Regional occurrence, high frequency but low diversity of mitochondrial DNA haplogroup d1 suggests a recent dog-wolf hybridization in Scandinavia

The domestic dog mitochondrial DNA (mtDNA)-gene pool consists of a homogenous mix of haplogroups shared among all populations worldwide, indicating that the dog originated at a single time and place. However, one small haplogroup, subclade d1, found among North Scandinavian/Finnish spitz breeds at frequencies above 30%, has a clearly separate origin. We studied the genetic and geographical diversity for this phylogenetic group to investigate where and when it originated and whether through independent domestication of wolf or dog-wolf crossbreeding. We analysed 582 bp of the mtDNA control region for 514 dogs of breeds earlier shown to harbour d1 and possibly related northern spitz breeds. Subclade d1 occurred almost exclusively among Swedish/Finnish Sami reindeer-herding spitzes and some Swedish/Norwegian hunting spitzes, at a frequency of mostly 60–100%. Genetic diversity was low, with only four haplotypes: a central, most frequent, one surrounded by two haplotypes differing by an indel and one differing by a substitution. The substitution was found in a single lineage, as a heteroplasmic mix with the central haplotype. The data indicate that subclade d1 originated in northern Scandinavia, at most 480–3000 years ago and through dog-wolf crossbreeding rather than a separate domestication event. The high frequency of d1 suggests that the dog-wolf hybrid phenotype had a selective advantage.

2 to Scandinavia. Furthermore, in order to detect potential additional diversity in the dog breeds under investigation, unregistered dogs were also collected and care was taken to obtain samples from unrelated dogs at least at the grandparent level.
Sampling included also several local breeds not recognized by all Kennel Clubs, dogs with no identity information other than the breed, and dogs of no particular breed belonging to indigenous Arctic peoples (Table 1; Table S2; Table S3). For calculating the "minimum number of female lineages" for a breed (Table 1, Table S2), individuals lacking lineage information, but having a haplotype differing from those of the identified lineages of the breed, were treated as an additional lineage in the analyses, but each haplotype counted as a single lineage even if found among several individuals. The samples were collected as buccal epithelial cells using FTAindicating cards according to the manufacturer's specifications (Whatman International, UK) or as EDTA-blood samples from which genomic DNA was extracted using a commercially available kit (Puregene, Gentra Systems, Minneapolis, MN).

D A sequence analysis
Amplification and DNA sequencing (the sequence determined in both forward and reverse direction for all nucleotide positions) of a 582 bp long fragment of the mtDNA control region was performed as earlier described (Angleby & Savolainen 2005). Sequencing of PCR products was performed with BigDye Terminator chemistry on ABI 377 and ABI 3700 instruments (Applied Biosystems). Sequences were aligned with BioEdit (Hall 1999) and checked by eye. Comparisons of sequences and identification of haplotypes were performed with DnaSP v5 (Librado & Rozas 2009).
(2009), where it was calculated from the average genetic distance between dog/wolf and coyote sequences in a phylogenetic tree (a maximum likelihood evaluated neighbor-joining tree), and the time since separation of dog/wolf and coyote according to the fossil record. The rate estimate has a broad range since there is no exact calibration point for the separation time of the dog/wolf and coyote lineages; the 3 split may have occurred 1.5-4.5 million years ago (Nowak 2003). The time since the introduction of subclade d1 into the dog population was calculated using the ρ statistic, the mean number of substitutions for a set of sequences to their common ancestral haplotype (Forster et al. 1996).

Haplotypes in subclade d1
Four haplotypes (D1-D4) belonging to subclade d1 were found in the study, all of which previously described (Savolainen et al. 2002). A fifth haplotype (D8) earlier reported for a single individual (Pang et al. 2009) was upon re-sequencing found to be a D3.

ew haplotypes identified in the study
Eight new haplotypes belonging to clades A, C, and E were found, one in Norrbottenspets and Finnish Spitz, one in Swedish Elkhound (white) and six in Arctic breeds. The sequences have been deposited at GenBank with accession numbers GQ896338 -GQ896345.

Heteroplasmy and dating of the origin of subclade d1
The date of origin from wolf for subclade d1 was calculated to 480-3,000 years ago based on the ρ statistic, the mean number of substitutions for a set of sequences to their common ancestral haplotype (Forster et al. 1996). This value was 1/63, because a single lineage out of 63 differed by a single substitution (defining the D4 haplotype) from the other 62 lineages, which were all identical ignoring indel variation.
However, the calculation of ρ was based on treating the single substitution as fixed, while it in reality occurred as a heteroplasmic mix with the wild-type. The estimated age of subclade d1 is therefore likely an overestimation. Furthermore, the heteroplasmic state of haplotype D4 (and also D3 which was heteroplasmic for the indel distinguishing it from D1) represents in itself an indication of a young age of the subclade, since it does not seem likely that the heteroplasmic state would have been transferred through the generations during thousands of years.
For a neutral mitochondrial heteroplasmic variant, the time to fixation has been estimated to approximately 200 generations in humans and chinook salmon (White et al. 2008). Thus, assuming a generation time of 3-5 years in dogs (Fuller et al. 2003) this would indicate an origin less than 1,000 years ago for the D3 and D4 mutations.