Search

Monitoring the demographics and genetics of reintroduced populations is critical to evaluating reintroduction success, but species ecology and the landscapes that they inhabit often present challenges for accurate assessments. If suitable habitats are restricted to hierarchical dendritic networks, such as river systems, animal movements are typically constrained and may violate assumptions of methods commonly used to estimate demographic parameters. Using genetic detection data collected via fecal sampling at latrines, we demonstrate applicability of the spatial capture–recapture (SCR) network distance function for estimating the size and density of a recently reintroduced North American river otter (Lontra canadensis) population in the Upper Rio Grande River dendritic network in the southwestern United States, and we also evaluated the genetic outcomes of using a small founder group (n = 33 otters) for reintroduction. Estimated population density was 0.23–0.28 otter/km, or 1 otter/3.57–4.35 km, with weak evidence of density increasing with northerly latitude (β = 0.33). Estimated population size was 83–104 total otters in 359 km of riverine dendritic network, which corresponded to average annual exponential population growth of 1.12–1.15/year since reintroduction. Growth was ≥40% lower than most reintroduced river otter populations and strong evidence of a founder effect existed 8–10 years post‐reintroduction, including 13–21% genetic diversity loss, 84%–87% genetic effective population size decline, and rapid divergence from the source population (F ST accumulation = 0.06/generation). Consequently, genetic restoration via translocation of additional otters from other populations may be necessary to mitigate deleterious genetic effects in this small, isolated population. Combined with non‐invasive genetic sampling, the SCR network distance approach is likely widely applicable to demogenetic assessments of both reintroduced and established populations of multiple mustelid species that inhabit aquatic dendritic networks, many of which are regionally or globally imperiled and may warrant reintroduction or augmentation efforts., Monitoring the demographics and genetics of reintroduced populations is critical to evaluating reintroduction success, but species ecology and the landscapes that they inhabit often present challenges for accurate assessments. Using genetic detection data collected via fecal sampling at latrines, we demonstrate applicability of the spatial capture–recapture (SCR) network distance function for estimating the size and density of a recently reintroduced North American river otter (Lontra canadensis) population in the Upper Rio Grande River dendritic network in the southwestern United States, and we also evaluated the genetic outcomes of using a small founder group for reintroduction. Combined with noninvasive genetic sampling, the SCR network distance approach is likely widely applicable to demogenetic assessments of both reintroduced and established populations of multiple mustelid species that inhabit aquatic dendritic networks, many of which are regionally or globally imperiled and may warrant reintroduction or augmentation efforts.

Genetic composition can influence host susceptibility to, and transmission of, pathogens, with potential population‐level consequences. In bighorn sheep (Ovis canadensis), pneumonia epidemics caused by Mycoplasma ovipneumoniae have been associated with severe population declines and limited recovery across North America. Adult survivors either clear the infection or act as carriers that continually shed M. ovipneumoniae and expose their susceptible offspring, resulting in high rates of lamb mortality for years following the outbreak event. Here, we investigated the influence of genomic composition on persistent carriage of M. ovipneumoniae in a well‐studied bighorn sheep herd in the Wallowa Mountains of Oregon, USA. Using 10,605 SNPs generated using RADseq technology for 25 female bighorn sheep, we assessed genomic diversity metrics and employed family‐based genome‐wide association methodologies to understand variant association and genetic architecture underlying chronic carriage. We observed no differences among genome‐wide diversity metrics (heterozygosity and allelic richness) between groups. However, we identified two variant loci of interest and seven associated candidate genes, which may influence carriage status. Further, we found that the SNP panel explained ~55% of the phenotypic variance (SNP‐based heritability) for M. ovipneumoniae carriage, though there was considerable uncertainty in these estimates. While small sample sizes limit conclusions drawn here, our study represents one of the first to assess the genomic factors influencing chronic carriage of a pathogen in a wild population and lays a foundation for understanding genomic influence on pathogen persistence in bighorn sheep and other wildlife populations. Future research should incorporate additional individuals as well as distinct herds to further explore the genomic basis of chronic carriage., Bighorn sheep populations impacted by infectious pneumonia may experience long‐term recovery complications due to chronically infected individuals shedding pathogen and suppressing lamb recruitment in years following outbreak events. We investigated genetic associations with the chronic carrier phenotype in bighorn sheep to better understand underlying dynamics influencing pathogen persistence in wild sheep populations.

Understanding the neutral (demographic) and adaptive processes leading to the differentiation of species and populations is a critical component of evolutionary and conservation biology. In this context, recently diverged taxa represent a unique opportunity to study the process of genetic differentiation. Northern and southern Idaho ground squirrels (Urocitellus brunneus – NIDGS, and U. endemicus - SIDGS, respectively) are a recently diverged pair of sister species that have undergone dramatic declines in the last 50 years and are currently found in metapopulations across restricted spatial areas with distinct environmental pressures. Here we genotyped single-nucleotide polymorphisms (SNPs) from buccal swabs with restriction site-associated DNA sequencing (RADseq). With these data we evaluated neutral genetic structure at both theinter- and intraspecific level, and identified putatively adaptive SNPs using population structure outlier detection and genotype-environment association (GEA) analyses. At the interspecific level, we detected a clear separation between NIDGS and SIDGS, and evidence for adaptive differentiation putatively linked to torpor patterns. At the intraspecific level, we found evidence of both neutral and adaptive differentiation. For NIDGS, elevation appears to be the main driver of adaptive differentiation, while neutral variation patterns match and expand information on the low connectivity between some populations identified in previous studies using microsatellite markers. For SIDGS, neutral substructure generally reflected natural geographic barriers, while adaptive variation reflected differences in land cover and temperature, as well as elevation. These results clearly highlight the roles of neutral and adaptive processes for understanding the complexity of the processes leading to species and population differentiation, which can have important conservation implications in susceptible and threatened species.

Loss of genetic diversity has serious conservation consequences (e.g., loss of adaptive potential, reduced population viability), but is difficult to evaluate without developing long‐term, multigenerational datasets. Alternatively, historical samples can provide insights into changes in genetic diversity and effective population size (N e). Kit foxes (Vulpes macrotis) are a species of conservation concern across much of their range. In western Utah, kit fox abundance has declined precipitously from historical levels, causing concern about population persistence. We analyzed genetic samples from museum specimens and contemporary scats to evaluate temporal changes in (a) genetic diversity and (b) N e for kit foxes in western Utah, and (c) discuss our findings with respect to population risk and conservation. The N e of kit foxes in western Utah has decreased substantially. When compared to established conservation thresholds for N e (e.g., the 50/500 rule), observed levels suggest the population may be at risk of inbreeding depression and local extinction. In contrast, we found no significant decrease in genetic diversity associated with declining N e. We detected evidence of low levels of immigration into the population and suspect genetic diversity may have been maintained by this previously undescribed gene flow from adjacent populations. Low or intermittent immigration may serve to temper the potential short‐term negative consequences of low N e. We recommend that kit fox conservation efforts focus on evaluating and maintaining landscape connectivity. We demonstrate how historical specimens can provide a baseline of comparison for contemporary populations, highlighting the importance of natural history collections to conservation during a period of declining funding and support.

