Horse Domestication and Conservation Genetics of Przewalski’s Horse Inferred
from Sex Chromosomal and Autosomal Sequences
Allison N. Lau,*1 Lei Peng,* Hiroki Goto,* Leona Chemnick, Oliver A. Ryder, and Kateryna
D. Makova*
*Department of Biology, The Pennsylvania State University, University Park; and Conservation and Research for Endangered
Species, San Diego Zoological Society
Despite their ability to interbreed and produce fertile offspring, there is continued disagreement about the genetic
relationship of the domestic horse (Equus caballus) to its endangered wild relative, Przewalski’s horse (Equus
przewalskii). Analyses have differed as to whether or not Przewalski’s horse is placed phylogenetically as a separate
sister group to domestic horses. Because Przewalski’s horse and domestic horse are so closely related, genetic data can
also be used to infer domestication-specific differences between the two. To investigate the genetic relationship of
Przewalski’s horse to the domestic horse and to address whether evolution of the domestic horse is driven by males or
females, five homologous introns (a total of ;3 kb) were sequenced on the X and Y chromosomes in two Przewalski’s
horses and three breeds of domestic horses: Arabian horse, Mongolian domestic horse, and Dartmoor pony. Five
autosomal introns (a total of ;6 kb) were sequenced for these horses as well. The sequences of sex chromosomal and
autosomal introns were used to determine nucleotide diversity and the forces driving evolution in these species. As
a result, X chromosomal and autosomal data do not place Przewalski’s horses in a separate clade within phylogenetic
trees for horses, suggesting a close relationship between domestic and Przewalski’s horses. It was also found that there
was a lack of nucleotide diversity on the Y chromosome and higher nucleotide diversity than expected on the X
chromosome in domestic horses as compared with the Y chromosome and autosomes. This supports the hypothesis that
very few male horses along with numerous female horses founded the various domestic horse breeds. Patterns of
nucleotide diversity among different types of chromosomes were distinct for Przewalski’s in contrast to domestic horses,
supporting unique evolutionary histories of the two species.
Introduction
Przewalski’s horse (Equus przewalskii) is an endangered species and is considered to be the only true wild
horse species alive today. Przewalski’s horse is the closest
relative to the domestic horse (Equus caballus) and can be
used to infer domestication-specific features. The karyotype
of the domestic horse (2n 5 64) differs from that of Przewalski’s horse (2n 5 66) by an extra chromosome pair either because of the fission of domestic horse chromosome 5
in Przewalski’s horse or fusion of Przewalski’s horse chromosomes 23 and 24 in the domestic horse (Benirschke et al.
1965; Bowling and Ruvinsky 2000; Myka et al. 2003; Yang
et al. 2003; Ahrens and Stranzinger 2005). In comparison,
the chromosomal differences between domestic horses and
zebras include numerous translocations, fusions, and inversions (Yang et al. 2003). Przewalski’s horse is known to
have the highest diploid chromosome number among all
equine species (Ryder et al. 1978).
Although the different chromosomal number genetically separates Przewalski’s horse from the domestic horse,
the two have been known to interbreed with each other and
to produce fertile offspring (Short et al. 1974). In contrast,
hybridizations of domestic horses with donkeys (Equus asinus) or with other species within the genus Equus usually
result in viable but infertile offspring due to multiple chromosomal differences (Zong and Fan 1989; Bowling 1996;
Yang et al. 2004). Skull measurements of Przewalski’s and
domestic horses often overlap (Forsten 1987; Eisenmann
1
Present address: Program in Biological and Biomedical Sciences,
Harvard University Medical School.
Key words: Przewalski’s horse, horse domestication, nucleotide
diversity, conservation genetics.
E-mail: [email protected].
Mol. Biol. Evol. 26(1):199–208. 2009
doi:10.1093/molbev/msn239
Advance Access publication October 17, 2008
Ó The Author 2008. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: [email protected]
and Baylac 2000), but differences in morphology are seen
in other skeletal features (Sasaki et al. 1999).
Przewalski’s horses became extinct in the wild due to
hunting by humans, heightened military activity within
their habitat, and competition for habitat with domesticated
livestock (Ryder 1993; Bouman I and Bouman J 1994).
