J Mol Evol (1995) 41:180-188

Iou..oMOLECULARIEVOLUTION© Springer-VedagNewYorkInc. 1995

Mitochondrial DNA Sequences of Various Species of the Genus Equus with
Special Reference to the Phylogenetic Relationship Between Przewalskii's
Wild Horse and Domestic Horse
Nobushige Ishida, 1 Tsendsuren Oyunsuren,2 Suguru Mashima, 3 Harutaka Mukoyama, 1 Naruya Saitou4
1 Laboratory of Molecular and Cellular Biology, Equine Research Institute, Japan Racing Association, 27-7, Tsurumaki 5-chome, Setagaya-ku,
Tokyo 154, Japan
2 Department of Molecular Biology, Institute of Biotechnology, Enkhtaivan ave 54, Ulaanbaatar 210351, Mongolia
3 Section of Molecular Genetics, Laboratory of Racing Chemistry, 4-37-6, Kamiyoga, Setagaya-ku, Tokyo 158, Japan
4 Laboratory of Evolutionary Genetics, National Institute of Genetics, Mishima 411, Japan
Received: 7 November 1994 / Accepted: 27 February 1995

Abstract. The noncoding region between tRNA Pr°
and the large conserved sequence block is the most variable region in the mammalian mitochondrial DNA
D-loop region. This variable region (ca. 270 bp) of four
species of Equus, including Mongolian and Japanese native domestic horses as well as Przewalskii's (or Mongolian) wild horse, were sequenced. These data werecompared with our recently published Thoroughbred
horse mitochondrial DNA sequences. The evolutionaryrate of this region among the four species of Equus wasestimated to be 2-4 x 10-8 per site per year. Phylogenetic
trees of Equus species demonstrate that Przewalskii's
wild horse is within the genetic variation among the domestic horse. This suggests that the chromosome number
change (probably increase) of the Przewalskii's wild
horse occurred rather recently.
Key words: Mitochondrial DNA - - Noncoding
D-loop region - - Phylogenetic tree - - Przewalskii's
wild horse - - Equus caballus - - Evolutionary rate
Introduction
Tracing the evolution of the horse family Equidae fromeohippus (genus Hyracotherium) to the modern genus
Equus is a classic achievement of paleontology (e.g.,

Correspondence to: N. Ishida

Simpson 1951; Groves and Willoughby 1981; MacFadden 1992). Most members of the horse family are now
extinct, and all the living Equidae species represent asingle genus, Equus. The phylogenetic relationshipwithin the species of this genus is, however, not wellunderstood. According to recent literature, Equus is classified into four subgenera with seven species: subgenus
Equus includes E. caballus (domestic horses and Przewalskii's or Mongolian wild horse); subgenus Asinus includes E. asinus (donkeys), E. hemionus (onagers), andE. kiang (kiang); subgenus DoIichohippus has only E.
grevyi (Grevy's zebra); and E. zebra (mountain zebra),
E. burchelli (Burchell' s or plains zebra), and the recently
extinct E. quagga (quagga) are classified into subgenus
Hippotigris (Nowak and Paradiso 1983).
Because its chromosome number (2n = 66) is different
from that (2n = 64) of domestic horses (Benirschke et al.
1965), some researchers classify Przewalskii's wild
horse as a distinct species, E. przewalskii (e.g., Ryder etal. 1978). It is generally considered that two pairs ofacrocentric chromosomes in the ancestral horse population (2n = 66; Przewalskii's wild horse type) changed to
one pair of submetacentric chromosomes by Robertsonian fusion, and this produced the modern horse chromosome number (2n = 64) (Benirschke et al. 1965; Ryder et al. 1978). By comparison of equine hemoglobinamino acid sequences (Clegg 1974) and serum protein
studies (Ryder et al. 1979; Kaminski 1979), a close relationship between domestic horses and Przewalskii's