Many species that undergo long breeding migrations, such as anadromous fishes, face highly heterogeneous environments along their migration corridors and at their spawning sites. These environmental challenges encountered at different life stages may act as strong selective pressures and drive local adaptation. However, the relative influence of environmental conditions along the migration corridor compared with the conditions at spawning sites on driving selection is still unknown. In this study, we performed genome–environment associations (GEA) to understand the relationship between landscape and environmental conditions driving selection in seven populations of the anadromous Chinook salmon (Oncorhynchus tshawytscha)—a species of important economic, social, cultural, and ecological value—in the Columbia River basin. We extracted environmental variables for the shared migration corridors and at distinct spawning sites for each population, and used a Pool‐seq approach to perform whole genome resequencing. Bayesian and univariate GEA tests with migration‐specific and spawning site‐specific environmental variables indicated many more candidate SNPs associated with environmental conditions at the migration corridor compared with spawning sites. Specifically, temperature, precipitation, terrain roughness, and elevation variables of the migration corridor were the most significant drivers of environmental selection. Additional analyses of neutral loci revealed two distinct clusters representing populations from different geographic regions of the drainage that also exhibit differences in adult migration timing (summer vs. fall). Tests for genomic regions under selection revealed a strong peak on chromosome 28, corresponding to the GREB1L/ROCK1 region that has been identified previously in salmonids as a region associated with adult migration timing. Our results show that environmental variation experienced throughout migration corridors imposed a greater selective pressure on Chinook salmon than environmental conditions at spawning sites., We assessed whether seven populations of ocean‐type lineage Chinook salmon in the Columbia River basin experience selection primarily driven by the migration corridor environment or by the spawning site environment. Results using whole‐genome resequencing suggest that environmental conditions at migration corridors may be more influential on Chinook salmon genetic variation compared with spawning site environmental conditions.

Harvest can affect vital rates such as reproduction and survival, but also genetic measures of individual and population health. Grey wolves (Canis lupus) live and breed in groups, and effective population size is a small fraction of total abundance. As a result, genetic diversity of wolves may be particularly sensitive to harvest. We evaluated how harvest affected genetic diversity and relatedness in wolves. We hypothesized that harvest would (a) reduce relatedness of individuals within groups in a subpopulation but increase relatedness of individuals between groups due to increased local immigration, (b) increase individual heterozygosity and average allelic richness across groups in subpopulations and (c) add new alleles to a subpopulation and decrease the number of private alleles in subpopulations due to an increase in breeding opportunities for unrelated individuals. We found harvest had no effect on observed heterozygosity of individuals or allelic richness at loci within subpopulations but was associated with a small, biologically insignificant effect on within-group relatedness values in grey wolves. Harvest was, however, positively associated with increased relatedness of individuals between groups and a net gain (+16) of alleles into groups in subpopulations monitored since harvest began, although the number of private alleles in subpopulations overall declined. Harvest likely created opportunities for wolves to immigrate into nearby groups and breed, thereby making groups in subpopulations more related over time. Harvest appears to affect genetic diversity in wolves at the group and population levels, but its effects are less apparent at the individual level given the population sizes we studied.

Demographic monitoring is required in threatened species management, yet effective and efficient monitoring is challenging for species that are difficult to capture or susceptible to capture stress. One possible monitoring approach for such species is non-invasive genetic sampling with capture–recapture methods (genetic capture–recapture). We evaluated the performance of genetic capture–recapture in a challenging model system, monitoring the threatened Sonoran pronghorn Antilocapra americana sonoriensis. In an effort to determine the best (i.e. efficient, accurate, precise, cost-effective) method for abundance estimation, we used simulations to examine the optimal genetic capture–recapture faecal sampling design for this population. We simulated encounter histories for 100–300 individuals, with 0.33–3.33 samples/individual/session, in 1–3 sampling sessions. We explored trade-offs between sample size, number of sessions and multi-session (MARK) versus single-session (capwire) closed capture–recapture abundance estimators, and an accurate and precise estimate. We also compared the cost between the genetic capture–recapture approaches and current aerial monitoring methods. Abundance was biased positively in capwire and negatively in MARK. Bias increased and precision decreased with fewer samples/individual/session. Annual genetic capture–recapture monitoring cost was nearly twice the cost of aerial surveys, although genetic capture–recapture methods provided much higher precision. However at the current estimated abundance (c. 200), the same level of precision achieved with aerial methods can be obtained by collecting 0.75 samples/individual in a single session, for an annual cost saving of > USD 4,000. This approach of comparing estimator performance and cost can easily be applied to other systems and is a useful evaluation for managers to implement prior to designing capture–recapture studies.

Admixture resulting from natural dispersal processes can potentially generate novel phenotypic variation that may facilitate persistence in changing environments or result in the loss of population-specific adaptations. Yet, under the US Endangered Species Act, policy is limited for management of individuals whose ancestry includes a protected taxon; therefore, they are generally not protected under the Act. This issue is exemplified by the recently re-established grey wolves of the Pacific Northwest states of Washington and Oregon, USA. This population was likely founded by two phenotypically and genetically distinct wolf ecotypes: Northern Rocky Mountain (NRM) forest and coastal rainforest. The latter is considered potentially threatened in southeast Alaska and thus the source of migrants may affect plans for their protection. To assess the genetic source of the re-established population, we sequenced a ~ 300 bp portion of the mitochondrial control region and ~ 5 Mbp of the nuclear genome. Genetic analysis revealed that the Washington wolves share ancestry with both wolf ecotypes, whereas the Oregon population shares ancestry with NRM forest wolves only. Using ecological niche modelling, we found that the Pacific Northwest states contain environments suitable for each ecotype, with wolf packs established in both environmental types. Continued migration from coastal rainforest and NRM forest source populations may increase the genetic diversity of the Pacific Northwest population. However, this admixed population challenges traditional management regimes given that admixture occurs between an adaptively distinct ecotype and a more abundant reintroduced interior form. Our results emphasize the need for a more precise US policy to address the general problem of admixture in the management of endangered species, subspecies, and distinct population segments.

Population density estimates are necessary to inform management and conservation, yet are difficult to obtain for cryptic species such as carnivores, and often require intensive sampling. We implemented a single-survey, closed session, scat sampling protocol to estimate bobcat density using fecal DNA and spatial capture-recapture at two sites over five sessions in Virginia, USA. We employed a Poisson encounter model to allow for multiple detections on scat transect segments over a single collection interval, and compared single session and multistrata (closed, multi-site, multisession) spatial capture-recapture (SCR) approaches to estimate density for each site and session. We found improved precision for most estimates using the multistrata SCR approach, sharing data on baseline detection and individual movement across sites and sessions. We suggest the summer session estimates are representative of the resident population, that differences in density between summer and winter are representative of potential net recruitment, and that differences between consecutive summer sessions are representative of the net recruitment realized for the population (dependent on survival and emigration). Finally, we assessed factors affecting precision in single session model estimates and provide recommendations to improve detection and reduce credible intervals that may be applicable across the bobcat range and to other carnivore species. The single survey transect methodology provides flexibility in monitoring carnivore populations specifically, or as part of concurrent monitoring for multiple carnivore species. This methodology has potential to dramatically increase the effectiveness of conservation and management dollars, improving our ability to monitor carnivore populations and assess conservation needs and actions.

Superspreading, the phenomenon where a small proportion of individuals contribute disproportionately to new infections, has profound effects on disease dynamics. Superspreading can arise through variation in contacts, infectiousness or infectious periods. The latter has received little attention, yet it drives the dynamics of many diseases of critical public health, livestock health and conservation concern. Here, we present rare evidence of variation in infectious periods underlying a superspreading phenomenon in a free-ranging wildlife system. We detected persistent infections of Mycoplasma ovipneumoniae, the primary causative agent of pneumonia in bighorn sheep (Ovis canadensis), in a small number of older individuals that were homozygous at an immunologically relevant genetic locus. Interactions among age-structure, genetic composition and infectious periods may drive feedbacks in disease dynamics that determine the magnitude of population response to infection. Accordingly, variation in initial conditions may explain divergent population responses to infection that range from recovery to catastrophic decline and extirpation.

