periods of time using ad hoc robust design. However, the non-invasive genetic sampling and spatial capture–recapture approach provides a useful methodology and framework for future research intended to estimate population dynamics for coyotes. This method will also be useful for other species that occur at low densities, over large spatial scales, and lack distinguishing marks for camera-trap surveys. Finally, we believe this method will allow for detection of population trends over greater periods of time, and we consider alternate sampling strategies and modeling approaches that may improve the ability to estimate demographic rates of change for coyote populations using noninvasive genetics and spatial capture–recapture. © 2016 The Wildlife Society.

Estimating Sonoran pronghorn abundance and survival with fecal DNA and capture-recapture methods.

Population abundance estimates are important for management but can be challenging to determine in low-density, wide-ranging, and endangered species, such as Sonoran pronghorn (Antilocapra americana sonoriensis). The Sonoran pronghorn population has been increasing; however, population estimates are currently derived from a biennial aerial count that does not provide survival or recruitment estimates. We identified individuals through noninvasively collected fecal DNA and used robust-design capture-recapture to estimate abundance and survival for Sonoran pronghorn in the United States from 2013 to 2014. In 2014 we generated separate population estimates for pronghorn gathered near 13 different artificial water holes and for pronghorn not near water holes. The population using artificial water holes had 116 (95% CI 102-131) and 121 individuals (95% CI 112-132) in 2013 and 2014, respectively. For all locations, we estimated there were 144 individuals (95% CI 132-157). Adults had higher annual survival probabilities (0.83, 95% CI 0.69-0.92) than fawns (0.41, 95% CI 0.21-0.65). Our use of targeted noninvasive genetic sampling and capture-recapture with Sonoran pronghorn fecal DNA was an effective method for monitoring a large proportion of the population. Our results provided the first survival estimates for this population in over 2 decades and precise estimates of the population using artificial water holes. Our method could be used for targeted sampling of broadly distributed species in other systems, such as in African savanna ecosystems, where many species congregate at watering sites.

Estimating population size using single-nucleotide polymorphism-based pedigree data.

Reliable population estimates are an important aspect of sustainable wildlife management and conservation but can be difficult to obtain for rare and elusive species. Here, we test a new census method based on pedigree reconstruction recently developed by Creel and Rosenblatt (2013). Using a panel of 96 single‐nucleotide polymorphisms (SNPs), we genotyped fecal samples from two Swedish brown bear populations for pedigree reconstruction. Based on 433 genotypes from central Sweden (CS) and 265 from northern Sweden (NS), the population estimates (N = 630 for CS, N = 408 for NS) fell within the 95% CI of the official estimates. The precision and accuracy improved with increasing sampling intensity. Like genetic capture–mark–recapture methods, this method can be applied to data from a single sampling session. Pedigree reconstruction combined with noninvasive genetic sampling may thus augment population estimates, particularly for rare and elusive species for which sampling may be challenging.

Noninvasive genetic sampling reveals intrasex territoriality in wolverines.

Due to its conspicuous manifestations and its capacity to shape the configuration and dynamics of wild populations, territorial behavior has long intrigued ecologists. Territoriality and other animal interactions insitu have traditionally been studied via direct observations and telemetry. Here, we explore whether noninvasive genetic sampling, which is increasingly supplementing traditional field methods in ecological research, can reveal territorial behavior in an elusive carnivore, the wolverine (Gulo gulo). Using the locations of genotyped wolverine scat samples collected annually over a period of 12years in central Norway, we test three predictions: (1) male home ranges constructed from noninvasive genetic sampling data are larger than those of females, (2) individuals avoid areas used by other conspecifics of the same sex (intrasexual territoriality), and (3) avoidance of same-sex territories diminishes or disappears after the territory owner's death. Each of these predictions is substantiated by our results: sex-specific differences in home range size and intrasexual territoriality in wolverine are patently reflected in the spatial and temporal configuration of noninvasively collected genetic samples. Our study confirms that wildlife monitoring programs can utilize the spatial information in noninvasive genetic sampling data to detect and quantify home ranges and social organization.

