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MultiplematernaloriginsofnativemodernandancienthorsepopulationsinChina

ArticleinAnimalGenetics·October2009

DOI:10.1111/j.1365-2052.2009.01950.x·Source:PubMed

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doi:10.1111/j.1365-2052.2009.01950.x

Multiple maternal origins of native modern and ancient horsepopulations in China

C. Z. Lei*, R. Su†, M. A. Bower‡, C. J. Edwards§,1, X. B. Wang*, S. Weining¶, L. Liu**, W. M. Xie*,

F. Li*, R. Y. Liu††, Y. S. Zhang*, C. M. Zhang* and H. Chen*,‡‡

*Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology, Northwest A & F University,

Yangling, Shaanxi 712100, China. †Henan Veterinary Medicine Engineering and Technology Center, Luoyang, Henan 471000, China.‡Glyn Daniel Laboratory for Archaeogenetics, McDonald Institute for Archaeological Research, Downing Street, Cambridge CB2 3ER, UK.§Smurfit Institute of Genetics, Trinity College, Dublin 2, Ireland. ¶College of Agronomy, Northwest A & F University, Yangling, Shaanxi

712100, China. **Archaeology Program, La Trobe University, Melbourne, Vic. 3086, Australia. ††College of Animal Science and

Technology, Guizhou University, Guiyang, Guizhou 550025, China. ‡‡Institute of Cellular and Molecular Biology, Xuzhou Normal

University, Xuzhou, Jiangsu 221116, China

Summary To obtain more knowledge of the origin and genetic diversity of domestic horses in China, this

study provides a comprehensive analysis of mitochondrial DNA (mtDNA) D-loop sequence

diversity from nine horse breeds in China in conjunction with ancient DNA data and evi-

dence from archaeological and historical records. A 247-bp mitochondrial D-loop sequence

from 182 modern samples revealed a total of 70 haplotypes with a high level of genetic

diversity. Seven major mtDNA haplogroups (A–G) and 16 clusters were identified for the 182

Chinese modern horses. In the present study, nine 247-bp mitochondrial D-loop sequences of

ancient remains of Bronze Age horse from the Chifeng region of Inner Mongolia in China (c.

4000–2000a BP) were used to explore the origin and diversity of Chinese modern horses and

the phylogenetic relationship between ancient and modern horses. The nine ancient horses

carried seven haplotypes with rich genetic diversity, which were clustered together with

modern individuals among haplogroups A, E and F. Modern domestic horse and ancient

horse data support the multiple origins of domestic horses in China. This study supports the

argument that multiple successful events of horse domestication, including separate intro-

ductions of wild mares into the domestic herds, may have occurred in antiquity, and that

China cannot be excluded from these events. Indeed, the association of Far Eastern mtDNA

types to haplogroup F was highly significant using Fisher�s exact test of independence

(P = 0.00002), lending support for Chinese domestication of this haplogroup. High diversity

and all seven mtDNA haplogroups (A–G) with 16 clusters also suggest that further work is

necessary to shed more light on horse domestication in China.

Keywords archaeology, Chinese horse, haplogroup, mitochondrial DNA D-loop, origin.

Introduction

The origin of the domestic horse has been a focus of

multi-disciplinary research for decades, but conclusions are

controversial. While osteoarchaeological data indicate a

restricted area in Central Asia for the earliest horse

domestication, genetic studies show widespread origins.

Horses have played a significant role in China�s political,

economic and military history, but the origin of domesti-

cated horses in China remains unclear. Literary sources

describe the importation of horses into China from different

parts of the world throughout history (Xie 1987), a situa-

tion that could have led to a complex mosaic pattern of

horse breeds. To explore this complexity, this study provides

a comprehensive analysis of mitochondrial DNA (mtDNA)

D-loop sequence diversity from nine native horse breeds in

China. The results of this genetic analysis are put into

Address for correspondence

H. Chen, Shaanxi Key Laboratory of Molecular Biology for Agriculture,

College of Animal Science and Technology, Northwest A & F University,

Yangling, Shaanxi 712100, China.

E-mail: chenhong1212@263.net

1Present address: RLAHA, University of Oxford, Dyson Perrins Building,

South Parks Road, Oxford OX1 3QY, UK

Accepted for publication 10 June 2009

� 2009 The Authors, Journal compilation � 2009 Stichting International Foundation for Animal Genetics, Animal Genetics, 40, 933–944 933

context within a framework of ancient DNA, archaeological

and historical records. As the evidence for horse domesti-

cation is complex and ephemeral, multiple lines of evidence

from interdisciplinary research need to be integrated to

achieve a better understanding of the origins of the domestic

horse in China.

Archaeological and historical background

Wild horse (Equus ferus Boddaert, 1785) remains occur

widely in Late Palaeolithic sites in China, particularly in the

north. Small numbers of horse bones have also been found

at some Neolithic sites (Olsen 1988; Yuan & Flad 2006),

although it is not possible, based on morphology alone, to

assign them as domestic or wild. Based on our current

understanding of the available archaeological evidence,

horses were almost certainly first domesticated in Central

Asia some 5000 years ago and later introduced to China

(Levine et al.2003; Mashkour 2006). However, it is essen-

tial to note that the archaeological record for these regions

is sparse and limited, and archaeozoological data collection

and analysis are rare.

