Genetic evidence for multiple events of hybridization between wolves and domestic dogs in the Iberian Peninsula

Hybridization between wild species and their domestic counterparts may represent a major threat to natural populations. However, high genetic similarity between the hybridizing taxa makes the detection of hybrids a difficult task and may hinder attempts to assess the impact of hybridization in conservation biology. In this work, we used a combination of 42 autosomal microsatellites together with Y-chromosome microsatellite-defined haplotypes and mtDNA sequences to investigate the occurrence and dynamics of wolf-dog hybridization in the Iberian Peninsula. To do this, we applied a variety of Bayesian analyses and a parallel set of simulation studies to evaluate (i) the differences between Iberian wolves and dogs, (ii) the frequency and geographical distribution of hybridization and (iii) the directionality of hybridization. First, we show that Iberian wolves and dogs form two well-differentiated genetic entities, suggesting that introgressive hybridization is not a widespread phenomenon shaping both gene pools. Second, we found evidence for the existence of hybridization that is apparently restricted to more peripheral and recently expanded wolf populations. Third, we describe compelling evidence suggesting that the dynamics of hybridization in wolf populations is mediated by crosses between male dogs and female wolves. More importantly, the observation of a population showing the occurrence of a continuum of hybrid classes forming mixed packs may indicate that we have underestimated hybridization. If future studies confirm this pattern, then an intriguing avenue of research is to investigate how introgression from free-ranging domestic dogs is enabling wolf populations to adapt to the highly humanized habitats of southern Europe while still maintaining their genetic differentiation.     

Bucking the Trend in Wolf-Dog Hybridization: First Evidence from Europe of Hybridization between Female Dogs and Male Wolves

Studies on hybridization have proved critical for understanding key evolutionary processes such as speciation and adaptation. However, from the perspective of conservation, hybridization poses a concern, as it can threaten the integrity and fitness of many wild species, including canids. As a result of habitat fragmentation and extensive hunting pressure, gray wolf (Canis lupus) populations have declined dramatically in Europe and elsewhere during recent centuries. Small and fragmented populations have persisted, but often only in the presence of large numbers of dogs, which increase the potential for hybridization and introgression to deleteriously affect wolf populations. Here, we demonstrate hybridization between wolf and dog populations in Estonia and Latvia, and the role of both genders in the hybridization process, using combined analysis of maternal, paternal and biparental genetic markers. Eight animals exhibiting unusual external characteristics for wolves - six from Estonia and two from Latvia - proved to be wolf-dog hybrids. However, one of the hybridization events was extraordinary. Previous field observations and genetic studies have indicated that mating between wolves and dogs is sexually asymmetrical, occurring predominantly between female wolves and male dogs. While this was also the case among the Estonian hybrids, our data revealed the existence of dog mitochondrial genomes in the Latvian hybrids and, together with Y chromosome and autosomal microsatellite data, thus provided the first evidence from Europe of mating between male wolves and female dogs. We discuss patterns of sexual asymmetry in wolf-dog hybridization.     

The systematic status of the Italian wolfCanis lupus

In the past, the gray wolfCanis lupus Linnaeus, 1758, has been recognized in Italy as either the subspecieslupus oritalicus. It has also been postulated that this population has undergone introgression from the domestic dogCanis familiaris. In order to clarify these issues, multistatistical analyses were made of 10 skull measurements of 34 full grown male wolves from the Italian Peninsula, 91 other male Eurasian wolves, and 20 domestic dogs. The analyses, together with other morphological evidence and prior genetic research, support recognition of the Italian wolf as a separate subspecies,Canis lupus italicus. The same evidence indicates that the subspecies has not been affected through hybridization with the domestic dog.     