Sample diversity adds value to non-invasive genetic assessment of a pine marten (Martes martes) population in Galloway Forest, southwest Scotland

The European pine marten (Martes martes) is a species of considerable conservation interest in Britain due to its rarity and status as a recovering native carnivore. In recent years, there has been increased application of non-invasive genetic sampling methods in population studies of Martes species. We investigated the effect of sample source (hair and faeces) in the non-invasive assessment of the distribution, population size and density of pine martens in the Fleet Basin in Galloway Forest, southwest Scotland. Fifty-two hair samples and 114 scats were collected during September and October 2014. Genetic analysis was used to identify the species, gender and individual genotype of samples. There was a significant difference in the genotyping success rate for hair samples (43 %) and scat samples (24 %). In total, 15 individual pine martens were identified; 7 males and 8 females. Capture-recapture programme Capwire produced a population size estimate of 18 individuals (95 % CI 15 to 25). Of the 15 individuals sampled, 14 (93 %) were detected from scat sampling and 5 (33 %) were detected from hair sampling. The population density estimate for a post-breeding population was 0.13 to 0.15 pine martens per square kilometre which is towards the lower limit of densities reported for pine martens elsewhere in Scotland. Data from the study highlight that future non-invasive studies aiming to determine pine marten population size and density should incorporate the collection of both hair and scat samples in order to detect as many individuals within the population as possible.

Feasibility of using Scent-Baited Hair Traps to Monitor Carnivore Populations in Peninsular Malaysia

Non-invasive genetic sampling is increasingly being used for monitoring mammalian carnivore populations. However, environmental conditions in the tropics challenge researchers' ability to collect samples. We present the results of a preliminary study on the feasibility of using scent-baited hair traps for population monitoring of mammalian carnivores in Peninsular Malaysia. Stations were baited using either fatty acid scent or male cologne applied to hair traps. Video camera traps were also used to monitor carnivore reactions to the scent stations. We recorded 19 visits by seven carnivore species over 764 camera trap nights. Cheek-rubbing and scent-marking behaviour was recorded only for single individuals of two species: the Malayan tiger ( Panthera tigris jacksoni) and clouded leopard ( Neofelis nebulosa). This study suggests that scent-baited hair traps hold some promise for ecological issues requiring DNA analysis in Peninsular Malaysia. Additional research is needed to develop its full potential for conservation monitoring of large carnivores.

Spider Web DNA: A New Spin on Noninvasive Genetics of Predator and Prey.

Noninvasive genetic sampling enables biomonitoring without the need to directly observe or disturb target organisms. This paper describes a novel and promising source of noninvasive spider and insect DNA from spider webs. Using black widow spiders (Latrodectus spp.) fed with house crickets (Acheta domesticus), we successfully extracted, amplified, and sequenced mitochondrial DNA from spider web samples that identified both spider and prey to species. Detectability of spider DNA did not differ between assays with amplicon sizes from 135 to 497 base pairs. Spider and prey DNA remained detectable at least 88 days after living organisms were no longer present on the web. Spider web DNA as a proof-of-concept may open doors to other practical applications in conservation research, pest management, biogeography studies, and biodiversity assessments.

ConGenR: rapid determination of consensus genotypes and estimates of genotyping errors from replicated genetic samples

ConGenR (available at http://www.uidaho.edu/cnr/research-outreach/facilities/leecg/publications-and-software ) is an R based conservation genetics script that facilitates rapid determination of consensus genotypes from replicated samples, determines overall (successful amplifications/amplification attempted) and individual sample level (proportion of samples with successful amplifications at n loci) amplification success rates, and quantifies genotyping error rates. ConGenR is intended for use with codominant, multilocus microsatellite data generated primarily through noninvasive genetic sampling and processed with a multi-tubes approach. ConGenR handles input that can be easily exported from GENEMAPPER, a program commonly used to score allele sizes. Amplification success and genotyping error rates can be evaluated by sample class (i.e., any identifiable and meaningful subdivision of samples; e.g., sex, season, region, or sample condition), offering insights into processes driving amplification success and genotyping error rates. Additionally, amplification success and genotyping error rates are calculated by locus, expediting the identification of problematic loci during pilot studies.

