INTRODUCTION

The dog, Canis familiaris, is the only member of the family Canidae, and is the "oldest" animal that can be fully domesticated in the world, since the historical evidence showing its strong connection with humans can be traced back to the far preagricultural age (Turnbell and Reed, 1974). During the history of dogs' domestication, more than 400 breeds with high morphological variations have been established by crosses between or within their ancestral stacks and also by artificial selection. Despite such a large number of extant breeds, only a few reports have been published about the genetic backgrounds or genetic variations among the breeds. For example, Tanabe et al. (1991) analyzed 25 blood protein loci, using approximately 3,000 individual dogs of 40 breeds, including Japanese, Asian, and European dog breeds, and showed that the dogs maintained low genetic variations among the populations. Those authors also suggested that most modern Japanese dogs have been affected by two major genetic components which were brought to Japan from Southeast Asia about 10,000 years ago and from the northwest through the Korean peninsula about 1,700 -- 2,300 years ago (Tanabe, 1990, 1991; Tanabe et al., 1991; Fujise et al, 1997). However, the low heterozygosity in the polymorphic biochemical loci did not allow further detailed investigations.

Although a high degree of morphological variation is present among the 400-plus breeds of dogs, the most widespread and accepted view, based mainly on morphological studies, places only one species, the wolf (Canis lupus), as the wild progenitor of domestic dogs (Lorenz, 1975; Zimen, 1981) However, the existence of more than 5 subspecies of wolves and the absence of detailed information on the genetic diversity among these subspecies underlie a fundamental question: From which of the wolf subspecies are domesticated dogs descended?

In the past two decades, polymorphisms of mitochondrial DNA (mtDNA) have been successfully applied in examination of the genetic relationships among populations of the same animal species and between closely related species (Avise, 1991; Wayne, 1993), since mtDNA evolves 5 – 10 times as much as do single-copy nuclear genes (Brown et al., 1979).The strictness of the maternal inheritance of mtDNA (Kaneda et al., 1995) and the lack of genetic re-combination in mtDNA also make mtDNA polymorphisms, in particular nucleotide substitutions, a useful tool for disclosing the evolutionary events in matriarchal Lineages of animal species. Since domesticated animals have generally been maintained through matriarchal lineages with the introduction of patarnal genes which are useful for the improvement of a breed, the mtDNA polymorphisms can be used to determine which wild species was the matriarchal ancestor of a domesticated animal of interest.

Using this molecular genetical analysis, Wayne et al. (1991, 1997) reported that the domestic dog is an extremely close relative of the gray wolf, differing from it by at most 0.2% of the DNA sequence. Okumura et al. (1996) made an extensive survey of the mtDNA D-loop region from 73 individual dogs of 24 breeds and found extensive polymorphisms in the mtDNA D-loop region even within breeds. Those authors also mentioned that they found no distinct correlations between the European and Japanese breeds of dogs, demonstrating that different aspects of the genetic relationships between dog breeds compared with allozyme analysis. However, they did not investigate the relationship between these dog breeds and wolves as their putative ancestor.

To address this issue, we performed a sequence comparison of the mtDNA D-loop region among 24 breeds of domestic dogs (34 individual dogs) and three subspecies of wolf (C. l. lupus. C. l. pallipes, and C. l. chanco), and found that the dogs were not genetically differentiated from the wolves. This result is the first evidence suggesting the dog has multiple domestication centers from wild wolves, and that extensive interbreedings occurred among the multiple matriarchal origins.

MATERIALS AND METHODS

DNA samples. Whole-blood samples were collected from 24 breeds of domestic dog (33 individuals) and 3 subspecies (19 individuals) of wolf European wolves (C. l. lupus) and Indiana wolves (C. l. pellipes) were captured in Yugoslavia and Afghanistan, respectively. All wild individuals of both subspecies were temporarily bred at Kiel University. Wild individual Chinese wolves (C. l. chanco) were captured near Ulan Bator in Mongolia. DNA samples from four foxes and a raccoon dog were also used as outgroups of the dogs when phylogenetic trees were constructed. All DNA samples used in this study are listed in Table l.