Detection of an East European wolf haplotype puzzles mitochondrial DNA monomorphism of the Italian wolf population

Southern European wolves suffered from reiterated population declines during glacial periods and historically due to human persecution. Differently from other European wolf populations, a single mitochondrial DNA (mtDNA) control region haplotype (W14) has been so far described in the Italian wolves, although no intensive genetic sampling has ever been conducted in historical source populations from central and southern Italy. Using non-invasive genetic techniques, we report the occurrence of an unexpected mtDNA haplotype (W16) in the wolf population of the Abruzzo, Lazio and Molise National Park (PNALM), central Italy. This haplotype, detected in three out of 90 faecal samples from the PNALM, was previously reported in wolves from the North Carpathians, Slovakia and the Balkans only. Microsatellite analysis and molecular sex determination confirmed that the W16 samples belonged to three distinct wolves. Although alternative explanations can be formulated for the origin of this mtDNA haplotype in the otherwise monomorphic Italian wolf population, assignment procedures indicated the likely admixed ancestry of one W16 sample with East European wolves. Anthropogenic introgression with dogs has been detected in the Italian wolf population using nuclear DNA microsatellites, but no population-wide genetic survey had previously reported a mtDNA control region variant in Italian wolves. Our findings strongly suggest that, in addition to wolf × dog hybridization, captive-released wolves or wolf × dog hybrids may successfully interbreed with wolves in the wild, and that human-mediated introgression may occur even in well established protected areas.     

A new mitochondrial haplotype confirms the distinctiveness of the Italian wolf (Canis lupus) population

In the past century the Italian wolf has been repeatedly indicated as a distinct subspecies, Canis lupus italicus, due to its unique morphology and its distinctive mtDNA control region (CR) monomorphism. However, recent studies on wolf x dog hybridization in Italy documented the presence of a second mtDNA CR haplotype (W16), previously found only in wolves from Eastern Europe, casting doubts on the genetic uniqueness of the Italian wolves. To test whether this second haplotype belongs to the Italian wolf population, we genotyped 92 wolf DNA samples from Italy, Slovenia, Greece and Bulgaria at four mtDNA regions (control-region, ATP6, COIII and ND4 genes) and at 39 autosomal microsatellites. Results confirm the presence of two mtDNA multi-fragment haplotypes (WH14 and WH19) in the Italian wolves, distinct from all the other European wolves. Network analyses of the multi-fragment mtDNA haplotypes identified two strongly differentiated clades, with the Italian wolf WH14 and WH19 multi-fragment haplotypes rooted together. Finally, Bayesian clustering clearly assigned all the wolves sampled in Italy to the Italian population, regardless of the two different multi-fragment haplotypes. These results demonstrate that the W16 CR haplotype is part of the genetic pool of the Italian wolf population, reconfirming its distinctiveness from other European wolves. Overall, considering the presence of unique mtDNA and Y-linked haplotypes, the sharply different frequencies of genome-wide autosomal alleles and the distinct morphological features of Italian wolves, we believe that this population should be considered a distinct subspecies.     

Hybridization between wolf and domestic dog: First evidence from an endangered population in central Portugal

Human population expansion has promoted contact between wildlife and domestic animals with severe ecological consequences, such as anthropogenic hybridization. In Portugal, Iberian wolf (Canis lupus signatus) populations are considered “Endangered” and co-habit with humans so the risks of hybridization with free-ranging dogs, and livestock depredation can be particularly high. Our aim was to report the occurrence of wolf-dog hybridization in an endangered Iberian wolf sub-population, located in the south of the Douro river, Portugal. We used mitochondrial DNA and microsatellite data to investigate putative hybrids between Iberian wolves and dogs. Here, we report for the first time a wolf-dog hybrid located in the south of the Douro river. This is the second hybrid found in Portugal, and even if hybridization cases are still considered rare, they can be particularly problematic in isolated, fragmented and endangered populations, such as the one studied here. Appropriate management and conservation measures are recommended.     