181
wild horses has been shown. Genetic material o f these
two species is so similar that their hybrids are fertile(Ryder et al. 1978; Trommerhausen-Smith et al. 1979).
Therefore, there is uncertainty on the species status of E.
przewalskii, and it is tentatively included into E. caballus
in the following.
Mammalian mitochondrial D N A (mtDNA) has a rateof nucleotide substitution five to ten times as high as
nuclear D N A (Brown et al. 1979). George and Ryder(1986) showed, on the basis of their several equine mitochondrial D N A restriction-site maps, that the sequence
divergence between Przewalskii's wild horse and domestic horse was within the intraspecific variation. George
and Ryder (1986) also examined five other Equus species, but the phylogenetic relationship among those
Equus species was not clarified.
Several studies have suggested that the most rapidly
evolving part of the mitochondrial genome is the noncoding control region which contains the D-loop (Walberg and Clayton 1981; Chang and Clayton 1985). The
substitution rate in the human D-loop region has been
estimated to be 2.8-5 times higher than the rate for theremainder of the mitochondrial genome (Aquadro andGreenberg 1982; Cann et al. 1984). Recently we sequenced the whole D-loop region (about 1,100 bp) for
three Thoroughbred horse m t D N A s (Ishida et al. 1994)
and found the noncoding region between t R N A r~° andthe large conserved sequence block was most variablea m o n g the three T h o r o u g h b r e d i n d i v i d u a l s . In thepresent study, we sequenced the c o r r e s p o n d i n g six
equine m t D N A D-loop regions and conducted a phylogenetic analysis of the genus Equus. Special reference
will be made to the phylogenetic relationship between
Przewalskii's (or Mongolian) wild horse and domestic
horse.

Materials and Methods
Isolation of GenomicDNA. The blood of E. zebra and E. asinus was
obtained through the courtesy of Yumemigasaki Zoological Park, Kawasaki, The blood of E. grevyi was a gift from Tama Zoological Park,Tokyo. The blood of a Przewalskii's wild horse (International Studbook Number 880) and a Japanese native horse were from the Equestrian Park of Japan Racing Association, Tokyo. The blood sample of a
Mongolian native horse was collected in Mongolia. Genomic DNA was
isolated from peripheral blood of those six individuals by using thestandard techniques (Sambrook et al. 1989). The chromosome numbers(2n) of those sampled individuals were 64, 66, 62, 46, and 32 forMongolian and Japanese native horses, Przewalskii's wild horse, E.
asinus, E. grevyi, and E. zebra, respectively (K. Hirota personal communication, 1993), compatible with previous studies (e.g., Ryder et al.
1978).
Sequencing. PCR primers were designed to amplify the region between tRNAr'r° and the large conserved sequence block on the basis ofthree Thoroughbreds' DNA sequences (Ishida et al. 1994). Primer sequences are 5'-gctgaaattctacttaaa-3' and 5'(biotin labeled)-agttggaagggttgctgatt-3'. PCR was performed using 600-ng genomic DNA, 1.2
gM of each primer in 75 ~tl 1 x PCR buffer containing 1.25 mM of eachdNTP, and 2.5U Amplitaq Polymerase (Perkin Elmer Cetus). Amplification conditions were 96°C for 2 min, followed by 35 cycles of 94°C
1 min, 58°C 1 min, and 72°C 1 rain, and final 10-rain extension at
72°C. PCR product was purified with MicroSpin Columns (Pharmacia). The alkali-denatured single-stranded DNA was collected with
Dynabeads (Dynal). Sequence analysis was done by the dideoxyribonucleotide chain termination method (Sanger et al. 1977), using theAutomated Laser Fluorescent (ALF) sequencer (Phannacia).

DataAnalysis. Raw sequence data were analyzed by using software
DNASIS-Mac (Hitachi Software Engineering), and CLUSTAL V (Higgins et al. 1992) was used for multiple sequence alignment. Phylogenetic trees were constructed by using the neighbor-joiningmethod (Saitou and Nei 1987) and the maximum parsimony method (Fitch 1977).
Computer packages MEGA (Kumar et al. 1993) and PAUP (Swofford
1993) were used for assisting the neighbor-joining and the maximum
parsimony analysis, respectively. Numbers of nucleotide substitutions
between sequences were estimated by using Tamura and Nei's (1993)
substitution model with a gamma distribution (c~ = 0.5), because thatmodel seems to be suitable for estimating the hypervariable region of
mitochondrial DNA D-loop (Kumar et al. 1993).

Results and Discussion
Nucleotide Differences
Approximately 270 bp of noncoding regions of m t D N A
for E. zebra, E. grevyi, E. asinus, and three E. caballus(Mongolian native horse, Japanese native horse, andPrzewalskii's wild horse) were sequenced using the PCR
direct sequencing method. Figure 1 presents the multiple-aligned sequences of this region for all studied individuals as well as the three Thoroughbreds of Ishida et al.
(1994). Five nucleotide positions (indicated by an asterisk at the bottom of each sequence block) were assumed
to contain at least one gap out of 274 positions. Nucleotide differences were more frequently observed near the
5' region (close to tRNA Pr°) of sequenced D N A thannear the 3' region, and a total of 60 nucleotide sites werevariable among the 269 positions without any gap. The
pairwise sequence differences (transitional and transversional differences among those 269 positions) are shownin Table 1. It is clear that transitions outnumbered transversions.
W h e n we restrict the comparison into the six E. caballus sequences (three Thoroughbreds, Mongolian native horse, Japanese native horse, and Przewalskii's wild
horse), only Przewalskii's wild horse has one insertion at
position 103, and 271 nucleotide positions do not haveany gap. Sequence data for these 271 positions were used
for the phylogenetic analysis of E. caballus sequences.
Nineteen sites were variable among those 271 positions,and a majority of the nucleotide differences were transitional ones.

