Mitochondrial DNA control region sequences were analyzed from 162 wolves at 27 localities worldwide and from 140 domestic dogs representing 67 breeds. Sequences from both dogs and wolves showed considerable diversity and supported the hypothesis that wolves were the ancestors of dogs. Most dog sequences belonged to a divergent monophyletic clade sharing no sequences with wolves. The sequence divergence within this clade suggested that dogs originated more than 100,000 years before the present. Associations of dog haplotypes with other wolf lineages indicated episodes of admixture between wolves and dogs. Repeated genetic exchange between dog and wolf populations may have been an important source of variation for artificial selection.

The archaeological record cannot resolve whether domestic dogs originated from a single wolf population or arose from multiple populations at different times (1, 2). However, circumstantial evidence suggests that dogs may have diverse origins (3). During most of the late Pleistocene, humans and wolves coexisted over a wide geographic area (1), providing ample opportunity for independent domestication events and continued genetic exchange between wolves and dogs. The extreme phenotypic diversity of dogs, even during the early stages of domestication (1, 3, 4), also suggests a varied genetic heritage. Consequently, the genetic diversity of dogs may have been enriched by multiple founding events, possibly followed by occasional interbreeding with wild wolf populations.

We sequenced portions of the mitochondrial DNA of wolves and domestic dogs. Initially, 261 base pairs (bp) of the left domain of the mitochondrial control region (5) were sequenced from 140 dogs representing 67 breeds and five cross-breeds and 162 wolves representing 27 populations from throughout Europe, Asia, and North America (Fig. 1) (6). Because all wild species of the genus Canis can interbreed (7) and thus are potential ancestors of the domestic dog, five coyotes (Canis latrans) and two golden, two black-backed, and eight Simien jackals (C. aureus, C. mesomelas, and C. simensis, respectively) were also sequenced.

Fig. 1. Substitutions and deletions ( -- ) observed in 261 bp of control region sequence from wolves (W) and dogs (D). The dog sequence D13 had the same sequence as D4 except for an insertion of a 67-bp tandem repeat. The numerals I, II, III, and IV indicate assignments to the four clades of dog sequences. Wolf localities: Bulgaria (n = 1, W7); Croatia (n = 5, W2); Estonia (n = 1, W10); France (n = 2, W4); Finland (n = 2, W10); Greece (n = 7; W2, W5, W8, and W9); Italy (n = 12, W4); Poland (n = 1, W3); Portugal (n = 19; W1 and W2); Romania (n = 4; W5 and W6); Russia (n = 3; W6, W10, and W26); Spain (n = 46; W1 and W3); Sweden (n = 2; W2 and W10); Afghanistan (n = 3, W18); China (n = 3; W14, W19, and W27); India (n = 1, W12); Iran (n = 6; W16 and W17); Israel (n = 16, W11); Saudi Arabia (n = 7; W7, W12, W13, W14, and W15]; Turkey (n = 2, W2); Alaska (n = 3, W20); Alberta (n = 1, W22); Labrador (n = 3, W22); Mexico (n = 5, W25); Montana (n = 1, W22); Northwest Territories (n = 3, W22); and Yukon (n = 3; W21, W23, and W24). Dog breeds: basenji (n = 1, D2); basset (n = 1, D6); boxer (n = 1, D4); Norwegian buhund (n = 1, D1); bulldog (n = 1, D6); Chinese crested (n = 2; D2 and D25); chow chow (n = 3; D1, 02, and D3); collie (n = 1, D1); border collie (n = 3; D1 and D5); wirehaired dachshund (n = 3; D5 and D10); Australian dingo (n = 4, D18); grey Norwegian elkhound (n = 9; D3 and D8); Eskimo dog (n = 1, D23); German shepherd (n = 8; D4, D5, D6, D7, and D19); greyhound (n = 1, D9); groenendael (n = 1, D6); Mexican hairless (n = 6; D3, D6, D21, and D26); hamilton-stovare (n = 1, D5); Afghanistan hound (n = 3, D6); Alaskan husky (n = 2; D4 and D7); Siberian husky (n = 3; D3, D7, and D18); jamthund (n = 3; D7 and D8); keeshond (n = 1, D5); kuvasz (n = 1, D4); Leonberger (n = 2; Dl and D4); Norwegian lundehund (n = 1, D16); Mareema (n = 1, D=6); Pyrenean mastiff (n = 1, D11); Newfoundland (n = 1, D4); otter hound (n = 1, D6); papillon (n = 2; D3 and 04); poodle (n = 1, D3); toy poodle (n = 1, D6); pug (n = 1, D26); Chesapeake Bay retriever (n = 1, D13); flat-coated retriever (n = 3; D4 and D10); golden retriever (n = 6; D4, D6, D15, and D24); labrador retriever (n = 6; D4 and D12); Rhodesian ridgeback (n = 1, D26); rottweiler (n = 2, D3); Samoyede (n = 3; D1, D4, and D5); St. Bernard (n = 1, D9); schipperke (n = 1, D4); giant schnauzer (n = 3; D4 and D7); miniature schnauzer (n = 1, D9); English setter (n = 4; D3 and D5); Irish setter (n = 3; D1 and D9); New Guinea singing dog (n = 2, D18); shar (n = 1, D26); Icelandic sheepdog (n = 1, D3); Old English sheepdog (n = 1, D5); shiba inu (n = 1, D20); Cavalier King Charles spaniel (n = 1, D17); Irish water spaniel (n = 1, D6); springer spaniel (n = 1, D3); Tibetan spaniel (n = 1, D6); spitz (n = 1, D22); Japanese spitz (n = 1, D3); airedale terrier (n = 1, D7); border terrier (n = 2, D3); fox terrier (n = 2; D3 and D14); Norfolk terrier (n = 2, D4); West Highland terrier (n = 2, D7); Tibetan terner (n = 2; D2 and  D9); wachtelhund (n = 1, 05); whippet (n = 1, D3); Irish wolfhound (n = 2, D11); and crossbreeds (n = 5; D1, D3, D4, D5, and D18).

