Genetic Variability of the Grey Wolf Canis lupus in the Caucasus in Comparison with Europe and the Middle East: Distinct or Intermediary Population?

Despite continuous historical distribution of the grey wolf (Canis lupus) throughout Eurasia, the species displays considerable morphological differentiation that resulted in delimitation of a number of subspecies. However, these morphological discontinuities are not always consistent with patterns of genetic differentiation. Here we assess genetic distinctiveness of grey wolves from the Caucasus (a region at the border between Europe and West Asia) that have been classified as a distinct subspecies C. l. cubanensis. We analysed their genetic variability based on mtDNA control region, microsatellite loci and genome-wide SNP genotypes (obtained for a subset of the samples), and found similar or higher levels of genetic diversity at all these types of loci as compared with other Eurasian populations. Although we found no evidence for a recent genetic bottleneck, genome-wide linkage disequilibrium patterns suggest a long-term demographic decline in the Caucasian population – a trend consistent with other Eurasian populations. Caucasian wolves share mtDNA haplotypes with both Eastern European and West Asian wolves, suggesting past or ongoing gene flow. Microsatellite data also suggest gene flow between the Caucasus and Eastern Europe. We found evidence for moderate admixture between the Caucasian wolves and domestic dogs, at a level comparable with other Eurasian populations. Taken together, our results show that Caucasian wolves are not genetically isolated from other Eurasian populations, share with them the same demographic trends, and are affected by similar conservation problems.

and Hadrut. Martakert region has an average elevation of 1,200-1,450 m a.s.l. and its natural vegetation varies from deciduous, coniferous and mixed forest to subalpine and alpine vegetation. This region is characterised with abundance of ungulates and carnivorous mammals, including wolves, jackals, bears, and lynx. Kashatakh region includes mountainous areas covered with forest, high mountain peaks of 3,000-3,500 m a.s.l., covered by snow, and semidesert and desert zones with average heights 550-660 m a.s.l. Hadrut region includes both mountainous and lowland areas, with average height in different parts varying from 620 to 1,050 m a.s.l. The north-east part of the region is mountainous, with vegetation zones from the forest to alpine vegetation, and highest peaks reaching 2,300-2,500 m a.s.l. In the southern part there are lowlands of the river Araks, covered by meadows and pastures, which attract wild ungulates and carnivores. It is the area with highest concentration of livestock farms in Nagorno-Karabakh. However, even in the lowland areas human density is relatively low, and there are large areas uninhabited by humans.

Null allele detection
To test for the presence of null alleles at the microsatelite loci analysed in this study, Therefore, we concluded that the observed pattern does not justify the exclusion of any locus from the data analysis due to the presence of putative null alleles.

A comment on the relationship between a population bottleneck and inbreeding
Populations that went through a bottleneck and then increased in size do not have to show signs of a recent inbreeding, i.e. there may be no cases of breeding between close kin, even though average relatedness in a population is high. This is the case of Italian wolves: the population went through a strong, long term bottleneck, but the genetic patterns suggest that now breeding between kin is rare there: the Italian population has a very extensive linkage disequilibrium, but low proportion of long

Differentiating between mtDNA haplotypes of grey wolves and domestic dogs (a comment to Table 2)
The issue of differentiating between wolf and dog haplotypes is problematic for the following reasons: -The split between the two species (or subspecies according to the present taxonomic classification) was very recent in the evolutionary timescale, and therefore they may still share a number of common ancestral haplotypes.
-It is likely that after the initial domestication event some level of gene flow was maintained between the two (sub)species, so mtDNA haplotypes could have been exchanged.
-Consistent with the two earlier points, phylogenetic studies on wolf and dog mtDNA haplotypes (e.g. Vilá et al. 1997, Savolainen et al. 2002, Verginelli et al. 2009 show that these two (sub)species are not reciprocally monophyletic in mtDNA, the dog clades include wolf haplotypes, and some haplotypes are shared between dogs and wolves.
-There are accounts from various parts of the word (including the Caucasus) of deliberate crossing of dogs with wolves by humans to "improve the breed" (Kopaliani et al. in press).
-Contemporary hybridisation has been documented in different regions of Eurasia (including the Caucasus), with backcrossing into both wolf and dog populations (see references in the main text). Therefore, mtDNA haplotypes could have been recently exchanged.
For these reasons, distinguishing between wolf and dog haplotypes is problematic.
However, in the case of the haplotypes found in the Caucasus, we identified only one GenBank match with the domestic dog haplotypes, so the remaining haplotypes may be assumed to derive from wolves.   Figure S4. Population structure in the southern Caucasus inferred from mtDNA haplotype distribution using the spatially explicit model implemented in GENELAND. Two genetic clusters were detected, and the figure represents spatial distribution of posterior probability for individuals to belong to Cluster 1.