Phylogenetic Tree of the Six E. caballus Sequences
Figure 2 is a phylogenetic tree of six E. caballus sequences. After the neighbor-joining tree was constructed182
1
Thoroughbred

I

Thoroughbred- ................

- .......

T .....

- ................

- .......

T .....

wild...................................

G ....................

T ............

- ................

- .......

T .....

native...................................

G ....................

T ............

- ................

- .......

T .....

...................................

G ....................

T ............

- ................

- .......

T .....

Thoroughbred
Przewalskli
MongolianJapanese

I00

50

ACAACAATTCACCCTCATGTGCTATGTCAGTATCAAATTATACCCCCACATAACACCATACCCACCTGA-CATGCAATATCTTATG-AATGGCCCATGTA

2........

3.....................................................................

nativeC.G........................

E.zebra.......

E.asinus.........

T.T..........

E.grevyi........

C..T........

CC .............

G ............

C ..........

T ....

C ..........

T...A..CA ............

T...A

T ....................

GG.GT...-..C..G......

........

G..C

GC.TTAT..A..-..CA.........

-T..C.GT.T

101

Thoroughbred
Thoroughbred
Thoroughbred....

T.AG.TC..A...A

2

.A-.

.........

A ......

CA............................................

.........

native.A-.

.........

AG.....................................

Japanese native..-..........

AG.....................................

E.zebra.A-.

.........

E.esinua..-..........

E.grevyi.T-.

.........

GA.....................................................
T ..............

A .............................

C...T

G ........

A ......................................

.......

C...C-....C..T

G ...............

A ................

G ...............

A ................

......

T ......

G .................

..........

.....
.....
.....

250

G ...............

G ....

G ...............

A ..........

A ................

274.........................................................
..........................................................................

T .................

......................................................

T ...................

....................

T ...................

C .................................

......................................................

T ...................

E.zebra......................................................

T ...................

E.asinus...........................................

E.grevyi......................................................

A ..........

Fig. 1.

An alignmentfor nine equinemitochondrialDNA sequences.
Three already-knownThoroughbred sequences (Thoroughbred 1, 2,
and 3) of Ishida et al. (1994) were included with the six sequences
determinedby the present study; Przewalskii'swild horse, Mongolian

Table 1.

C..T

CCA~GTCAAATCATTTCCAGTCAACACGCATATCACAACCCATGTTCCACGAGCCTAATCACCAAGCCGCGGGA

3
wild

....

A ...............................
T...-

2

Mongolian n a t i v eJapanese n a t i v e

CTC°,.TT

A ...............................................

A ..................................

201

Przewalskii

C..T

G .................................

.................................................................................................

..C

1- ....

200

CG-TCGTGCATTAGATTGTTTGCCCCATGAAT~TAAGCATGTACATAATATCATTTATCTTACATAAGTACATTATATTATTGATCGTGCATACCcCAT..-

Thoroughbred
Thoroughbred
Thoroughbred

....

150

1

3

Mongolian

G..C

.....

.... TTAG.TCA.-...Awild

Przewalskii....

T ...................
T ...................

native horse, Japanese native horse, E. zebra, E. asinus, and E. grevyi.
Sequence identity is indicated by a dot, and gap is indicated by ahyphen. Five nucleotidepositionsthat include gaps are indicatedwith
asterisks at the bottom.

Nucleotidedifferences among the nine equine mtDNA sequencesa

Sequence name(abbr.)