DNA sequencing and sequence alignment. Before the analysis of the DNA samples mentioned above, we determined the complete sequence of the D-loop region of mtDNA, which was purified from the livers of two mongrel dogs by the method described by Yonekawa et al. (1981). Genomic DNAs were then extracted from the blood samples of all other dogs as described by Maniatis et at. (1982). Based on the sequence, four primers for direct sequencing were synthesized. The names and sequences of the primers were: 5'-tgtaaaacgacggccagtgctcttgctccaccatca-3' (DL14: a region within the tRNA{Pro}), 5'-caggaaacagctatggccccccttgatttttatgcg-3' (DH6: a region within the D-loop), 5'-tgtaaaacgacggccagtatcactcatctacgaccg-3' (DL10; a region within the D-loop) and 5'-caggaaacagctatggcccgtgcgactcatcttggc-3' (DH7; a region within the tRNA{Phe}).
 

 
Table I. List of source of DNA samples used in this study
 

Breed or population	Locality	No. of samples
Hokkaido dog	Japan	2
Akita dog	Japan	2
Kai dog	Japan	2
Kishu dog	Japan	2
Shiba dog (Shinshu)	Japan	2
Shiba dog (Mino)	Japan	2
Mikawa dog	Japan	2
Shikoku dog	Japan	2
Ryukyu dog	Japan	2
Korean Chejudo	Korea	2
Mongolian native dog	Mongolia	2
Indonesian native dog	Kalimantan	2
German Shephard	bred in Japan	2
Shetland Sheepdog	bred in Japan	1
Pointer	bred in Japan	1
Beagle	bred in Japan	1
Dachshund	bred in Japan	1
Doberman Pinscher	bred in Japan	1
English Setter	bred in Japan	1
Maltese	bred in Japan	1
Siberian Husky	bred in Japan	1
European Wolf (C. l. lupus)	* Yugoslavia	7
Indian Wolf (C. l. pallipes)	* Afghanistan	5
Chinese Wolf (C. l. chanco)	* Mongol	7
Red Fox (V. u. japonica)	Japan	2
Red Fox (V. u. vulpes)	Russia	2
Raccoon dog (N. p. viverrinis)	Japan	1
Total		58

* none of the individual wolves had been bred.
 

The sequence of the mtDNA D-loop regions were determined by direct sequencing with semi-nested polymerace chain reaction (PCR) (Kikkawa et al., 1997) using the four primers, followed by an automated method. We then aligned the nucleotide sequences of the D-loop region among the dogs, wolves, foxes, and raccoon dog.

Phylogenetic Analysis. The sequence data were analyzed using distances corrected for multiple hits by the two-parameter method of Kimura (1980) with the DNADIST of PHYLIP program (Ver. 3.5c; Felsenstein, 1993). Phylogenetic trees were constructed by the neighbor-joining (NJ) method (Saitou and Nei, 1987) and by the UPGMA (Sneath and Sokal, 1973)incorporated into the NEIGHBOR program. The sequences of the foxes and raccoon dog were used for outgroups to construct phylogenetic trees. The phylogenetic analysis of character-state matrices using the parsimony method was performed with the DNAPARS, with a majority-rule consensus tree produced by the CONSENSE; a majority-rule consensus tree based on 100 or 1,000 boot-strap replicates was produced using the SEQBOOT.

RESULTS

Nucleotide substitutions and length variations in the D-loop region. We determined the complete sequences of the mtDNA D-loop region for domestic dogs, wolves, foxes and a raccoon dog. By aligning these sequences, we found that the D-loop region was divided into three parts; the 5'-highly polymorphic segment (672-4 bp), the repetitive sequence (from 200 to 300 bp), and the 3'- conserved segment (294 bp). Of the 52 nucleotide substitutions found in the D-loop region, 51 were restricted in the 5'-side segment to the repetitive sequence (674 bp), and only one substitution and 3 deletions/insertions could he observed in the 3'-side segment of the repetitive sequence (Fig. 1).

The dogs and wolves both had the repetitive sequence with two repetitive units of TACACGT([A/G])CG. The foxes and raccoon dog also had sequences with the TACACATACG, TACACACA([C/T])ACG and the TAC([A/G])CACG units, respectively(Fig. 1). The sequence of the units found in the dogs and wolves differed from three of the other two species, and thus the repetitive sequences were species specific. Among the dogs and wolves, the repetitive sequence caused extensive length variations and intra-individual heteroplasmy in the D-loop. We found no specific arrays of the units nor specific length variations among the breeds of domestic dogs or subspecies of wolf (data not shown).

