Search

Aquatic ectotherms are predicted to harbour genomic signals of local adaptation resulting from selective pressures driven by the strong influence of climate conditions on body temperature. We investigated local adaptation in redband trout (Oncorhynchus mykiss gairdneri) using genome scans for 547 samples from 11 populations across a wide range of habitats and thermal gradients in the interior Columbia River. We estimated allele frequencies for millions of single nucleotide polymorphism loci (SNPs) across populations using low-coverage whole genome resequencing, and used population structure outlier analyses to identify genomic regions under divergent selection between populations. Twelve genomic regions showed signatures of local adaptation, including two regions associated with genes known to influence migration and developmental timing in salmonids (GREB1L, ROCK1, SIX6). Genotype-environment association analyses indicated that diurnal temperature variation was a strong driver of local adaptation, with signatures of selection driven primarily by divergence of two populations in the northern extreme of the subspecies range. We also found evidence for adaptive differences between high-elevation desert vs. montane habitats at a smaller geographical scale. Finally, we estimated vulnerability of redband trout to future climate change using ecological niche modelling and genetic offset analyses under two climate change scenarios. These analyses predicted substantial habitat loss and strong genetic shifts necessary for adaptation to future habitats, with the greatest vulnerability predicted for high-elevation desert populations. Our results provide new insight into the complexity of local adaptation in salmonids, and important predictions regarding future responses of redband trout to climate change.

The rate of global climate change is projected to outpace the ability of many natural populations and species to adapt. Assisted migration (AM), which is defined as the managed movement of climate‐adapted individuals within or outside the species ranges, is a conservation option to improve species' adaptive capacity and facilitate persistence. Although conservation biologists have long been using genetic tools to increase or maintain diversity of natural populations, genomic techniques could add extra benefit in AM that include selectively neutral and adaptive regions of the genome. In this review, we first propose a framework along with detailed procedures to aid collaboration among scientists, agencies, and local and regional managers during the decision‐making process of genomics‐guided AM. We then summarize the genomic approaches for applying AM, followed by a literature search of existing incorporation of genomics in AM across taxa. Our literature search initially identified 729 publications, but after filtering returned only 50 empirical studies that were either directly applied or considered genomics in AM related to climate change across taxa of plants, terrestrial animals, and aquatic animals; 42 studies were in plants. This demonstrated limited application of genomic methods in AM in organisms other than plants, so we provide further case studies as two examples to demonstrate the negative impact of climate change on non‐model species and how genomics could be applied in AM. With the rapidly developing sequencing technology and accumulating genomic data, we expect to see more successful applications of genomics in AM, and more broadly, in the conservation of biodiversity.

Ocelots (Leopardus pardalis) are a wide-ranging felid species, occurring from southern United States to northern Argentina. They occupy various habitats and are usually considered the most abundant wild cat species in the Neotropics. However, genetic studies that include free-ranging Mesoamerican ocelots are rare and generally based on small sample sizes. This is the first conservation genetics study on ocelots in Costa Rica and the second one in Mesoamerica that has conducted a genetic assessment of the species at a countrywide scale. We evaluated genetic diversity and population structure of ocelots using 15 microsatellite loci in 28 successfully genotyped individuals from throughout the country. We also compared genetic diversity of Costa Rican ocelots with that of jaguars (Panthera onca) and pumas (Puma concolor) in the country, and with ocelots in Belize. Genetic diversity of ocelots in Costa Rica was relatively high as measured by rarified allelic richness (AR = 5.50 ± 1.36) and expected heterozygosities (HE = 0.79 ± 0.08). We did not detect patterns of genetic substructure, suggesting high levels of gene flow throughout the country and no strong barriers to movement. As expected, genetic diversity of Costa Rican ocelots was higher than co-occurring jaguars and pumas. Additionally, levels of genetic diversity were slightly higher in Costa Rican ocelots when compared with their counterparts in Belize, confirming the south to north decrease in genetic diversity reported in other studies. Our study provides critical baseline information to understand the status of wild ocelot populations in Costa Rica. Future studies on ocelots and other threatened or keystone species should also integrate genetic monitoring and conservation genetics analysis to properly inform management decisions, guarantee their long-term survival, and improve the resilience of ecosystems.