However, they have been reintroduced into their natural
steppe habitat in parts of Mongolia and China (e.g., Ryder
1993). Przewalski’s horses are known to have once lived in
the Dzungarian Basin (an area which is now part of Mongolia), Kazakhstan, and the Xinjiang-Uygur Autonomous
Region of China and probably once lived in a broader range
throughout Europe and Asia (Ryder 1993). The current
population of Przewalski’s horses existing in zoos and
wildlife preserves descends from only 13 horses (eight females and five males; Volf et al. 1991). However, only four
Przewalski’s horse maternal lineages have survived from
this population; the other females were either domestic
horses or domestic/Przewalski’s horse hybrids (Volf
et al. 1991). Since that time, conservation efforts in zoos
and wildlife parks have been successful in breeding over
1,500 Przewalski’s horses (Geyer et al. 1989).
Determining the genetic relationship between Przewalski’s and domestic horses is expected to inform conservation
efforts aimed at preserving the genetic diversity of the former
species. Because the population of Przewalski’s horses is so
small and went through a recent bottleneck, the genetic effects of inbreeding within this species should be considered.
Inbreeding has been known to lead to reduced genetic diversity, high mortality, and short life span within small captive
populations in general (Ralls and Ballou 1983) and also
within the captive population of Przewalski’s horse in particular (Bouman and Bos 1979). Analysis of variation in
class II major histocompatibility complex alleles revealed
that Przewalski’s horse has diminished variation in this region as compared with domestic horses. This may play a role
200 Lau et al.
in the horses’ ability to recognize parasites (Hedrick et al.
1999). Interestingly, examination of blood groups showed
that despite their small, inbred population, Przewalski’s
horses had an average heterozygosity similar to that of domestic horses, possibly due to breeding with domestic horses
(Bowling and Ryder 1987; Bowling et al. 2003).
Previous studies have used different DNA markers to
determine the genetic relationship between Przewalski’s and
domestic horses and have led to contradictory results. Bowling et al. (2003) found that autosomal and X chromosomal
microsatellite data place Przewalski’s horse as an outgroup
to domestic horse, and Wallner et al. (2003) found that Przewalski’s and domestic horse were sister taxa based on Y
chromosomal haplotypes. In contrast, studies of mitochondrial DNA (mtDNA) provided evidence that domestic horse
and Przewalski’s horse do not form separate clades (Ishida
et al. 1995; Oakenfull and Ryder 1998; Oakenfull et al.
2000). Thus, there is still disagreement about whether Przewalski’s horse and the domestic horse represent two separate
phylogenetic clades or a single one. Possible explanations
for the differences in findings are gene flow from domestic
horse into the Przewalski’s horse population, different effective population sizes of males and females, an unequal ratio
of female to male founders (Wallner et al. 2003), or different
mutation rates for different markers. It is also possible that
these discrepancies are due to sampling of different mitochondrial lineages of Przewalski’s horses. There are four
maternal lineages in the current Przewalski’s horse population (Ryder 1994); however, sequencing of the mitochondrial control region revealed only two mtDNA haplotypes
in these lineages (Oakenfull and Ryder 1998).
In addition, it is of great interest to know whether domestic horse evolution is driven by males or females. This
can be informative in determining how domestication and
the formation of various horse breeds occurred. Although
there is considerable variation in maternally inherited mitochondrial DNA in domestic horses (Bowling et al.
2000; Vilà 2001; Aberle et al. 2007), there is little variation
in the Y chromosome (Lindgren et al. 2004). This view is
consistent with the breeding practices of most horse breeds,
in which one male is bred with many different females
(McDonnell 2005). However, studies of horse domestication that include data from the X chromosome along with
other genetic markers are currently lacking. Investigations
of domestication in other mammals have revealed a low
level of genetic diversity on the Y chromosome as compared with other genetic markers in domesticated dogs
(Sundqvist et al. 2006), cattle (Hellborg and Ellegren
2004), and sheep (Meadows et al. 2004). This suggests that
this bias in genetic diversity is a result of domestication.