Detecting introgressive hybridization between free-ranging domestic dogs and wild wolves (Canis lupus) by admixture linkage disequilibrium analysis

Occasional crossbreeding between free-ranging domestic dogs and wild wolves (Canis lupus) has been detected in some European countries by mitochondrial DNA sequencing and genotyping unlinked microsatellite loci. Maternal and unlinked genomic markers, however, might underestimate the extent of introgressive hybridization, and their impacts on the preservation of wild wolf gene pools. In this study, we genotyped 220 presumed Italian wolves, 85 dogs and 7 known hybrids at 16 microsatellites belonging to four different linkage groups (plus four unlinked microsatellites). Population clustering and individual assignments were performed using a Bayesian procedure implemented in structure 2.1, which models the gametic disequilibrium arising between linked loci during admixtures, aiming to trace hybridization events further back in time and infer the population of origin of chromosomal blocks. Results indicate that (i) linkage disequilibrium was higher in wolves than in dogs; (ii) 11 out of 220 wolves (5.0%) were likely admixed, a proportion that is significantly higher than one admixed genotype in 107 wolves found previously in a study using unlinked markers; (iii) posterior maximum-likelihood estimates of the recombination parameter r revealed that introgression in Italian wolves is not recent, but could have continued for the last 70 (+/- 20) generations, corresponding to approximately 140-210 years. Bayesian clustering showed that, despite some admixture, wolf and dog gene pools remain sharply distinct (the average proportions of membership to wolf and dog clusters were Q(w) = 0.95 and Q(d) = 0.98, respectively), suggesting that hybridization was not frequent, and that introgression in nature is counteracted by behavioural or selective constraints.     

Mitochondrial DNA restriction-fragment-length monomorphism in the Italian wolf (Canis lupus) population

Mitochondrial-DNA (mtDNA) restriction patterns were studied in 22 wolves (Canis lupus) sampled in central-northern Italy. A total of 60 restriction sites were detected, encompassing about 2 % of the mitochondrial genome of canids. All wolves showed the same restriction pattern. Therefore, a single mtDNA haplotype was detected in the Italian wolf population. Historical information on peninsular isolation and demographic decline suggest that low genetically effective population size and random drift may have strongly reduced the mtDNA variability of wolves in Italy over the last 100–200 years. A different mtDNA restriction pattern in feral dogs sampled from a wolf range in central Italy was detected. These findings suggest that the hybridization and introgression of female dog genomes into the Italian wolf population may be rare or absent.     

Combined use of maternal,paternal and bi-parental genetic markers for the identification of wolf-dog hybrids

The identification of hybrids is often a subject of primary concern for the development of conservation and management strategies, but can be difficult when the hybridizing species are closely related and do not possess diagnostic genetic markers. However, the combined use of mitochondrial DNA (mtDNA), autosomal and Y chromosome genetic markers may allow the identification of hybrids and of the direction of hybridization. We used these three types of markers to genetically characterize one possible wolf-dog hybrid in the endangered Scandinavian wolf population. We first characterized the variability of mtDNA and Y chromosome markers in Scandinavian wolves as well as in neighboring wolf populations and in dogs. While the mtDNA data suggested that the target sample could correspond to a wolf, its Y chromosome type had not been observed before in Scandinavian wolves. We compared the genotype of the target sample at 18 autosomal microsatellite markers with those expected in pure specimens and in hybrids using assignment tests. The combined results led to the conclusion that the animal was a hybrid between a Scandinavian female wolf and a male dog. This finding confirms that inter-specific hybridization between wolves and dogs can occur in natural wolf populations. A possible correlation between hybridization and wolf population density and disturbance deserves further research.     