Within China, horse remains began to be more common

throughout the Bronze Age (2nd and 1st millennia BC). Horse

remains, incorporated in human burials, have been reported

from almost all the Bronze Age Qijia culture sites in the Upper

Yellow River region, northwest China, dating around

2000 BC. Such mortuary practice has been interpreted as

evidence of horse domestication; however, this is inconclu-

sive. If this claim is plausible, domestic horses were used only

for limited purposes, primarily for food and burial offerings

(Linduff 2003; Yuan & Flad 2003; Flad et al. 2007).

In the Middle and Lower Yellow River regions, domesti-

cated horses did not appear in the archaeological record

until the Late Shang period (c. 1300–1046 BC) (Linduff

2003; Yuan & Flad 2005, 2006). If these domestic horses

were imported into China, it is not clear through which

routes they came at this time, nor is it certain if the Qijia

horses found in the Upper Yellow River were related to those

from the Late Shang period. Horses may have been intro-

duced together with chariots to the Central Plains, and were

primarily symbols of status. For example, large numbers of

horses appeared as prestige sacrificial items in the Shang

royal ritual ceremonies at the capital city in Anyang,

Henan, whereas similar horse sacrifices at small scales were

also practised in other areas, such as Laoniupo in Shaanxi

and Qianzhangda in Shandong (Linduff 2003; Yuan & Flad

2005, 2006). It seems that horses had special relationships

with the Shang royal elite at this time, as oracle-bone

inscriptions recorded that even the Shang king fed horses in

the stables (Linduff 2003; Yuan & Flad 2005, 2006).

The practice of using horses and chariots in elite burials

continued during the next millennium, but diminished as

the millennium drew to a close. At the same time, however,

against the background of the development of equine

pastoral nomadism, horses gained an increasingly impor-

tant military role throughout the dynastic eras in Chinese

history, as demonstrated in the archaeological record and

ancient documents (Yuan & Flad 2005, 2006). In the

Western Zhou period (1045–771 BC), raising good breeding

horses was an important task, which was sometimes

directly overseen by the kings. It is believed that the

ancestors of the Qin dynasty specialized in raising horses for

the Western Zhou kings in the northwestern region of

China (Hsu & Linduff 1988). During the Spring and

Autumn period (770–476 BC), the military power of a state

was often measured by the number of horse-chariot units it

possessed. Early evidence for horseback riding in East Asia,

as depicted on a buckle-like bronze fitting, is from the Upper

Xiajiadian culture (1000–300 BC) in Inner Mongolia (Inner

Mongolia Team 1975). Numerous life-sized terracotta

horses along with chariots and soldiers arranged in military

formations were entombed in the Qin (221–207 BC) Em-

peror�s mausoleum near Xi�an, to show the political, mili-

tary and symbolic power of the Emperor. The demand for

the horses increased dramatically during the Han dynasty

(206 BC–AD 220) as the Chinese heartland was constantly

facing military threats from mounted nomads in the

northern steppes. The Eurasian steppes were seen as the

region where the finest horses were produced. Throughout

the Chinese history, dynastic rulers adopted various

strategies, such as marriage, warfare, tribute and trade, to

ensure a plentiful supply of horses from this region. In the

late Tang dynasty (around the 9th century AD), for example,

China paid the exorbitant price of a million bolts of silk per

year for 100 000 steppe horses (Mair 2003).

In south China, the earliest evidence for domesticated

horses is from bronze figures from the Dian culture (4th

century BC–AD 1st century) in Yunnan (Zhang 1998). Horses

were depicted as being of short built and as being used

primarily as a riding animal by the elite. Based on their

stature and morphology, these horses may have been related

to Mongolian ponies from the Central Asiatic steppes

(Chiou-Peng 2004). Starting in the Tang dynasty, horses

became an indispensable commodity in the ancient trade

system known as chama gudao, the Ancient Tea-Horse Road,

in south-west China, mainly involving the bilateral trade

between Tibet (producing horses) and the Yunnan-Sichuan

region (producing tea). The horses from Yunnan, which

were originated in other areas, became highly desirable for

the imperial court in the Central Plains (Zhang 1998, 2006).

From an economic perspective, the horse has been an

essential component of Chinese society because of its

importance for riding, draught and transportation. It was

ranked at the top of the six traditional domestic animals

(horse, pig, cattle, sheep and goat, dog and chicken) (Xie

1987). However, the demand for horses for transport and

agriculture has rapidly decreased in recent decades and as a

result, some horse breeds are today on the brink of extinc-

tion. Nevertheless, the horse is still an important source of

� 2009 The Authors, Journal compilation � 2009 Stichting International Foundation for Animal Genetics, Animal Genetics, 40, 933–944

Lei et al.934

animal power in mountainous and pastoral regions in

northern and south-western parts of China such as Xinji-

ang, Tibet, Inner Mongolia, Gansu, Qinghai, Yunnan and

Guizhou.

