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.

Copyright:  Hindrikson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Funding: This work was supported by grants from the Environmental Investment Centre, the Estonian Ministry of Education and Sciences (target financing project SF0180122s08), the Estonian Science Foundation (grant 7040) and from the European Union through the European Regional Development Fund (Centre of Excellence FIBIR), and from the Estonian Doctoral School of Ecology and Environmental Sciences. These funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. AK is employed by Wildlife Park Kadzidlowo and, therefore, Wildlife Park Kadzidlowo played a role in the data collection and analysis of the manuscript.

The steady growth of human populations worldwide has resulted in the expansion of human-occupied territory, a decrease in suitable habitats for wildlife, and closer proximity between humans and wild animals. These trends, coupled with the increasing number of domesticated animals accompanying humans, mean that the potential for hybridization between wild and closely related domestic animals is increasing, which is especially relevant for domesticated dogs and wild canids.

In this study we used combined genetic analysis of mtDNA control region (1134 bp), 11 autosomal and 7 Y chromosome microsatellite loci to investigate putative hybrids, wolves and dogs in Estonia and Latvia. With these data, we aimed to document the occurrence of wolf-dog hybrids and the role of females and males of both species in the hybridization process.

The hybrids were identified on the basis of external characteristics. The six animals from Estonia exhibited coat coloration that was atypical of wolves: four of them were unusually dark, and two were yellow. All individuals were juveniles, i.e. less than one year old (based on teeth and skull development) and were shot from the same large wolf pack. From this pack, two adult male wolves were also shot (these individuals clustered genetically with wolves; see Fig. 2) and one possibly subadult wolf was sighted, but escaped during the hunt. The pack territory was situated in an area of low wolf density in Western-Estonia. The two putative hybrids shot in Latvia were also juveniles from a single wolf pack and exhibited unusual yellow coats with curly fur. During the same hunt, two stray dogs were also shot nearby, though it is not clear whether they belonged to the same pack as the hybrids (these samples were not available for current study). The location of the Latvian hybrids was not far from the area used by another litter of hybrid pups in 1999 [16]. Wolf samples (animals displaying wolf-like morphology according to information from hunters) were collected at localities throughout Estonia and Latvia, including both areas where the hybrids were hunted (Fig. 1). The dog samples used included the following breeds: pedigree German Shepherd Dogs, Greyhounds, English Springer Spaniels, West Siberian Laikas and Siberian Huskies.

The presence of null alleles was analyzed with Micro-Checker [34]. Eighteen randomly chosen samples (15.5% of all samples) were genotyped a second time using the autosomal microsatellite loci, while eight randomly chosen male samples (17.8% of all analysed males) were genotyped a second time using the Y chromosome microsatellite loci. The results were analysed using the software Gimlet [35] to estimate the rate of different types of errors: allelic dropouts, false alleles, double errors and complete errors, as defined in Valiere [35].

A minimum spanning network was calculated with Network 4.510 using a median-joining approach [55]. The network was based on partial mtDNA control region (the final length after alignment and trimming was 1134 bp) and included wolves, dogs and hybrids from this study (from Estonia and Latvia; Table S3).

Allele frequencies at 11 autosomal microsatellite loci recorded for all 103 individuals demonstrated that eight wolf-dog hybrids carried several alleles that were also found in wolves but were absent in dogs used in this study; for example, allele 133 at locus FH2001 and allele 222 at locus FH2010. Conversely, two alleles (167 at locus FH2054 and 105 at locus M-CPH2) were present in hybrid and dog samples but absent in Estonian and Latvian wolves (see these and other cases in Fig. S1).

In the FCA analysis, wolves, dogs and eight Estonian and Latvian hybrids could be clearly distinguished from each other based on the distributions of allele frequencies at 11 microsatellite loci (Fig. 2). The wolf-dog hybrids from Estonia and Latvia were assigned into two clusters according to their geographic location, and all eight hybrids were placed between dogs and wolves.

According to the kinship analysis, some of the Estonian hybrids were full siblings, while others were more distantly related (Table S2). Therefore, it is possible that Estonian hybrids were not descendants of same parents. The Latvian hybrids were full siblings.