The genetic legacy of extirpation and re-colonization in Vancouver Island wolves

Hybridization between wild and domestic species is of conservation concern because it can result in the loss of adaptations and/or disappearance of a distinct taxon. Wolves from Vancouver Island, British Columbia (Canada), have been subject to several eradication campaigns during the twentieth century and were considered virtually extirpated between 1950 and 1970. In this study, we use control region mitochondrial DNA sequences and 13 autosomal microsatellite loci to characterize Vancouver Island wolves as well as dogs from British Columbia. We observe a turnover in the haplotypes of wolves sampled before and after the 1950–1970 period, when there was no permanent wolf population on the island, supporting the probable local extinction of wolves on Vancouver Island during this time, followed by re-colonization of the island by wolves from mainland British Columbia. In addition, we report the presence of a domestic dog mtDNA haplotype in three individuals eliminated in 1986 that were morphologically identified as wolves. Here we show that Vancouver Island wolves were also identified as wolves based on autosomal microsatellite data. We attribute the hybridization event to the episodically small size of this population during the re-colonization event. Our results demonstrate that at least one female hybrid offspring, resulting from a cross of a male wolf and a female dog or a female hybrid pet with dog mtDNA, successfully introgressed into the wolf population. No dog mtDNA has been previously reported in a population of wild wolves. Genetic data show that Vancouver Island wolves are distinct from dogs and thus should be recognized as a population of wild wolves. We suggest that the introgression took place due to the Allee effect, specifically a lack of mates when population size was low. Our findings exemplify how small populations are at risk of hybridization.     

Molecular genetics of the most endangered canid: the Ethiopian wolf Canis simensis

The world's most endangered canid is the Ethiopian wolf Canis simensis , which is found in six isolated areas of the Ethiopian highlands with a total population of no more than 500 individuals. Ethiopian wolf populations are declining due to habitat loss and extermination by humans. Moreover, in at least one population, Ethiopian wolves are sympatric with domestic dogs, which may hybridize with them, compete for food, and act as disease vectors. Using molecular techniques, we address four questions concerning Ethiopian wolves that have conservation implications. First, we determine the relationships of Ethiopian wolves to other wolf-like canids by phylogenetic analysis of 2001 base pairs of mitochondrial DNA (mtDNA) sequence. Our results suggest that the Ethiopian wolf is a distinct species more closely related to gray wolves and coyotes than to any African canid. The mtDNA sequence similarity with gray wolves implies that the Ethiopian wolf may hybridize with domestic dogs, a recent derivative of the gray wolf. We examine this possibility through mtDNA restriction fragment analysis and analysis of nine microsatellite loci in populations of Ethiopian wolves. The results imply that hybridization has occurred between female Ethiopian wolves and male domestic dogs in one population. Finally, we assess levels of variability within and between two Ethiopian wolf populations. Although these closely situated populations are not differentiated, the level of variability in both is low, suggesting long-term effective population sizes of less than a few hundred individuals. We recommend immediate captive breeding of Ethiopian wolves to protect their gene pool from dilution and further loss of genetic variability.     

Characterization of 59 canine single nucleotide polymorphisms in the Italian wolf (Canis lupus) population

We characterized 59 canine single nucleotide polymorphisms (SNPs) in the endangered Italian wolf (Canis lupus) population, which were discovered by resequencing sequence‐tagged‐site (STS) DNA sequences that are known to contain SNPs in domestic dogs. Dog SNPs were usually found also in wolves. Additional SNPs unique in dogs or wolves were discovered, which is important for detecting hybrids between dogs and wolves. We developed new primer sets and analysed 15 SNPs by Pyrosequencing. The characterized SNPs will provide an important addition to the genetic markers that are currently available for studying wild populations of canids.     

Assessment of coyote–wolf–dog admixture using ancestry‐informative diagnostic SNPs

The evolutionary importance of hybridization as a source of new adaptive genetic variation is rapidly gaining recognition. Hybridization between coyotes and wolves may have introduced adaptive alleles into the coyote gene pool that facilitated an expansion in their geographic range and dietary niche. Furthermore, hybridization between coyotes and domestic dogs may facilitate adaptation to human‐dominated environments. We genotyped 63 ancestry‐informative single‐nucleotide polymorphisms in 427 canids to examine the prevalence, spatial distribution and the ecology of admixture in eastern coyotes. Using multivariate methods and Bayesian clustering analyses, we estimated the relative contributions of western coyotes, western and eastern wolves, and domestic dogs to the admixed ancestry of Ohio and eastern coyotes. We found that eastern coyotes form an extensive hybrid swarm, with all our samples having varying levels of admixture. Ohio coyotes, previously thought to be free of admixture, are also highly admixed with wolves and dogs. Coyotes in areas of high deer density are genetically more wolf‐like, suggesting that natural selection for wolf‐like traits may result in local adaptation at a fine geographic scale. Our results, in light of other previously published studies of admixture in Canis, revealed a pattern of sex‐biased hybridization, presumably generated by male wolves and dogs mating with female coyotes. This study is the most comprehensive genetic survey of admixture in eastern coyotes and demonstrates that the frequency and scope of hybridization can be quantified with relatively few ancestry‐informative markers.     

