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.     

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.     

Y‐chromosome evidence supports widespread signatures of three‐species Canis hybridization in eastern North America

There has been considerable discussion on the origin of the red wolf and eastern wolf and their evolution independent of the gray wolf. We analyzed mitochondrial DNA (mtDNA) and a Y‐chromosome intron sequence in combination with Y‐chromosome microsatellites from wolves and coyotes within the range of extensive wolf–coyote hybridization, that is, eastern North America. The detection of divergent Y‐chromosome haplotypes in the historic range of the eastern wolf is concordant with earlier mtDNA findings, and the absence of these haplotypes in western coyotes supports the existence of the North American evolved eastern wolf (Canis lycaon). Having haplotypes observed exclusively in eastern North America as a result of insufficient sampling in the historic range of the coyote or that these lineages subsequently went extinct in western geographies is unlikely given that eastern‐specific mtDNA and Y‐chromosome haplotypes represent lineages divergent from those observed in extant western coyotes. By combining Y‐chromosome and mtDNA distributional patterns, we identified hybrid genomes of eastern wolf, coyote, gray wolf, and potentially dog origin in Canis populations of central and eastern North America. The natural contemporary eastern Canis populations represent an important example of widespread introgression resulting in hybrid genomes across the original C. lycaon range that appears to be facilitated by the eastern wolf acting as a conduit for hybridization. Applying conventional taxonomic nomenclature and species‐based conservation initiatives, particularly in human‐modified landscapes, may be counterproductive to the effective management of these hybrids and fails to consider their evolutionary potential.     

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.     

Y-chromosome microsatellite variation in Italian wolves: A contribution to the study of wolf-dog hybridization patterns

One major concern in wolf (Canis lupus) conservation is the risk of genetic contamination due to crossbreeding with domestic dogs. Although genetic monitoring of wolf populations has become widely used, the behavioural mechanisms involved in wolf-dog hybridization and the detrimental effects of genetic introgression are poorly known. In this study we analysed Y-chromosome microsatellite variation in the recovering Italian wolf population and detected strikingly different allele frequencies between wolves and dogs. Four Y haplotypes were found in 74 analysed male wolves, and all of them were present in a focus wolf population in the Apennines. On the other hand, only 1 haplotype was found in the recolonizing wolf population from the Western Alps. The most common haplotype in a sample of domestic dogs, was also found in 5 wolves, 2 of which revealing a signature of recent hybridization. Moreover, another suspect hybrid carried a private haplotype of possible canine origin. These results give support to the idea that female wolves can breed with male stray dogs in the wild. The Y-chromosome variation in Italian wolves contrasts with the previously observed lack of mitochondrial variation. Further investigations are needed to clarify at what extent historical or recent wolf-dog hybridization events may have contributed to the observed haplotype diversity. In conclusion, the two molecular markers employed in this study represent effective means to trace directional genetic introgression into the wolves male lineage and have the noteworthy advantage of being suitable for analyses on low-quality DNA samples.     

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.     

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.     

On the path to extinction: Inbreeding and admixture in a declining grey wolf population

Allee effects reduce the viability of small populations in many different ways, which act synergistically to lead populations towards extinction vortexes. The Sierra Morena wolf population, isolated in the south of the Iberian Peninsula and composed of just one or few packs for decades, represents a good example of how diverse threats act additively in very small populations. We sequenced the genome of one of the last wolves identified (and road‐killed) in Sierra Morena and that of another wolf in the Iberian Wolf Captive Breeding Program and compared them with other wolf and dog genomes from around the world (including two previously published genome sequences from northern Iberian wolves). The results showed relatively low overall genetic diversity in Iberian wolves, but diverse population histories including past introgression of dog genes. The Sierra Morena wolf had an extraordinarily high level of inbreeding and long runs of homozygosity, resulting from the long isolation. In addition, about one‐third of the genome was of dog origin. Despite the introgression of dog genes, heterozygosity remained low because of continued inbreeding after several hybridization events. The results thus illustrate the case of a small and isolated wolf population where the low population density may have favoured hybridization and introgression of dog alleles, but continued inbreeding may have resulted in large chromosomal fragments of wolf origin completely disappearing from the population, and being replaced by chromosomal fragments of dog origin. The latest population surveys suggest that this population may have gone extinct.     

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.     

Variability of canine microsatellites within and between different dog breeds

Polymorphic animal microsatellites have proved valuable genetic markers. For this project, the variability of 19 canine microsatellite loci was examined within and between three pure breeds of dog: Greyhounds, Labradors, and German Shepherds. The number of alleles, absolute and relative frequencies, and the statistics that express polymorphism within a breed were determined. The evolutionary relationships among these closely related dog breeds were estimated by genetic distance measures developed for use with microsatellite loci. According to the pairwise genetic distances, Greyhounds and German Shepherds had longer diverse evolutionary histories than Greyhounds and Labradors or Labradors and German Shepherds. Although a few breed-specific alleles were observed, the significant differences between breeds are in their relative frequencies and distribution of the alleles across a locus. None of the three pure dog breeds corresponds to Hardy-Weinberg equilibrium. A considerable reduction in intrapopulation variation was observed within three pure breeds, compared with the population of individuals belonging to 15 dog breeds. This reduction was especially pronounced in the Greyhound breed, which expressed the lowest degree of variation. Intrapopulation variations of Labradors and German Shepherds did not differ significantly, that of Labradors being only slightly higher. The intra-species variation of dogs is lower than in humans, mouse, or rat, but similar to that in domestic animals, probably reflecting similarly high inbreeding coefficients. However, some highly informative loci were common to all dog breeds tested so far. Such population data are necessary for mapping studies and linkage analysis in dogs. Received: 31 July 1996 / Accepted: 21 October 1996     

