Worldwide patterns of genomic variation and admixture in gray wolves.

The gray wolf (Canis lupus) is a widely distributed top predator and ancestor of the domestic dog. To address questions about wolf relationships to each other and dogs, we assembled and analyzed a data set of 34 canine genomes. The divergence between New and Old World wolves is the earliest branching event and is followed by the divergence of Old World wolves and dogs, confirming that the dog was domesticated in the Old World. However, no single wolf population is more closely related to dogs, supporting the hypothesis that dogs were derived from an extinct wolf population. All extant wolves have a surprisingly recent common ancestry and experienced a dramatic population decline beginning at least ∼30 thousand years ago (kya). We suggest this crisis was related to the colonization of Eurasia by modern human hunter-gatherers, who competed with wolves for limited prey but also domesticated them, leading to a compensatory population expansion of dogs. We found extensive admixture between dogs and wolves, with up to 25% of Eurasian wolf genomes showing signs of dog ancestry. Dogs have influenced the recent history of wolves through admixture and vice versa, potentially enhancing adaptation. Simple scenarios of dog domestication are confounded by admixture, and studies that do not take admixture into account with specific demographic models are problematic.

Natural re-colonization and admixture of wolves (Canis lupus) in the US Pacific Northwest: challenges for the protection and management of rare and endangered taxa.

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.

Hypoxia adaptations in the grey wolf (Canis lupus chanco) from Qinghai-Tibet Plateau.

The Tibetan grey wolf (Canis lupus chanco) occupies habitats on the Qinghai-Tibet Plateau, a high altitude (>3000 m) environment where low oxygen tension exerts unique selection pressure on individuals to adapt to hypoxic conditions. To identify genes involved in hypoxia adaptation, we generated complete genome sequences of nine Chinese wolves from high and low altitude populations at an average coverage of 25× coverage. We found that, beginning about 55,000 years ago, the highland Tibetan grey wolf suffered a more substantial population decline than lowland wolves. Positively selected hypoxia-related genes in highland wolves are enriched in the HIF signaling pathway (P = 1.57E-6), ATP binding (P = 5.62E-5), and response to an oxygen-containing compound (P≤5.30E-4). Of these positively selected hypoxia-related genes, three genes (EPAS1, ANGPT1, and RYR2) had at least one specific fixed non-synonymous SNP in highland wolves based on the nine genome data. Our re-sequencing studies on a large panel of individuals showed a frequency difference greater than 58% between highland and lowland wolves for these specific fixed non-synonymous SNPs and a high degree of LD surrounding the three genes, which imply strong selection. Past studies have shown that EPAS1 and ANGPT1 are important in the response to hypoxic stress, and RYR2 is involved in heart function. These three genes also exhibited significant signals of natural selection in high altitude human populations, which suggest similar evolutionary constraints on natural selection in wolves and humans of the Qinghai-Tibet Plateau.

Interactions between demography, genetics, and landscape connectivity increase extinction probability for a small population of large carnivores in a major metropolitan area.

The extinction vortex is a theoretical model describing the process by which extinction risk is elevated in small, isolated populations owing to interactions between environmental, demographic, and genetic factors. However, empirical demonstrations of these interactions have been elusive. We modelled the dynamics of a small mountain lion population isolated by anthropogenic barriers in greater Los Angeles, California, to evaluate the influence of demographic, genetic, and landscape factors on extinction probability. The population exhibited strong survival and reproduction, and the model predicted stable median population growth and a 15% probability of extinction over 50 years in the absence of inbreeding depression. However, our model also predicted the population will lose 40-57% of its heterozygosity in 50 years. When we reduced demographic parameters proportional to reductions documented in another wild population of mountain lions that experienced inbreeding depression, extinction probability rose to 99.7%. Simulating greater landscape connectivity by increasing immigration to greater than or equal to one migrant per generation appears sufficient to largely maintain genetic diversity and reduce extinction probability. We provide empirical support for the central tenet of the extinction vortex as interactions between genetics and demography greatly increased extinction probability relative to the risk from demographic and environmental stochasticity alone. Our modelling approach realistically integrates demographic and genetic data to provide a comprehensive assessment of factors threatening small populations.