We report a chromosomal-level genome assembly of a male North American wolverine (Gulo gulo luscus) from the Kugluktuk region of Nunavut, Canada. The genome was assembled directly from long-reads, comprising: 758 contigs with a contig N50 of 36.6 Mb; contig L50 of 20; base count of 2.39 Gb; and a near complete representation (99.98%) of the BUSCO 5.2.2 set of 9,226 genes. A presumptive chromosomal-level assembly was generated by scaffolding against two chromosomal-level Mustelidae reference genomes, the ermine and the Eurasian river otter, to derive a final scaffold N50 of 144.0 Mb and a scaffold L50 of 7. We annotated a comprehensive set of genes that have been associated with models of aggressive behavior, a trait which the wolverine is purported to have in the popular literature. To support an integrated, genomics-based wildlife management strategy at a time of environmental disruption from climate change, we annotated the principal genes of the innate immune system to provide a resource to study the wolverine’s susceptibility to new infectious and parasitic diseases. As a resource, we annotated genes involved in the modality of infection by the coronaviruses, an important class of viral pathogens of growing concern as shown by the recent spillover infections by severe acute respiratory syndrome coronavirus-2 to naïve wildlife. Tabulation of heterozygous single nucleotide variants in our specimen revealed a heterozygosity level of 0.065%, indicating a relatively diverse genetic pool that would serve as a baseline for the genomics-based conservation of the wolverine, a rare cold-adapted carnivore now under threat.

A correction to this paper has been published: https://doi.org/10.1007/s10592-021-01376-9

Climate change can have particularly severe consequences for high-elevation species that are well-adapted to long-lasting snow conditions within their habitats. One such species is the wolverine, Gulo gulo, with several studies showing a strong, year-round association of the species with the area defined by persistent spring snow cover. This bioclimatic niche also predicts successful dispersal paths for wolverines in the contiguous United States, where the species shows low levels of genetic exchange and low effective population size. Here, we assess the influence of additional climatic, vegetative, topographic, and anthropogenic, variables on wolverine genetic structure in this region using a multivariate, multiscale, landscape genetic approach. This approach allows us to detect landscape-genetic relationships both due to typical, small-scale genetic exchange within habitat, as well as exceptional, long-distance dispersal among habitats. Results suggest that a combination of snow depth, terrain ruggedness, and housing density, best predict gene flow in wolverines, and that the relative importance of variables is scale-dependent. Environmental variables (i.e., isolation-by-resistance, IBR) were responsible for 79% of the explained variation at small scales (i.e., up to ~230 km), and 65% at broad scales (i.e., beyond ~420 km). In contrast, a null model based on only space (i.e., isolation-by-distance, IBD) accounted only for 17% and 11% of the variation at small and broad scales, respectively. Snow depth was the most important variable for predicting genetic structures overall, and at small scales, where it contributed 43% to the variance explained. At broad spatial scales, housing density and terrain ruggedness were most important with contributions to explained variation of 55% and 25%, respectively. While the small-scale analysis most likely captures gene flow within typical wolverine habitat complexes, the broad-scale analysis reflects long-distance dispersal across areas not typically inhabited by wolverines. These findings help to refine our understanding of the processes shaping wolverine genetic structure, which is important for maintaining and improving functional connectivity among remaining wolverine 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.