As a result of the limited findings of early horse remains

and because of the long history of transporting and trading

horses across the vast area of China, the origins and early

history of domesticated horses in China remain a mystery.

Population genetics can, therefore, offer an important line of

evidence to help us address this question.

Genetic research

Mitochondrial DNA D-loop sequence diversity has been an

important tool for understanding the origin of domesticated

horses (Kim et al. 1999; Lister et al. 2001; Vila et al. 2001;

Hill et al. 2002; Jansen et al. 2002; Cozzi et al. 2004; Royo

et al. 2005; Luıs et al. 2006; McGahern et al. 2006a;

Glazewska et al. 2007). Vila et al. (2001) suggested that the

mtDNA data from domesticated horses fell into six different

maternal haplogroups (A–F). Jansen et al. (2002) indicated

that seven maternal haplogroups (A–G) could additionally

be grouped into 17 distinct phylogenetic clusters. McGahern

et al. (2006a) also defined two new clusters A7 and F3, so

that the seven haplogroups (A–G) were further grouped into

19 distinct phylogenetic clusters. These results are generally

regarded as evidence for multiple occurrences of horse

domestication. However, the mtDNA results can also be

explained as mares from multiple separate lineages

contributing to the modern genetic pool. Levine (2006)

has suggested that as the original domesticated population

expanded, horses from wild populations were introduced

into the domestic herds over time.

China has rich horse genetic resources; the number of

Chinese domestic horses was 7.639 million in 2004 (Statistic

report in China, 2004). Horses are widespread throughout

14 provinces of North-west, South-west and North-east

China, in addition to Hubei province of Central China. Fifteen

native domestic horse breeds remaining as several separate

populations are recorded in China today (Hendricks 1995).

Based on the origin, habitats and body size, the 15 Chinese

breeds can be grouped into five horse types: Mongolian,

South-western, Hequ, Kazahk and Tibetan (Xie 1987). In

addition, China has pony or small horse populations dis-

tributed mainly in the Shaanxi, Sichuan, Guangxi, Guizhou

and Yunnan provinces. In China, an adult Equus caballus

individual with a height at the withers less than or equal to

106 cm is classified as a pony, while the one with a height

greater than 106 cm is called a common horse (Xie 1987).

Despite the presence of diverse native horse breeds and

populations, research on equine genetics is not well devel-

oped in China and only a limited number of reports on the

mtDNA diversity of Chinese domestic horses are available.

Previously, mtDNA of three short horses and three common

horses in Yunnan was digested by 20 restriction endonuc-

leases and results indicated high genetic diversity and

multiple maternal origins (Wang et al. 1994). mtDNA

D-loop sequences have also been analysed for four Mongo-

lian individuals, seven Tibetan individuals and 10 Guan-

shan individuals (Mang et al. 2005; McGahern et al. 2006a;

Xu et al. 2007). Furthermore, a 262-bp mtDNA D-loop

sequence from nine Chinese ancient horse remains indicates

multiple maternal origins (Cai et al. 2007a). Modern and

ancient mtDNA sequence analysis has provided critical

evidence on the origins and dispersal routes of horse, pig,

cattle, goat and sheep (Jung et al. 2002; Anderung et al.

2005; Keyser-Tracqui et al. 2005; Larson et al. 2005,

2007a,b; Beja-Pereira et al. 2006; Fernandez et al. 2006;

McGahern et al. 2006b; Cai et al. 2007b). In this study,

mtDNA D-loop sequences of 182 animals (48 from Gen-

Bank) from nine modern Chinese domestic horse breeds and

of nine previously published ancient horse specimens were

analysed to cast new light on the origin and genetic diver-

sity of domestic horses in China.

Materials and methods

Specimen collection and DNA extraction

All 134 blood samples representing five local domestic horse

breeds were collected in China. The genomic DNA was

extracted from blood by a standard phenol–chloroform

method. In addition, 48 mtDNA D-loop sequences from

five Chinese local domestic horse breeds were available from

GenBank (AF014413–AF014417, AF056071, DQ297622–

DQ297633, DQ297634–DQ297635, DQ297637–DQ297638,

DQ327838–327847, DQ986464–DQ986479) and nine mtDNA

D-loop sequences from Bronze Age Chinese horse remains were

also obtained from GenBank (DQ900922–DQ900930). The

distributions of all nine Chinese domestic horse breeds and the

sites of the ancient horse remains are shown in Fig. 1a.

Geographical locations and number of horse samples are shown

in Table 1.

PCR amplification and sequencing

To amplify the 247-bp hypervariable region I sequence of

the equine mtDNA D-loop region (McGahern et al. 2006a)

between positions 15 494 and 15 740 of reference

sequence X79547 (Xu & Arnason 1994), a pair of primers

were used according to the published sequences, HA:

5¢-AGTCTCACCATCAACACCCAAAGC-3¢ and HB: 5¢-CCT

GAAGTAGGAACCAGATG-3¢ (Ivankovic et al. 2002). PCR

amplifications were conducted in a 50-ll volume containing

5 ll of 10· buffer, 1.5 mM MgCl2, 0.25 mM dNTPs, 0.2 lM

each primer, 1.5 U Taq DNA polymerase (TaKaRa Biosys-

tems) and approximately 50 ng genomic DNA. The PCR

was carried out using a standard program with 4 min

denaturation at 95 �C, 35 cycles for 30 s at 94 �C, 60 s at

55 �C, and 90 s at 72 �C, and final extension for 10 min at

� 2009 The Authors, Journal compilation � 2009 Stichting International Foundation for Animal Genetics, Animal Genetics, 40, 933–944

The origins of Chinese horses 935

72 �C. PCR products were purified using a Watson PCR

Purification Kit (Watson BioTechnologies) according to the

manufacturer�s instructions. Sequencing was performed

using an ABI model 377 automated sequencer (PE Applied

Biosystems).