The expansion of coyotes (Canis latrans) into the eastern United States has had major consequences for ecological communities and wildlife managers. Despite this, there has been little investigation of the genetics of coyotes across much of this region, especially outside of the northeast. Understanding patterns of genetic structure and interspecific introgression would provide insights into the colonization history of the species, its response to the modern environment, and interactions with other canids. We examined the genetic characteristics of 121 coyotes from the mid-Atlantic states of West Virginia and Virginia by genotyping 17 polymorphic nuclear DNA microsatellite loci. These genotypes were compared with those from other canid populations to evaluate the extent of genetic introgression. We conducted spatial clustering analyses and spatial autocorrelation to assess genetic structure among sampled coyotes. Coyotes across the 2 states had high genetic diversity, and we found no evidence of genetic structure. Six to sixteen percent of individuals displayed some evidence of genetic introgression from other species depending on the method and criteria used, but the population possessed predominantly coyote ancestry. Our findings suggested introgression from other canid populations has played less of a role in shaping the genetic character of coyotes in these states compared with populations closer to the Canadian border. Coyotes appear to display a panmictic population structure despite high habitat heterogeneity and heavy human influence in the spatial environment, underscoring the adaptability of the species.

Monitoring demographic and genetic parameters of reintroduced populations of endangered species is essential for evaluating and informing conservation strategies to maximize the chances of a successful recovery. We applied noninvasive genetic monitoring using fecal DNA to evaluate demographic and genetic parameters as they relate to the recovery of the endangered Columbia Basin pygmy rabbit (Brachylagus idahoensis) in central Washington, United States, during the initial 3 years of a renewed reintroduction effort. We quantified post-release dispersal, apparent survival, and reproduction in the wild, and monitored the genetic diversity and composition of the released cohorts, wild surviving population, and breeding population held in large enclosures. During this study, we reintroduced 1,206 pygmy rabbits into the wild, and detected a total of 176 individuals living on or near the release area during surveys the following winters. Median dispersal distance of juveniles was 776 m, compared to 471 m for adults, and dispersal distances for juveniles decreased for rabbits released later in the release season. Survival of juveniles differed across years and was positively associated with release date, release weight (a surrogate for age), and heterozygosity. Survival of adults was similarly influenced by release day, with some evidence for a positive effect of heterozygosity. Only 14 wild-born individuals were detected during the study. Genetic monitoring was an effective way to evaluate the demographic and genetic status of the reintroduced population within a limited study area, to inform changes to the conservation strategy, and to generate a data set to address long-term research and recovery goals.

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. DBK USAID [FOG AID-367-G-11-00001]; United States Agency for International DevelopmentUnited States Agency for International Development (USAID) Funded by DBK USAID FOG AID-367-G-11-00001 United States Agency for International Development.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Knowledge of genetic diversity and population structure is critical for conservation and management planning at the population level within a species’ range. Many brown bear populations in Central Asia are small and geographically isolated, yet their phylogeographic relationships, genetic diversity, and contemporary connectivity are poorly understood. To address this knowledge gap, we collected brown bear samples from the Gobi Desert (n = 2360), Altai, Sayan, Khentii, and Ikh Khyangan mountains of Mongolia (n = 79), and Deosai National Park in the Himalayan Mountain Range of Pakistan (n = 5) and generated 927 base pairs of mitochondrial DNA (mtDNA) sequence data and genotypes at 13 nuclear DNA microsatellite loci. We documented high levels of mtDNA and nDNA diversity in the brown bear populations of northern Mongolia (Altai, Sayan, Buteeliin nuruu and Khentii), but substantially lower diversity in brown bear populations in the Gobi Desert and Himalayas of Pakistan. We detected 3 brown bear mtDNA phylogeographic groups among bears of the region, with clade 3a1 in Sayan, Khentii, and Buteeliin nuruu mountains, clade 3b in Altai, Sayan, Buteeliin nuruu, Khentii, and Ikh Khyangan, and clade 6 in Gobi and Pakistan. Our results also clarified the phylogenetic relationships and divergence times with other brown bear mtDNA clades around the world. The nDNA genetic structure analyses revealed distinctiveness of Gobi bears and different population subdivisions compared to mtDNA results. For example, genetic distance for nDNA microsatellite loci between the bears in Gobi and Altai (FST = 0.147) was less than that of the Gobi and Pakistan (FST = 0.308) suggesting more recent male-mediated nuclear gene flow between Gobi and Altai than between Gobi and the Pakistan bears. Our results provide valuable information for conservation and management of bears in this understudied region of Central Asia and highlight the need for special protection and additional research on Gobi brown bears.

Connectivity is important for population persistence and can reduce the potential for inbreeding depression. Connectivity between populations can also facilitate disease transmission; respiratory diseases are one of the most important factors affecting populations of bighorn sheep (Ovis canadensis). The mechanisms of connectivity in populations of bighorn sheep likely have implications for spread of disease, but the behaviors leading to connectivity between bighorn sheep groups are not well understood. From 2007–2012, we radio-collared and monitored 56 bighorn sheep in the Salmon River canyon in central Idaho. We used cluster analysis to define social groups of bighorn sheep and then estimated connectivity between these groups using a multi-state mark-recapture model. Social groups of bighorn sheep were spatially segregated and linearly distributed along the Salmon River canyon. Monthly probabilities of movement between adjacent male and female groups ranged from 0.08 (±0.004 SE) to 0.76 (±0.068) for males and 0.05 (±0.132) to 0.24 (±0.034) for females. Movements of males were extensive and probabilities of movement were considerably higher during the rut. Probabilities of movement for females were typically smaller than those of males and did not change seasonally. Whereas adjacent groups of bighorn sheep along the Salmon River canyon were well connected, connectivity between groups north and south of the Salmon River was limited. The novel application of a multi-state model to a population of bighorn sheep allowed us to estimate the probability of movement between adjacent social groups and approximate the level of connectivity across the population. Our results suggest high movement rates of males during the rut are the most likely to result in transmission of pathogens among both male and female groups. Potential for disease spread among female groups was smaller but non-trivial. Land managers can plan grazing of domestic sheep for spring and summer months when males are relatively inactive. Removal or quarantine of social groups may reduce probability of disease transmission in populations of bighorn sheep consisting of linearly distributed social groups. © 2016 The Wildlife Society.

Wildlife managers require knowledge of population demographics, yet for low-density, wide-ranging species procuring demographic information is challenging. While accurate abundance estimates can be costly and difficult to obtain, recruitment and survival trends can be used as an alternative indicator of a population's trajectory. Physical capture has been the traditional practice for obtaining these demographic parameters, yet capture-related stress can lead to reduced levels of fitness, impaired locomotion, or even mortality for some species. Thus, noninvasive sampling methods may provide an alternative to physical capture. Population monitoring of endangered Sonoran pronghorn Antilocapra americana sonoriensis is critical for assessing the success of recovery efforts, and monitoring annual survival and recruitment by age class would provide information on the trajectory of population growth. We measured noninvasively collected Sonoran pronghorn fecal pellets collected post-fawning in Arizona, USA and mat...

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.

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 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.

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.

