esj020 107..113


Iberian Origins of New World Horse
Breeds
CRISTINA LUÍS, CRISTIANE BASTOS-SILVEIRA, E. GUS COTHRAN, AND MARIA DO MAR OOM

From the Centro de Biologia Ambiental, Departamento de Biologia Animal, Faculdade de Ciências, Universidade de Lisboa,
Edifı́cio C2-Piso 3, Campo Grande, 1749-016 Lisboa, Portugal (Luı́s, Bastos-Silveira, and Oom); and the Department of
Veterinary Science, University of Kentucky, Lexington, KY 40546-0099 (Cothran). E. G. Cothran is now at the Department
of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University,
College Station, TX 77843.

Address correspondence to C. Luı́s at the address above, or e-mail: cmluis@fc.ul.pt.

Abstract

Fossil records, archaeological proofs, and historical documents report that horses persisted continuously in the Iberian
Peninsula since the Pleistocene and were taken to the American continent (New World) in the 15th century. To investigate
the variation within the mitochondrial DNA (mtDNA) control region of Iberian and New World horse breeds, to analyze
their relationships, and to test the historical origin of New World horses, a total of 153 samples, representing 30 Iberian and
New World breeds, were analyzed by sequencing mtDNA control region fragments. Fifty-four haplotypes were found and
assigned to seven haplogroups. Reduced levels of variation found for the Menorquina, Sorraia, and Sulphur Mustang breeds
are consistent with experienced bottlenecks or limited number of founders. For all diversity indices, Iberian breeds showed
higher diversity values than South American and North American breeds. Although, the results show that the Iberian and New
World breeds stem from multiple origins, we present a set of genetic data revealing a high frequency of Iberian haplotypes in
New World breeds, which is consistent with historical documentation.

The genus Equus evolved in North America, and during
the first major glaciations of the late Pliocene (2–3 million
years ago), some species crossed to Eurasia. North American
and South American Equus species became extinct about
10,000 years ago from causes that are not fully understood,
probably due to a combination of overhunting by humans
and environmental causes, such as climatic changes (e.g.,
Clutton-Brock 1996).

However, horses persisted continuously on the Iberian
Peninsula since the Pleistocene (1.8 million years ago) even
during the Mesolithic, when the horse became extinct north
of the Pyrenees (Gonzaga 2004).

Gene flow between horse populations of Iberia and
North Africa occurred at multiple times throughout history.
Exchanges between these two regions were particularly fre-
quent during the long period of occupation of the Peninsula
by the Moors (AD 711–1492), who brought Barb horses
from North Africa (e.g., Oom 1992).

Horses only returned to the American continent (the New
World) in 1493, with the navigator Christopher Columbus
and during the subsequent Spanish colonization period (Bort
2004; Primo 2004). Those stallions and mares were bought in
Seville’s province, mainly from the peasant stock bred in the
islands and salt marshes of the Guadalquivir River (Bort 2004).

Historical records report the presence of around 70 horses
on the first colony of La Española (Dominican Republic and
Haiti) by the year 1503. Subsequently, horses were brought
into Panama (1514), Mexico (1524), Brazil (1531), Peru
(1532), Argentina (1535), and Florida (1538) (Digard 1994).
By 1553, there were some 10,000 free-roaming horses in
the area of Queretaro (Mexico) that spread throughout North
and South America (Clutton-Brock 1992).

Analysis of the mitochondrial DNA (mtDNA) control
region sequence diversity has been an important tool for
understanding the origin and diversification of domestic
horses. Studies by Jansen et al. (2002), Lister et al. (1998),
and Vilà et al. (2001) all point to extensive variation within
and among breeds, with little congruence of haplotype
assignment to breed or geographic region. These studies
suggest that the domestic horse arose from several distinct
wild horse populations and distributed over a moderately
extensive geographic region large enough to contain con-
siderable preexisting haplotype diversity and that there was
considerable mixing of these haplotypes after domestication.

Here we compare the mtDNA control region variation
in Iberian and New World horse breeds in order to under-
stand their relationships and test the accuracy of historical
documentation of New World horse origins.

Journal of Heredity 2006:97(2):107–113
doi:10.1093/jhered/esj020
Advance Access publication February 17, 2006

ª The American Genetic Association. 2006. All rights reserved.
For permissions, please email: journals.permissions@oxfordjournals.org.