The control region of wolves and dogs was highly polymorphic (Fig. 1). We found 27 wolf haplotypes that differed on average by 5.31 +/- 0.11 (+/- SE) substitutions (2.10 +/- 0.04%), with a maximum of 10 substitutions (3.95%). The distribution of wolf haplotypes demonstrated geographic specificity, with most localities containing haplotypes unique to a particular region (Fig. 1). Four haplotypes (W2, W7, W14, and W22) had a widespread distribution. In dogs, 26 haplotypes were found. Only haplotype D6 also occurred in some gray wolves from western Russia and Romania (W6). Sequence divergence among dogs was similar to that found among wolves. Dog haplotypes differed by an average of 5.30 +/- 0.17 substitutions

Several methods of phylogenetic analysis, including maximum likelihood (9), maximum parsimony (10), minimum spanning networks (1 I), and statistical parsimony (12), were used to investigate relationships among sequences. All analyses supported a grouping of dog haplotypes into four distinct clades, although the topology within and among clades differed among trees (13). As exemplified by the neighbor-joining analysis (Fig. 2A), three of the four monophyletic clades defined a larger clade containing all but three dog haplotypes and a subset of wolf haplotypes (W4 and W5). Clade I included 19 of the 26 dog haplotypes. This group contained representatives of many common breeds as well as ancient breeds such as the dingo, New Guinea singing Jog, African basenji, and greyhound (14). Clade II included dog haplotype D8, from two Scandinavian breeds (elkhound and jamthund), and was closely related to two wolf haplotypes found in Italy, France, Romania, and Greece (W4 and W5). Clade III contained three dog haplotypes (D7, D19, and D21) found in a variety of breeds such as the German shepherd, Siberian husky, and Mexican hairless. Finally, clade IV contained three haplotypes (D6, D10, and D24) that were identical or very similar to a wolf haplotype (W6) found in Romania and western Russia, which suggests recent hybridization between dogs and wolves. Many breeds contained representatives of more than one dog haplotype grouping (Fig. 1).

Because the overall bootstrap support for many of the internodes in Fig. 2A was low, 1030 bp of the control region were sequenced for 24 canids, including representatives of the four dog clades (15). Although the association of clades was different, the analyses of the longer sequences provided stronger support for the tour monophyletic groupings of dog haplotypes (Fig. 2B) (13). A Wilcoxon signed-rank test was used to assess the monophyly of dog clades (16). Monophyly of all dog haplotypes can be rejected, and monophyly of clades I, II, and III is marginally rejected (P = 0.0004 and P = 0.053, respectively). In both trees, dog haplotype clakes II and IV are most closely related to wolf sequences from eastern Europe (Greece, Italy, Romania, and western Russia).