The decreasing cost and increasing scope and power of emerging genomic technologies are reshaping the field of molecular ecology. However, many modern genomic approaches (e.g., RAD‐seq) require large amounts of high‐quality template DNA. This poses a problem for an active branch of conservation biology: genetic monitoring using minimally invasive sampling (MIS) methods. Without handling or even observing an animal, MIS methods (e.g., collection of hair, skin, faeces) can provide genetic information on individuals or populations. Such samples typically yield low‐quality and/or quantities of DNA, restricting the type of molecular methods that can be used. Despite this limitation, genetic monitoring using MIS is an effective tool for estimating population demographic parameters and monitoring genetic diversity in natural populations. Genetic monitoring is likely to become more important in the future as many natural populations are undergoing anthropogenically driven declines, which are unlikely to abate without intensive adaptive management efforts that often include MIS approaches. Here, we profile the expanding suite of genomic methods and platforms compatible with producing genotypes from MIS, considering factors such as development costs and error rates. We evaluate how powerful new approaches will enhance our ability to investigate questions typically answered using genetic monitoring, such as estimating abundance, genetic structure and relatedness. As the field is in a period of unusually rapid transition, we also highlight the importance of legacy data sets and recommend how to address the challenges of moving between traditional and next‐generation genetic monitoring platforms. Finally, we consider how genetic monitoring could move beyond genotypes in the future. For example, assessing microbiomes or epigenetic markers could provide a greater understanding of the relationship between individuals and their environment.

Recent advances in DNA sequencing and genotyping technologies are rapidly building our capacity to address ecological, evolutionary, and conservation questions for wildlife species. However, a large portion of wildlife genetic research relies on samples containing low quantities and quality of DNA, such as non-invasive, archival, and environmental DNA (eDNA) samples. These samples present unique methodological challenges that are largely responsible for a lag in the adoption of new genetic technologies for many areas of wildlife research. Nonetheless, steady progress is being made as researchers test and refine laboratory protocols and bioinformatic methods tailored to low-quality samples. Here we provide an overview of the progress toward low-quality sample applications for amplicon sequencing, single nucleotide polymorphism (SNP) genotyping, DNA capture, mitogenome sequencing, restriction site-associated DNA sequencing (RADseq), and whole-genome sequencing. We also review methods for generating DNA sequence data from samples comprised of multiple individuals and species, such as eDNA or fecal samples, including metagenome sequencing, metabarcoding, metagenome skimming, and metatranscriptomics. The implementation of these approaches has provided insight into a wide range of questions such as modern and historic population genetic structure and diversity, adaptation, inbreeding, ancient hybridization, occupancy, diet composition, microbiome composition, and many more. As the development of methods tailored for low-quality DNA sources continues to advance over the coming years, we expect these samples to provide unprecedented insight into the ecology, evolution, and conservation of wildlife species.

With fewer than 200 tigers (Panthera tigris tigris) left in Nepal, that are generally confined to five protected areas across the Terai Arc Landscape, genetic studies are needed to provide crucial information on diversity and connectivity for devising an effective country-wide tiger conservation strategy. As part of the Nepal Tiger Genome Project, we studied landscape change, genetic variation, population structure, and gene flow of tigers across the Terai Arc Landscape by conducting Nepal's first comprehensive and systematic scat-based, non-invasive genetic survey. Of the 770 scat samples collected opportunistically from five protected areas and six presumed corridors, 412 were tiger (57%). Out of ten microsatellite loci, we retain eight markers that were used in identifying 78 individual tigers. We used this data set to examine population structure, genetic variation, contemporary gene flow, and potential population bottlenecks of tigers in Nepal. We detected three genetic clusters consistent with three demographic sub-populations and found moderate levels of genetic variation (H-e = 0.61, A(R) = 3.51) and genetic differentiation (F-ST = 0.14) across the landscape. We detected 3-7 migrants, confirming the potential for dispersal-mediated gene flow across the landscape. We found evidence of a bottleneck signature likely caused by large-scale land-use change documented in the last two centuries in the Terai forest. Securing tiger habitat including functional forest corridors is essential to enhance gene flow across the landscape and ensure long-term tiger survival. This requires cooperation among multiple stakeholders and careful conservation planning to prevent detrimental effects of anthropogenic activities on tigers. USAID [FOG AID-367-G-11-00001]; Katheryn Fuller Fellowship (WWF US); Wildlife Conservation Society (WCS); Virginia Tech (VT) This work was supported by USAID FOG AID-367-G-11-00001. Netra Sharma and Bronwyn Llewellyn were involved with the project and helped in conceptualization. The Katheryn Fuller Fellowship (WWF US), Wildlife Conservation Society (WCS), and Virginia Tech (VT) supported Kanchan Thapa.

Tiger (Panthera tigris) populations are in danger across their entire range due to habitat loss, poaching and the demand for tiger parts. The Bengal tiger (Panthera tigris tigris) is an endangered apex predator with a population size estimated to be less than 200 in Nepal. In spite of strict wildlife protection laws, illegal trade of tiger parts is increasing; and Nepal has become one of the major sources and transit routes for poached wildlife parts. Identification of wildlife parts is often challenging for law enforcement officials due to inadequate training and lack of available tools. Here, we describe a molecular forensic approach to gain insight into illegally trafficked tiger parts seized across Nepal. We created Nepal's first comprehensive reference genetic database of wild tigers through the Nepal Tiger Genome Project (2011-2013). This database has nuclear DNA microsatellite genotype and sex profiles, including geo-spatial information, of over 60% (n = 120) of the wild tigers of Nepal. We analyzed 15 putative cases of confiscated poached tiger parts and all were confirmed to be of tiger. Ten samples were identified as male and five were female. We determined probable geo-source location for 9 of the 14 samples with 6-8 nuclear DNA microsatellite loci using inferences from four different statistical assignment methods. Six samples were assigned to Bardia National Park and one of these was an exact match to a female tiger previously profiled in our fecal DNA reference database. Two tiger samples were assigned to Shuklaphanta Wildlife Reserve and one to Chitwan National Park. We are unable to definitively assign five tiger samples which could be offspring dispersers or might have come from tiger population outside of Nepal. Our study revealed that the western region, particularly Bardia National Park, is a poaching hotspot for illegal tiger trade in Nepal. We present feasibility of using molecular forensic based evidence to incriminate criminals in a court of law in the fight against wildlife crime.

Isolation of environmental DNA (eDNA) is becoming a valuable tool for detecting presence of rare or secretive aquatic species. The recent use of quantitative PCR (qPCR) with eDNA sampling presents the possibility of using this method to infer population abundance and status. This approach would be especially useful for species such as the Eastern hellbender (Cryptobranchus alleganiensis alleganiensis), a declining, secretive, aquatic salamander that requires intense field survey effort to study. In 2012, we conducted eDNA sampling at sites across the range of the species in North Carolina. Our objectives were to assess presence across 61 sites, test for a correlation of abundance and biomass with eDNA estimates at a subset of 23 sites, and sample at multiple spatial and temporal scales in three river systems. Overall, we detected hellbender eDNA at 33 sites, including all sites with 2012 hellbender records, 71% of all recent or historic sites with hellbender presence, and at nine sites that lack species occurrence records. We did not find a correlation between eDNA estimates and field survey counts of individuals or biomass. We detected a strong temporal increase in eDNA during the September breeding period, but no consistent evidence of a spatial relationship with eDNA. Overall, our results demonstrate the efficacy of eDNA for detecting hellbender populations. Furthermore, the potential utility of qPCR to assess population status in hellbenders requires further study and testing, although it may be promising for determining population reproductive status.