Statistical analysis

All 182 mtDNA D-loop sequences determined in this study

were edited using the DNASTAR 5.0 package (DNASTAR).

All mtDNA D-loop sequences were aligned with the

CLUSTALX package (Thompson et al. 1997) and truncated

to 247 bp. Insertions/deletions in the aligned sequences

were excluded from the analyses. Identical sequences were

considered as the same haplotype. Haplotype diversity (h)

and nucleotide diversity (p) for breed samples with sample

size larger than five were estimated using DNASP 4.0

software (Rozas et al. 2003). Mismatch distribution

was generated using the NETWORK 4.1 program

(http://www.fluxus-engineering.com). All sequences were

(a)

(b)

(c)

Figure 1 (a) Geographical and mitochondrial

DNA (mtDNA) haplogroup distributions in

Chinese horses, see Table 1 for details.

Geographical locations for Chinese horse

populations are denoted by the position of the

individual pie charts. The size of each circle is

proportional to the number of sequences

sampled from that region. The colour indicates

haplogroup (Vila et al. 2001). (b) Mitochon-

drial DNA haplogroup distributions in global

horse populations (BI, n = 264, EUR, n = 646;

MEA, n = 248, FE, n = 380). The size of each

circle is proportional to the number of

sequences sampled from that region. (c) A

median joining phylogenetic network

constructed for 1538 horse mtDNA sequences

using 247 bp of the control region to show the

relationships within Eurasian horse populations

of the main haplogroups, A–G, and their sub-

types. Circles represent sequence haplotypes,

the area being proportional to the frequency of

the haplotype. The positions of the new

Chinese horse mtDNA sequences are shown as

purple pie slices.

� 2009 The Authors, Journal compilation � 2009 Stichting International Foundation for Animal Genetics, Animal Genetics, 40, 933–944

Lei et al.936

Tab

le1

Sourc

ean

dgen

etic

div

ersi

tyin

dex

of

the

Chin

ese

hors

esa

mple

s.

Bre

eds

Codes

Geo

gra

phic

loca

tion

nk

Clu

ster

(no.

sam

ple

)H

aplo

gro

up

Hap

loty

pe

div

ersi

ty

Nucl

eotide

div

ersi

tySo

urc

e

Nin

gqia

ng

NQ

Shaa

nxi

27

16

A1(2

),A

4(2

),A

5(2

),A

6(9

),

B2(1

),D

2(2

),D

3(1

),F1

(1),

F2(4

),F3

(3)

A,B

,D,F

0.9

43

±0.0

27

0.0

236

±0.0

096

This

study

Kaz

ahk

KK

Xin

jiang

18

15

A4(2

),A

6(4

),A

7(1

),B2(1

),

C1(2

),D

1(2

),D

2(1

),D

3(2

),

F1(1

),F2

(1),

G(1

)

A,B

,C,D

,F,

G0.9

80

±0.0

24

0.0

245

±0.0

136

This

study

Guiz

hou

GZ

Guiz

hou

62

26

A3(2

),A

4(1

4),

A5(1

),A

6(4

),

A7(2

),B2(1

),C

1(3

),D

2(1

1),

D3(1

2),

E(2),

F1(1

),F2

(3),

F3(4

),G

(2)

A,B

,C,D

,E,

F,G

0.9

26

±0.0

19

0.0

229

±0.0

087

This

study

Guan

zhong

GU

Shaa

nxi

27

9A

5(1

),A

7(1

),C

1(9

),D

2(6

),

D3(1

),F1

(7),

G(2

)

A,C

,D,F

,G0.8

42

±0.0

41

0.0

219

±0.0

076

This

study

Mongolia

MG

Inner

Mongolia

19

18

A1(4

),A

4(1

),A

6(2

),B2(3

),

D2(2

),F1

(4),

F3(3

)

A,B

,D,F

0.9

94

±0.0

19

0.0

293

±0.0

128

Gen

Ban

kA

F014413–A

F014415,

AF0

56071,

Q291622–D

Q291633,

DQ

297635,

DQ

297637,

DQ

297638

Tib

etTB

Tib

et16

14

A1(2

),A

6(3

),A

7(1

),B2(2

),

D2(3

),E(

3),

F3(2

)

A,B

,D,E

,F0.9

83

±0.0

28

0.0

261

±0.0

105

Gen

Ban

kD

Q986464–D

Q986479

Guan

shan

GS

Shaa

nxi

10

8A

4(1

),A

6(1

),D

1(1

),D

2(2

),

D3(2

),F2

(1),

F3(1

),G

(1)

A,D

,F,G

0.9

56

±0.0

59

0.0

244

±0.0

118

Gen

Ban

kD

Q327838–D

Q327847

from

McG

aher

net

al.