The coyote and wolf have a sequence divergence of 0.075 +/- 0.002 (17) and diverged about one million years ago, as estimated from the fossil record (18). Consequently, because the sequence divergence between the most different genotypes in clade I (the most diverse group of dog sequences) is no more than 0.010, this implies that dogs could have originated as much as 135,000 years ago (19). Although such estimates may be inflated by unobserved multiple substitutions at hypervariable sites (20), the sequence divergence within clade I clearly implies an origin more ancient than the 14,000 years before the present suggested by the archaeological record (21). Nevertheless, bones of wolves have been found in association with those of hominids from as early as the middle Pleistocene, up to 400,000 years ago (1, 22). The  ancient dates for domestication based on the control region sequences cannot be explained by the retention of ancestral wolf lineages, because clade I is exclusively monophyletic with respect to dog sequences and thus the separation between dogs and wolves has been long enough for coalescence to have occurred. To explain the discrepancy in dates, we hypothesize that early domestic dogs may not have been morphologically distinct from their wild relatives. Conceivably, the change around 10,000 to 15,000 years ago from nomadic hunter-gatherer societies to more sedentary agricultural population centers may have imposed new selective regimes on dogs that resulted in marked phenotypic divergence from wild wolves (23).

Although individual breeds show uniformity with respect to behavior and morphology, most breeds show evidence of a genetically diverse heritage because they contain different haplotypes. Moreover, dog sequences cluster with different groups of wolf haplotypes. Therefore, after the origin of dogs from a wolf ancestor, dogs and wolves may have continued to exchange genes. Backcrossing events could have provided part of the raw material for artificial selection and for the extraordinary degree of phenotypic diversity in the domestic dog. Domestic species of plants and animals whose wild progenitors are extinct cannot be enriched through periodic interbreeding, and change under artificial selection may be more limited. Consequently, the preservation of wild progenitors may be a critical issue in the continued evolution of domestic plants and animals.

REFERENCES AND NOTES

1. J. Glutton-Brock, in The Domestic Dog, Its Evolution. Behaviour and Interactions with People, J. Serpell, Ed. (Cambridge Univ. Press, Cambridge, 1995), pp. 7-20.

2. S. J. Olsen and J. W. Olsen, Science 197, 533 (1977).

3. J. Glutton-Brock, A Natural History of Domesticated Mammals (Cambridge Univ. Press. British Museum of Natural History, Cambridge. 1987).

4. F. E. Zeuner, A History of Domesticated Animals (Harper & Row, New York. 1963); H. Epstein, The
Origin of the Domestic Animals of Africa (Africana Publishing, New York, 1971); I. L. Brisbin Jr., Am.
0 Kennel Gaz. January 1976, p. 23.

5 C. Saccone, M. Attimonelli, E. Sbisa, J. Mal. Evol. 26, 205 (1987).

6. DNA from blood, tissue, or hair was isolated by cell lysis followed by organic solvent purification. A
261-bp control region fragment was sequenced from a slightly larger (394 bp) fragment amplified with primers L15910 and H16498 [R. DeSalle, A. K. Williams, M. George, Methods Enzymol. 224, 176
(1993)). See polymerase chain reaction and sequencing conditions and protocols in J. E. Maldonado, F. O. Davila, B. S. Stewart, E. Geffen, R. K. Wayne. Mar. Mamm. Sci. 11, 46 (1995).

7. A. P. Gray, Mammalian Hybrids. a Check-list with Bibliography (Farnham Royal, Bucks. Common-
wealth Agricultural Bureaux, UK, 1954).

8. D. Gottelli et al., Mol. Ecol. 3, 301 (1994); J. Garcia-Moreno, M. D. Matocq, M. S. Roy, E. Geffen, R. K. Wayne, Conserv. Biol. 10, 376 (1996); P.W. Hedrick, P. S. Miller, E. Geffen, R. K. Wayne, Zoo Bio/. 16, 47 (1997).

9. K. Strimmer and A. von Haessler, Mol. Biol. Evo/. 13, 964 (1996).

10. D. L. Swofford, PAUP: Phylogenetic Analysis Using Parsimony, Version 3.0 (Illinois Natural History Survey, Champaign, IL. 1990).