Specimens from natural history collections (NHCs) are increasingly being used for genetic studies and can provide information on extinct populations, facilitate comparisons of historical and contemporary populations, produce baseline data before environmental changes, and elucidate patterns of change. Destructive sampling for DNA may be in disagreement with NHC goals of long-term care and maintenance. Differentiating quality among sample sources can direct destructive sampling to the source predicted to yield the highest quality DNA and most reliable data, potentially reducing damage to specimens, laboratory costs, and genotyping errors. We used the kit fox (Vulpes macrotis) as a model species and evaluated the quality and reliability of genetic data obtained from carnivoran specimens via three different sample sources: cranial bones, nasal bones, and toepads. We quantified variation in microsatellite amplification success and genotyping error rates and assessed the reliability of source-specific genic data. Toepads had the highest amplification success rates and lowest genotyping error rates. Shorter loci had higher amplification success and lower allelic dropout rates than longer loci. There were substantial differences in the reliability of resulting multilocus genotypes. Toepads produced the most reliable data, required the fewest replicates, and therefore, had the lowest costs to achieve reliable data. Our results demonstrate that the quality of DNA obtained from specimens varies by sample source and can inform NHCs when evaluating requests for destructive sampling. Our results suggest that prior to large-scale specimen sampling, researchers should conduct pilot studies to differentiate among source-specific data reliability, identify high performing loci, reduce costs of analyses, and minimize destructive sampling.

ispartof: CONSERVATION GENETICS vol:22 issue:4 pages:533-536 status: published

The most remarkable feature of our planet is the diversity of its life forms, ranging from viruses and nanobacteria to blue whales and giant sequoias to satanic leaf‐tailed geckos and leafy seadragons (look them up!). Life is found in essentially all environments on earth, and the number of species living on our planet is many times greater than we could have imagined a century ago. A well‐regarded estimate pegs the number of eukaryotic species on earth at 8.7 million (±1.3 million), of which fewer than 15% are currently described (Mora et al., 2011). The diversity of prokaryotes is less clear (and highly controversial), but an analysis of 1.6 billion 16S ribosomal RNA sequences estimated that 0.8–1.6 million prokaryotic operational taxonomic units exist globally (Louca et al., 2019). While we do not know how many species are currently extant, or have existed in the past, we do know that this biodiversity is valuable, providing food, fibre and medicine, furnishing ecosystem services such as water and air purification, nutrient cycling, pollination and carbon uptake, and contributing to technological innovations ranging from biotechnology to robotics to material science. Moreover, biodiversity underlies the cultural identity of human populations and is important to human health and well‐being. Geographically, species richness increases from the Polar Regions to the tropics in terrestrial and surface marine ecosystems. Thus, some countries, especially those in tropical and subtropical regions, are endowed with much greater biodiversity than others. Unfortunately, benefits arising from the access and utilization of this biodiversity have been unequally shared, with (paradoxically) biodiversity‐poor countries often accruing the lion's share of economic gains. There can be imbalances within countries as well, wherein some segments of the population obtain greater economic benefits from biodiversity and associated traditional knowledge than indigenous peoples. The “Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization,” which came into force in 2014, is an international agreement designed to ensure that the benefits arising from biodiversity are shared equitably (https://www.cbd.int/abs/). However, few scientific journals require compliance with the Nagoya Protocol or the reporting of benefits from biodiversity research. In this editorial, we (the editors of Molecular Ecology and Molecular Ecology Resources) express support for the Nagoya Protocol and the principle of benefit sharing. We believe that scientific journals publishing research on biodiversity can play an important role in implementing the Nagoya Protocol and in reporting on benefits generated from such research. Below, we provide background on the Nagoya Protocol, discuss the kinds of benefits that may arise from biodiversity research, describe the rationale for reporting on these benefits and introduce changes to the journals’ Data Accessibility Statements to incorporate the requirements and goals of the Nagoya Protocol.

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.

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.

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.

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.

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.

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.

An understanding of a species’ diet is required to make sound conservation and management decisions. Traditionally, morphological analyses of undigested hard parts from food items remaining in scats have been used to assess diets. More recently, molecular analyses of scats have been used to identify plant and prey species’ DNA, but no studies have compared morphological and molecular diet analyses for large, terrestrial carnivores. We used molecular tools to determine the percentage of black bear and coyote scats that contained 3 common prey species (caribou, moose, and snowshoe hares) in Newfoundland and compared the results to a traditional morphological analysis. We found that a ranking of relative prey frequencies was consistent between the 2 methods, but molecular methods tended to detect prey species in a greater percentage of scats for all prey species. However, there were individual scats in which a prey species was detected by morphological methods only, and we provide evidence that molecular methods could result in false negatives if prey DNA is not uniformly distributed throughout a scat or as a result of PCR inconsistency. We also found that the per sample cost comparison between morphological and molecular analyses was dependent upon whether or not a molecular test was needed to identify scats to the predator species, the cost of developing molecular methods, and the number of samples being processed. We recommend that controlled feeding studies be performed to validate molecular methods and investigate the utility of molecular techniques to estimate the proportions of food items consumed.