Admixture mapping identifies introgressed genomic regions in North American canids.

Hybrid zones typically contain novel gene combinations that can be tested by natural selection in a unique genetic context. Parental haplotypes that increase fitness can introgress beyond the hybrid zone, into the range of parental species. We used the Affymetrix canine SNP genotyping array to identify genomic regions tagged by multiple ancestry informative markers that are more frequent in an admixed population than expected. We surveyed a hybrid zone formed in the last 100 years as coyotes expanded their range into eastern North America. Concomitant with expansion, coyotes hybridized with wolves and some populations became more wolflike, such that coyotes in the northeast have the largest body size of any coyote population. Using a set of 3102 ancestry informative markers, we identified 60 differentially introgressed regions in 44 canines across this admixture zone. These regions are characterized by an excess of exogenous ancestry and, in northeastern coyotes, are enriched for genes affecting body size and skeletal proportions. Further, introgressed wolf-derived alleles have penetrated into Southern US coyote populations. Because no wolves currently exist in this area, these alleles are unlikely to have originated from recent hybridization. Instead, they probably originated from intraspecific gene flow or ancient admixture. We show that grey wolf and coyote admixture has far-reaching effects and, in addition to phenotypically transforming admixed populations, allows for the differential movement of alleles from different parental species to be tested in new genomic backgrounds.

Genome-wide SNP and haplotype analyses reveal a rich history underlying dog domestication.

Advances in genome technology have facilitated a new understanding of the historical and genetic processes crucial to rapid phenotypic evolution under domestication. To understand the process of dog diversification better, we conducted an extensive genome-wide survey of more than 48,000 single nucleotide polymorphisms in dogs and their wild progenitor, the grey wolf. Here we show that dog breeds share a higher proportion of multi-locus haplotypes unique to grey wolves from the Middle East, indicating that they are a dominant source of genetic diversity for dogs rather than wolves from east Asia, as suggested by mitochondrial DNA sequence data. Furthermore, we find a surprising correspondence between genetic and phenotypic/functional breed groupings but there are exceptions that suggest phenotypic diversification depended in part on the repeated crossing of individuals with novel phenotypes. Our results show that Middle Eastern wolves were a critical source of genome diversity, although interbreeding with local wolf populations clearly occurred elsewhere in the early history of specific lineages. More recently, the evolution of modern dog breeds seems to have been an iterative process that drew on a limited genetic toolkit to create remarkable phenotypic diversity.

A genome-wide perspective on the evolutionary history of enigmatic wolf-like canids.

High-throughput genotyping technologies developed for model species can potentially increase the resolution of demographic history and ancestry in wild relatives. We use a SNP genotyping microarray developed for the domestic dog to assay variation in over 48K loci in wolf-like species worldwide. Despite the high mobility of these large carnivores, we find distinct hierarchical population units within gray wolves and coyotes that correspond with geographic and ecologic differences among populations. Further, we test controversial theories about the ancestry of the Great Lakes wolf and red wolf using an analysis of haplotype blocks across all 38 canid autosomes. We find that these enigmatic canids are highly admixed varieties derived from gray wolves and coyotes, respectively. This divergent genomic history suggests that they do not have a shared recent ancestry as proposed by previous researchers. Interspecific hybridization, as well as the process of evolutionary divergence, may be responsible for the observed phenotypic distinction of both forms. Such admixture complicates decisions regarding endangered species restoration and protection.

Identification of recent hybridization between gray wolves and domesticated dogs by SNP genotyping.