No significance in the sequence divergence values among dogs and wolves. Using a pairwise method, we compared the nucleotide sequences in the 5'- highly polymorphic segments among dogs and wolves and calculated the intra- and inter-species sequence divergences. The values of sequence divergence within domestic dogs varied from 0% to 3.19%, and those within wolf subspecies varied from 0% to 2.88%. The sequence divergence was calculated to be 0.30 -- 3.35% between dogs and wolves (Table 2). These results showed that there were no significant differences among the intraspecific or interspecific divergence values. These results also showed that the domestic dogs tested maintain a large degree of mtDNA polymorphisms, suggesting that the polymorphisms had been introduced by the wolves. It is thus concluded that the wolf is the strongest candidate for the matriarchal ancestor of the dog.

We also calculated the sequence divergences between domestic dogs and foxes, between domestic dogs and the raccoon dog, and between foxes and the raccoon dog to be from 18.37% to 21.35%(the average value, 19.71%), 19.81% to 21.88% (the average value, 21.01%), and from 19.81% to 20.43% (the average value, 20.28%), respectively (Table 2).

Phylogenetic analysis and nucleotide substitution patterns. We constructed phylogenetic trees by the NJ method using the sequence divergence values. Two distinct clades. Clade A and Clade B, appeared in the N J trees, but no clades specific for any particular dog breeds were observed, consistent with the results reported by Okumura et al. (1986). It should be noted that five breeds of Asiatic native dog, i.e., Shiba (Mino), Kai, Atita, Korean Chejudo, and Mongolian native dog shared Clade A and Clade B, although the sample size was very small; namely, we examined only two individuals in each of these breeds (Fig. 2). These results suggest that most breeds of Asiatic native dog possess two major genetic components in their matriarchal lineages. The Asiatic wolves showed clade specificity among the subspecies; namely, Chinese wolves (C. l. chanco) were limited to Clade A, whereas Indian wolves (C.l. pallipes) were limited to Clade B. European wolves (C. l. lupus) shared both clades. We then tested the significance of tree topology by bootstrapping using the wolves, foxes, and raccoon dog. Domestic dogs were excluded from this test, since we considered that the exclusion would eliminate any bias caused by artificial intra- and interbreeding in the dog populations. The existence of two clades was confirmed to be significant by the bootstrapping values (Fig. 3).

After construction of the NJ tree, we compared the nucleotide substitutions within and between the clades. As mentioned above, 53 substitutions were restricted in the 5'- highly polymorphic segment. We found 6 transversions, 43 transitions, 2 transitions/transversions and 2 insertions/deletions in the segment. Five substitutions were specific for the Clade B which contains Indian wolves (Fig. 1). In particular, one substitution, which is located at the 27th nucleotide position from the beginning of the D-loop (in the 5' segment) and changed from A to G, occurred specifically in the Indian wolves, being a good diagnostic marker for the subspecies. However, no substitutions were specific for the Clade A.

DISCUSSION

The close relationships between the domestic dog and wolves. Based on the molecular analysis of mtDNA between domestic dogs and wolves, we present here direct molecular evidence that the ancestor of the domestic dog is the wolf. We compared their nucleotide substitutions in the mtDNA D-loop region. and found no differences in the specificity for the substitution between the two species (Fig. 1 and Table 2). Moreover, the clades of the individual domestic dogs were completely overlapped with those of the individual wolves, showing that the domestic dog was descended from the wolf.

From the morphological point of view, domesticated dogs were suggested to be descended from wolves (Wayne, 1986). This was supported by the results from the studies of behavior (Zimen. 1981), vocalization (Zimen, 1981) and molecular biology (Wayne et el., 1987a, b). Wayne et al (1991) also showed in a mtDNA restriction fragment length polymorphisms (RFLP) analysis that the domestic dog is an extremely close relative of the gray wolf, differing from it by at most 0.2% of the mtDNA sequence. Our results indicate that the mtDNA polymorphisms in the domestic dog are not distinguishable from those in the wolf. This slight difference may be caused by the difference of collection localities between our study and that of Wayne et al. (1991). We collected the wolf samples from an Old continent, whereas Wayne et al. (1991) had collected from a New continent. There may be a slight difference in mtDNA polymorphisms between the wolves from the two continents.