T1

T2

T3

Pr

Mo

Jp

Zb

As

Gv

Thoroughbred 1 (T1)
Thoroughbred 2 (T2)
Thoroughbred 3 (T3)
Przewalskii wild (Pr)Mongoliannative (Mo)Japanese native (Jp)
E. zebra (Zb)
E. asinus (As)E. grevyi (Gv)

-10
1
8
11
6
24
22
22

1
-9
12
13
10
24
28
25

0
1
-7
10
5
23
21
20

0
1
0
-5
4
26
24
20

0
1
0
0
-4
25
26
20

1
2
1
1

7
8
7
7

5
6
5
5

6
7
6
6

1

7

5

6

-23
22
21

8
-25
21

4
8
-19

7
9
3
--

a Numbersbelow and above diagonalare transitionaland transversionaldifferences, respectively,amongthe 269 nucleotidepositionswithout gaps
(see Fig. 1)
using the original pairwise distances, branch lengths(number of nucleotide substitutions per site) were multiplied the number of nucleotides was compared (271 in
the present case), and integer values were obtained afterrounding. These integer-value branch lengths were compared with those estimated by using the m a x i m u m parsimony principle applied to the neighbor-joining tree,
and the final branch lengths were determined. This procedure follows that of Nerurker et al. (1993). A total of
24 nucleotide substitutions were estimated in the tree ofFig. 2 by applying this procedure. The location of theroot was assumed to be equal to that of Fig. 3, in which
three other E q u u s species sequences were also used.
Numbers above the three internal branches are boot-strap probabilities (in %) based on 1,000 bootstrappedneighbor-joining trees. Relatively high bootstrap probabilities of 90% and 88% were observed for the clusteringof Thoroughbred 1 and 3 sequences and that of three
Thoroughbred sequences, respectively, while the clustering of Przewalskii's wild horse and Mongolian native
horse showed a 61% probability.
We found a unique most parsimonious tree by using
the branch-and-bound option of PAUP, and the topologyof that tree was identical with that of the neighborjoining tree (Fig. 2). The maximum parsimony tree required 22 nucleotide substitutions, two substitutions lessthan that for the neighbor-joining tree. Numbers in parentheses below the three internal branches of the tree of

183

90

[

Thoroughbred 1(88) ~Thoroughbred 3....

Thoroughbred 2
J a p a n e s e native horse

88

(86)

]

.

.

.

61

,I

(66)

I

Fig. 2. A phylogenetic tree of the six Equus
caballus sequences constructed by using theneighbor-joining method. Figures above andbelow (in parentheses) the three internalbranches are bootstrap probabilities (in %)based on 1,000 bootstrapped trees when theneighbor-joining method and the maximum
parsimony method were used, respectively.

P r z e w a l s k i i ' s w i l d horse...... M o n g o l i a n native horse

.

I

0

.

.

.

.

I

5

10

No. nucleotide substitutions~

Thoroughbred 1
roughbred 3.....Thoroughbred 2anese native horse(59) 1 5 8Przewalskii's wild horse(58)
Mongolian native horse

98
(97)

E. asinus(59)[

E. grevyi
E. zebra

I
0

;

'

'

'

I
5

.

.

.

.

I
10

.

.

.

.

I
15

.

.

.

.

I
20

.

.

.

.

I
25

.

.

.

.

I
30

No. nucleotide substitutions
Fig. 3. A phylogenetictree of nine Equus sequences constructedby using the neighbor-joiningmethod. See text for the rooting procedure. Figures
above and below (in parentheses) the three internal branches are bootstrap probabilities (in %) based on 1,000 bootstrapped trees when theneighbor-joining method and the maximum parsimony method were used, respectively.

Fig. 2 are bootstrap probabilities (in %) based on 1,000
bootstrapped maximum parsimony trees. The bootstrap
values are more or less the same as those for the neighbor-joining tree.
Table 2 shows a list of estimated change in nucleotides mapped to the tree of Fig. 2. Two substitutions outof 24 were nonparsimonious estimation. Three nucleotide substitutions are assumed at position 153, and all ofthem are transversions, while the most parsimonious explanation for the nucleotide configuration of this site
under the tree of Fig. 2 requires two substitutions involving one transversion (shown in parentheses in Table 2).
Another nonparsimonious substitution is assumed at thebranch going to the Thoroughbred 2 sequence. (Its position is designated by " ? " in Table 2.) This extra substitution was estimated apparently because of the use of
Tamura and Nei's (1993) substitution model with agamma distribution. If this is a real one, it was probably
a transition. Under this assumption, four out of 24 substitutions are transversions, or if we choose the parsimonious explanation at position 153, two (C ~ A at position 153 and A ~ T at position 257) out of 23 aretransversions. Depending on either case, transitions oc-