The ability to detect recent hybridization between dogs and wolves is important for conservation and legal actions, which often require accurate and rapid resolution of ancestry. The availability of a genetic test for dog-wolf hybrids would greatly support federal and legal enforcement efforts, particularly when the individual in question lacks prior ancestry information. We have developed a panel of 100 unlinked ancestry-informative SNP markers that can detect mixed ancestry within up to four generations of dog-wolf hybridization based on simulations of seven genealogical classes constructed following the rules of Mendelian inheritance. We establish 95 % confidence regions around the spatial clustering of each genealogical class using a tertiary plot of allele dosage and heterozygosity. The first- and second-backcrossed-generation hybrids were the most distinct from parental populations, with >90 % correctly assigned to genealogical class. In this article we provide a tool kit with population-level statistical quantification that can detect recent dog-wolf hybridization using a panel of dog-wolf ancestry-informative SNPs with divergent allele frequency distributions.

Intraspecific morphological and genetic variation of common species predicts ranges of threatened ones.

Predicting where threatened species occur is useful for making informed conservation decisions. However, because they are usually rare, surveying threatened species is often expensive and time intensive. Here, we show how regions where common species exhibit high genetic and morphological divergence among populations can be used to predict the occurrence of species of conservation concern. Intraspecific variation of common species of birds, bats and frogs from Ecuador were found to be a significantly better predictor for the occurrence of threatened species than suites of environmental variables or the occurrence of amphibians and birds. Fully 93 per cent of the threatened species analysed had their range adequately represented by the geographical distribution of the morphological and genetic variation found in seven common species. Both higher numbers of threatened species and greater genetic and morphological variation of common species occurred along elevation gradients. Higher levels of intraspecific divergence may be the result of disruptive selection and/or introgression along gradients. We suggest that collecting data on genetic and morphological variation in common species can be a cost effective tool for conservation planning, and that future biodiversity inventories include surveying genetic and morphological data of common species whenever feasible.

Genome sequence, comparative analysis and haplotype structure of the domestic dog.

Here we report a high-quality draft genome sequence of the domestic dog (Canis familiaris), together with a dense map of single nucleotide polymorphisms (SNPs) across breeds. The dog is of particular interest because it provides important evolutionary information and because existing breeds show great phenotypic diversity for morphological, physiological and behavioural traits. We use sequence comparison with the primate and rodent lineages to shed light on the structure and evolution of genomes and genes. Notably, the majority of the most highly conserved non-coding sequences in mammalian genomes are clustered near a small subset of genes with important roles in development. Analysis of SNPs reveals long-range haplotypes across the entire dog genome, and defines the nature of genetic diversity within and across breeds. The current SNP map now makes it possible for genome-wide association studies to identify genes responsible for diseases and traits, with important consequences for human and companion animal health.

A novel assessment of population structure and gene flow in grey wolf populations of the Northern Rocky Mountains of the United States.

The successful re-introduction of grey wolves to the western United States is an impressive accomplishment for conservation science. However, the degree to which subpopulations are genetically structured and connected, along with the preservation of genetic variation, is an important concern for the continued viability of the metapopulation. We analysed DNA samples from 555 Northern Rocky Mountain wolves from the three recovery areas (Greater Yellowstone Area, Montana, and Idaho), including all 66 re-introduced founders, for variation in 26 microsatellite loci over the initial 10-year recovery period (1995-2004). The population maintained high levels of variation (H(O) = 0.64-0.72; allelic diversity k=7.0-10.3) with low levels of inbreeding (F(IS) < 0.03) and throughout this period, the population expanded rapidly (n(1995) =101; n(2004) =846). Individual-based Bayesian analyses revealed significant population genetic structure and identified three subpopulations coinciding with designated recovery areas. Population assignment and migrant detection were difficult because of the presence of related founders among different recovery areas and required a novel approach to determine genetically effective migration and admixture. However, by combining assignment tests, private alleles, sibship reconstruction, and field observations, we detected genetically effective dispersal among the three recovery areas. Successful conservation of Northern Rocky Mountain wolves will rely on management decisions that promote natural dispersal dynamics and minimize anthropogenic factors that reduce genetic connectivity.

Mapping the risk of avian influenza in wild birds in the US.