11. L. Excoffier, P. E. Smouse, J. M. Quattro, Genetics 131, 479 (1992).

12. A. R. Templeton, K. A. Crandall, C. F. Sing, ibid. 132, 619(1992).

13. The coyote was used as an outgroup on the basis of morphologic and mitochondrial DNA sequence analyses (R. M. Nowak, North American Quaternary Canis (Monograph of the Museum of Natural History, University of Kansas, N 6, Lawrence, KS, 1979); R. K. Wayne. Trends Genet. 6, 218 (1993); C. Vila et al., unpublished data). Trees retaining the four indicated clades resulted from neighbor-joining searches with Tamura-Nei, Kimura 2-parameter, Kimura 3-parameter, and HKY85 models of evolution; parsimony searches with equal, 2:1, and 10:1 weights of transversions to transitions; and maximum likelihood analyses with the HKY85 model of evolution, taking into account differences in nucleotide frequencies and transition and transversion biases.

14. R. Coppinger and R. Schneider, in The Domestic Dog, Its Evolution, Behaviour and Interactions with People, J. Serpell, Ed. (Cambridge Univ. Press, Cambridge, 1995), pp. 21-47.

15. We amplified the entire canid control region with primers in (6) and the following primers: L16462
(5'-CATACTAACGTGGGGGTTAC-3'), H222 (5'-A-AACTATATGTCCTGAAACC-3'), L296 (5'-ATA-CAAACCCCCCTTACC-3'), and H652 (5'-AAG-GCTAGGACCAAACCT-3'). The primer numbering refers to their approximate location in the human mitochondrial genome [S. Anderson et al., Nature 290, 457 (1981)J. The 1030-bp sequence that we report includes the entire control region except for a hypervariable region close to the 3' end that encompasses an imperfect 20-bp repeat having a variable number of units [A. R. Hoelzel, J. V. Lopez, G. A. Dover, S. J. O'Brien, J. Mol. Evo/. 39, 191 (1994)}.

16. A. R. Templeton, Evolution 37, 221 (1983).

17. Sequence divergence values and trees were based on a gamma correction [K. Tamura and M. Nei, Mol. Biol. Evo/. 10, 513 (1993)) with the empirically determined parameter a = 0.18 for the 261-bp sequences and a = 0.22 for the 1030-bp sequences [S. Kumar, K. Tamura, M. Nei, MEGA: Molecular Evolutionary Genetic Analysis, Version 1.01 (Pennsylvania State University. University Park, PA, 1993)).

18. R. K. Wayne, B. Van Valkenburgh, S. J. O'Brien, Mol. Biol. Evo/. 8, 297 (1991).

19. A relative rate test indicated that substitution rates were similar for all wolf and dog sequences [F. Tajima, Genetics 135. 599 (1993)).

20. T. J, Parsons et al., Nature Genet. 15. 363 (1997).

21. G. Nobis, Umshau 19, 610 (1979); S. J. Qlsen, Origins of the Domestic Dog (University of Arizona
Press. Tucson, AZ. ] 985).

22. M. E. Thurston, The Lost History of the Canine Race. Our 15,00O-Year Love Affair with Dogs (Andrews & McMeel, Kansas City, KS, 1996).

23. D. F. Morey, Am. Sci. 82, 336 (1994).

24. We thank P. Taberlet for providing wolf sequences from France and Poland and J. Castroviejo, Grupo Lobo-Portugal, E. Geffen, and many others for their help in obtaining samples. C.V. was supported by a
postdoctoral fellowship from the Spanish Ministerio de Educacion y Ciencia, and I.R.A, was supported
by a fellowship from Junta Nacional de Investigasao Cientifica e Tecnologica, Portugal. This research was
supported in part by NSF grants to R.L.H. (DEB-9208022) and R.K.W. (BSR-9020282) and by a
Sloan Young Investigator Award to K.A.C.
 

P. Savolainen and J. Lundeberg, Department of Bio-chemistry, Royal Institute of Technology, S 100 44 Stockholm, Sweden.

J. E. Rice and R. L. HoneyCutt, Faculty at Genetics and Department of Wildlife and Fisheries Sciences, Texas A&M University. College Station, TX 77843, USA.

K A. Crandall, Department of Zoology and M. L. Bean Museum, Brigham Young University, Provo. UT 84602, USA.

To whom correspondence should be addressed E-mail: rwayne@ucla.edu

SCIENCE,  VOL. 276  13 JUNE 1997

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