In the present study, sequences from Y chromosomal, X
chromosomal, and autosomal introns are used to test the hypothesis that Przewalski’s horse represents a well-isolated
clade on a horse phylogenetic tree for each of these genetic
markers and to determine whether genetic variation in horse
breeds is derived primarily from males or females. Introns
were chosen because most of their sequences are selectively
neutral. Przewalski’s horses belonging to two different mitochondrial lineages were utilized in this study. Unraveling
the relationship between Przewalski’s and domestic horses
may be important for establishing future breeding plans
Table 1
Introns Selected
Chromosome
X and Y
Gene
UTX/UTY
ZFX/ZFY
DBX/DBY
All in concatenation
Autosome
IFNG
SMF
COL9A2
All in concatenation
X
Y
X
Y
X
Y
X
Y
X
Y
X
Y
Intron
Length (bp)
23
18
Next to last
Next to last
Last
Last
6
4
9
7
424
475
404
403
538
516
729
783
934
785
3,029
2,962
1,321
1,137
1,164
1,226
1,137
5,985
3
5
15
2
16
and conservation efforts for Przewalski’s horse as well as
for elucidating the history of horse domestication.
Materials and Methods
DNA Samples
Three male domestic horses (E. caballus) representing
three different breeds were analyzed—Arabian (OR535),
Dartmoor Pony (KB11923), and Mongolian domestic horse
(KB7754). Three Przewalski’s horses (E. przewalskii)
were also analyzed—two males (OR525, studbook number
381, Bertland, maternal lineage Bijsk B, and KB4064, studbook number 615, Kuporovitch, maternal lineage Bijsk 2)
for the analysis of the Y introns and one male (OR525, studbook number 381, Bertland, maternal lineage Bijsk B) and
one female (OR383, studbook number 319, Belina, maternal lineage Staraja 2) for the analysis of X and autosomal
introns. Sequences of onager (Equus hemionus onager;
OR68) were also obtained for use as an outgroup. DNA
samples were provided by the Zoological Society of
San Diego’s Conservation and Research for Endangered
Species.
Introns Sequenced
Two introns were each amplified from ZFX/ZFY and
DBX/DBY and one from UTX/UTY (table 1). All sequences
were deposited in GenBank (accession numbers FJ153229
and FJ222471–FJ222549). These introns are located in
nonrecombining regions of the X and Y chromosomes.
X- or Y-chromosome–specific amplification primers were
designed using Oligo Lite V.6.66 (Molecular Biology Insights, Inc., Cascade, CO), based on human and horse X
and Y sequences available (GenBank accession numbers
NT_079573.3, NC_000024, DQ519372, DQ520642,
DQ520647,
DQ520652,
DQ520657,
DQ520662,
DQ520667, DQ520672, DQ520677, and DQ520682).
Among autosomal introns, primers for two introns from
SMF (horse chromosome 14), two introns from COL9A2
Diversity of Domestic and Przewalski’s Horses 201
(horse chromosome 2), and one intron from IFNG (horse
chromosome 6) were designed based on horse sequences from
GenBank (accession numbers AJ698944, AM182498, and
A11777). Autosomal introns were amplified in onager for
use as an outgroup; onager X and Y chromosome sequences
were obtained from Goetting-Minesky and Makova (2006)
(accession numbers DQ519373, DQ520643, DQ520648,
DQ520653,
DQ520658,
DQ520663,
DQ520668,
DQ520673, DQ520678, and DQ520683).
All amplifications were carried out on a Gene Amp
PCR System 9700 (Applied Biosystems, Foster City,
CA) or a PTC-200 DNA Engine Peltier Thermal Cycler
(MJ Research, Waltham, MA) and contained 0.4 lM
chromosome-specific forward and reverse primers, 1
polymerase chain reaction (PCR) buffer, 0.2 mM
deoxynucleoside triphosphates, 1.5 mM MgCl2, 0.5 U
Taq DNA polymerase, and 30 ng genomic DNA in
25 lL reactions. The amplification conditions were as follows: 94 °C for 2 min followed by 30 cycles of 94 °C for
30 s, a primer-specific annealing temperature for 35 s, and
72 °C for a length-dependent extension time, followed by
72 °C for 7 min (the details of PCR conditions are given in
Supplemental Table 1, supplementary material online).
PCR products were then purified from the gel using a kit
from Qiagen, Inc., Valencia, CA.