curred 5-10.5 times more than transversions. This transition/transversion ratio is slightly lower than that (ca.
15) observed in the human mtDNA D-loop region (Vigilant et al. 1991; Tamura and Nei 1993). Among the 19(or 20) transitions, 7 (or 8) are changes between pyrimidines (T ( ~ C) and the remaining 12 are those between purines (A < ~ G).
Phylogenetic Tree o f the Nine Equus Sequences
Figure 3 is a phylogenetic tree of nine Equus sequences
constructed by using the neighbor-joining method. The
branch-length estimation procedure is the same as above,
except that the estimates among the six E. caballus sequences were taken from those of Fig. 2. This is because
the branch-length estimation is generally more accurate
when only closely related sequences are compared. A
total of 97 nucleotide substitutions were estimated in thetree of Fig. 3. The root was located under the assumptionof the molecular clock and the unrooted tree obtainedby the neighbor-joining method. More specifically,branches of a pair of neighbors are successively averaged
to find the root. For example, the height of the nodeconnecting Thoroughbred 1 and 3 sequences is calcu-

184
Table 2. Estimated substitution events on the phylogenetic tree ofFig. 2
Nucleotide
positiona

Nucleotidechangeb9T(8) --+ C(2)11

A(8) -+ G(2)

36
49
57
95
102
113
114
115
121
122
153

G(8) --~ A(7)C(8) ~ T(2)C(8) ~-~ T(10)T(7) ~ C(1)G(8) ---)A(2), G(9) --~ A(5)A(9) ~ G(4)A(8) -+ G(7)A(9) ~ G(5), A(10) -+ G(6)T(8) --~ C(2)G(8) --~ A(2)A(9) ~ C(4), A(9) --+ T(5), C(8) ~-) A(10) [orC(10) ~ A(6), C(9) -~ T(5)]A(8) -+ G(2)A(10) ---r6(9)G(10) -+ A(9)T(9) --~ C(5)T(8) +-~ C(10)A(8) --~ T(2)?(8) ~ ?(2)

167
168
184
221
255
257
?

a Nucleotide positions of Fig. 1. The last row with "?" indicates one
unknown change inferred in the tree of Fig. 2
b Numbers in parentheses designate sequences and internal nodes asfollows: 1 = T1, 2 = T2, 3 = T3, 4 = Pr, 5 = Mo, 6 = Jp, 7 = nodeconnecting T1 and T3, 8 = node connectingT2 and node 7, 9 = nodeconnecting Pr and Mo, and 10 = node connecting Jp and node 9.
Species abbreviations follow those of Table 1. An arrow "---~" indicates the direction of change, and a double arrow "+-#' indicatesambiguity of direction. At position 153, two possibilities are presented.
The last row indicates an extra substitution out of estimated nine at thebranch going to the Thoroughbred2 (T2) sequence
lated to be (1 + 0)/2 = 0.5. Then the height of the nodeconnecting the former node and Thoroughbred 2 becomes [(2 + 0.5) + 9]/2 = 5.75. This averaging procedure
is continued until the largest height (the root) is found.
The root thus determined was located at 22.34 substitutions apart from E. zebra. Therefore, the point that divides the branch to E. zebra into 22 and 3 was determined as the root of the neighbor-joining tree (Fig. 3).
This procedure is different from the usual midpoint rooting, in which only a pair of sequences that show thelargest patristic distance is used. We also tried to use thebovine sequence (Anderson et al. 1982) as an outgroup to
locate the root. However, the multiple alignment was noteasy, and we did not incorporate the bovine sequence forcomparison.
Numbers above the six internal branches are bootstrap
probabilities (in %) based on 1,000 bootstrap trees. Six
Equus caballus sequences are monophyletic with a highbootstrap probability (98%), and the clustering of E. asinus and E. grevyi is moderately supported (73% of bootstrap probability). Because of the inclusion of three othersequences, bootstrap probabilities within the six Equus
caballus sequences were slightly lower than those of
Fig. 2.