BACKGROUND: Avian influenza virus (AIV) is an important public health issue because pandemic influenza viruses in people have contained genes from viruses that infect birds. The H5 and H7 AIV subtypes have periodically mutated from low pathogenicity to high pathogenicity form. Analysis of the geographic distribution of AIV can identify areas where reassortment events might occur and how high pathogenicity influenza might travel if it enters wild bird populations in the US. Modelling the number of AIV cases is important because the rate of co-infection with multiple AIV subtypes increases with the number of cases and co-infection is the source of reassortment events that give rise to new strains of influenza, which occurred before the 1968 pandemic. Aquatic birds in the orders Anseriformes and Charadriiformes have been recognized as reservoirs of AIV since the 1970s. However, little is known about influenza prevalence in terrestrial birds in the order Passeriformes. Since passerines share the same habitat as poultry, they may be more effective transmitters of the disease to humans than aquatic birds. We analyze 152 passerine species including the American Robin (Turdus migratorius) and Swainson's Thrush (Catharus ustulatus). METHODS: We formulate a regression model to predict AIV cases throughout the US at the county scale as a function of 12 environmental variables, sampling effort, and proximity to other counties with influenza outbreaks. Our analysis did not distinguish between types of influenza, including low or highly pathogenic forms. RESULTS: Analysis of 13,046 cloacal samples collected from 225 bird species in 41 US states between 2005 and 2008 indicates that the average prevalence of influenza in passerines is greater than the prevalence in eight other avian orders. Our regression model identifies the Great Plains and the Pacific Northwest as high-risk areas for AIV. Highly significant predictors of AIV include the amount of harvested cropland and the first day of the year when a county is snow free. CONCLUSIONS: Although the prevalence of influenza in waterfowl has long been appreciated, we show that 22 species of song birds and perching birds (order Passeriformes) are influenza reservoirs in the contiguous US.

Landscape genetics of California mule deer (Odocoileus hemionus): the roles of ecological and historical factors in generating differentiation.

Landscape genetics is an emerging discipline that utilizes environmental and historical data to understand geographic patterns of genetic diversity. Niche modelling has added a new dimension to such efforts by allowing species-environmental associations to be projected into the past so that hypotheses about historical vicariance can be generated and tested independently with genetic data. However, previous approaches have primarily utilized DNA sequence data to test inferences about historical isolation and may have missed very recent episodes of environmentally mediated divergence. We type 15 microsatellite loci in California mule deer and identify five genetic groupings through a Structure analysis that are also well predicted by environmental data. We project the niches of these five deer ecotypes to the last glacial maximum (LGM) and show they overlap to a much greater extent than today, suggesting that vicariance associated with the LGM cannot explain the present-day genetic patterns. Further, we analyse mitochondrial DNA (mtDNA) sequence trees to search for evidence of historical vicariance and find only two well-supported clades. A coalescence-based analysis of mtDNA data shows that the genetic divergence of the mule deer genetic clusters in California is recent and appears to be mediated by ecological factors. The importance of environmental factors in explaining the genetic diversity of California mule deer is unexpected given that they are highly mobile species and have a broad habitat distribution. Geographic differences in the timing of reproduction and peak vegetation as well as habitat choice reflecting natal origin may explain the persistence of genetic subdivision.

Coquillettidia (Culicidae, Diptera) mosquitoes are natural vectors of avian malaria in Africa.

BACKGROUND: The mosquito vectors of Plasmodium spp. have largely been overlooked in studies of ecology and evolution of avian malaria and other vertebrates in wildlife. METHODS: Plasmodium DNA from wild-caught Coquillettidia spp. collected from lowland forests in Cameroon was isolated and sequenced using nested PCR. Female Coquillettidia aurites were also dissected and salivary glands were isolated and microscopically examined for the presence of sporozoites. RESULTS: In total, 33% (85/256) of mosquito pools tested positive for avian Plasmodium spp., harbouring at least eight distinct parasite lineages. Sporozoites of Plasmodium spp. were recorded in salivary glands of C. aurites supporting the PCR data that the parasites complete development in these mosquitoes. Results suggest C. aurites, Coquillettidia pseudoconopas and Coquillettidia metallica as new and important vectors of avian malaria in Africa. All parasite lineages recovered clustered with parasites formerly identified from several bird species and suggest the vectors capability of infecting birds from different families. CONCLUSION: Identifying the major vectors of avian Plasmodium spp. will assist in understanding the epizootiology of avian malaria, including differences in this disease distribution between pristine and disturbed landscapes.