All sequencing reactions contained 1 lMchromosomespecific forward or reverse sequencing primer and 35 fmol
purified PCR product. BigDye Terminator Version 3.1
was used for those sequences analyzed on an ABI Hitachi
3730XL (Applied Biosystems), whereas 2 lL DTCS Quick
Start Master Mix (Beckman Coulter, Inc., Fullerton, CA) was
used for those sequences analyzed on a CEQ8000 (Beckman
Coulter, Inc.). The conditions for thermocycling were 96 °C
for 20 s, 50 °C for 20 s, and 60 °C for 4 min for 40 cycles.
Ethanol precipitation of the sequencing products was used for
purification. All sequence differences were confirmed by
reamplification and resequencing. To confirm the presence
of indels, PCR products were cloned into the pCR2.1-TOPO
vector with the TOPO TA cloning kit (Invitrogen Life
Technologies, Carlsbad, CA). Sequences obtained from
clones were compared with direct sequencing data.
Computational Analysis
Intron- and horse-specific contigs were assembled using SeqManII from the Lasergene sequence analysis software (DNASTAR, Inc., Madison, WI). Alignments of each
chromosome-specific intron were assembled using ClustalW (Thompson et al. 1994) in the MegAlign program
from the Lasergene software. Alignments were then
adjusted manually.
Nucleotide diversity (p; Nei and Li 1979) was determined using the DnaSP software, version 4.10 (Rozas et al.
2003). Heterozygous sites were resolved by randomly
choosing one of the bases for each sequence and creating
two pseudosequences, a and b, as in Makova and Li (2002).
Autosomal haplotypes and X-chromosome haplotypes
from a female were not determined. Phylogenetic trees were
constructed by two methods implemented in the MEGA 3.1
Software (Kumar et al. 2004) with 1,000 bootstrap repli-
cates: the Neighbor-Joining method (Saitou and Nei
1987) using the Kimura two-parameter model (Kimura
1980) and the maximum parsimony method (Nei and
Kumar 2000).
Tajima’s D test was performed using DnaSP (Rozas
et al. 2003) on all introns to determine if the sequences were
evolving under selection (Tajima 1989). Sequences
were also analyzed by hand to ascertain whether SNPs were
in Hardy–Weinberg equilibrium (Hartl and Clark 1997).
Results
Introns Sequenced
Sex chromosome–specific and autosomal introns
were sequenced for Przewalski’s horses (E. przewalskii)
and domestic horses (E. caballus). Przewalski’s horses selected, Bertland (male), Kuporovitch (male), and Belina
(female), belonged to three different maternal lineages
(Bijsk B, Bijsk 2, and Staraja 2, respectively; Volf et al.
1991). The domestic horses represented the following
breeds: Arabian, Dartmoor pony, and Mongolian domestic.
The breeds selected are diverse (Bjornstad and Roed 2001;
Jansen et al. 2002) and thus capture the general trends
of domestic horse diversity observed in other studies
(see Discussion).
For each of the five horses (two male Przewalski’s
horses for Y introns and one male and one female Przewalski’s horses for X introns as well as three male domestic
horses), ;3 kb of total intronic sequence from each of
the X and Y chromosomes (3,029 bp and 2,962 bp, respectively) were sequenced. In general, introns were sequenced
because they are likely to evolve neutrally (see results of
neutrality test below) and also because they can be efficiently amplified by placing conserved primers in exons.
To avoid the influence of nucleotide composition on mutation rates, homologous X and Y introns were amplified and
sequenced. These included two introns each from ZFX/ZFY
and DBX/DBY and one from UTX/UTY (table 1). These particular introns were sequenced because their sequences for
another Equus species, onager (E. h. onager), were already
available. A total of 5,985 bp of autosomal intronic sequence was also obtained for the five horses for which
X intronic sequences were obtained. Two autosomal introns
were amplified from SMF, two introns from COL9A2, and
one intron from IFNG (table 1).
The sequences of X chromosomal and autosomal introns differed among the five sequenced horse samples
(tables 2 and 3); however, the Y intronic sequences were
identical among domestic and Przewalski’s horses. A total
of six nucleotide sites were polymorphic in the X intronic
sequence, whereas 28 nucleotide sites and 2 indel sites were
polymorphic in the autosomal sequence. No fixed differences were identified between Przewalski’s and domestic
horses on either chromosome X or autosomes.