Table 3 presents the two kinds of estimated numbersof nucleotide substitution. Numbers below the diagonal
are pairwise evolutionary distances computed by using
Tamura and Nei's (1993) substitution model with agamma distribution (c~ = 0.5). These pairwise distances
are larger than those when Kimura's (1980) method wasused (data not shown). The increase was 5-14% within
the six E. caballus sequences, while it was 18-48%
among the Equus sequences. Those above the diagonal
are patristic distances reconstructed from the tree of Fig.
3. Though some of those patristic distances are smaller
than the corresponding original distances below the diagonal, both of them are larger than the nucleotide difference shown in Table 1. For example, the number ofnucleotide differences between E. zebra and E. grevyi
was 30 (21 transitional and 9 transversional differences;
see Table 1), while the corresponding original and patristic distances are 41.5 and 43, respectively (Table 3).
Because of these large differences, estimation of substitution events, involving different Equus species, was impossible.
Three equally parsimonious trees, requiring 81 nucleotide substitutions, were found by using the branch-andbound option of PAUP. The topology of one of those,tree 1, was identical with that of the neighbor-joining tree
(Fig. 3). Numbers in parentheses below the six internalbranches of Fig. 3 are bootstrap probabilities (in %)based on 1,000 bootstrapped maximum parsimony trees.
Those probabilities are slightly lower than those for theneighbor-joining trees except for one for the Thoroughbred 1 and 3 cluster (Fig. 3). The topological difference
between the tree of Fig. 3 and the other two equallyparsimonious trees (trees 2 and 3; not shown) resides in
the position of the root for the six Equus caballus sequences; the root at the branch separating the cluster ofThoroughbred 1 and 3 and the remaining four sequences
for tree 2, and that at the branch separating Thoroughbred 2 and the remaining five sequences for tree 3. Nucleotide positions 255, 36, and 9 supported trees 1, 2, and3, respectively. Although the root (the position of thenearest common ancestor) for the six Equus sequences is
not clear, the root is estimated to be always between
some Thoroughbred horse(s) and the other horses.

Reanalysis of Restriction-Site Data
George and Ryder (1986) studied restriction-site variation of mtDNA for several Equus species. We reanalyzed
their data in the following way. The maximum likelihood
method of Nei and Tajima (1983) was used for estimating numbers of nucleotide substitution for all pairs ofsequences, based on the restriction maps of Figs. 1 and 3of George and Ryder (1986). Evolutionary distances thus
obtained (Table 4) were not much different from thosepresented by George and Ryder (1986), who used Nei
and Li's (1979) method. Figure 4 shows a neighbor-

185
Table 3. Estimatednumber of nucleotidesubstitutionsamong the nine equine mtDNA sequencesa
Sequence name (abbr.)

T1

T2

T3

Pr

Mo

Jp

Zb

As

Gv

Thoroughbred 1 (T1)
Thoroughbred 2 (T2)
Thoroughbred 3 (T3)
Przewalskii wild (Pr)Mongoliannative (Mo)Japanese native (Jp)
E. zebra (Zb)
E. asinus (As)E. grevyi (Gv)

-12.8
1.0
9.1
12.9
7.6
43.8
37.0
38.6

13
-11.7
15.6
16.7
13.7
45.4
51.7
46.8

1
12
-8.0
11.7
6.5
41.8
35.1
36.6

10
16
9
-5.4
5.6
49.0
41.3
34.8

12
18
11
6
-5.4
46.5
45.8
34.8

7
12
6
5
7
-43.7
35.5
38.2

43
49
42
45
47
42
-47.9
41.5

39
45
38
41
43
38
46
-29.1

36
42
35
38
40
35
43
29
--

a Numbersbelow and above diagonalare originaland patristic (underthe tree of Fig. 3) numberof nucleotidesubstitutions,respectively,within the269 nucleotidepositionscompared

Table 4. Estimatednumbers of nucleotidesubstitutionper site among the nine mtDNA sequence: data of George and Ryder (1986) were useda
Eb
Eg
Ez
Ea
Eh
Ecl
Ec2
Ec3

0.0725(0.0135)
0.0728(0.0132)
0.0743(0.0138)
0.0684(0.0126)
0.0818(0.0147)0.0045(0.0027)
0.0029(0.0021)0.0044(0.0026)
Ep

0.0341(0.0079)
0.0567(0.0113)0.0640(0.0120)0.0722(0.0134)
0.0710(0.0133)
0.0711(0.0132)
0.0725(0.0133)
Eb

0.0500(0.0101)
0.0532(0.0104)
0.0683(0.0126)
0.0713(0.0131)0.0714(0.0129)0.0728(0.0131)Eg

0.0615(0.0117)0.0695(0.0130)
0.0683(0.0129)0.0728(0.0134)
0.0742(0.0135)Ez

0.0639(0.0120)0.0669(0.0125)
0.0671(0.0124)0.0685(0.0125)Ea0.0756(0.0140)0.0755(0.0138)
0.0770(0.0139)Eh0.0045(0.0026)
0.0059(0.0030)Ec 1