Wayne (1993) stated that dogs are gray wolves, despite their diversity in size and population, and that the wide variation in their adult morphology probably the result of simple changes in the developmental rats and timing. Recently, Wayne and his colleagues carried out extensive survey of DNA sequence polymorphisms in the mtDNA D-loop region among domestic dogs and wild wolves (Vila et al., 1997). They concluded that 1) wolves were the ancestors of dogs, and 2) dogs had multiple origins, based on extensive polymorphisms shown in dog populations. We also obtained the same conclusion by extensive survey of mtDNA D-loop sequences mainly among Japanese dogs and Asitatic wild wolves. The lack of difference between our conclusion and theirs suggests two possibilities on  the domestication processes of dogs: 1) Asia is the secondary domestication center for the dog and the ancestors of Asiatic dogs had maintained the extensive polymorphisms from the original ancestors of dogs, and 2) human migrations frequently occurred during the domestication of dogs and consequently modern dogs exhibit the extensive polymorphisms. Wayne and his colleagues (Vila et al., 1997) also suggests that dogs originated more than 100,000 years before the present, based on the experimental evidence that most dog sequences belonged to a divergent monophyletic clade sharing no sequences with wolves. However, we could not confirm this evidence because our phylogenetic tree shows no clades belonged to only dogs (Fig. 2).

Intraspecific mtDNA polymorphisms in the domestic dog. When we analyzed the phylogenetic relationships among domestic dog breeds based on the sequence polymorphisms of the mtDNA D-loop region, we found extensive polymorphisms We also observed two major clades in the breeds, showing that the domestic dogs possess two major genetic components. However, we found no clades or haplotypes specific for certain dog breeds, consistent with the results of Okumura et al. [1996]. Tanabe et al. (1991) suggested that Japanese dogs were derived from influxes from two distinct routes; one from Southwest Asia, and the other from the northwest through the Korean peninsula. Our present results are not consistent with this suggestion, because Japanese native dog breeds could not be delimited as distinct breeds. This discrepancy may be elucidated by the difference of the inheritance manner between nuclear-coded genes and mtDNA-coded genes. All of the genes that were investigated by Tanabe et al. (1991) are encoded by the nuclear genome, and they are biparentally transmitted. In contrast, mtDNA is maternally transmitted in mammals, and the maternal transmission is quite strict (Kaneda et al., 1995). When the genetic status of domesticated animals is to he improved by breeding, the most common method is that useful genes are introduced from males. Since domestic dogs have been improved in this way the results reported by Tanabe et al. (1991) may reflect the male effect. It is thus necessary to investigate male-specific genes much as unique genes encoded on the Y-chromosome.

Intraspecific mtDNA polymorphisms in the wolf. We obtained experimental evidence that Asiatic wolves have subspecies-specific clads in their phylogenetic trees. This suggests that the Asiatic subspecies of wolf are genetically differentiated from each other. Our phylogenetic analysis showed that the European subspecies of wolf shared two clades: one clade contains the Indian subspecies, and the other contains the Chinese subspecies. Two possibilities underlying this result can be posited. One possibility is that the European subspecies is the ancestor of the Asiatic subspecies; the other is that the haplotypes found in the Asiatic subspecies were introduced in the European subspecies. From the archeological point of view, historical remains of wolf bones have been found exclusively in the New Continent. This suggests that the ancestor of the wolf had arisen there and moved to the Old Continent. If this is true, the migration routes should be from the Old continent to the European Continent through the Asian Continent. Thus, the latter possibility might be more likely.

Wayne (1993) reported that European wolves possessed, with one exception, a single haplotype specific for each population. He suggested that the mtDNA genotypes were randomly fixed to be monomorphic because of the decreasing and fracturing of available habits for wolves and the decreases in their populations. Our results support his idea. We found only two haplotypes in the European subspecies, and the same was true for the Indian subspecies (Figs. 2 and 3). Not only in Europe but also in India, the wolves' habitats may have become smaller and fractured. In sharp contrast to these subspecies, the Chinese subspecies showed, as did the domestic dogs, extensive polymorphisms in their mtDNA. This suggests that the Chinese subspecies still maintain populations large enough to keep such polymorphisms, and that these wolves can move freely through their territories.