To test for natural selection, we performed Tajima’s
D test (Tajima 1989). None of the X chromosomal and autosomal introns had Tajima’s D values significantly different from neutral expectations (Supplemental Table 2,
supplementary material online); thus, the sequences likely
evolved neutrally. Hardy–Weinberg equilibrium could not
202 Lau et al.
Table 2
Polymorphic Sites on X Chromosome
UTX, Intron 23
ZFX, Last Intron
DBX, Intron 6
476
387
582
106
499
648
T
A
A
A
A
A
T
T
C
T
C
T
A
A
A
A
G
A
G
G
A
G
G
G
C
C
T
C
C
C
T
C
C
T
C
C
Przewalski male (Bertland)
Przewalski female (Belina)
Arabian
Mongolian domestic
Dartmoor Pony
Onager
be rejected for each of the polymorphic sites (Supplemental
Table 3, supplementary material online).
Phylogenetic Analysis
Because there was variation in the X chromosomal and
autosomal introns, these sequences were concatenated in
each case and used to construct separate phylogenetic trees
using the Neighbor-Joining and maximum parsimony
methods (figs. 1 and 2). Onager (E. h. onager) sequences,
obtained here for autosomal introns and available for sex
chromosomal introns from Goetting-Minesky and Makova
(2006), were used as an outgroup.
The two autosomal trees indicated different topologies
depending on the method chosen (fig. 1). The NeighborJoining tree placed one of the Przewalski’s horse male
(Bertland) sequences as an outgroup to the Dartmoor pony
and Arabian horse sequences, whereas the maximum parsimony tree placed one of Bertland’s sequences between the
two Dartmoor pony sequences. The X-chromosome trees
differed depending on the phylogenetic method utilized
as well (fig. 2). In the Neighbor-Joining tree, the Przewalski’s horse female (Belina) is located outside of the cluster
of Przewalski’s horse male (Bertland) and Mongolian domestic horse sequences and outside of the cluster of the
Arabian horse and Dartmoor pony sequences. In the maximum parsimony tree, the Przewalski’s horse female (Belina) comes in between these two clusters. These alternative
groupings had relatively low bootstrap support values.
In all cases, Przewalski’s and domestic horses were
intermixed in these trees and did not form separate clades.
Bootstrap support was low in all branches that distinguish
relationships between the domestic and Przewalski’s
horses. Arabian horse and Dartmoor pony were usually
more closely grouped, as were Mongolian domestic horse
and Przewalski’s horse. This may be due to interbreeding
between Przewalski’s and Mongolian domestic horses,
whose range has historically overlapped. Phylogenetic
analysis revealed that Przewalski’s horse and different
breeds of domestic horse are very closely related.
Nucleotide Diversity Analysis
The nucleotide diversity (p) of the sequences was
compared for X, Y, and autosomes for domestic and Przewalski’s horses separately (fig. 3). An unusual pattern of
genetic diversity was observed in domestic horses: a higher
DBX, Intron 9
than expected amount of relative diversity on the X chromosome and no diversity on the Y. The ratio of autosomalto-X diversity is expected to be 4:3 (;1.33) based on
population size: Autosomes are present in two copies in
both males and females, whereas the X is present in two
copies only in females and in one copy in males. The ratio
of autosomal-to-X diversity obtained from domestic horses
was 1.2 10 3 (standard deviation [SD] 2.2 10 4) to
1.1 10 3 (SD 3.1 10 4), or 1.09, lower than expected
(however not significantly so, based on overlapping 95%
confidence intervals [CIs]). There were no sequence differences in the Y introns of any of the horses sequenced; however, by introducing one artificial nucleotide change, the
maximum genetic diversity of the Y sequence was estimated to be 2.3 10 4 (SD 1.1 10 4). In the past, coalescent models have been used to determine the expected
level of diversity in sequences with no variation (Dorit et al.
1995), but the accuracy of this method has been debated
(Donnelly et al. 1996; Fu and Li 1996; Rogers et al.
1996; Weis and von Haeseler 1996). Based on chromosome
population size, the ratio of diversity on the X chromosome
and autosomes compared with the Y chromosome is expected to be 3:1 and 4:1, respectively. The ratios for domestic horses obtained using the maximum diversity estimates
for Y were ;4.78 and ;5.17, higher than the mere population size would suggest (however not significantly so,
based on overlapping 95% CIs).