Extensive population genetic structure in the giraffe.

BACKGROUND: A central question in the evolutionary diversification of large, widespread, mobile mammals is how substantial differentiation can arise, particularly in the absence of topographic or habitat barriers to dispersal. All extant giraffes (Giraffa camelopardalis) are currently considered to represent a single species classified into multiple subspecies. However, geographic variation in traits such as pelage pattern is clearly evident across the range in sub-Saharan Africa and abrupt transition zones between different pelage types are typically not associated with extrinsic barriers to gene flow, suggesting reproductive isolation. RESULTS: By analyzing mitochondrial DNA sequences and nuclear microsatellite loci, we show that there are at least six genealogically distinct lineages of giraffe in Africa, with little evidence of interbreeding between them. Some of these lineages appear to be maintained in the absence of contemporary barriers to gene flow, possibly by differences in reproductive timing or pelage-based assortative mating, suggesting that populations usually recognized as subspecies have a long history of reproductive isolation. Further, five of the six putative lineages also contain genetically discrete populations, yielding at least 11 genetically distinct populations. CONCLUSION: Such extreme genetic subdivision within a large vertebrate with high dispersal capabilities is unprecedented and exceeds that of any other large African mammal. Our results have significant implications for giraffe conservation, and imply separate in situ and ex situ management, not only of pelage morphs, but also of local populations.

Whole-genome sequence analysis shows that two endemic species of North American wolf are admixtures of the coyote and gray wolf.

Protection of populations comprising admixed genomes is a challenge under the Endangered Species Act (ESA), which is regarded as the most powerful species protection legislation ever passed in the United States but lacks specific provisions for hybrids. The eastern wolf is a newly recognized wolf-like species that is highly admixed and inhabits the Great Lakes and eastern United States, a region previously thought to be included in the geographic range of only the gray wolf. The U.S. Fish and Wildlife Service has argued that the presence of the eastern wolf, rather than the gray wolf, in this area is grounds for removing ESA protection (delisting) from the gray wolf across its geographic range. In contrast, the red wolf from the southeastern United States was one of the first species protected under the ESA and was protected despite admixture with coyotes. We use whole-genome sequence data to demonstrate a lack of unique ancestry in eastern and red wolves that would not be expected if they represented long divergent North American lineages. These results suggest that arguments for delisting the gray wolf are not valid. Our findings demonstrate how a strict designation of a species under the ESA that does not consider admixture can threaten the protection of endangered entities. We argue for a more balanced approach that focuses on the ecological context of admixture and allows for evolutionary processes to potentially restore historical patterns of genetic variation.

Disease and freeways drive genetic change in urban bobcat populations.

Urbanization profoundly impacts animal populations by causing isolation, increased susceptibility to disease, and exposure to toxicants. Genetic effects include reduced effective population size, increased population substructure, and decreased adaptive potential. We investigated the influence that urbanization and a disease epizootic had on the population genetics of bobcats (Lynx rufus) distributed across a highly fragmented urban landscape. We genotyped more than 300 bobcats, sampled from 1996 to 2012, for variation at nine neutral and seven immune gene-linked microsatellite loci. We found that two freeways are significant barriers to gene flow. Further, a 3-year disease epizootic, associated with secondary anticoagulant rodenticide exposure, caused a population bottleneck that led to significant genetic differentiation between pre- and post-disease populations that was greater than that between populations separated by major freeways for >60 years. However, balancing selection acted on immune-linked loci during the epizootic, maintaining variation at functional regions. Conservation assessments need to assay loci that are potentially under selection to better preserve the adaptive potential of populations at the urban-wildland interface. Further, interconnected regions that contain appropriate habitat for wildlife will be critical to the long-term viability of animal populations in urban landscapes.

Genome-wide Evidence Reveals that African and Eurasian Golden Jackals Are Distinct Species.