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mailto:cmluis@fc.ul.pt
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Materials and Methods
DNA Samples

We analyzed 90 samples, representing 3 Iberian, 4 North
American, and 12 South American breeds (Table 1). When
pedigree information was available, we selected unrelated
individuals.

DNA was extracted from (1) fresh whole-blood samples
after a high-salt extraction procedure (Montgomery and Sise
1990), (2) frozen whole-blood samples using the QIAmp

Mini Blood Kit (Quiagen Inc., Valencia, CA), (3) frozen
blood lysates using the Puregene Genomic DNA Isolation
Kit (Gentra Systems Inc., Minneapolis, MN), and (iv) hair
roots using Chelex-100 (Walsh et al. 1991).

DNA Sequencing

The universal primers L15926 and H16498 (Kocher et al.
1989) were used to amplify 360- to 442-bp fragments be-
tween sites 15411 and 15852 according to the horse reference
sequence X79547 (Xu and Árnason 1994), including the

Table 1. Number of samples sequenced in this study and gathered from GenBank and the respective main geographic location, breed,
breed’s code, and GenBank accession numbers

Number of samples

Geographic location Breed Code This study GenBank Accession number

Iberia Asturcon AST — 6 AY519872, AY519875–76,
AY519879–81

Caballo de Corro CCO — 6 AY519884–86, AY519888,
AY519890, AY519897–98

Cartujano CTJ — 6 AY519897–98, AY519900,
AY519902, AY519904, AY519906

Garrano GR 3 3 AF516500–01, AY997193,
AY246231, AY246233, AY246235

Lusitano LUS 6 — AF516502–05, AY997194–95
Losino LOS — 6 AY519924–25, AY519928,

AY519930, AY519932, AF466009
Mallorquina MA — 2 AF466013–14
Marismeño MAR — 6 AY519934, AY519936,

AY519938, AY519940,
AY519942, AY519944

Menorquina ME — 2 AF466015–16
Potoka POT — 6 AY519958–60, AY519963,

AY519967, AF4666012
Pura Raza Española PRE 6 — AY917165–66, AY917168,

AF516509–11
Sorraia SOR — 2 AF447764–65
Barb BA — 6 AJ413658, AJ413661,

AJ413664–66, AJ413668
North America Florida Cracker FC 3 — AY997150–51, AY997192

Kiger Mustang KM 6 — AF516489–90, AY997152–55
Spanish Mustang SM 4 — AY997178–81
Sulphur Mustang SUL 6 — AF516494–95, AY997187,

AY997200–02
Mustang MU — 6 AJ413753, AJ413797,

AJ413802, AJ413804,
AJ413807, AJ413817

South America Argentine Criollo AC 1 5 AF465986–90, AY997128
Brazilian Criollo CR 6 — AF516496, AF516498,

AY997145, AY997147,
AY997149, AY997190

Campolina CMP 6 — AY997139–44
Chilean Criollo CC 4 — AY997131–34
Chilote CH 4 — AY997135–38
Mangalarga BM 5 — AF516506–08, AY997129–30
Mangalarga Marchador MM 4 — AY997156–59
Pantaneiro PN 6 — AY997160–64, AY997199
Paso Fino PF 6 — AF516491–93, AY997197–99
Peruvian Paso Fino PVP 5 1 AF465993, AY997169–73
Puerto Rican Paso Fino RP 4 — AY997174–77
Venezuelan Spanish VS 5 — AY997182–86

The accession numbers in bold are new ones generated by this study.

Journal of Heredity 2006:97(2)

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tRNA
pro

and hyper variable region I of the mtDNA control
region.

The polymerase chain reaction (PCR) cycle sequencing
reactions were performed on both strands, twice for each
sample, using the CycleReader� Auto DNA Sequencing Kit
(MBI Fermentas GmbH, St. Leon-Rot, Germany), with infra-
red dye 800–labeled PCR primers used as sequencing primers
and with the ABI� Prism BigDye� Terminator Cycle
Sequencing Ready Reaction Kit (Perkin Elmer Inc., Boston,
MA). Sequences were determined with a Li-Cor� 4200S
Sequencer and an ABI 377 DNA Sequencer and were ana-
lyzed, respectively, with Li-Cor Image Analysis� software and
Sequencing Analysis Software� v3.4.1 with free Factura�.