Ancient wolf lineages in India

All previously obtained wolf (Canis lupus) and dog (Canis familiaris) mitochondrial (mt) DNA sequences fall within an intertwined and shallow clade (the 'wolf-dog' clade). We sequenced mtDNA of recent and historical samples from 45 wolves from throughout lowland peninsular India and 23 wolves from the Himalayas and Tibetan Plateau and compared these sequences with all available wolf and dog sequences. All 45 lowland Indian wolves have one of four closely related haplotypes that form a well-supported, divergent sister lineage to the wolf-dog clade. This unique lineage may have been independent for more than 400,000 years. Although seven Himalayan wolves from western and central Kashmir fall within the widespread wolf-dog clade, one from Ladakh in eastern Kashmir, nine from Himachal Pradesh, four from Nepal and two from Tibet form a very different basal clade. This lineage contains five related haplotypes that probably diverged from other canids more than 800,000 years ago, but we find no evidence of current barriers to admixture. Thus, the Indian subcontinent has three divergent, ancient and apparently parapatric mtDNA lineages within the morphologically delineated wolf. No haplotypes of either novel lineage are found within a sample of 37 Indian (or other) dogs. Thus, we find no evidence that these two taxa played a part in the domestication of canids.     

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.     

Y‐chromosome evidence supports asymmetric dog introgression into eastern coyotes

Hybridization has played an important role in the evolutionary history of Canis species in eastern North America. Genetic evidence of coyote–dog hybridization based on mitochondrial DNA (mtDNA) is lacking compared to that based on autosomal markers. This discordance suggests dog introgression into coyotes has potentially been male biased, but this hypothesis has not been formally tested. Therefore, we investigated biparentally, maternally, and paternally inherited genetic markers in a sample of coyotes and dogs from southeastern Ontario to assess potential asymmetric dog introgression into coyotes. Analysis of autosomal microsatellite genotypes revealed minimal historical and contemporary admixture between coyotes and dogs. We observed only mutually exclusive mtDNA haplotypes in coyotes and dogs, but we observed Y‐chromosome haplotypes (Y‐haplotypes) in both historical and contemporary coyotes that were also common in dogs. Species‐specific Zfy intron sequences of Y‐haplotypes shared between coyotes and dogs confirmed their homology and indicated a putative origin from dogs. We compared Y‐haplotypes observed in coyotes, wolves, and dogs profiled in multiple studies, and observed that the Y‐haplotypes shared between coyotes and dogs were either absent or rare in North American wolves, present in eastern coyotes, but absent in western coyotes. We suggest the eastern coyote has experienced asymmetric genetic introgression from dogs, resulting from predominantly historical hybridization with male dogs and subsequent backcrossing of hybrid offspring with coyotes. We discuss the temporal and spatial dynamics of coyote–dog hybridization and the conditions that may have facilitated the introgression of dog Y‐chromosomes into coyotes. Our findings clarify the evolutionary history of the eastern coyote.     

Assessing the prevalence of hybridization between sympatric Canis species surrounding the red wolf (Canis rufus) recovery area in North Carolina

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.     

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 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 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.     

DLA-DRB1, DQA1, and DQB1 alleles and haplotypes in North American Gray Wolves

The canine major histocompatibility complex contains highly polymorphic genes, many of which are critical in regulating immune response. Since domestic dogs evolved from Gray Wolves (Canis lupus), common DLA class II alleles should exist. Sequencing was used to characterize 175 Gray Wolves for DLA class II alleles, and data from 1856 dogs, covering 85 different breeds of mostly European origin, were available for comparison. Within wolves, 28 new alleles were identified, all occurring in at least 2 individuals. Three DLA-DRB1, 8 DLA-DQA1, and 6 DLA-DQB1 alleles also identified in dogs were present. Twenty-eight haplotypes were identified, of which 2 three-locus haplotypes, and many DLA-DQA1/DQB1 haplotypes, are also found in dogs. The wolves studied had relatively few dog DLA alleles and may therefore represent a remnant population descended from Asian wolves. The single European wolf included carried a haplotype found in both these North American wolves and in many dog breeds. Furthermore, one wolf DQB1 allele has been found in Shih Tzu, a breed of Asian origin. These data suggest that the wolf ancestors of Asian and European dogs may have had different gene pools, currently reflected in the DLA alleles present in dog breeds.     

The genetic structure of the Lithuanian wolf population

Lithuanian wolves form part of the larger Baltic population, the distribution of which is continuous across the region. In this paper, we evaluate the genetic diversity of the Lithuanian wolf population using mitochondrial DNA analysis and 29 autosomal microsatellite loci. Analysis of the mtDNA control region (647 bp) revealed 5 haplotypes distributed among 29 individuals and high haplotype diversity (0.658). Two haplotypes were distributed across the country, whilst the others were restricted to eastern Lithuania. Analysis of microsatellites revealed high heterozygosity (H_E=0.709) and no evidence for a recent bottleneck. Using detection of first generation migrants, four individuals appeared to assign better with populations genetically differentiated from those resident in Lithuania. These immigrants were males carrying rare mitochondrial haplotypes and were encountered in the eastern part of the country, this indicates that Lithuania is subject to immigration from differentiated populations. Additionally, we did not detect any signs of recent hybridisation with dogs.