Hybridization presents a unique challenge for conservation biologists and managers. While hybridization is an important evolutionary process, hybridization is also a threat formany native species. The endangered species recovery effort for the red wolf Canis rufus is a classic system for understanding and addressing the challenges of hybridization. From 1987?1993, 63 red wolves were released from captivity in eastern North Carolina, USA, to establish a free-ranging, non-essential experimental population. By 1999, managers recognized hybridization with invasive coyotes Canis latrans was the single greatest threat to successful recovery, and an adaptive management plan was adopted with innovative approaches for managing the threat of hybridization. Here we review the application and results of the adaptive management efforts from 1993 to 2013 by comparing: (1) the numbers of wolves, coyotes, and hybrids captured, (2) the numbers of territorial social groups with presumed breeding capabilities, (3) the number of red wolf and hybrid litters documented each year and (4) the degree of coyote introgression into the wild red wolf gene pool. We documented substantial increases in the number of known red wolves and red wolf social groups from 1987–2004 followed by a plateau and slight decline by 2013.The number of red wolf litters exceeded hybrid litters each year and the proportion of hybrid litters per year averaged 21%. The genetic composition of the wild red wolf population is estimated to include < 4% coyote ancestry from recent introgression since reintroduction. We conclude that the adaptive management plan was effective at reducing the introgression of coyote genes into the red wolf population, but population recovery of red wolves will require continuation of the current management plan, or alternative approaches, for the foreseeable future. More broadly, we discuss the lessons learned from red wolf adaptive management that could assist other endangered species recovery efforts facing the challenge of minimizing hybridization.

Endangered red wolves (Canis rufus) receive intense conservation efforts in the United States, and to date, population recovery has been challenged by hybridization with closely related coyotes (C. latrans) and illegal human-caused mortality. Ongoing review of the red wolf program in the single recovery area in North Carolina prompted us to compare demography (survival, recruitment) and cause of death of red wolves and coyotes/hybrids. In most respects, canids had similar demographic rates, although sterilization was effective in controlling coyote reproduction. Comparison of previous (1999-2007) to contemporary (2009-2014) causes of death revealed that shooting mortality consistently accounted for ∼25% of wolf mortality. As evidenced by the lack of coyote deaths from strife with wolves, and stationary/declining wolf numbers during the last 15 years, current conditions are inadequate to establish a viable self-sustaining wolf population. Accordingly, the program review should determine whether: (1) banning coyote hunting will sufficiently benefit wolf survival or recruitment; (2) the wolf population should be considered conservation-reliant under revised recovery goals; or (3) the recovery program in North Carolina should be abandoned.

Knowledge about recruitment in a population can be critical when making conservation decisions, particularly for harvested species. Harvest can affect population demography in complex ways and this may be particularly true for cooperatively breeding species whose successful reproduction is often linked with complex social dynamics. We currently have a poor understanding of how harvest affects recruitment in cooperatively breeding species. We used non-invasive genetic sampling and a natural experiment to estimate recruitment in a population of gray wolves Canis lupus before and after harvest in the northern Rocky Mountains, US (2008–2013). We hypothesized that recruitment would decline after hunting and trapping began and that the decline in recruitment would be attributable to the harvest of pups and not to the subtler mechanisms associated with group dynamics and reduced reproductive success. We collected fecal samples from wolves in 10 packs for 6 consecutive years, extracted DNA and genotyped 154 individual pups across 18 microsatellite loci. Population harvest rates averaged 23.8% (sd = 9.2). Our hypothesis that recruitment would decline was supported; survival from 3 to 15 months of age decreased from 0.60 [95% confidence interval (CI): 0.48–0.72] without harvest to 0.38 (95% CI: 0.28–0.48) with harvest and recruitment declined from 3.2 (95% CI: 2.1–4.3) to 1.6 (95% CI: 1.1–2.1) pups per pack after harvest was initiated. We cannot unequivocally dismiss other factors that could have reduced recruitment, however, an increase in recruitment when harvest temporarily ceased lends support to our conclusion that harvest reduced recruitment. We attributed just 18–38% of pup mortality directly to harvest and suggest that there are indirect effects of harvest on recruitment that may be associated with changes in group size and structure. Models that do not include both direct and indirect effects of harvest on recruitment may underestimate the potential impact of harvest on population growth in social species.

The persistence of small populations is influenced by genetic structure and functional connectivity. We used two network-based approaches to understand the persistence of the northern Idaho ground squirrel (Urocitellus brunneus) and the southern Idaho ground squirrel (U. endemicus), two congeners of conservation concern. These graph theoretic approaches are conventionally applied to social or transportation networks, but here are used to study population persistence and connectivity. Population graph analyses revealed that local extinction rapidly reduced connectivity for the southern species, while connectivity for the northern species could be maintained following local extinction. Results from gravity models complemented those of population graph analyses, and indicated that potential vegetation productivity and topography drove connectivity in the northern species. For the southern species, development (roads) and small-scale topography reduced connectivity, while greater potential vegetation productivity increased connectivity. Taken together, the results of the two network-based methods (population graph analyses and gravity models) suggest the need for increased conservation action for the southern species, and that management efforts have been effective at maintaining habitat quality throughout the current range of the northern species. To prevent further declines, we encourage the continuation of management efforts for the northern species, whereas conservation of the southern species requires active management and additional measures to curtail habitat fragmentation. Our combination of population graph analyses and gravity models can inform conservation strategies of other species exhibiting patchy distributions.

The Sonoran pronghorn (Antilocapra americana sonoriensis) is a subspecies of pronghorn found exclusively in the Sonoran Desert of Arizona (USA) and Mexico. Sonoran pronghorn persist at low densities and are geographically isolated from other pronghorn populations. Numbers have declined in recent decades, but the population has rebounded from a low of fewer than 50 animals in 2003 to an estimated 159 individuals in 2012; however, little is known about population demographics beyond abundance estimates. We developed a species identification test that uses mitochondrial DNA (mtDNA) species-specific primers to distinguish between sympatric Sonoran pronghorn and mule deer (Odocoileus hemionus) using DNA extracted from fecal pellets. We accurately identified each species in 100% of the blood and tissue reference samples. We also evaluate the rate of DNA degradation in pronghorn fecal samples ranging from 1 day to 124 days old and document that mtDNA species identification success rates were 100% through day 14. Success rates dropped to 95% by day 21, 50% on day 60, and 10% by day 124. This new test will be a valuable tool for documenting the presence of Sonoran pronghorn across their current range and can also be used for other pronghorn populations. © 2014 The Wildlife Society.

In natural populations, the expression and severity of inbreeding depression can vary widely across taxa. Describing processes that influence the extent of inbreeding and inbreeding depression aid in our understanding of the evolutionary history of mating systems such as cooperative breeding and nonrandom mate selection. Such findings also help shape wildlife conservation theory because inbreeding depression reduces the viability of small populations. We evaluated the extent of inbreeding and inbreeding depression in a small, re-introduced population of red wolves (Canis rufus )i n North Carolina. Since red wolves were first re-introduced in 1987, pedigree inbreeding coefficients (f) increased considerably and almost every wild born wolf was inbred (average f = 0.154 and max f = 0.383). The large inbreeding coefficients were due to both background relatedness associated with few founders and numerous close relative matings. Inbreeding depression was most evident for adult body size and generally absent for direct fitness measures such as reproductive success and survival; no lethal equivalents (LE = 0.00) were detected in juvenile survival. The lack of strong inbreeding depression in direct measures of fitness could be due to a founder effect or because there were no outbred individuals for comparison. Our results highlight the variable expression of inbreeding depression across traits and the need to measure a number of different traits when evaluating inbreeding depression in a wild population.