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.

The development of high-throughput sequencing technologies is dramatically increasing the use of single nucleotide polymorphisms (SNPs) across the field of genetics, but most parentage studies of wild populations still rely on microsatellites. We developed a bioinformatic pipeline for identifying SNP panels that are informative for parentage analysis from restriction site-associated DNA sequencing (RADseq) data. This pipeline includes options for analysis with or without a reference genome, and provides methods to maximize genotyping accuracy and select sets of unlinked loci that have high statistical power. We test this pipeline on small populations of Mexican gray wolf and bighorn sheep, for which parentage analyses are expected to be challenging due to low genetic diversity and the presence of many closely related individuals. We compare the results of parentage analysis across SNP panels generated with or without the use of a reference genome, and between SNPs and microsatellites. For Mexican gray wolf, we conducted parentage analyses for 30 pups from a single cohort where samples were available from 64% of possible mothers and 53% of possible fathers, and the accuracy of parentage assignments could be estimated because true identities of parents were known a priori based on field data. For bighorn sheep, we conducted maternity analyses for 39 lambs from five cohorts where 77% of possible mothers were sampled, but true identities of parents were unknown. Analyses with and without a reference genome produced SNP panels with ≥95% parentage assignment accuracy for Mexican gray wolf, outperforming microsatellites at 78% accuracy. Maternity assignments were completely consistent across all SNP panels for the bighorn sheep, and were 74.4% consistent with assignments from microsatellites. Accuracy and consistency of parentage analysis were not reduced when using as few as 284 SNPs for Mexican gray wolf and 142 SNPs for bighorn sheep, indicating our pipeline can be used to develop SNP genotyping assays for parentage analysis with relatively small numbers of loci.

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.

The Great Plains region is fragmented by natural and anthropogenic sources, yet the interaction between habitat fragmentation and genetic structure in this region has received limited study. Swift fox (Vulpes velox) are closely associated with short and mixed grass ecosystems, providing an opportunity to study patterns of gene flow, diversity and genetic structure in this area. We collected 589 samples throughout the species’ distribution in the United States and analyzed these samples using 15 microsatellite loci and a 250 base pair sequence of the mitochondrial DNA control region. We detected three levels of spatial genetic structure using microsatellite markers and identified six mitochondrial haplotypes, five of which showed spatial clustering. Differentiation between groups was significant while genetic diversity within groups was generally high. Anthropogenic influences, particularly agriculture, appear to reducing gene flow, especially in the central portion of the species’ range. Conservation measures should be taken to remediate these impacts and to maintain future gene flow in light of expected agricultural expansion in the Great Plains. Potential evolutionary significant units are identified, although further investigation using ecological indicators and adaptive loci is recommended to characterize the adaptive distinctiveness of swift fox populations.

There is a great need to develop efficient, noninvasive genetic sampling methods to study wild populations of multiple, co-occurring, threatened felids. This is especially important for molecular scatology studies occurring in challenging tropical environments where DNA degrades quickly and the quality of faecal samples varies greatly. We optimized 14 polymorphic microsatellite loci for jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) and assessed their utility for cross-species amplification. Additionally, we tested their reliability for species and individual identification using DNA from faeces of wild felids detected by a scat detector dog across Belize in Central America. All microsatellite loci were successfully amplified in the three target species, were polymorphic with average expected heterozygosities of HE = 0.60 ± 0.18 (SD) for jaguars, HE = 0.65 ± 0.21 (SD) for pumas and HE = 0.70 ± 0.13 (SD) for ocelots and had an overall PCR amplification success of 61%. We used this nuclear DNA primer set to successfully identify species and individuals from 49% of 1053 field-collected scat samples. This set of optimized microsatellite multiplexes represents a powerful tool for future efforts to conduct noninvasive studies on multiple, wild Neotropical felids.