The golden jackal of Africa (Canis aureus) has long been considered a conspecific of jackals distributed throughout Eurasia, with the nearest source populations in the Middle East. However, two recent reports found that mitochondrial haplotypes of some African golden jackals aligned more closely to gray wolves (Canis lupus), which is surprising given the absence of gray wolves in Africa and the phenotypic divergence between the two species. Moreover, these results imply the existence of a previously unrecognized phylogenetically distinct species despite a long history of taxonomic work on African canids. To test the distinct-species hypothesis and understand the evolutionary history that would account for this puzzling result, we analyzed extensive genomic data including mitochondrial genome sequences, sequences from 20 autosomal loci (17 introns and 3 exon segments), microsatellite loci, X- and Y-linked zinc-finger protein gene (ZFX and ZFY) sequences, and whole-genome nuclear sequences in African and Eurasian golden jackals and gray wolves. Our results provide consistent and robust evidence that populations of golden jackals from Africa and Eurasia represent distinct monophyletic lineages separated for more than one million years, sufficient to merit formal recognition as different species: C. anthus (African golden wolf) and C. aureus (Eurasian golden jackal). Using morphologic data, we demonstrate a striking morphologic similarity between East African and Eurasian golden jackals, suggesting parallelism, which may have misled taxonomists and likely reflects uniquely intense interspecific competition in the East African carnivore guild. Our study shows how ecology can confound taxonomy if interspecific competition constrains size diversification.

Individual behaviors dominate the dynamics of an urban mountain lion population isolated by roads.

Large carnivores can be particularly sensitive to the effects of habitat fragmentation on genetic diversity [1, 2]. The Santa Monica Mountains (SMMs), a large natural area within Greater Los Angeles, is completely isolated by urban development and the 101 freeway to the north. Yet the SMMs support a population of mountain lions (Puma concolor), a very rare example of a large carnivore persisting within the boundaries of a megacity. GPS locations of radio-collared lions indicate that freeways are a near-absolute barrier to movement. We genotyped 42 lions using 54 microsatellite loci and found that genetic diversity in SMM lions, prior to 2009, was lower than that for any population in North America except in southern Florida, where inbreeding depression led to reproductive failure [3-5]. We document multiple instances of father-daughter inbreeding and high levels of intraspecific strife, including the unexpected behavior of a male killing two of his offspring and a mate and his son killing two of his brothers. Overall, no individuals from the SMMs have successfully dispersed. Gene flow is critical for this population, and we show that a single male immigrated in 2009, successfully mated, and substantially enhanced genetic diversity. Our results imply that individual behaviors, most likely caused by limited area and reduced opportunities to disperse, may dominate the fate of small, isolated populations of large carnivores. Consequently, comprehensive behavioral monitoring can suggest novel solutions for the persistence of small populations, such as the transfer of individuals across dispersal barriers.

Batrachochytrium dendrobatidis shows high genetic diversity and ecological niche specificity among haplotypes in the Maya Mountains of Belize.

The amphibian pathogen Batrachochytrium dendrobatidis (Bd) has been implicated in amphibian declines around the globe. Although it has been found in most countries in Central America, its presence has never been assessed in Belize. We set out to determine the range, prevalence, and diversity of Bd using quantitative PCR (qPCR) and sequencing of a portion of the 5.8 s and ITS1-2 regions. Swabs were collected from 524 amphibians of at least 26 species in the protected areas of the Maya Mountains of Belize. We sequenced a subset of 72 samples that had tested positive for Bd by qPCR at least once; 30 samples were verified as Bd. Eight unique Bd haplotypes were identified in the Maya Mountains, five of which were previously undescribed. We identified unique ecological niches for the two most broadly distributed haplotypes. Combined with data showing differing virulence shown in different strains in other studies, the 5.8 s - ITS1-2 region diversity found in this study suggests that there may be substantial differences among populations or haplotypes. Future work should focus on whether specific haplotypes for other genomic regions and possibly pathogenicity can be associated with haplotypes at this locus, as well as the integration of molecular tools with other ecological tools to elucidate the ecology and pathogenicity of Bd.