Estimating densities of cougar (Puma concolor) is important for managing cougars and their prey but remains challenging because of cougar's elusive and solitary behavior. To evaluate a non-invasive, genetic capture–recapture method to estimate cougar population size and density, we surveyed a 220-km2 area using conservation detection dogs trained to locate scat over a 4-week sampling period in northeast Oregon. We collected 272 scat samples and conducted DNA analysis on 249 samples from which we determined individual identification from 73 samples that represented 21 cougars (9 males and 12 females). We evaluated 4 models to estimate cougar densities: Huggins closed population capture–recapture (Huggins), CAPWIRE, multiple detections with Poisson (MDP), and spatially explicit capture–recapture (SECR). Population estimates for cougars using our study area were 26 (95% CI = 22–35, 9 males and 17 females) from Huggins models, 24 (95% CI = 21–30, 9 males and 15 females) from CAPWIRE, and 27 (95% CI = 24–42, 9 males and 18 females) from the MDP model. We accounted for the edge effect in density estimates caused by individuals whose home ranges included only a portion of the survey grid by buffering the study area using the mean home range radius of 8 cougars equipped with global positioning system collars on or near the study area. We estimated densities of 4.6 cougars/100 km2 (95% CI = 3.8–8.3) for the Huggins model, 4.8 cougars/100 km2 (95% CI = 4.2–7.8) for the MDP model, 4.2 cougars/100 km2 (95% CI = 3.3–5.3) for the CAPWIRE model, and 5.0 cougars/100 km2 (95% CI = 3.2–7.7) for the SECR model. Our results suggested estimating cougar densities using scat detection dogs could be feasible at a broader scale with less effort than other methods currently being used. © 2014 The Wildlife Society.

Various monitoring methods have been developed for large carnivores, but not all are practical or sufficiently accurate for long-term monitoring over large spatial scales. From 2009 to 2010, we used a predictive habitat model to locate gray wolf rendezvous sites in 4 study areas in Idaho, USA and conducted noninvasive genetic sampling (NGS) of scat and hair found at the sites. We evaluated species and individual identification PCR success rates across the study areas, and estimated population size with a single-session population estimator using 2 different recapture-coding methods. We then compared NGS population estimates to estimates generated concurrently from telemetry data. We collected 1,937 scat and 166 hair samples and identified 193 unique individuals over 2 years. For fecal DNA samples, species identification success rates were consistently high (>92%) across areas. Individual identification success rates ranged from 78% to 80% in the drier study areas and dropped to 50% in the wettest study area. The degree of agreement between NGS- and telemetry-derived population estimates varied by recapture-coding method with considerable variability in 95% confidence intervals. Population estimates derived from NGS methods were most influenced by the average number of detections per individual. We demonstrate how changes in field effort and recapture-coding method can affect population estimates in a widely used single-session population estimation model. Our study highlights the need to further develop reliable population estimation tools for single-session NGS data, especially those with large differences in capture frequencies among individuals stemming from severe capture heterogeneity (i.e., overdispersion). © 2014 The Wildlife Society.

Drylands typically have strong seasonal variation in rainfall and primary productivity. This study examines the effects of seasonal change in grass-derived resource availability on the base of the food chain of a mammalian predator. Seasonal changes in live grass cover were measured in two vegetation types at the Sevilleta National Wildlife Refuge in central New Mexico, USA. Non-invasive genetic sampling of scat was used to identify individuals in the local coyote (Canis latrans) population. Stable carbon and nitrogen isotope analysis of hair removed from scats of 45 different coyotes was used to assess seasonal variation in the diet of mammalian coyote prey that came from C4 grasses. Live grass cover increased from the spring to the summer and fall; contribution of C4 grasses to the diet of mammalian coyote prey increased from the summer to the fall and was higher in grassland areas. There were significant differences in the seasonal patterns in the prey diet between grassland and shrubland areas.

The behavioral patterns and large territories of large carnivores make them challenging to monitor. Occupancy modeling provides a framework for monitoring population dynamics and distribution of territorial carnivores. We combined data from hunter surveys, howling and sign surveys conducted at predicted wolf rendezvous sites, and locations of radiocollared wolves to model occupancy and estimate the number of gray wolf (Canis lupus) packs and individuals in Idaho during 2009 and 2010. We explicitly accounted for potential misidentification of occupied cells (i.e., false positives) using an extension of the multi-state occupancy framework. We found agreement between model predictions and distribution and estimates of number of wolf packs and individual wolves reported by Idaho Department of Fish and Game and Nez Perce Tribe from intensive radiotelemetry-based monitoring. Estimates of individual wolves from occupancy models that excluded data from radiocollared wolves were within an average of 12.0% (SD = 6.0) of existing statewide minimum counts. Models using only hunter survey data generally estimated the lowest abundance, whereas models using all data generally provided the highest estimates of abundance, although only marginally higher. Precision across approaches ranged from 14% to 28% of mean estimates and models that used all data streams generally provided the most precise estimates. We demonstrated that an occupancy model based on different survey methods can yield estimates of the number and distribution of wolf packs and individual wolf abundance with reasonable measures of precision. Assumptions of the approach including that average territory size is known, average pack size is known, and territories do not overlap, must be evaluated periodically using independent field data to ensure occupancy estimates remain reliable. Use of multiple survey methods helps to ensure that occupancy estimates are robust to weaknesses or changes in any 1 survey method. Occupancy modeling may be useful for standardizing estimates across large landscapes, even if survey methods differ across regions, allowing for inferences about broad-scale population dynamics of wolves. © 2014 The Wildlife Society.

Genetic data are increasingly used to describe the structure of wildlife populations and to infer landscape infl uences on functional connectivity. To accomplish this, genetic structure can be described with a multitude of methods that vary in their assumptions, advantages and limitations. While some methods discriminate distinct subpopulations separated by sharp genetic boundaries (i.e. barrier detection or clustering methods), other methods estimate gradient genetic structures using individual-based genetic distances. We present an analytical framework based on individual ancestry values that combines these diff erent approaches and can be used to a) test for local barriers to gene fl ow and b) evaluate eff ects of landscape gradients through individual-based genetic distances that account for hierarchical genetic structure. We illustrate the approach with a data set of 371 cougars Puma concolor from a 217 000 km 2 study area in Idaho and western Montana (USA) that were genotyped at 12 microsatellite loci. Results suggest that cougars in the region show a complex, hierarchical genetic structure that is infl uenced by a local barrier to gene fl ow (an urban population cluster connected by high traffi c volumes), diff erent landscape features (the Snake River Plain, forested habitat), and geographic distance. Our novel approach helped to elucidate the relative infl uence of these factors on diff erent hierarchical levels of population structure, which was not possible when using either clustering methods or standard genetic distances. Results obtained with our analytical framework highlight the need for multi-scale management of cougars in the region and show that landscape heterogeneity can create complex genetic structures, even in generalist species with high dispersal capabilities. Genetic data are increasingly used in a landscape-genetics framework to assess the structure of wildlife populations and identify landscape features that infl uence functional connectivity. To accomplish this, patterns of spatial genetic

Individual behavior promotes genetic structure within many mammalian populations, yet few studies have explored coarse- and fine-scale structure associated with migration. Fewer still have considered the conservation implications of such structure in at-risk populations. Pronghorn ( Antilocapra americana ) inhabiting Yellowstone National Park are partially migratory, and strong adult fidelity to migratory strategy and breeding areas may promote social and genetic structure within this population. We used 18 nuclear DNA microsatellite loci and fecal samples from 47 individuals to quantify group divergence and pairwise relatedness of Yellowstone pronghorn. The genetics of this population are characterized by individual isolation by distance ( P = 0.009). Evidence for fine-scale social and genetic structure was strong, with mean relatedness between individuals declining rapidly with geographic distance (0–3 km) within areas selected by both migrants and non-migrants. On average, females sampled within social groups were related at the level of first cousins (mean R = 0.105 ± 0.192 SD). We found low differentiation of the population by migratory strategy ( F ST = 0.019), moderate differentiation among some summer use areas ( F ST ⩾ 0.033), and an excess of heterozygotes within all migrant groups ( F IS ⩽ −0.017). Weak and inconsistent substructure was detected using spatial and aspatial Bayesian clustering methods. Our results are the first to document fine-scale social and genetic structure in pronghorn, most likely organized along matrilines. Access to a majority of the total summer range available to this population is maintained by social inheritance and individual fidelity to areas of use. The maintenance and reestablishment of migratory routes may therefore hinge on the retention of experienced individuals, the strength of natal and adult philopatry, and the accessibility of seasonal habitat to pioneering females.