For Przewalski’s horse, a different pattern of diversity
was noticed, although again there was no variation on the Y
chromosome (and its maximum diversity was estimated to
be 3.4 10 4, SD 1.7 10 4). The autosomal nucleotide
diversity in Przewalski’s horses was over three times
the value from the X chromosome (2.0 10 3, SD
4.1 10 4, vs. 6.6 10 4, SD 3.1 10 4); this is in
contrast to domestic horses, in which the autosomal-to-X
chromosome diversity ratio is about 1.09. This indicates
that the two groups have evolved differently potentially
due to domestication influencing evolution of domestic
horses. The estimates for X:Y and autosomes:Y in
Przewalski’s horses were 1.94 and 5.91, respectively.
The autosomal nucleotide diversity values were higher
for Przewalski’s horses (2.0 10 3, SD 4.1 10 4) than
for domestic horses (1.2 10 3, SD 2.2 10 4). However, the values of X chromosomal nucleotide diversity were
higher for the domestic horses (1.1 10 3, SD 3.1 10 4)
than for Przewalski’s horses (6.6 10 4, SD 3.1 10 4).
Although these comparisons were not statistically significant
(likely due to a relatively limited number of polymorphisms
Y
C
C
C
C
C
T
C
Y
C
found and according to overlapping 95% CIs), they suggest
different evolutionary histories of the two populations.
Discussion
C
Y
C
C
Y
C
—
T/—
—
M
C
C
C
Y
C
C
C
C
G
G
—
Y
Y
T
C
Y
C
A
A
A
C
C
C
A
A
T
R
G
G
C
C
A
T
T
C
G
G
A
A
A
A
C
C
C
G
G
G
Y
C
C
Y
C
T
S
C
G
A
A
A
C
C
C
NOTE.—represents a deletion.
Y
C
C
R
G
G
R
R
A
C
C
R
G
R
A
C
C
M
A
C
G
C
T
C
T
R
G
Y
C
R
A
R
G
Y
T
M
C
G
A
W
A
Y
C
R
A
C
C
T
T
G
G
Y
C
C
C
C
C
T/—
—
C
T
C
T
Indels
C
COL9A2, Intron 16
Y
Y
Y
T/—
C
C
Y
R
Y
C
R
Y
W
G
M
Y
R
R
Y
R
C
C
C
A
Y
R
C
R
Przewalski male
(Bertland)
Przewalski female
(Belina)
Arabian
Mongolian
domestic
Dartmoor Pony
Onager
772
613
COL9A2, Intron 2
212
792
293
189
SMF, Intron 15
SMF, Intron 5
IFNG, Intron 3
264 303 341 619 773 1090 1219 1303 22 110 213 369 598 647 664 744 864 1004 1035
Table 3
Polymorphic Sites on Autosomes
108 149 405–408 535 671
Diversity of Domestic and Przewalski’s Horses 203
This study of Przewalski’s horse is the first that includes all three types of chromosomes: X, Y, and autosomes. Including markers from different chromosome
types is important in developing a more complete picture
of the genetics of horse domestication and Przewalski’s
horse evolution. Four major findings emerged as a result
of this analysis.
The first finding was that the data presented here
(figs. 1 and 2) indicate that Przewalski’s and domestic
horses are not genetically separated into monophyletic
clades based on both the X chromosome and autosomes.
We also observed no fixed difference and many shared
polymorphisms (8 of 14 polymorphic sites of Przewalski’s
horse) between Przewalski’s and domestic horses. Therefore, the first finding can be explained by introgression
of the domestic horse to Przewalski’s horse. In support
of this explanation, there are pedigree records indicating recent interbreeding of Przewalski’s horses with domestic
horses (Volf et al. 1991). However, there are Przewalski’s
horses without documented domestic horse ancestry, often
called ‘‘A-line,’’ one of which was used in this study (studbook number 381, Bertland; Volf et al. 1991). An alternative explanation for the first finding is that the time of
domestic horse formation might be insufficient for nucleotide substitutions to be fixed. In this spirit, our results contradict the results of Bowling et al. (2003) who found that
autosomal and X chromosomal microsatellite data place
Przewalski’s horse as an outgroup to the domestic horse;
sequence data presented here and microsatellite data (Bowling et al. 2003) might not agree due to different mutation
rates between sequence and microsatellite markers.