Origin and status of the Great Lakes wolf

An extensive debate concerning the origin and taxonomic status of wolf-like canids in the North American Great Lakes region and the consequences for conservation politics regarding these enigmatic predators is ongoing. Using maternally, paternally and biparentally inherited molecular markers, we demonstrate that the Great Lakes wolves are a unique population or ecotype of gray wolves. Furthermore, we show that the Great Lakes wolves experienced high degrees of ancient and recent introgression of coyote and western gray wolf mtDNA and Y-chromosome haplotypes, and that the recent demographic bottleneck caused by persecution and habitat depletion in the early 1900s is not reflected in the genetic data.     

Widespread occurrence of a domestic dog mitochondrial DNA haplotype in southeastern US coyotes

Sequence analysis of the mitochondrial DNA control region from 112 southeastern US coyotes (Canis latrans) revealed 12 individuals with a haplotype closely related to those in domestic dogs. Phylogenetic analyses grouped this new haplotype in the dog/grey wolf (Canis familiaris/Canis lupus) clade with 98% bootstrap support. These results demonstrate that a male coyote hybridized with a female dog, and female hybrid offspring successfully integrated into the coyote population. The widespread distribution of this haplotype from Florida to West Virginia suggests that the hybridization event occurred long ago before the southeastern USA was colonized by coyotes. However, it could have occurred in the southeastern USA before the main front of coyotes arrived in the area between male coyotes released for sport and a local domestic dog. The introgression of domestic dog genes into the southeastern coyote population does not appear to have substantially affected the coyote's genetic, morphological, or behavioural integrity. However, our results suggest that, contrary to previous reports, hybridization can occur between domestic and wild canids, even when the latter is relatively abundant. Therefore, hybridization may be a greater threat to the persistence of wild canid populations than previously thought.     

The evolutionary history of grey wolf Y chromosomes

Analyses of Y chromosome haplotypes uniquely provide a paternal picture of evolutionary histories and offer a very useful contrast to studies based on maternally inherited mitochondrial DNA (mtDNA). Here we used a bioinformatic approach based on comparison of male and female sequence coverage to identify 4.7 Mb from the grey wolf (Canis lupis) Y chromosome, probably representing most of the male‐specific, nonampliconic sequence from the euchromatic part of the chromosome. We characterized this sequence and then identified ≈1,500 Y‐linked single nucleotide polymorphisms in a sample of 145 resequenced male wolves, including 75 Finnish wolf genomes newly sequenced in this study, and in 24 dogs and eight other canids. We found 53 Y chromosome haplotypes, of which 26 were seen in grey wolves, that clustered in four major haplogroups. All four haplogroups were represented in samples of Finnish wolves, showing that haplogroup lineages were not partitioned on a continental scale. However, regional population structure was indicated because individual haplotypes were never shared between geographically distant areas, and genetically similar haplotypes were only found within the same geographical region. The deepest split between grey wolf haplogroups was estimated to have occurred 125,000 years ago, which is considerably older than recent estimates of the time of divergence of wolf populations. The distribution of dogs in a phylogenetic tree of Y chromosome haplotypes supports multiple domestication events, or wolf paternal introgression, starting 29,000 years ago. We also addressed the disputed origin of a recently founded population of Scandinavian wolves and observed that founding as well as most recent immigrant haplotypes were present in the neighbouring Finnish population, but not in sequenced wolves from elsewhere in the world, or in dogs.     