Swift fox (Vulpes velox) were historically dis- tributed in southwestern South Dakota including the region surrounding Badlands National Park (BNP). The species declined during the mid-1800s, largely due to habitat loss and poisoning targeted at wolves (Canis lupus) and coyotes (Canis latrans). Only a small population of swift foxes near Ardmore, which is located in Fall River County, South Dakota, persisted. In 2003, a reintroduction program was initiated at BNP with swift foxes translocated from Colo- rado and Wyoming. Foxes released in the years 2003, 2004 and 2005 were translocated from Colorado (BNP-Colo- rado) whereas in 2006, released foxes were translocated from Wyoming (BNP-Wyoming). Our objective was to evaluate genetic diversity and structure of the restored swift fox population in the area surrounding BNP com- pared to source fox populations in an area of Colorado and Wyoming, as well as the local swift fox population neighboring BNP near Ardmore in Fall River County, South Dakota. A total of 400 swift foxes (28 released in 2003, 28 released in 2004, 26 released in 2005, 26 released in 2006, 252 wild-born foxes, 40 individual foxes from the Ardmore area of South Dakota) was genotyped using twelve microsatellite loci. We report mean gene diversity values of 0.778 (SD = 0.156) for the BNP-Colorado pop- ulation, 0.753 (SD = 0.165) for the BNP-Wyoming pop- ulation, 0.751 (SD = 0.171) for the BNP population, and 0.730 (SD = 0.166) for the Fall River population. We also obtained Fst values ranging from 0.014 to 0.029 for pair- wise comparisons of fox populations (BNP, Fall River, BNP-Wyoming, BNP-Colorado). We conclude that the reintroduced fox population around BNP has high genetic diversity comparable to its source populations in Colorado and Wyoming. Although genetic diversity indicates that the reintroduction was successful, additional time is necessary to fully evaluate long-term genetic maintenance and in- terconnectivity among these populations.

Bayesian clustering methods have emerged as a popular tool for assessing hybridization using genetic markers. Simulation studies have shown these methods perform well under certain conditions; however, these methods have not been evaluated using empirical data sets with individuals of known ancestry. We evaluated the performance of two clustering programs, baps and structure, with genetic data from a reintroduced red wolf (Canis rufus) population in North Carolina, USA. Red wolves hybridize with coyotes (C. latrans), and a single hybridization event resulted in introgression of coyote genes into the red wolf population. A detailed pedigree has been reconstructed for the wild red wolf population that includes individuals of 50-100% red wolf ancestry, providing an ideal case study for evaluating the ability of these methods to estimate admixture. Using 17 microsatellite loci, we tested the programs using different training set compositions and varying numbers of loci. structure was more likely than baps to detect an admixed genotype and correctly estimate an individual's true ancestry composition. However, structure was more likely to misclassify a pure individual as a hybrid. Both programs were outperformed by a maximum-likelihood-based test designed specifically for this system, which never misclassified a hybrid (50-75% red wolf) as a red wolf or vice versa. Training set composition and the number of loci both had an impact on accuracy but their relative importance varied depending on the program. Our findings demonstrate the importance of evaluating methods used for detecting admixture in the context of endangered species management.

Understanding reproduction and mating systems is important for managers tasked with conserving vulnerable species. Genetic tools allow biologists to investigate reproduction and mating systems with high resolution and are particularly useful for species that are otherwise difficult to study in their natural environments. We conducted parentage analyses using 19 nuclear DNA microsatellite loci to assess the influence of population density, genetic diversity, and ancestry on reproduction, and to examine the mating system of pygmy rabbits (Brachylagus idahoensis) bred in large naturalized enclosures for the reintroduction and recovery of the endangered distinct population in central Washington, USA. Reproductive output for females and males decreased as population density and individual homozygosity increased. We identified an interaction indicating that male reproductive output decreased as genetic diversity declined at high population densities, but there was no effect at low densities. Males with high amounts (> 50%) of Washington ancestry had higher reproductive output than the other ancestry groups, while reproductive output was decreased for males with high northern Utah/Wyoming ancestry and females with high Oregon/Nevada ancestry. Females and males bred with an average of 3.8 and 3.6 mates per year, respectively, and we found no evidence of positive or negative assortative mating with regards to ancestry. Multiple paternity was confirmed in 81% of litters, and we report the first documented cases of juvenile breeding by pygmy rabbits. This study demonstrates how variation in population density, genetic diversity, and ancestry impact fitness for an endangered species being bred for conservation. Our results advance understanding of basic life history characteristics for a cryptic species that is difficult to study in the wild and provide lessons for managing populations of vulnerable species in captive and free-ranging populations.

Understanding the genetics of a population is a critical component of developing conservation strategies. We used archived tissue samples from golden eagles (Aquila chrysaetos canadensis) in three geographic regions of western North America to conduct a preliminary study of the genetics of the North American subspecies, and to provide data for United States Fish and Wildlife Service (USFWS) decision-making for golden eagle management. We used a combination of mitochondrial DNA (mtDNA) D-loop sequences and 16 nuclear DNA (nDNA) microsatellite loci to investigate the extent of gene flow among our sampling areas in Idaho, California and Alaska and to determine if we could distinguish birds from the different geographic regions based on their genetic profiles. Our results indicate high genetic diversity, low genetic structure and high connectivity. Nuclear DNA Fst values between Idaho and California were low but significantly different from zero (0.026). Bayesian clustering methods indicated a single population, and we were unable to distinguish summer breeding residents from different regions. Results of the mtDNA AMOVA showed that most of the haplotype variation (97%) was within the geographic populations while 3% variation was partitioned among them. One haplotype was common to all three areas. One region-specific haplotype was detected in California and one in Idaho, but additional sampling is required to determine if these haplotypes are unique to those geographic areas or a sampling artifact. We discuss potential sources of the high gene flow for this species including natal and breeding dispersal, floaters, and changes in migratory behavior as a result of environmental factors such as climate change and habitat alteration. Our preliminary findings can help inform the USFWS in development of golden eagle management strategies and provide a basis for additional research into the complex dynamics of the North American subspecies.

With increasing anthropogenic impact and landscape change, terrestrial carnivore populations are becoming more fragmented. Thus, it is crucial to genetically monitor wild carnivores and quantify changes in genetic diversity and gene flow in response to these threats. This study combined the use of scat detector dogs and molecular scatology to conduct the first genetic study on wild populations of multiple Neotropical felids coexisting across a fragmented landscape in Belize, Central America. We analyzed data from 14 polymorphic microsatellite loci in 1053 scat samples collected from wild jaguars (Panthera onca), pumas (Puma concolor), and ocelots (Leopardus pardalis). We assessed levels of genetic diversity, defined potential genetic clusters, and examined gene flow for the three target species on a countrywide scale using a combination of individual- and population-based analyses. Wild felids in Belize showed moderate levels of genetic variation, with jaguars having the lowest diversity estimates (HE = 0.57 ± 0.02; AR = 3.36 ± 0.09), followed by pumas (HE = 0.57 ± 0.08; AR = 4.20 ± 0.16), and ocelots (HE = 0.63 ± 0.03; AR = 4.16 ± 0.08). We observed low to moderate levels of genetic differentiation for all three target species, with jaguars showing the lowest degree of genetic subdivision across the country, followed by ocelots and pumas. Although levels of genetic diversity and gene flow were still fairly high, we detected evidence of fine-scale genetic subdivision, indicating that levels of genetic connectivity for wild felids in Belize are likely to decrease if habitat loss and fragmentation continue at the current rate. Our study demonstrates the value of understanding fine-scale patterns of gene flow in multiple co-occurring felid species of conservation concern, which is vital for wildlife movement corridor planning and prioritizing future conservation and management efforts within human-impacted landscapes. Published version