The larger phylogeny (Fig. 6) which included homologous wolf and dog sequences from GenBank (Table S3) revealed two large clades: one specific to dogs and another that included both wolves and dogs. Both hybrids from Latvia were positioned in the dog-specific clade, while the six hybrids from Estonia were positioned in the wolf-dog clade.

In the whole sample set only two hybrids were males (both from Estonia). Network analysis demonstrated that they were more closely related to dog than wolf haplotypes, suggesting that their paternal lineage was most likely of dog origin (Fig. 7).

While this is the first time that hybridization between gray wolves and dogs has been confirmed in Estonia, it is the second time in Latvia: wolf-dog hybridization was previously reported in Latvia in 1999 and subsequently verified using genetic analysis [16]. The appearance of wolf-dog hybrids in both countries can most likely be explained by the combined effect of two factors: severe and continuous hunting pressure on wolf populations, together with the abundance of stray dogs.

Intensive hunting may have the dual effects of reducing wolf population density and destroying the structure of wolf packs [64], [65]. Some wolves may encounter difficulty of finding a conspecific mate due to the low wolf population density - the Allee effect [66]. If one animal from the alpha pair is removed before mating occurs (the peak of wolf hunting coincides with the wolf mating season in both countries), the remaining animal has to seek another partner in order to reproduce. Where wolf density is low and stray dogs are present, the probability that such a scenario results in hybridization with stray dogs may be increased.

Despite high hunting pressure, the presence of stray dogs and hybridization, this study shows that wolf population has largely remained genetically distinct from dogs in both countries, suggesting that introgressive hybridization in nature might be counteracted by selection or behavioural factors.

The Structure analysis indicated that ten wolves (three from Estonia and seven from Latvia) exhibited lower membership coefficients of belonging to the wolf cluster than other wolves in this study. Additional attempts to assign these ten animals with Structure and Newhybrids gave inconclusive results, not allowing to make a firm verdict about their filial status and consequently of possible introgression (data not shown). Therefore, analysis with higher number of loci and animals is needed in future studies to investigate introgression.

While autosomal microsatellite data allowed the existence of wolf-dog hybrids in Estonia and Latvia to be established, gender specific genetic markers were used to evaluate the direction of hybridization. Based on mitochondrial control region data representing the maternal lineage, all six hybrids from Estonia were placed into the wolf haplogroup, carrying sequences identical to a major wolf haplotype (Fig. 5). This suggests that for hybrids collected in Estonia, hybridization took place according to the common pattern, i.e. between female wolf and male dog. On the other hand, mtDNA haplotypes found in two Latvian hybrids grouped with dogs, representing an extremely rare case of hybridization between a female dog and a male wolf. This result provides a rare example that violates the general rule of sexual asymmetry in mating between wolves and dogs and it is the first confirmed case from Europe to demonstrate that hybridization has occurred between female dog and male wolf. Although it has been shown that dog haplotypes occasionally fall into the same haplogroup as wolves [e.g. 13], [15], the power to distinguish wolf and dog haplotypes in these cases has been limited by the very short length of mtDNA control region sequences used. Here our results, based on longer mtDNA control region sequences (1134 bp), demonstrate that wolves and dogs form reciprocally monophyletic haplogroups at a local geographic scale, i.e. when only wolves and dogs from Latvia and Estonia were represented, and that mtDNA haplotypes of Latvian hybrids clearly group together with dogs (Fig. 5), suggesting that their mother was most likely a dog. On the other hand, when homologous wolf and dog sequences from various other regions were added from GenBank, two clades appeared, one specific to dogs, but in the other both wolves and dogs were present (Fig. 6; note that this is not a definitive phylogenetic network for wolves and dogs, but represents only animals for which homologous sequence of 1134 bp from the mtDNA control region was available in GenBank). In this phylogeny, hybrids from Latvia were placed into the dog-specific clade and hybrids from Estonia into a well-supported subclade of wolves. Thus, despite the lack of reciprocal monophyly, this result also suggests that the mother of Latvian hybrids was a dog, whereas for Estonian hybrids the mother was wolf. e1438dca32