A New Method for Noninvasive Genetic Sampling of Saliva in Ecological Research.

Noninvasive samples for genetic analyses have become essential to address ecological questions. Popular noninvasive samples such as faeces contain degraded DNA which may compromise genotyping success. Saliva is an excellent alternative DNA source but scarcity of suitable collection methods makes its use anecdotal in field ecological studies. We develop a noninvasive method of collection that combines baits and porous materials able to capture saliva. We report its potential in optimal conditions, using confined dogs and collecting saliva early after deposition. DNA concentration in saliva extracts was generally high (mean 14 ng μl-1). We correctly identified individuals in 78% of samples conservatively using ten microsatellite loci, and 90% of samples using only eight loci. Consensus genotypes closely matched reference genotypes obtained from hair DNA (99% of identification successes and 91% of failures). Mean genotyping effort needed for identification using ten loci was 2.2 replicates. Genotyping errors occurred at a very low frequency (allelic dropout: 2.3%; false alleles: 1.5%). Individual identification success increased with duration of substrate handling inside dog’s mouth and the volume of saliva collected. Low identification success was associated with baits rich in DNA-oxidant polyphenols and DNA concentrations <1 ng μl-1. The procedure performed at least as well as other noninvasive methods, and could advantageously allow detection of socially low-ranked individuals underrepresented in sources of DNA that are involved in marking behaviour (faeces or urine). Once adapted and refined, there is promise for this technique to allow potentially high rates of individual identification in ecological field studies requiring noninvasive sampling of wild vertebrates.

Genetic and Photographic Detections Document Sierra Nevada Red Fox in the Northern Cascades of Oregon

Abstract The red fox (Vulpes vulpes) exhibits a high degree of ecological plasticity, but the indigenous Sierra Nevada red fox (SNRF; V. v. necator) belongs to an evolutionarily divergent complex of montane red foxes, apparently specialized to subalpine habitats. Mountain red foxes historically occurred among isolated sky islands of the major mountain ranges of the western U.S., with the SNRF occurring along the Pacific Crest of Oregon and California. Little is known about the current distribution and status of SNRF in Oregon, which complicates regulatory decisions and impedes conservation of the subspecies. We conducted a survey in the northern Oregon Cascade Range during 2012–2014 using baited ground-level camera stations and noninvasive genetic sampling. Our objectives were to document red fox occurrences in our study area and investigate their genetic affinities using mitochondrial DNA sequences to indicate maternal haplotype. We detected red foxes at 11 of 41 camera stations and collected DNA from 18...

Compensatory immigration counteracts contrasting conservation strategies of wolverines (Gulo gulo) within Scandinavia

In wide ranging species, portions of the same population often fall within different administrative jurisdictions; where different regulations apply. The same species can be fully protected or heavily harvested on different sides of a border. This can generate a source–sink dynamic from the areas with lower to those with higher mortality, a process known as compensatory immigration. We tested this hypothesis on the wolverine (Gulo gulo) population of southern Scandinavia, which is shared between two countries: Sweden and Norway. Wolverines are fully protected in Sweden, but subject to intensive population regulation in Norway. Using non-invasive genetic sampling and capture–recapture modeling, we analyzed the dynamics of wolverine survival and emigration patterns between 2002 and 2013. Wolverines in Norway experienced a lower survival than in Sweden. Migration across the national border was directed towards movements from Sweden to Norway. There was a functional relationship between harvest rate in Norway and emigration rates across the national border, both at the individual and population level, thus confirming the compensatory immigration hypothesis. Contrasting management regimes within the same population can generate undesired demographic and spatial dynamics, jeopardize conservation goals on the two sides of a border, and reduce the efficiency of management actions. This calls for the adoption of a coordinate population approach in large carnivore conservation and management. Failing to do so can cause a waste of the already limited resources allocated for large carnivore conservation, and it might hinder effective conflict mitigation.