The sequences obtained in this study were deposited in
GenBank database, and their accession numbers are shown
in Table 1.

We also included in this study mtDNA control region
sequences available from GenBank for 14 other Iberian
and New World horse breeds (see Table 1). The Barb horse
sequences were included as part of the Iberian group due to
the historical link between this breed and the Iberian breeds.

Sequences were aligned using the CLUSTALW software
(Thompson et al. 1994) and truncated to 288 bp, between
positions 15483 and 15770, according to the horse reference
sample X75947 (Xu and Árnason 1994), allowing the com-
parison with published sequences.

Data Analysis

Nucleotide diversity and haplotype (gene) diversity were ob-
tained with the DNASP 4.00.5 software (Rozas et al. 2003),
while the mean number of pairwise differences (MNPD) was
obtained using the ARLEQUIN 2000 software (Schneider
et al. 2000).

Median-joining network (Bandelt et al. 1999) was gener-
ated using NETWORK 4.1.0.8 software (available at http://
www.fluxus-engineering.com).

Results
mtDNA Lineages in Iberian and New World Horse Breeds

We sequenced 90 individuals and identified 26 haplotypes
of which 10 were new, namely, Hap_36, Hap_37, Hap_39,
Hap_42, Hap_43, Hap_44, Hap_45, Hap_47, Hap_49,
and Hap_50 (accession numbers AY997128, AF516507,
AY997138, AY997131, AF516503, AY997158, AY997159,
AF516492, AY997176, and AY997177, respectively). These
haplotypes belonged to one Iberian and seven South American
breeds (Table 1, Figure 1).

Our analysis was based on a sample set composed of
90 sequences from this study and 63 available from Gen-
Bank. The 153 mtDNA control region sequences yielded
54 different haplotypes defined by 44 polymorphic sites:
43 transitions and 1 transversion. For each breed, we iden-
tified from two to six haplotypes, differing from the reference
sequence (GenBank X79547) by 1–11 sites within the 288 bp
analyzed (the table with all this information is available from
the authors on request).

Fifteen haplotypes (27.8%) were detected more than once,
and 39 (72.2%) were singletons: 21 (53.8%) from Iberia
(corresponding to 10 breeds), 2 (5.1%) from North America
(one breed), and 16 (41%) from South America (eight breeds).
Potoka is the breed with the highest percentage of singletons
(100%) followed by Puerto Rican Paso Fino and Argentine
Criollo, with 75% and 67%, respectively.

The 54 haplotypes (26 from the present study) could
be assigned to five (D1, D2, D3, C2, and A4) out of the
17 major mtDNA lineages defined by Jansen et al. (2002).
Cluster D1 is considered, by these authors, as representative
of Iberian and North African breeds.

We further analyzed our sequences, and according to
the presence of specific point mutations, we defined a total
of seven major haplogroups. Their names indicate the mu-
tation position and nucleotide used as diagnostic of the hap-
logroups. As these point mutations were also present in
clusters defined by Jansen et al. (2002), we decided to incor-
porate the corresponding letters of these clusters into the
haplogroup names. The sequences were assigned to the haplo-
groups, and results are presented in Table 2.

Figure 1 shows a median-joining network relating the
mtDNA sequences of the analyzed breeds and the hap-
logroups defined here.

Haplogroup D494C,496G,534T,603C,649G is composed of indi-
viduals from all breeds (Iberia, 44%; North America, 60%;
South America, 48%) except from Menorquina and Sorraia.
Included in this haplogroup is the modal sequence Hap_1
(D1 from Jansen et al. 2002) seen in high frequency in the
Iberian and New World horse breeds analyzed here and hav-
ing the highest overall frequency in all the geographic regions
(Iberia, 26%; North America, 44%; South America, 29%).

Haplogroup A542C,666A was the second most common
with higher frequency of sequences in our sample set (12.4%)
being found in all geographic regions (Iberia, 10.6%; North
America, 24%; South America, 9.7%).

Haplogroup A542T,666A almost exclusively comprised
Iberian breeds, the exception was the North American Sul-
phur Mustang breed. Haplogroup C617C comprised only
South American breeds, and C601C was predominantly rep-
resented by breeds from that region.