Introgression between an endangered wild species and
a close domesticated relative has been observed in the
North American bison (Bison bison) with domesticated cattle (Bos taurus) (Ward et al. 1999) and in captive markhors
(Capra falconeri) with domesticated goat (Capra hircus)
(Hammer et al. 2008). Based on mtDNA haplotypes, it
was estimated that over 35% of markhors from zoos were
introgressed by domestic goats (Hammer et al. 2008),
whereas about 5% of North American bison were hybrids
(Ward et al. 1999). The genetic analysis of bison, yaks, and
cattle resulted in a phylogeny similar to the one seen in this
study: wild species were not monophyletic but intermingled
with the domestic species (Ward et al. 1999). In both markhor and bison, the endangered wild animals had historically been purposefully hybridized with domesticated
relatives to ensure the survival of the former animals. In
a situation similar to Przewalski’s horse (Treus 1962; Mohr
1971), the small founding population of bison that served as
a founding stock for most current existing populations was
exposed to hybridization with domesticated animals (Ward
et al. 2001).
The second finding was that in domestic horses, the
levels of nucleotide diversity on the X chromosome were
higher than expected relative to autosomes and the Y chromosome. Because the X chromosomal diversity was higher
204 Lau et al.
FIG. 1.—Phylogenetic trees based on autosomal sequence using the Neighbor-Joining (A) and maximum parsimony (B) methods. Heterozygous
sites were resolved by randomly choosing one of the bases for each sequence and creating two pseudosequences, a and b, as in Makova and Li (2002).
in comparison to other chromosomes, it can be inferred that
females had a greater role (more females participated) in
forming the various horse breeds. Our results support the
hypothesis that there were very few male horses that
founded the various domestic horse breeds and that male
horses bred with many different females (as is customary
in agricultural domestic horse breeding; McDonnell
2005). Additionally, archaeological data show that domesticated male animals were preferentially hunted for food
over females (Zeder and Hesse 2000). This indicates that
females contribute most variation in the domestic horse
breeds. The fact that this pattern was not as apparent in
Przewalski’s horse suggests that it is specific to domestic
horses and may be a result of domestication.
The hypothesis that few male horses were used in the
formation of horse breeds is supported by other genetic
analysis indicating that horse breed genetic diversity comes
mostly from females. For instance, studies of mitochondrial
variation within horse breeds found high levels of variation
within and between breeds possibly due to a larger female
effective population size (Bowling et al. 2000; Vilà 2001).
Examination of 15 horse breeds also resulted in greater nucleotide diversity on the X chromosome than the Y chromosome (Lindgren et al. 2004). Pedigree analysis combined
with microsatellite diversity showed that one founder
stallion was responsible for 95% of the paternal lineages
within thoroughbred horses, whereas 10 founder females
represented 72% of the maternal lineages (Cunningham
et al. 2001).
The third finding was that autosomal diversity was
higher and X chromosomal diversity was lower in Przewalski’s horses than in domestic horses. These differences suggest that the Przewalski’s horse population was not formed
in the same way that the domestic horse population was
formed (e.g., females did not play such a prominent role).
A population bottleneck in female Przewalski’s horses
could account for the observed pattern. Alternatively, this
pattern could be due to the small number of individuals examined for Przewalski’s horses. If substantial interbreeding
between domestic and Przewalski’s horses has occurred, we
would expect similar levels of diversity on their X chromosomes as well as on their autosomes.
The fourth finding was that there were no nucleotide
differences in any of the Y-chromosome sequences for both
domestic and Przewalski’s horses. We can provide several
explanations for this absence of variation. First, for domestic
horses, this can be explained by a small male founding
population. Similar results have been found for other
Diversity of Domestic and Przewalski’s Horses 205
FIG. 2.—Phylogenetic trees based on X chromosomal sequence using Neighbor-Joining (A) and maximum parsimony (B) methods. Only one
sequence is used for Przewalski’s horse female (Belina) because its two X chromosomal sequences at the studied loci were identical.
domesticated animals, including dogs, cattle, and sheep
(Hellborg and Ellegren 2004; Meadows et al. 2006; Sundqvist et al. 2006). Comparison of Y chromosomal, mitochondrial, and autosomal DNA markers shows fewer Y
chromosome than mitochondrial DNA haplotypes in domesticated dog breeds (Sundqvist et al. 2006). This pattern was
not seen in wolves; in fact, more Y haplotypes were found
than mitochondrial haplotypes. As observed with horses,
there may have been fewer males than females that contributed to the formation of domesticated dog breeds. Cattle have
also shown a bias in nucleotide diversity on the sex chromosomes. In a study of some of the same genetic markers, no
segregating sites were found on the Y chromosomal regions
of cattle, whereas high levels of variation were found on X
(Hellborg and Ellegren 2004). Additionally, this pattern of
low nucleotide diversity on the Y chromosome (Meadows
et al. 2004) along with high rates of Y-chromosome dispersal
(Meadows et al. 2006) have been seen in sheep.