Detecting rare introgression of domestic dog genes into wild wolf (Canis lupus) populations by Bayesian admixture analyses of microsatellite variation

Hybridization with free-ranging dogs isthought to threat the genetic integrity ofwolves in Europe, although available mtDNA dataevidenced only sporadic cases of crossbreeding.Here we report results of population assignmentand genetic admixture analyses in 107wild-living Italian wolves, 95 dogs including30 different breeds and feral dogs, andcaptive-reared wolves of unknown or hybridorigins, which were genotyped at 18microsatellites. Two Italian wolves showedunusually dark coats (``black wolves'), and oneshowed a spur in both hindlegs (``fifth fingerwolf'), suggesting hybridization. Italianwolves showed significant deficit ofheterozygotes, positive F_IS values anddeviations from Hardy-Weinberg equilibrium.Genetic variability was significantlypartitioned between groups, suggesting thatwolves and dogs represent distinct gene pools.Multivariate ordination of individual genotypesand clustering of inter-individual geneticdistances split wolves and dogs into twodifferent clusters congruent with the priorphenotypic classification, but hybrids andwolves of unknown origin were not identifiedfrom genetic information alone. By contrast, aBayesian admixture analysis assigned all theItalian wolves and dogs to two differentclusters, independent of any prior phenotypicinformation, and simultaneously detected theadmixed gene composition of the hybrids, whichwere assigned to more than one cluster.Captive-reared wolves of unknown origin wereprevalently assigned to the Italian wolfpopulation. Admixture analyses showed that one``black wolf' had mixed ancestry in the dog genepool and could be a hybrid, while the other twowolves with unusual phenotypes were assigned tothe Italian wolf population.     

Intense harvesting of eastern wolves facilitated hybridization with coyotes

Despite ethical arguments against lethal control of wildlife populations, culling is routinely used for the management of predators, invasive or pest species, and infectious diseases. Here, we demonstrate that culling of wildlife can have unforeseen impacts that can be detrimental to future conservation efforts. Specifically, we analyzed genetic data from eastern wolves (Canis lycaon) sampled in Algonquin Provincial Park (APP), Ontario, Canada from 1964 to 2007. Research culls in 1964 and 1965 killed the majority of wolves within a study region of APP, accounting for approximately 36% of the park's wolf population at a time when coyotes were colonizing the region. The culls were followed by a significant decrease in an eastern wolf mitochondrial DNA (mtDNA) haplotype (C1) in the Park's wolf population, as well as an increase in coyote mitochondrial and nuclear DNA. The introgression of nuclear DNA from coyotes, however, appears to have been curtailed by legislation that extended wolf protection outside park boundaries in 2001, although eastern wolf mtDNA haplotype C1 continued to decline and is now rare within the park population. We conclude that the wolf culls transformed the genetic composition of this unique eastern wolf population by facilitating coyote introgression. These results demonstrate that intense localized harvest of a seemingly abundant species can lead to unexpected hybridization events that encumber future conservation efforts. Ultimately, researchers need to contemplate not only the ethics of research methods, but also that future implications may be obscured by gaps in our current scientific understanding.     

Genetic variation and subspecific status of the grey wolf (Canis lupus) in Saudi Arabia

This work represents the most extensive genetic study of the grey wolf (Canis lupus Linnaeus, 1758) in Arabia and the first considering genetic data from multiple locations within Saudi Arabia. Previous suggestion of the occurrence of two subspecies of wolves in Arabia is not supported by this study. The genetic evidence suggests that the wolves of Saudi Arabia are genetically variable and more closely related to the Eurasian wolf Canis lupus group (dog included) than to the Indian wolf C. l. pallipes. The genetic diversity observed for C. lupus in Saudi Arabia indicates that the subspecific status C. l. arabs should be retained for the Arabian wolf. What remains unclear is the degree to which genetic introgression from domestic dogs has influenced the composition and integrity of C. lupus in Saudi Arabia.