(2006a)

Yunnan

YN

Yunnan

21

D2(2

)D

//

Gen

Ban

kA

F014416–A

F014417

Guan

gxi

pony

GX

Guan

gxi

11

A6(1

)A

//

Gen

Ban

kD

Q297634

Chin

ese

anci

ent

hors

eC

AIn

ner

Mongolia

97

A1(1

),A

3(1

),A

7(3

),E(

1),

F1(1

),F2

(1),

F3(1

)

A,E

,F0.9

17

±0.0

92

0.0

243

±0.0

119

Gen

Ban

kD

Q900922–D

Q900930

n,

sam

ple

size

;k,

num

ber

of

hap

loty

pes

.

� 2009 The Authors, Journal compilation � 2009 Stichting International Foundation for Animal Genetics, Animal Genetics, 40, 933–944

The origins of Chinese horses 937

deposited in Genbank (accession nos AY136785–

AY136786, EU203727–EU203858).

In addition, the 247-bp region was examined in a total

dataset of 1538 horses (the 134 new sequences recorded

here and 1404 GenBank accessions) sampled from 119

populations from three broad geographical regions: Europe

(EUR), Middle East and Africa (MEA), and the Far East (FE).

Diversity values were estimated using the ARLEQUIN program

(Schneider et al. 2000) and an analysis of molecular vari-

ance (AMOVA) was performed to investigate the partitioning

of mtDNA molecular variance among these three geo-

graphical groups. To test the concordance of haplogroup

F with Far Eastern populations, Fisher�s exact test of inde-

pendence (Sokal & Rohlf 1995) was performed in a 2 · 2

contingency table featuring EUR/MEA populations vs. FE

populations, with counts of haplogroup F vs. non-haplo-

group F sequences. Median joining networks (Bandelt

et al.1999) of the total dataset and subsets of the data

according to the geographical regions defined above were

drawn in NETWORK 4.1. Star contraction pre-processing was

performed (Forster et al.2001). Parameters and down

weighting of hypervariable sites were according to Jansen

et al. (2002). Maximum parsimony calculations were per-

formed on the output networks (Polzin & Daneschmand

2003). Haplotype data were plotted geographically using

ARCGIS 9 (Esri).

Results

Sequence variation

A 247-bp equine mtDNA D-loop region sequence was

examined in 182 Chinese domestic horses. Seventy mtDNA

haplotypes were identified and defined by 47 polymorphic

nucleotide sites: 43 transitions, two transversions and two

coexistence sites of transition and transversion, suggesting

high polymorphisms and a strong bias towards transitions

(Table 2). The mismatch analysis performed for Chinese

horse breeds (Fig. 2) shows a monomodal distribution with

a major peak at six mutational differences. Sequence dif-

ferences ranged from one to 14 base pairs. Among the 70

haplotypes, 27 haplotypes (38.6%) were detected more than

once and were thus shared by individuals among breeds or

within breeds. The largest haplotype consisted of 18 indi-

viduals, with another two haplotypes containing 15 and 12

individuals respectively, and 23 haplotypes possessing two

to nine individuals each. Forty-three haplotypes (61.4%)

were singletons: 10 (23.3%) from the Guizhou breed, nine

(20.9%) from the Mongolia breed, eight (18.6%) from the

Kazahk breed, seven (16.3%) from the Tibetan breed, four

(9.3%) from the Guanzhong breed, three (7.0%) from the

Ningqiang breed and two (4.6%) from the Guanshan breed.

The number of haplotypes identified in each breed (except

the Yunnan and Guangxi breeds) ranged from eight to 26,

and haplotype diversity values ranged from 0.842 ± 0.041

in the Guanzhong breed to 0.994 ± 0.019 in the Mongolia

breed. Similarly, the Mongolia breed showed the highest

nucleotide diversity value (0.02926 ± 0.01282), whereas

the Guanzhong breed displayed the lowest nucleotide

diversity value (0.02194 ± 0.00763), indicating abundant

genetic diversity in Chinese domestic horse breeds (Table 1).

The Chinese breed nucleotide diversity values compare

favourably with those estimated from the broad geo-

graphical regions (EUR = 0.029123 ± 0.01511; MEA =

0.02576 ± 0.01356; FE = 0.02594 ± 0.01363).

Nine ancient DNA sequences of 247 bp of the D-loop

region cited from GenBank carried seven haplotypes. Three

(DQ900922, DQ900923, DQ900925) out of these nine

ancient sequences shared the same haplotype. Interestingly,

one modern haplotype was identical to the ancient sequence

DQ900929, one to DQ900924, and another to DQ900927.

The nine ancient sequences were differentiated by a total of

17 polymorphic nucleotide sites, all of which were transi-

tions. These nine ancient DNA sequences were shown to

display high level of haplotype diversity (0.917 ± 0.092)

and nucleotide diversity (0.0243 ± 0.0119) (Table 1).