0.0014(0.0014)Ec2

a Numbers in parentheses are standard errors. Ep = Equus przewalskii (=Prezewalskii'swild horse); Eb = Equus burchelli antiquorum; Eg = Equus
grevyi; Ez = Equus zebrahartmannae; Ea = Equus africanus somalicus (=Equusasinus); Eh = Equus hemionus, Ecl = Arabianhorses; Ec2 = quarterand Peruvian paso horses; and Ec3 = Morgan horsejoining tree based on the distance matrix of Table 4. The
rooting procedure is the same as that used in Fig. 3.
Sequence variation within the E. caballus (including
the Przewalskii's wild horse) is much smaller than thatbetween various Equus species. This confirms the resultof George and Ryder (1986), and this situation is similar
to the tree of Fig. 3. The topological relationship within
the four horse sequences is identical with the result ofGeorge and Ryder (1986), while the branching pattern
among the six equine species of Fig. 4 corresponds totree c of Fig. 4 of George and Ryder (1986), which
required one additional change compared to the two
m a x i m u m parsimonious trees. Because the branch goingto the cluster of E. asinus (=E. africanus somalicus in
their nomenclature) and E. hemionus is quite short (seeFig. 4), tree c is not much different from trees a and b.
Four species (E. caballus, E. asinus, E. zebra, and E.
grevyi) are shared between trees of Figs. 3 and 4, but theunrooted topological relationship is different from eachother. E. asinus and E. grevyi are clustered in Fig. 3,
while E. zebra and E. grevyi are clustered in Fig. 4. The
bootstrap probability (73% and 59% for the neighbor-joining and the m a x i m u m parsimony trees, respectively)
for the branch clustering E. asinus and E. grevyi is nothigh in Fig. 3, and the standard errors of evolutionary
distances (Table 4) used to produce the tree of Fig. 4 are
relatively large. Apparently, more sequence data are necessary to resolve the branching order among different
Equus species.

Evolutionary Rates
The root of the phylogenetic tree shown in Fig. 3 was
located on the assumption of the molecular clock, and apoint within the branch going to E. zebra was chosen as
the root. If we consider E. zebra as the outgroup, anapproximate constancy seems to hold for the remaining
part of the tree of Fig. 3. Therefore, we estimated the rate(~,) of nucleotide substitution using the equation d = 2~,t,where d is the number of nucleotide substitutions per site
between a pair of sequences, and t is the divergence time.
Higuchi et al. (1987) considered 2.0-3.3 million years

186
horse

A•mQhuarter
Morgan horse
Przewalskii's wild horse
ian horses

E. asinus

E. hemionus
E. zebra

E. burchelli' 0.005't

E. grevyi

for the divergence between horse (E. caballus) and zebras (E. zebra and E. burchelli). In our tree of Fig. 3,
however, the lineage of E. zebra was considered to diverge first. Lindsay et al. (1980) suggested that the ancestral Equus species migrated from North America toEurasia at around 2.5-3.0 million years ago, and MacFadden (1992) wrote that "Equus was essentially monospecific in both North American and Eurasia during the
Pliocene." In any case, there is always uncertainty on thecorrespondence between the fossil record and the molecular data. Therefore, we used a wider, more conservative
range of divergence time. The time (t) of the root of thetree of Fig. 3 was assumed to correspond to 2-4 million
years ago. Because the largest divergence had a mean
evolutionary distance of 44 nucleotide substitutions
among the 269 sites (see Fig. 3), d = 0.164 (=44/269).
Thus the rate 0~ = d/2t) becomes approximately 2-4 x
10-8 per nucleotide site per year.
Higuchi et al. (1987) found about 5-7% difference
between horse and zebras for the coding regions of cytochrome oxidase I and NADH dehydrogenase I genes. If
we assume the same divergence time estimate (2--4 million years ago) used in the present study, the evolutionary rate for that region becomes approximately 0.6-1.8 x
10-8 per nucleotide site per year, lower than our estimate
for the D-loop region. (Note that the definition of thisevolutionary rate is different from Higuchi et al.'s (1987)"the mean rate of divergence,' ' - - t h a t is, twice this rate.)
This difference occurred apparently because of the functional differences of the coding and noncoding sequencesof mtDNA, and this supports the notion that the noncoding region mtDNA is more variable than its coding sequences (Aquadro and Greenberg 1982; Cann et al.
1984).
Reanalysis of the restriction-site data of George andRyder (1986) gives the estimate for the largest divergence to be 0.072 (Fig. 4). Therefore, the evolutionaryrate becomes 0.9-1.8 x 10.8 per nucleotide site per year,
assuming the divergence time range adopted in theFig. 4. A neighbor-joiningtree of thenine Equus mtDNA sequences based on
the evolutionarydistance matrix ofTable 4. Original data were from
George and Ryder (1986). E. asinus
and Przewalskii'swild horse are E.
africanus somalicus and E. przewalskii
in their nomenclature.