Second, the absence of variation on the Y chromosome in Przewalski’s horses may be explained by other factors than for domestic horse. Many mammalian species
have very low levels of nucleotide diversity on the Y chromosome (Hellborg and Ellegren 2004), which may indicate
that selection has been important in the evolution of nucleotide diversity on this chromosome; thus, our results might
be influenced by forces acting specifically on this chromosome. Furthermore, it is also possible that there was little Y
variation in the common ancestor of domestic and Przewalski’s horses. Fixed nucleotide differences between Przewalski’s and domestic horses have been discovered on the Y
chromosome (Wallner et al. 2003); analysis of a greater
length of sequence could yield similar results.
Moreover, the identical Y chromosomal sequences observed in Przewalski’s horse could be accounted for by unidirectional hybridization with the domestic and Przewalski’s
horse populations if interbreeding occurred only between
Przewalski’s horse males and domestic horse females. Such
hybridization was seen in two closely related species of African elephants—the African forest elephants (Loxodonta cyclotis) and African savanna elephants (Loxodonta africana;
Roca et al. 2005). This type of unidirectional hybridization
has also been observed in populations of American bison
and domesticated cattle (Ward et al. 2001). It has been suggested that extensive backcrossing of wild males and domesticated females has occurred in many domesticated mammals
(Vilà et al. 2005). This could explain why Przewalski’s
horses show such low genetic diversity on their Y chromosomes: Most Y chromosomes originally came from one population. Studbook records indicate that a female domestic
horse and female Przewalski’s/domestic hybrid were included as part of the 13-member Przewalski’s horse founder
population but that all males were Przewalski’s horses (Volf
et al. 1991).
To summarize, our results indicate that the domestic
and Przewalski’s horses are very closely related; Przewalski’s horses do not form a separate monophyletic group.
The lack of variation on the Y chromosome and the relative
diversity between X chromosome and autosomes provide
information about the history of horse domestication and
formation of the Przewalski’s horse population. In the future, it would be beneficial to use large-scale sequencing
techniques to sequence both the mitochondrial and nuclear
genomes of the Przewalski’s horse and to compare them
with the genomes of the domestic horse in order to study
diversity in more detail and to further elucidate the evolutionary relationships between the two species. This will further test a hypothesis that there was a constant flux of
genetic information between Przewalski’s and domestic
horses through their interbreeding. When formulating
breeding plans for the endangered Przewalski’s horse, it
is important for conservation geneticists to consider the
level of interbreeding or introgression by domestic horse,
especially when the goal is eventual reintroduction into
a wild or semiwild environment. Conservation of the
206 Lau et al.
Fund for Research Grants to A.N.L. and L.P. from Penn
State University.
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FIG. 3.—Nucleotide diversity with SD in the domestic horse (A) and
Przewalski’s horse (B). Values were calculated using DnaSP software
version 4.10 (Rozas et al. 2003).
endangered Przewalski’s horse has been successful in the
past, and the study of more genetic data of this species
would only support these efforts.
Supplementary Material
Supplementary tables are available at Molecular Biology
and Evolution online (http://www.mbe.oxfordjournals.org/).
Acknowledgments
We would like to thank M. Paula Goetting-Minesky
for providing the initial Przewalski’s horse and onager sequences. We would also like to thank Stephen Schaeffer for
his advice and critical reading of the manuscript. The Zoological Society of San Diego supplied the DNA samples
used in this study. Funding was provided by start-up funds
from the Eberly College of Science at Penn State University
to K.D.M. This study was also supported by an Undergraduate Summer Discovery Grant to A.N.L. and President’s
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Naoko Takezaki, Associate Editor
Accepted October 13, 2008