Phylogenetic analysis

Previous research grouped equine mtDNA haplotypes into

seven major haplogroups (A–G) (Vila et al. 2001; Jansen

et al. 2002) and 19 clusters (Jansen et al. 2002; McGahern

et al. 2006a). mtDNA haplotypes in Chinese domestic

horses demonstrated the presence of these seven major

haplogroups (A–G) that included 16 out of the 19 clusters,

except clusters A2, B1 and C2 (Fig. 1a,b and Table 1). A, D

and F were the predominant haplogroups, comprising

35.7%, 18.6% and 22.9% of the 70 haplotypes respectively,

whereas haplogroups B, C, E and G comprised 7.1%, 4.3%,

7.1% and 4.3% respectively. Furthermore, among the 182

individuals, haplogroups A–G showed similar distribution

patterns (Table 3). The results indicated that the majority

of sequences in Chinese domestic horse breeds belong to

haplogroups A, D and F. Different horse breeds displayed

distinctive profiles of the previously reported seven major

haplogroups A–G and only 16 of the 17 clusters (Vila et al.

2001; Jansen et al. 2002) (Table 1). At least four major

haplogroups and seven clusters were observed in each of the

seven Chinese domestic horse breeds. All of the seven major

haplogroups in addition to 14 clusters were observed in the

Guizhou breed (Table 1). These data are consistent with

multiple maternal origins in living native Chinese domestic

horses.

Mitochondrial data from the living Chinese horse popu-

lations were grouped with 1404 previously published

sequences. A median-joining network of the complete

dataset (1538 horses) was drawn to determine the rela-

tionships of the seven main haplogroups, A–G (Fig. 1c,

purple pie slices represent the relative positions of the new

Chinese sequences). The network confirms the position of

� 2009 The Authors, Journal compilation � 2009 Stichting International Foundation for Animal Genetics, Animal Genetics, 40, 933–944

Lei et al.938

Tab

le2

Var

iable

nucl

eotide

posi

tions

ina

247-b

pre

gio

nof

the

mitoch

ondrial

D-l

oop

of

Chin

ese

nat

ive

hors

esco

mpar

edw

ith

refe

rence

sequen

ceG

enBan

kac

cess

ion

num

ber

X79547.

Gen

ban

k

acce

ssio

n

num

ber

Var

iable

nucl

eotide

posi

tion

Haplotype

15494

15495

15496

15507

15514

15521

15525

15526

15528

15534

15538

15540

15542

15585

15595

15596

15597

15598

15600

15602

15603

15604

15615

15616

15617

15635

15649

15650

15651

15653

15659

15666

15667

15700

15703

15709

15720

15722

15726

15728

15735

15737

15740

X79547

TT

AC

–G

AT

CC

AA

CG

AA

AT

GC

TG

AA

TC

AA

GA

TG

AC

TC

GC

GT

GT

An

Freq

uen

cy

AY

136785

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C.

..

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10.0

07

AY

136786

C.

C.

..

..

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..

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11

0.0

82

EU203728

A.

C.

..

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A.

..

..

T.

..

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G.

A.

..

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..

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10.0

07

EU203730

F.

C.

..

..

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.G

..

T.

..

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50.0

37

EU203732

DC

CG

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A.

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70.0

52

EU203735

C.

C.

..

.-

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..

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..

..

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T.

..

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10.0

07

EU203737

C.

C.

T.

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10.0

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EU203739

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10.0

07

EU203751

D.

C.

..

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T.

..

A.

..

..

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..

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..

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.A

..

..

..

10.0

07

EU203752

B.

C.

..

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A.

..

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T.

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10.0

07

EU203753

A.

C.

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..

..

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T.

..

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..

10.0

07

EU203754

A.

C.

..

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A.

..

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70.0

52

EU203755

A.

C.

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15

0.1

12

EU203756

A.

C.

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10.0

07

EU203757

DC

CG

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A.

..

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TC

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60.0

45

EU203759

A.

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10.0

07

EU203767

A.

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30.0

22

EU203768

DC

CG

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50.0

37

EU203776

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C.

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10.0

07

EU203778

A.

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A.

..

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30.0

22

EU203779

F.

C.

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10.0

07

EU203781

E.

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10.0

07

EU203782

DC

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20.0

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EU203785

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10.0

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EU203787

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70.0

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30.0

22

� 2009 The Authors, Journal compilation � 2009 Stichting International Foundation for Animal Genetics, Animal Genetics, 40, 933–944

The origins of Chinese horses 939

Tab

le2

Continued

.

Gen

ban

k

acce

ssio

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num

ber

Var

iable

nucl

eotide

posi

tion

Haplotype

15494

15495

15496

15507

15514

15521

15525

15526

15528

15534

15538

15540

15542

15585

15595

15596

15597

15598

15600

15602

15603

15604

15615

15616

15617

15635

15649

15650

15651

15653

15659

15666

15667

15700

15703

15709

15720

15722

15726

15728

15735

15737

15740

EU203795

AA

C.