The recent co-occurrence of red wolves (Canis rufus) and coyotes (Canis latrans) in eastern North Carolina provides a unique opportunity to study prey partitioning by sympatric canids. We collected scats from this region and examined them for prey contents. We used fecal DNA analysis to identify which taxa deposited each scat and multinomial modeling designed for mark–recapture data to investigate diets of sympatric red wolves and coyotes. Diets of red wolves and coyotes did not differ, but the proportion of small rodents in the composite scats of both canids was greater in the spring than in the summer. White-tailed deer (Odocoileus virginianus), rabbits (Sylvilagus spp.), and small rodents were the most common diet items in canid scats. The similarity of diet between red wolves and coyotes suggests that these 2 species may be affecting prey populations similarly.

Environmental DNA (eDNA) methods for detecting and estimating abundance of aquatic species are emerging rapidly, but little is known about how processes such as secretion rate, environmental degradation, and time since colonization or extirpation from a given site affect eDNA measurements. Using stream-dwelling salamanders and quantitative PCR (qPCR) analysis, we conducted three experiments to assess eDNA: (i) production rate; (ii) persistence time under different temperature and light conditions; and (iii) detectability and concentration through time following experimental introduction and removal of salamanders into previously unoccupied streams. We found that 44-50 g individuals held in aquaria produced 77 ng eDNA/h for 2 h, after which production either slowed considerably or began to equilibrate with degradation. eDNA in both full-sun and shaded treatments degraded exponentially to

Predator species identification is an important step in understanding predator-prey interactions, but predator identifications using kill site observations are often unreliable. We used molecular tools to analyse predator saliva, scat and hair from caribou calf kills in Newfoundland, Canada to identify the predator species, individual and sex. We sampled DNA from 32 carcasses using cotton swabs to collect predator saliva. We used fragment length analysis and sequencing of mitochondrial DNA to distinguish between coyote, black bear, Canada lynx and red fox and used nuclear DNA microsatellite analysis to identify individuals. We compared predator species detected using molecular tools to those assigned via field observations at each kill. We identified a predator species at 94% of carcasses using molecular methods, while observational methods assigned a predator species to 62.5% of kills. Molecular methods attributed 66.7% of kills to coyote and 33.3% to black bear, while observations assigned 40%, 45%, 10% and 5% to coyote, bear, lynx and fox, respectively. Individual identification was successful at 70% of kills where a predator species was identified. Only one individual was identified at each kill, but some individuals were found at multiple kills. Predator sex was predominantly male. We demonstrate the first large-scale evaluation of predator species, individual and sex identification using molecular techniques to extract DNA from swabs of wild prey carcasses. Our results indicate that kill site swabs (i) can be highly successful in identifying the predator species and individual responsible; and (ii) serve to inform and complement traditional methods.

The discipline of molecular ecology has undergone enormous changes since the journal bearing its name was launched approximately two decades ago. The field has seen great strides in analytical methods development, made groundbreaking discoveries and experienced a revolution in genotyping technology. Here, we provide brief perspectives on the main subdisciplines of molecular ecology, describe key questions and goals, discuss common challenges, predict future research directions and suggest research priorities for the next 20 years., We thank the participants in the Molecular Ecology Symposium and Online Forum for many of the ideas put forward in this article.

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.