Diversity of Iberian and New World Breeds

Results from MNPD, nucleotide diversity, and haplotype
diversity showed a similar pattern in all breeds (Table 3).
The Sulphur Mustang presented the lowest values and
Mallorquina the highest for all diversity indices, with the ex-
ception of haplotype diversity where several breeds had the
maximum value. For all diversity indices, Iberian breeds
showed higher diversity values followed by South American
and North American breeds (Table 4).

Discussion

From the 12 Iberian breeds studied, we provide new infor-
mation for three: Garrano, Lusitano, and Pura Raza Española.
Among the Iberian breeds, Menorquina and Sorraia showed

Luı́s et al. � Iberian Origins of New World Horse Breeds

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the lowest diversity values. Menorquina (from an island of
Spain with the same name) was established as a breed in
the early 1980s (Gallardo PP and Andres Cara DF, unpub-
lished). Approximately 70 horses were selected based on
phenotypic characteristics and, since 1994, have been bred
in a closed system. At the time of this breed’s establishment,
a bottleneck effect surely occurred; however, we cannot
exclude the sample size as a cause for this low diversity value.
For the Sorraia horse, the lower diversity found was in ac-
cordance with previous studies using different genetic mark-
ers, namely, blood groups and biochemical polymorphisms
(Oom and Cothran 1994), mtDNA (Luı́s et al. 2002a), micro-

satellites (Luı́s et al. 2002b), and major histocompatibility
complex genes (Luı́s et al. 2005). This Portuguese horse
breed was recovered in 1937 from 12 founders (five males
and seven females), and only two maternal lineages are pres-
ently represented in the living population (Luı́s et al. 2002a).

Of the five North American breeds analyzed, the Florida
Cracker, Spanish Mustang, and Sulphur Mustang were tested
for the first time. The North American samples showed
lower numbers of haplotypes and haplotype diversity com-
pared to the Iberian and South American breeds. All the
North American populations in this analysis represent horses
derived from feral populations. The Kiger, Florida Cracker,

Figure 1. Median-joining network relating the mitochondrial DNA D-loop sequences observed in Iberian and New
World horse breeds. Black represents Iberian Peninsula individuals, dark gray represents North American individuals, and light

gray represents South America individuals. Circle size is proportional to sequence frequency. Free-form shapes represent

haplogroups defined by certain point mutations. D1, D2, D3, C2, and A4 are clusters previously defined by Jansen et al. (2002).

See Table 2 for haplogroup characteristics.

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Sulphur Mustang, and Spanish Mustang groups have recently
become represented by breed registries. The first three are
from single, isolated populations in Oregon, Florida, and
Utah, respectively. The Spanish Mustang breed was formed
with horses that originated from feral or Native American
stock from all over North America. All were selected based
on a phenotype that was believed to represent Spanish an-
cestry. The Mustang group also is a collection of horses
with feral origins and presumed Spanish physical character-
istics. It is likely that the Kiger, Florida Cracker, and Sulphur
Mustang breeds experienced bottlenecks or a limited num-
ber of founders, resulting in lower diversity. The Sulphur
Mustang has the lowest diversity with only two haplotypes
found among six individuals. The Spanish Mustang and
Mustang groups have the highest diversity of the North
American breeds, comparable to the higher values for the
other groups, which reflects their more diverse origins.

From the 12 South American breeds considered in our
study, 10—Brazilian Criollo, Campolina, Chilean Criollo,
Chilote, Mangalarga, Mangalarga Marchador, Pantaneiro, Paso
Fino, Puerto Rican Paso Fino, and Venezuelan Spanish—are
analyzed for the first time. The higher variability of the South
American breeds compared with the North American ones
may be explained by the founding of North American breeds
with horses coming from Mexico and the Caribbean. The
South American breeds also have been selected for a greater
diversity of forms and uses when compared to the North
American horses, and some have been crossed to other
non-Iberian type horses (Hendricks 1995).

In comparison with the New World breeds, the Iberian
samples showed the highest values for the diversity param-
eters analyzed (including the frequency of singletons). This

finding supports the historical documentation that Iberia

was the source of much of the original stock that was used
to populate the New World with horses. Also Iberia experi-
enced an active interchange of horses with other breeding
countries, such as the Pontic-Caspian steppes, Gaul, Italy,
Macedonia, and Greece (Gonzaga 2004), that might have in-
creased the variability of the Iberian horses. Therefore, the
diversity in this region would be expectedly higher. The
low variation in the New World breeds may be an indication
of founder effect or a bottleneck during their establishment,
an hypothesis previously suggested by Mirol et al. (2002) in
their work with Argentinean Criollo.