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EU203804

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10.0

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50.0

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10.0

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EU203852

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10.0

07

� 2009 The Authors, Journal compilation � 2009 Stichting International Foundation for Animal Genetics, Animal Genetics, 40, 933–944

Lei et al.940

the two new haplogroups A7 and F3 observed by McGahern

et al. (2006a). In addition, the data were partitioned into

regional subsections: Far East (FE), including the newly

sequenced populations, Middle East and African (MEA),

European (EUR) and British Isles (BI) sequences. Individual

median joining networks were then constructed (data not

shown, Fig. 1b). In each case, a majority of sequences were

found in haplogroups A and D. Haplogroups A and F were

found more frequently in Eastern populations (FE:

A = 47%, F = 17%; MEA: A = 40%, F = 15%; EUR:

A = 35%, F = 9%; BI: A = 26%, F = 0%), while a relatively

small proportion of Far Eastern sequences were found in

haplogroup D (17%) compared with other geographical

regions (MEA: 26%, EUR: 34%, BI: 34%). Geographical

mapping of the regional haplotype data clearly demon-

strates the increase of haplogroup F in a west–east direction,

and a decrease of haplotype D in an east–west direction

(Fig. 1b). Comparing three broad geographical regions of all

Old World horses (EUR, MEA and FE), an AMOVA analysis

found the majority of sequence diversity to be within pop-

ulations (97.89%, P = 0.00000). A Fisher�s exact test

(Sokal & Rohlf 1995) established that this concordance of

haplogroup F with the Far Eastern (FE) population origin

was highly significant (P = 0.00002).

Nine ancient DNA sequences were compared with 70

haplotypes from Chinese native horses. The results showed

that the nine ancient DNA sequences were similar to

modern day equine mtDNA, clustering in haplogroups A, E,

and F (Fig. 1a). Furthermore, the nine ancient DNA

sequences were grouped into clusters A1 (DQ900928), A3

(DQ900926), A7 (DQ900922, DQ900923, DQ900925), E

(DQ900927), F1 (DQ900924), F2 (DQ900929) and F3

(DQ900930) based on the known 19 clusters for equine

mtDNA (Table 1).

Discussion

Using mtDNA analysis, 70 different haplotypes were found

in Chinese native horses. These 70 haplotypes could be

grouped into all of the seven major haplogroups and 16 out

of the 19 clusters defined by Vila et al. (2001), Jansen et al.

(2002) and McGahern et al. (2006a). The results demon-

strate that living Chinese native horses possess abundant

mtDNA diversity and may have multiple maternal origins.

Our results are in accordance with those obtained for

Yunnan and Guanshan horses, which indicate high genetic

diversity (Wang et al. 1994; McGahern et al. 2006a). The

Guanzhong breed shows the lowest haplotype and nucleo-

tide diversity (Table 1). Only four singleton haplotypes were

identified in this breed, which is the lowest among the

Chinese breeds, except for the Yunnan and Guangxi breeds,

of which too few individuals have been sampled for com-

parison. This may be partly because all the individuals of

the Guanzhong breed were raised on one farm (Horse

Genetic Resource Conservation Farm) and probably had close

genetic relationships among them. However, the Guanzhong

breed has recently undergone a severe bottleneck. Today, the

total number of individuals in the Guanzhong breed is no

more than 50 (data not shown). Although the Ningqiang

breed appears more genetically diverse than the Guanzhong

and Guizhou breeds (Table 1), two different populations were

sampled for this study (including common horses and short

horses). The Ningqiang breed has undergone a similar recent

bottleneck. The number of individuals of the Ningqiang breed

is reduced to 250, with only 20 short horses left (data not

shown). Therefore, it is not surprising that these endangered

breeds (Guanzhong and Ningqiang) demonstrate the lowest

genetic diversity.

Of the seven Chinese horse breeds analysed, the Kazahk,

Tibet, and Mongolia breeds displayed abundant genetic

diversity (Table 1). The Kazahk, Tibet and Inner Mongolia

regions have vast, widespread grasslands, where large-scale

animal husbandry is still sustained. It appears natural that

higher genetic diversity is found in horses from these areas,

although more extensive sampling would be needed to

confirm this. The Guizhou breed, however, was shown to

have all seven major haplogroups, although this may be a

misrepresentation as the sample size for the Guizhou breed

was greater compared to other Chinese breeds (Fig. 1a).

Table 3 Haplogroup frequency of the Chinese modern horse samples.

Haplogroups

No.

haplotypes

Frequency

of haplogroups1

(%)

No.

samples

Frequency of

haplogroups2

(%)

A 25 35.7 63 34.6

B 5 7.1 8 4.4

C 3 4.3 14 7.7

D 13 18.6 50 27.5

E 5 7.1 5 2.7

F 16 22.9 36 19.8

G 3 4.3 6 3.3

1, number of haplotypes/total haplotypes; 2, number of samples/total

samples.

Figure 2 Mismatch distribution of mitochondrial DNA sequences in

Chinese horses.

� 2009 The Authors, Journal compilation � 2009 Stichting International Foundation for Animal Genetics, Animal Genetics, 40, 933–944

The origins of Chinese horses 941

Other native horse breeds in China might have similar

genetic diversity levels if sufficient samples were sequenced.