Agricultural intensification in tropical landscapes poses a new threat to the ability of biological corridors to maintain functional connectivity for native species. We use a landscape genetics approach to evaluate impacts of expanding pineapple plantations on two widespread and abundant frugivorous bats in a biological corridor in Costa Rica. We hypothesize that the larger, more mobile Artibeus jamaicensis will be less impacted by pineapple than the smaller Carollia castanea. In 2012 and 2013, we sampled 735 bats in 26 remnant forest patches surrounded by different proportions of forest, pasture, crops, and pineapple. We used 10 microsatellite loci for A. jamaicensis and 16 microsatellite loci for C. castanea to estimate genetic diversity and gene flow. Canonical correspondence analyses indicate that land cover type surrounding patches has no impact on genetic diversity of A. jamaicensis. However, for C. castanea, both percentage forest and pineapple surrounding patches explained a significant proportion of the variation in genetic diversity. Least-cost transect analyses (LCTA) and pairwise G”st suggest that for A. jamaicensis, pineapple is more permeable to gene flow than expected, while as expected, forest is the most permeable land cover for gene flow of C. castanea. For both species, LCTA indicate that development may play a role in inhibiting gene flow. The current study answers the call for landscape genetic research focused on tropical and agricultural landscapes, highlights the value of comparative landscape genetics in biological corridor design and management, and is one of the few studies of biological corridors in any ecosystem to implement a genetic approach to test corridor efficacy. This article is protected by copyright. All rights reserved.

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

Non-invasive genetic sampling is an increasingly popular approach for investigating the demographics of natural populations. This has also become a useful tool for managers and conservation biologists, especially for those species for which traditional mark-recapture studies are not practical. However, the consequence of collecting DNA indirectly is that an individual may be sampled multiple times per sampling session. This requires alternative statistical approaches to those used in traditional mark-recapture studies. Here we present the R package capwire, an implementation of the population size estimators of Miller et al. (Molecular Ecology 2005; 14: 1991), which were designed to deal specifically with this type of sampling. The aim of this project is to enable users across platforms to easily manipulate their data and interact with existing R packages. We have also provided functions to simulate data under a variety of scenarios to allow for rigorous testing of the robustness of the method and to facilitate further development of this approach.

Allocation of evolutionary divergence into species level versus subspecies ranks is critical for conservation and management. Urocitellus brunneus (Idaho ground squirrel; formerly Spermophilus brunneus) is currently apportioned into 2 subspecies, U. b. brunneus and U. b. endemicus, but recent studies have suggested elevation to distinct species based on differences in pelage, bacular morphology, genetic data, timing of life-history cycle, and behavior. Following recent movements toward integrated taxonomy, we use the cohesion species concept to test whether both genetic and ecological data support species-level classification of U. b. brunneus and U. b. endemicus. Eight microsatellite loci and mitochondrial DNA (mtDNA) sequence data were generated for 339 individuals from 14 localities for U. b. brunneus and 312 individuals from 11 localities for U. b. endemicus .T he null hypothesis of genetic interchangeability was tested with 2 independent data sets. First, we estimated an mtDNA gene tree from control region and cytochrome-b sequences via parsimony, maximum-likelihood, and Bayesian analyses. We then tested for evidence of recent migration using Bayesian clustering and coalescence analyses of microsatellite data. The 2nd null hypothesis, that of ecological exchangeability, was tested using an ecological niche-model analysis and a review of the literature based on morphology, habitat characteristics, and behavior. Although divergence of mtDNA sequences between the subspecies was modest (,1%), there are no haplotypes shared between the 2 taxa. There is strong support for monophyly of mtDNA haplotyes of U. b. endemicus (posterior probability 5 0.94), with those from U. b. brunneus forming a basal grade. No evidence of recent gene flow was detected; the Bayesian clustering algorithms of mutlilocus genotype data indicated separate ancestry for both U. b. brunneus and U. b. endemicus. The ecological-niche model showed a nonoverlapping niche for each taxon, allowing for the possibility of differential adaptation. We reject both null hypotheses based on the data, which supports elevation of U. b. brunneus and U. b. endemicus as distinct species.

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.