present study. This rate is again lower than that for the
D-loop region.
Tamura and Nei (1993) estimated the rate of nucleotide substitution for the entire control (D-loop) regionof human mtDNA to be 2.5-15 × 10-8 per site per year
(95% lower and upper bounds). Our estimate for equine
species (2-4 × 10-8) overlaps with that for human, but it
is smaller than the modal rate (7.5 x 10-8) for human(Tamura and Nei 1993). Because we used only a ca.
270-bp variable region out of the 1,100-bp control region
found by Ishida et al. (1994), it is possible that the substitution rate for the entire D-loop region of the equine
mtDNA may be much smaller than that for the human
mtDNA.
Under the tree of Fig. 3, we can map six insertions ordeletions on the 0.27-kb sequence data. (Gaps of E. zebra
and E. grevyi at position 47 [see Fig. 1] are considered tooccur independently according to Fig. 3.) Because thetotal number of nucleotide substitutions was 97 for thattree, it corresponds to 8.8-17.6 million years (=[97/22]/[2-4 million years]). Therefore, if we use method 1 of
Saitou and Ueda (1994), the rate of insertion and deletion
becomes 1.3-2.5 per kilobase per million year (=[6/0.27
kb]/[8.8-17.6 million years]). Interestingly, this is ingood agreement with the corresponding rate (ca. 2.0 per
kilobase per million year) for the primate noncoding
mtDNA (Saitou and Ueda 1994).

Emergence of the Przewalskii's Wild Horse Population
Finally, we would like to discuss the evolutionary historyof the Przewalskii's wild horse population. It is noteworthy that the lineage of the Przewalskii' s wild horse is not
located at the deepest branching among the E. caballus
sequences in either Fig. 3 or 4. This pattern contradicts
with the hypothesis that the domestic horse was derived
from the Przewalskii's wild horse lineage. In this context, Benirschke et al.'s (1965) hypothesis on the evolution of horse karyotype (see Introduction) should be re-187considered. If we accept the branching pattern of Figs. 3
and 4 regarding the position of the Przewalskii's wild
horse, its chromosome type (2n = 66) seems to be derived from the chromosome type (2n = 64) of the domestic horse.
Of course, a final clarification of this point waits formore accumulation of genetic polymorphism data of
Equus caballus. If this is the case, however, when did
this chromosomal change occur? The largest divergencewithin the six E. caballus sequences had a mean evolutionary distance of 11.5 nucleotide substitutions amongthe 271 sites (Fig. 2), and the d = 0.042 (=11.5/271).
Under the assumption of the molecular clock, the corresponding divergence time (t = d/2~) becomes approximately 0.5-1 million years ( = 0 . 0 4 2 / [ 4 - 8 x 10-8]). It isinteresting to note that this "coalescence time" for E.
caballus seems to be somewhat larger than that for human but comparable to that for common chimpanzee(Tamura and Nei 1993). In any case, emergence of thePrzewalskii's wild horse population should be much later
than this coalescence, according to the topology of thetree of Fig. 2. Therefore, the change of chromosome typeon the lineage of the Przewalskii's wild horse, probably
the increase of the chromosome number from 64 to 66,
seemed to happen within a half-million years. Such aswift change of chromosome number is in fact a common
feature for the genus Equus (Bush et al. 1977).
The above argument solely relies on the genealogy of
mtDNA. However, that may be different from the genealogy of nuclear DNA, as observed in Japanese wild
mice (Yonekawa et al. 1988). Przewalskii's wild horse
was clustered with the Mongolian native horse in the tree
of Fig. 2. Thus it is not excluded that a certain degree ofgene flow occurred between the ancestral populations of
the Przewalskii's wild horse and the Mongolian native
horse. In fact, a past distribution of the Przewalskii's
wild horse overlapped with the area where the Mongolian native horses are distributed now (Mohr 1971;
BOktnyiu 1974). It also should be mentioned that theJapanese native horses are considered to have derived
from the Mongolian native horses and have been kept as
breeds (Nozawa 1992). Therefore, detailed examinationof nuclear DNA at the molecular level is necessary toaccess our hypothesis on the emergence of the Przewalskii's wild horse population.

Acknowledgments. In collecting blood samples, we thank Masao
Saito of Tama Zoological Park in Tokyo, Yasunori Iwamoto of
YumemigasakiZoologicalPark in Kawasaki, and Eigi Taneda of The
Equestrian Park, JRA, in Tokyo. This study was partially supported by
a Grant-in-Aidfor Scientific Research on Priority Areas (Molecular
Evolution) of Ministry of Education, Science and Culture (Japan)
to N.S.

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