Estimation of divergence time. The average sequence divergence values were 19.71% (dogs and wolves - foxes), 20.28% (foxes - raccoon dog) and 21.01% (dogs and wolves - raccoon dog). The rate of sequence divergence of mtDNA in mammals is estimated to be 2-4% per million years (Brown et el., 1979). Based on this value, the divergence of the 3  genera occurred approximately 6 -- 10 million years ago. Wayne et al. (1987a.b) reported that the fossil record and genetic distances indicate that the division of canines began about 7 -- 10 million years ago, which is consistent with our results.

Did extensive interbreeding occur in the domestic dogs? As mentioned above, the Asiatic subspecies of the wolf showed subspecies-specific mtDNA polymorphisms. Though the European subspecies showed a much smaller degree of mtDNA polymorphisms, these wolves showed the existence of two genetic components in their mtDNA. In contrast, the domestic dogs exhibited extensive polymorphisms in their mtDNA, and these polymorphisms were not specific for any breed or for a geographical distribution (Fig. 2 and Table 4). Okumura et el. (1996) investigated 28 individuals of one Japanese native breed, the Shiba, and they found that at least 7 mtDNA haplotypes exist in the Shiba breed. The haplotypes are also found in the Ryukyu, an old Japanese native breed. Many haplotypes are shared among different dog breeds, not only Japanese native breeds but also non- Japanese breeds. These results suggest that extensive interbreeding occurred in the ancestral stocks of domestic dogs. We inferred, therefore, that the domestication from wolves to dogs occurred in several (at least two) places, and that the domestic dog may be derived from two wolf populations separated geographically (north and south).

There are several hypotheses regarding the domestication of dogs. The most widespread and accepted view regards the wolf as the ancestor of the domestic dog (Zeuner, 1963; Scott and Fuller, 1965; Lorenz, 1975; Herre and Rohrs, 1977). At present, a leading hypothesis is that the ancestor of dogs is the Arabian wolf [C. l. arabs) (Clutton-Brock, 1995). However, the report on skeletal anatomy, the dingo, C. familialis dingo, closely resembles the Indian wolf and the pariah dogs of Southeast Asia. It is probable that the dingo is a direct descendant of dogs that were originally domesticated from tamed Indian wolves (C. l. pallipes) (Corbett, 1985). We suggest that findings and ours indicate plural origins of the domestic dog. It is possible that the dogs were domesticated in a single place, mated with wolves along a migration route of humans. We are not able to conclude whether the domestication occurred in a single place or several places.

Repetitive sequences. We found that the repetitive sequences appeared in the identical positions near the 3' end of the mtDNA D-loop region among dogs, wolves, foxes, and a raccoon dog. We also found such repetitive sequences in the dhole (Cuon alpinus), bush dog (Speothos vanaticus), and marten (Martes melampus) (data not shown). Repetitive sequences in the mtDNA D-loop region have also been reported in other mammalian species, i.e., the white sturgeon (Buroker et al., 1990), evening bat (Wilkinson and Chapman, 1991), rabbit (Biju-Duval et al., 1990; Mignotte et al, 1990), harbor seal (Arnason and Johnsson, 1992), elephant seals (Hoelzel et al., 1993a), pig (Ghivizzani et al., 1993), and equine (Ishida et al., 1994). Our present result indicated the following characteristics of the repetitive sequence; the sequence of the repeat units is species specific, although the array and the length of the repetitive units vary among individuals in the same species. However, one exception occurs between dogs and wolves. In conclusion, our finding contain strong experimental evidence that dogs and wolves are members of the same species.

We thank H. Tumennasan, Mongolian Academy of Sciences. Ulanbator, and E. Haase, University of Keel, for their collection of wolf blood samples, and I. M. Mastika. Udayana University, Dempasar, Bali. for specimens from native Indonesian dogs. We also thank H. Suzuki, Hokkaido University, for specimens of fox DNA samples, and S. Yachimori for specimens of raccoon dog DNA samples.

* Corresponding author.
 

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