Beetles comprise nearly one quarter of described species and show high levels of morphological and ecological diversification. Because of their wide distribution, ease of detection, and correlation of species richness patterns with other taxa, tiger beetles have been recommended for use as a global indicator of regional biodiversity, requiring accurate taxonomic designations. The Bruneau Dune tiger beetle (Cicindela waynei), whose habitat consists of an isolated dune field in southern Idaho, was recently described as a distinct species from the St. Anthony Dunes tiger beetle (C. arenicola) based on morphological characteristics. While these characteristics include distinct differences in genital morphology that could indicate intrinsic reproductive isolation, morphological characteristics have sometimes been misleading in tiger beetle taxonomy. To evaluate genetic support for this species designation, we analyzed 1,751 base pairs of mitochondrial DNA sequence from 147 tiger beetles collected throughout the range of both C. arenicola and C. waynei. Maximum-likelihood and Bayesian phylogenetic analyses indicated monophyly for C. waynei on a well-supported, short branch nested within C. arenicola. Bayesian species delimitation analyses strongly supported C. waynei as a distinct species (speciation probability = 1.0) with the estimated time of divergence ca. 14,500–67,000 years ago. This lack of reciprocal monophyly and recency of speciation is consistent with C. waynei as a member of an evolutionary front where speciation has occurred at a rapid rate. Mitochondrial sequence data supports the species designation of C. waynei, emphasizing the need to determine appropriate management for this species and its restricted habitat.

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.

Although promiscuity is believed to be common among leporids, little is known about the mating system of free-ranging pygmy rabbits (Rrachylagus idahoensis). We used 16 polymorphic microsatellite markers and DNA extracted from tissue samples of 10 rabbits from 2 litters collected in east central Idaho to evaluate paternity. We incorporated maternal genotypes for better resolution and employed an exclusion approach to detect multiple paternity. Our results demonstrated multiple paternity at 6 of the 16 loci tested for each litter. These findings support the expectation that pygmy rabbits have a promiscuous mating system. It has been proposed that multiple paternity could lead to higher effective population sizes and help small populations maintain diversity. Given that pygmy rabbits persist in small and fragmented populations, a promiscuous mating system might be important for maintaining genetic diversity in free-ranging populations and could be advantageous for the captive breeding program.

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

Predicting spatial patterns of hybridization is important for evolutionary and conservation biology yet are hampered by poor understanding of how hybridizing species can interact. This is especially pertinent in contact zones where hybridizing populations are sympatric. In this study, we examined the extent of red wolf (Canis rufus) colonization and introgression where the species contacts a coyote (C. latrans) population in North Carolina, USA. We surveyed 22,000km(2) in the winter of 2008 for scat and identified individual canids through genetic analysis. Of 614 collected scats, 250 were assigned to canids by mitochondrial DNA (mtDNA) sequencing. Canid samples were genotyped at 6-17 microsatellite loci (nDNA) and assigned to species using three admixture criteria implemented in two Bayesian clustering programs. We genotyped 82 individuals but none were identified as red wolves. Two individuals had red wolf mtDNA but no significant red wolf nDNA ancestry. One individual possessed significant red wolf nDNA ancestry (approximately 30%) using all criteria, although seven other individuals showed evidence of red wolf ancestry (11-21%) using the relaxed criterion. Overall, seven individuals were classified as hybrids using the conservative criteria and 37 using the relaxed criterion. We found evidence of dog (C. familiaris) and gray wolf (C. lupus) introgression into the coyote population. We compared the performance of different methods and criteria by analyzing known red wolves and hybrids. These results suggest that red wolf colonization and introgression in North Carolina is minimal and provide insights into the utility of Bayesian clustering methods to detect hybridization.

We describe a method to determine the species of pinniped from faeces collected from sympatric Steller sea lion (Eumetopias jubatus) and northern fur seal (Callorhinus ursinus) rookeries using newly developed species-specific primers that amplify a 667-669-base pair segment from the mitochondrial DNA (mtDNA) cytochrome B (cytB) gene region. The primers yielded the correct species in 100% of tissue samples from 10 known animals and 100% of faecal samples from 13 known animals. Species could be identified unequivocally for 87.7% of faecal samples from 122 unknown individuals. The ability to differentiate between scats of sympatrically breeding Steller sea lions and northern fur seals will contribute to the range-wide knowledge of the foraging strategies of both species as well as allow researchers to examine the niche partitioning and potential resource competition between the two predators.