McGahern et al. (2006a), in their study of 962 horses

from 72 populations, revealed a significant biogeographical

association of eastern equine mtDNA types with haplogroup

F and a significant association of European equine mtDNA

types with haplogroup D. Using an increased dataset of

1538 horses from 119 populations, we confirmed that

haplotype F occurred more frequently in Eastern popula-

tions (17%) when compared with other Eurasian popula-

tions (MEA 15%, EUR 9%, BI 0%), whereas a relatively

small proportion of Far Eastern sequences were found in

haplogroup D (17%) compared with other geographical

regions (MEA 26%, EUR 34%, BI 34%). This biogeograph-

ical association of Far Eastern equine mtDNA types with

haplogroup F was highly significant (P = 0.00002), sug-

gesting that there has been a degree of genetic isolation and

differentiation of eastern and western horse populations and

lending support to the idea that haplogroup F may have

been taken into the domestic gene pool in the Far East.

Based on our data, we cannot exclude the possibility that

an early horse domestication event may have taken place

independently in China. Neither can we conclusively dem-

onstrate that the post-domestication inclusion of native

Chinese wild mares into the domestic horse breeding pop-

ulation is responsible for the patterns we observed in our

data. It is probable that multiple processes, which we may

never disentangle, have resulted in the observed modern

haplotype descriptions. For example, it is likely that the

constant procurement of horses from many regions by the

imperial court and the high mobility of horses and their

military utilization in past millennia, as described above,

resulted in considerable mixing of horse populations (Xie

1987), including input from native Chinese breeds. This

could result in the abundant genetic diversity we observed

in our data.

We compared our living native horse genetic data with

sequences from ancient horses dating to the Bronze Age

(c. 4000–2000a BP) from the Chifeng archaeological sites in

Inner Mongolia, northeast China (Cai et al. 2007a).

The majority of ancient samples (five) were shown to

possess haplogroup A, whereas haplogroup E was found in

one specimen (Table 1). Three ancient samples were char-

acterized by mtDNA haplogroup F, which was strongly

associated with Eastern breeds (McGahern et al. 2006a).

This demonstrates that haplotypes A, E and F were present

in horse populations in China between 4000 and 2000 BP.

However, without additional analyses (e.g. stable isotope

analysis), it is not possible to state whether the Bronze Age

horses were Chinese native horses, or whether they had

been imported from a distance.

Unlike the situation for many other large domesticated

animal species that demonstrate remarkably clear geo-

graphical patterning (MacHugh & Bradley 2001), caution

must be used when inferring population structure from

equine mtDNA data. Discrete horse populations share

mtDNA sequence with other populations despite geo-

graphical distance (McGahern et al. 2006b). For example, in

the Chifeng Bronze Age horses, the most common sequence

haplogroup (five out of nine individuals) was also the most

common globally, with 38% out of 1538 sequences repre-

sented in haplogroup A. The other two haplotypes repre-

sented in the Bronze Age data are of interest, as they are

more rare, with haplotype F occurring at 11% of the total

global population sequenced to date, and more significantly

with haplotype E, occurring at only 3% in living horse

populations. The likelihood of randomly sampling haplotype

E from a wild population is extremely low; therefore, it is

probable that haplotype E was more commonly occurring in

past populations.

Conclusions

Archaeological and osteological evidence has, thus far,

pointed to Central Asia as the region where the earliest

horse domestication took place, but genetic data, including

those in this study, show that the domestication of horses

was not a single discrete event. Multiple horse domestica-

tion events (i.e. sampling and re-sampling of wild horse

populations) may have occurred later in other regions

including China. Far Eastern domestic horses, like those in

Eurasia, originated from a diverse founder population,

sampled during multiple domestication events and/or via

trade and exchange over short and/or long geographical

distances, or else resulting from repeated post-domestication

resampling of native wild mare populations. Our data

demonstrate that much of this genetic diversity has been

preserved in domestic native horse populations across Chi-

na, particularly in the Inner Mongolian and south-western

regions. This finding is consistent with the scenario attested

in the archaeological and historical records that horses were

continuously brought into dynastic centres from the

northern steppes and south-western China. It is hoped that

further archaeogenetic study of ancient horse remains

within a multidisciplinary approach (including archaeozo-

ology and bioarchaeology) will provide more insights into

the origins and history of domestic horses in China.

Acknowledgements

This work is supported by Programme for New Century

Excellent Talents in University of 2007 (NCET-07-0699),

Research Program of Northwest A&F University (07ZR004).

MAB is supported by the Leverhulme Trust and the Isaac

Newton Trust; CJE is supported by the Irish Research

Council for Science Engineering and Technology (Basic

Research Grant Scheme project number SC/202/510).

Special thanks to Dr E.W. Hill (University College Dublin,

Ireland), Dr Richard Meadow (Harvard University, USA)

and Dr W.-S. Liu (The Pennsylvania State University, USA)

� 2009 The Authors, Journal compilation � 2009 Stichting International Foundation for Animal Genetics, Animal Genetics, 40, 933–944

Lei et al.942

for their valuable suggestions and English editing, which

improved the manuscript.

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