The high diversity of the mtDNA control region within
the studied horse breeds confirms a differentiated ancestry,
previously indicated by several authors (e.g., Hill et al. 2002;
Jansen et al. 2002; Keyser-Tracqui et al. 2005; Kim et al. 1999;
Lister et al. 1998; Lopes et al. 2005; Mirol et al. 2002; Vilà et al.
2001). However, some haplotypes have been identified as
corresponding to specific breeds/geographic areas, namely,
D1, first identified by Jansen et al. (2002), and further em-
phasized by Lopes et al. (2005), as being well represented
in Iberian breeds. Haplotype Hap_1, from our work, corre-
sponds to D1 and was found in high frequency not only in
the Iberian breeds but also in the New World ones. Besides
Hap_1, the high frequency of Iberian and New World sam-
ples belonging to haplogroup D494C,496G,534T,603C,649G (48%)
is striking. This haplogroup has been considered represen-
tative of the ancestral Iberian horse population (Royo
et al. 2005). These two findings support the documented
role of the Iberian breeds in the origin of New World horse
populations.

The second haplogroup with more representatives of
Iberian and New World horse breeds is A542C,666A. This hap-
logroup includes Marismeño horses (stripped horses from

Table 2. Characterization of the haplogroups defined in this study with indication of mutation points, diagnostic nucleotide, and
percentage of total samples from each geographic region assigned to each haplogroup

Percentage of total samples in
each haplogroup (%)

Jansen et al.
(2002) lineagesHaplogroup name Mutation points

Diagnostic
nucleotide Iberia South America North America

A542T,666A 15542 T 12 — 8 A1 and A2
15666 A

A542C,666A 15542 C 11 10 24 A3
15666 A

B538G,709T 15538 G 12 3 — B1 and B2
15709 T

C601C 15601 C 1.5 15 — C2
C617C 15617 C — 5 — C1
D494C,496G,534T,603C,649G 15494 C 44 48 60 D1, D2, and D3

15496 G
15534 T
15603 C
15649 G

F740G 15740 G 1.5 3 — F2
Others

a
— — 18 16 8 A4

We also indicate lineages defined by Jansen et al. (2002) that were screened (bold) and lineages that despite not being found in this sample set would belong to

our defined haplogroups because they have the diagnostic point mutations.
a

Haplotypes that are not assigned to the defined major haplogroups.

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the Guadalquivir salt marshes) that, like the Sorraia, are con-
sidered a primitive Iberian equine type (Andrade 1954; Bort
2004) and therefore might have been extensively used for
breeding in Iberia. The high frequency of New World horses
in this haplogroup may be explained by historical records stat-
ing that mares taken to the American continent by Christopher
Columbus and during the subsequent Spanish colonization
period were bought mainly from the stock bred in the islands
and salt marshes of the Guadalquivir River (Bort 2004).

Of the three geographic regions studied, South America
is the only one having sequences that belong to haplogroup
C617C, and it is also the region having almost the exclusive
representation in haplogroup C601C. Indeed, the only non–
South American samples that belong to this latter haplogroup

are two individuals from the Caballo de Corro breed, a Celtic
origin pony from Asturias. These two haplogroups named
‘‘C’’ share some mutation points with cluster C1 from Jansen
et al. (2002), who consider this as distinctive for northern
European ponies, known to have Celtic origin. These findings
may indicate common matrilineal ancestors between Celtic
ponies and South American breeds, a result that is in accor-
dance with historical records because in 1508 the Spanish
crown authorized the transport of 40 Celtic type horses (small
and resistant) in the expedition organized by Alonso Ojeda
and Diego Nicuesa to Panama (Mirol et al. 2002).