Genetic monitoring has rarely been used for wildlife translocations despite the potential benefits this approach offers, compared to traditional field-based methods. We applied genetic monitoring to the reintroduced brown bear population in northern Italy. From 2002 to 2008, 2781 hair and faecal samples collected noninvasively plus 12 samples obtained from captured or dead bears were used to follow the demographic and geographical expansion and changes in genetic composition. Individual genotypes were used to reconstruct the wild pedigree and revealed that the population increased rapidly, from nine founders to >27 individuals in 2008 (lambda=1.17-1.19). Spatial mapping of bear samples indicated that most bears were distributed in the region surrounding the translocation site; however, individual bears were found up to 163 km away. Genetic diversity in the population was high, with expected heterozygosity of 0.74-0.79 and allelic richness of 4.55-5.41. However, multi-year genetic monitoring data showed that mortality rates were elevated, immigration did not occur, one dominant male sired all cubs born from 2002 to 2005, genetic diversity declined, relatedness increased, inbreeding occurred, and the effective population size was extremely small (Ne=3.03, ecological method). The comprehensive information collected through genetic monitoring is critical for implementing future conservation plans for the brown bear population in the Italian Alps. This study provides a model for other reintroduction programmes by demonstrating how genetic monitoring can be implemented to uncover aspects of the demography, ecology and genetics of small and reintroduced populations that will advance our understanding of the processes influencing their viability, evolution, and successful restoration.

Landscape genetics has seen rapid growth in number of publications since the term was coined in 2003. An extensive literature search from 1998 to 2008 using keywords associated with landscape genetics yielded 655 articles encompassing a vast array of study organisms, study designs and methodology. These publications were screened to identify 174 studies that explicitly incorporated at least one landscape variable with genetic data. We systematically reviewed this set of papers to assess taxonomic and temporal trends in: (i) geographic regions studied; (ii) types of questions addressed; (iii) molecular markers used; (iv) statistical analyses used; and (v) types and nature of spatial data used. Overall, studies have occurred in geographic regions proximal to developed countries and more commonly in terrestrial vs. aquatic habitats. Questions most often focused on effects of barriers and/or landscape variables on gene flow. The most commonly used molecular markers were microsatellites and amplified fragment length polymorphism (AFLPs), with AFLPs used more frequently in plants than animals. Analysis methods were dominated by Mantel and assignment tests. We also assessed differences among journals to evaluate the uniformity of reporting and publication standards. Few studies presented an explicit study design or explicit descriptions of spatial extent. While some landscape variables such as topographic relief affected most species studied, effects were not universal, and some species appeared unaffected by the landscape. Effects of habitat fragmentation were mixed, with some species altering movement paths and others unaffected. Taken together, although some generalities emerged regarding effects of specific landscape variables, results varied, thereby reinforcing the need for species-specific work. We conclude by: highlighting gaps in knowledge and methodology, providing guidelines to authors and reviewers of landscape genetics studies, and suggesting promising future directions of inquiry.

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

Among the key issues determining success of a study employing molecular genetics tools in wildlife monitoring or research is a large enough set of highly informative genetic markers and a reliable, cost effective method for their analysis. While optimized commercial genotyping kits have been developed for humans and domestic animals, such protocols are rare in wildlife research. We developed a highly optimized multiplex PCR that genotypes 12 microsatellite loci and a sex determination locus in brown bear (Ursus arctos) faecal samples in a single multiplex PCR and a single sequencer run. We used this protocol to genotype 1053 faecal samples of bears from the Dinaric population, and obtained useful genotypes for 88% of the samples, a very high success rate. The new protocol outperformed the multiplex pre-amplification strategy used in a previous study of 473 faecal samples with a 78.4% success rate. On a subset of 182 samples we directly compared the performance of both approaches, and found no advantage of the multiplex pre-amplification. While pre-amplification protocols might still improve PCR success and reliability on a small fraction of low-quality samples, the higher costs and workload do not justify their use when analysing reasonably fresh non-invasive material. Moreover, the high number of multiplexed loci in the new protocol makes it comparable to commercially developed genotyping kits developed for domestic animals and humans.