There is ample evidence that inbreeding, or mating between relatives, can lead to increased homozygosity and decreased fitness. However, some animals have evolved mechanisms to avoid inbreeding. In a previous study of the National Bison Range, Montana, pronghorn Antilocapra americana, we detected moderate levels of inbreeding, as well as inbreeding depression, following a bottleneck. Here, we evaluated whether there was genetic evidence of inbreeding avoidance in pronghorn. We found that females were more related to all males in the population than they were to their mates, suggesting that pronghorn can avoid inbreeding. However, relatedness between females and the males they sampled prior to estrus did not differ from relatedness between females and all males, nor from relatedness between females and their mates. Inbreeding avoidance appears to occur during the female estrus period rather than during the female mate sampling period. Further work is needed to discover the sensory cues that female pronghorn use to avoid mating with relatives.

The existence of a direct link between intensity of sexual selection and mating-system type is widely accepted. However, the quantification of sexual selection has proven problematic. Several measures of sexual selection have been proposed, including the operational sex ratio (OSR), the breeding sex ratio (BSR), and the opportunity for sexual selection (I(mates)). For a wild population of pronghorn (Antilocapra americana), we calculated OSR and BSR. We estimated I(mates) from census data on the spatial and temporal distribution of receptive females in rut and from a multigenerational genetic pedigree. OSR and BSR indicated weak sexual selection on males, but census and pedigree I(mates) suggested stronger sexual selection on males than on females. OSR and BSR correlated with census but not pedigree estimates of I(mates), and census I(mates) did not correlate with pedigree estimates. This suggests that the behavioral mating system, as deduced from the spatial and temporal distribution of females, does not predict the genetic mating system of pronghorn. The differences we observed between estimators were primarily due to female mate sampling and choice and to the sex ratio. For most species, behavioral data are not perfectly accurate and therefore will be an insufficient alternative to using multigenerational pedigrees to quantify sexual selection.

Among cooperatively breeding species, delayed dispersal provides an opportunity for older siblings to help rear young. There is considerable variation in the extent to which cooperative breeders delay dispersal, however, and while delayed dispersal may be a consequence of adaptive evolution favouring family living, evidence for a genetic basis is lacking. Furthermore, the extent to which dispersal behaviour itself is under direct selection, or evolves indirectly in response to selection on correlated life-history traits, remains unclear. We tested for evidence of inter- and intraspecific quantitative genetic variation in age at first dispersal in a reintroduced population of the red wolf, Canis rufus , that naturally hybridizes with the coyote, Canis latrans , which delays dispersal to a lesser extent than the red wolf. Using analyses based on individual relationships to population founders and sire–offspring regressions, we found evidence for intraspecific genetic variation in dispersal age in male, but not female, red wolves. We also found evidence for interspecific genetic variation, in that red wolves had later dispersal age than coyote–red wolf hybrids. Intraspecific variation in dispersal age was not explained by variation in social and environmental factors nor alternative targets of selection such as body mass or age at first reproduction. Thus, selection may act directly on dispersal-related behaviours. However, life-history traits may continue to be influential in creating interspecific differences in dispersal age, as hybrids also showed earlier reproduction. Our findings suggest that evolution of delayed dispersal may be due to both direct and indirect selection on existing quantitative genetic variation, and that genetic and environmental mechanisms underlying dispersal may differ between the sexes.

The effective population size (N(e) ) could be the ideal parameter for monitoring populations of conservation concern as it conveniently summarizes both the evolutionary potential of the population and its sensitivity to genetic stochasticity. However, tracing its change through time is difficult in natural populations. We applied four new methods for estimating N(e) from a single sample of genotypes to trace temporal change in N(e) for bears in the Northern Dinaric Mountains. We genotyped 510 bears using 20 microsatellite loci and determined their age. The samples were organized into cohorts with regard to the year when the animals were born and yearly samples with age categories for every year when they were alive. We used the Estimator by Parentage Assignment (EPA) to directly estimate both N(e) and generation interval for each yearly sample. For cohorts, we estimated the effective number of breeders (N(b) ) using linkage disequilibrium, sibship assignment and approximate Bayesian computation methods and extrapolated these estimates to N(e) using the generation interval. The N(e) estimate by EPA is 276 (183-350 95% CI), meeting the inbreeding-avoidance criterion of N(e) > 50 but short of the long-term minimum viable population goal of N(e) > 500. The results obtained by the other methods are highly consistent with this result, and all indicate a rapid increase in N(e) probably in the late 1990s and early 2000s. The new single-sample approaches to the estimation of N(e) provide efficient means for including N(e) in monitoring frameworks and will be of great importance for future management and conservation.

For cooperatively breeding groups composed of close relatives, whether and how a group avoids inbreeding are questions of key evolutionary and conservation importance. A number of strategies for inbreeding avoidance may be employed by cooperative breeders, including extrapair reproduction, reproductive suppression, and juvenile dispersal. However, population-wide information on the prevalence of different strategies is difficult to obtain. We investigated the prevalence of inbreeding and potential mechanisms for inbreeding avoidance in a reintroduced population of the red wolf. Using long-term data on individuals of known pedigree, we determined that inbreeding among first-degree relatives was rare. Potential mechanisms for inbreeding avoidance included low levels of philopatric reproduction in spite of delayed dispersal, and reproductive suppression prior to dispersal. Inbreeding avoidance among siblings may have been further facilitated by independent dispersal trajectories, as many young wolves spent time alone or in small nonbreeding packs composed of unrelated individuals. The dominant pattern of breeding-pair formation involved the union of 2 unrelated individuals in a new home range. Replacement of 1 or both members of an existing breeding pair involved new immigrants to a pack or, in a small number of cases, ascendance of either resident offspring or adopted pack members to vacant breeding positions. Extrapair reproduction was rare, suggesting that it was not a major mechanism for outbreeding. We conclude that there are several prevalent behavioral strategies within the red wolf population that may work together to minimize inbreeding and any associated fitness costs, helping make cooperative breeding an evolutionarily viable strategy. Key wor ds: Canis r ufus, competition, lone wolves, monogamy, nonbreeding packs, pair formation. [Behav Ecol]

Canis rufus (Red Wolf) is critically endangered, with the only wild population consisting of

Although inbreeding depression affects survival, fitness and population viability, the extent of inbreeding depression in wild populations remains unclear. We examined inbreeding depression in the small, isolated National Bison Range (NBR), MT, pronghorn (Antilocapra americana) population following a bottleneck. We have studied the National Bison Range pronghorn extensively since 1981, and we have detailed birth, survival and mate choice data. We genotyped all animals in the population between 1999 and 2010 at 19 microsatellite loci, assigned paternities to all fawns based on genotype and constructed a genetic-based pedigree to calculate inbreeding coefficients (f). We found an increase in the frequency of inbreeding following the bottleneck. We detected evidence of inbreeding depression on fawn survival to weaning, birth mass, foot length and condition. We estimated the number of diploid lethal equivalents on survival to weaning as 24.17-28.72. Standardized heterozygosity (H) had a relatively small influence on survival, mass, length and condition compared with f, and H was not correlated with f. We conclude that for pronghorn, H was not a good predictor of pedigree-estimates of f.