The sharing of haplotypes between Iberia and New
World, and especially of those belonging to the haplogroup
considered as representative of the ancestral Iberian horse

Table 3. Diversity indices in the analyzed breeds

Geographic location Breed n nh MNPD SE Nucl. diver. SE Hd SE

Iberia Asturcon 6 4 6.67 3.67 0.023 0.015 0.800 0.172
Caballo de Corro 6 3 3.80 2.22 0.013 0.009 0.733 0.155
Cartujano 6 4 7.73 4.20 0.027 0.017 0.800 0.172
Garrano 6 4 6.27 3.47 0.022 0.014 0.867 0.129
Lusitano 6 6 7.80 4.24 0.027 0.017 1.000 0.096
Losino 6 4 3.93 2.29 0.014 0.009 0.800 0.172
Mallorquina 2 2 9.00 6.71 0.031 0.033 1.000 0.500
Marismeño 6 4 6.20 3.43 0.021 0.014 0.867 0.129
Menorquina 2 2 3.00 2.45 0.010 0.012 1.000 0.500
Potoka 6 6 7.00 3.83 0.024 0.015 1.000 0.096
Pura Raza Española 6 5 5.67 3.16 0.020 0.013 0.933 0.122
Sorraia 2 2 3.00 2.45 0.104 0.012 1.000 0.500
Barb 6 5 6.53 3.60 0.023 0.014 0.933 0.122

South America Argentine Criollo 6 6 7.33 4.00 0.026 0.016 1.000 0.096
Brazilian Criollo 6 4 3.20 1.92 0.011 0.008 0.867 0.129
Campolina 6 4 4.33 2.49 0.015 0.010 0.867 0.129
Chilean Criollo 4 4 5.50 3.34 0.019 0.014 1.000 0.177
Chilote 4 3 5.50 3.34 0.019 0.014 0.833 0.222
Mangalarga 5 5 6.60 3.76 0.023 0.015 1.000 0.126
Mangalarga Marchador 4 4 5.50 3.34 0.019 0.014 1.000 0.177
Pantaneiro 6 5 5.40 3.03 0.019 0.012 0.933 0.122
Paso Fino 6 3 5.33 3.00 0.019 0.012 0.600 0.215
Peruvian Paso Fino 6 3 4.40 2.53 0.015 0.010 0.600 0.215
Puerto Rican Paso Fino 4 4 5.83 3.53 0.020 0.015 1.000 0.177
Venezuelan Spanish 5 3 5.20 3.03 0.018 0.012 0.700 0.218

North America Florida Cracker 3 2 5.33 3.53 0.019 0.015 0.667 0.314
Kiger Mustang 6 3 3.27 1.95 0.011 0.008 0.733 0.155
Spanish Mustang 4 3 7.00 4.17 0.024 0.017 0.833 0.222
Sulphur Mustang 6 2 0.33 0.38 0.001 0.002 0.333 0.215
Mustang 6 5 6.47 3.57 0.023 0.015 0.933 0.122

n, number of individuals; nh, number of haplotypes found; MNPD, mean number of pairwise differences; Nucl. diver., nucleotide diversity; Hd, haplotype

(gene) diversity.

Table 4. Diversity indices in the analyzed geographic regions

Geographic location Total nh MNPD SE Nucl. diver. SE Hd SE

Iberia 66 34 6.67 3.19 0.023 0.012 0.924 0.025
South America 62 27 5.52 2.69 0.019 0.010 0.898 0.030
North America 25 10 5.00 2.52 0.018 0.010 0.793 0.075

Total, number of individuals; nh, number of haplotypes found; MNPD, mean number of pairwise differences; Nucl. diver., nucleotide diversity; Hd, haplotype

(gene) diversity.

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population (D494C,496G,534T,603C,649G), supports the widely
accepted view of Iberian ancestry in American livestock
(e.g., Miretti et al. 2004; Primo 2004).

Although extensive migrations in the past make it difficult
to find clear connections between mtDNA haplotypes and
geographic groups, we present a set of genetic data revealing
that New World breeds have a high frequency of haplotypes
of Iberian origin and represent a subset of the diversity found
in Iberia. Therefore, this study supports the historically docu-
mented Iberian origins of New World horses.

Acknowledgments
To Lyn Ennis, Pam Henney, Dr. Rytis Juras, and Dr. Kathryn Graves, at

the Equine Parentage Verification Laboratory, for laboratory help and sug-

gestions. C.L. was supported by a PhD grant (SFRH/BD/3318/2000) from

the Portuguese Foundation for Science and Technology (FCT/MCT), and

C.B.-S. was supported by a postdoctoral grant (SFRH/BPD/116/2002) from

the Portuguese Foundation for Science and Technology (FCT/MCT). The

authors thank an anonymous referee for very constructive comments.

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Received June 23, 2005
Accepted December 21, 2005

Corresponding Editor: Ernest Bailey

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