Phylogenetic relationships of Argentinean Creole horses and other South American and Spanish breeds inferred from mitochondrial DNA sequences

Phylogenetic relationships of Argentinean Creole horses and otherSouth American and Spanish breeds inferred from mitochondrialDNA sequences

P. M. Mirol*, P. Peral Garcıa*, J. L. Vega-Pla† and F. N. Dulout**CIGEBA, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, La Plata, Argentina. †Laboratorio de Grupos Sanguıneos,

Servicio de Cria Caballar, Cordoba, Spain

Summary South American horses constitute a direct remnant of the Iberian horses brought to the

New World by the Spanish conquerors. The source of the original horses was Spain,

and it is generally assumed that the animals belonged to the Andalusian, Spanish

Celtic, Barb or Arabian breeds. In order to establish the relationship between Argen-

tinean and Spanish horses, a portion of the mitochondrial D-loop of 104 animals

belonging to nine South American and Spanish breeds was analysed using SSCP and

DNA sequencing. The variability found both within and between breeds was very high.

There were 61 polymorphic positions, representing 16% of the total sequence obtained.

The mean divergence between a pair of sequences was 2.8%. Argentinean Creole

horses shared two haplotypes with the Peruvian Paso from Argentina, and the com-

monest haplotype of the Creole horses is identical to one of the Andalusian horses. Even

when there was substantial subdivision between breeds with highly significant

Wright’s Fixation Index (FST), the parsimony and distance-based phylogenetic ana-

lyses failed to show monophyletic groups and there was no clear relationship in the

trees between the South American and any of the other horses analysed. Although this

result could be interpreted as mixed ancestry of the South American breeds with

respect to the Spanish breeds, it is probably indicating the retention of very ancient

maternal lineages in the breeds analysed.

Keywords D-loop, horse, mtDNA, phylogeny.

Introduction

The Argentinean Creole horse constitutes a direct remnant

of the Iberian horses brought to the New World by the

Spanish conquerors during the 15th century. The source of

the original horses was Spain, and this was at a time when

the Spanish horse was being used for improvement of horse

breeding throughout Europe. On the basis of historical

records, at least 250 horses where shipped to the continent,

from the second voyage of Columbus in 1493 to Nunez de

Cabeza’s trip in 1540 (Rodero et al. 1992). These horses, as

all other domestic animals transported, were quickly dis-

persed and became very well adapted to the new environ-

ment. At that time, Spanish exports arrived directly from

the ports of Spain to their final destination, with a stop on

the Canary Islands or the Antilles. There were in Spain

three main morphological equine types: the Celtic type of

tarpanic origin in the north and west region of the penin-

sula; the Spanish type, descendent from the African Barb

horse, in the south and east; and finally, in the central area,

the hybrid between both of them. As Seville, Cadiz and other

southern ports monopolized the navigation to America, it

could be assumed that horses taken to America were mainly

of the Spanish type or Andalusian (Rodero et al. 1992).

Address for correspondence

P. M. Mirol, School of Biological Sciences, Queen Mary, University of

London, Mile End Road, London E1 4NS, UK.

E-mail: [email protected]

Accepted for publication 29 March 2002

� 2002 International Society for Animal Genetics, Animal Genetics, 33, 356–363

However, the possibility of taking to America other equine

types cannot be ruled out. During his second trip to the New

World, Columbus complained to the Spanish Crown that the

excellent horses shown to him before the departure had

been changed by cheaper animals (Tudela 1987). In 1508

the Spanish Crown authorized the transport of 40 horses

from Castilla in the expedition organized by Alonso Ojeda

and Diego de Nicuesa to Panama. The horses were of the

Celtic type, small and resistant. Furthermore, many animals

died during the 2-month trip, and other animals from the

intermediate ports, Canary Islands or Antilles, could have

replaced them.

The advent of mitochondrial DNA analysis in population

genetics during the 1970s produced a revolutionary change

regarding historical, biogeographic and phylogenetic per-

spectives on intra- and interspecific genetic structure (Avise

1994). Since then, it has been widely used to infer intra- and

interspecific phylogenetic relationships. Mitochondrial DNA

studies in horses have proved useful to characterize intra-

breed variation (Kavar et al. 1999; Kim et al. 1999; Bowling

et al. 2000), although retention of ancestral polymorphism

makes phylogenetic inference difficult (Vila et al. 2001).

In this context we analyse the mitochondrial DNA vari-

ation of Argentinean Creole horses and some other South

American and Spanish horses. The characterization of the

Creole breed has been traditionally based on morphology

and behaviour, and it was only during the last few years

that analysis based on molecular genetic markers have been

introduced (Peral Garcıa et al. 1996). Knowledge of the

South American breeds is also important to conservation

genetics of domestic horses, as the New World varieties are

probably closer in type to the historical horse of Spain than

are the current horses in Iberia, which over the last

500 years have interbred with other breeds.

Materials and methods

DNA extraction, PCR amplification and SSCPof the D-loop

Total DNA was extracted from blood samples using the

DNAZOL purification kit (Gibco Life Technologies, Rockville,

MD, USA) following the manufacturer instructions. Samples

included 45 Argentinean Creole horses (ARC), 30 Peruvian

Paso from Argentina (PPA), 18 Arabian (16 from Argen-

tina – AR, and two from Spain – ARSP) and 11 Spanish

horses belonging to the breeds Asturcon (AST), Losino (LO),

Potoka (PO), Mallorquina (MA), Menorquina (ME) and

Spanish Pure Breed or Andalusian (AND). When pedigree

information was available, we selected individuals that have

not shared a common ancestor for at least three genera-

tions. The polymerase chain reaction (PCR) primers used

were: L-strand: 5¢-AGGACTATCAAAGGAGAAGCTCTA-3¢(P1; Ishida et al. 1994) and H-strand: 5¢-CCTGAAGTA-

GGAACCAGATG-3¢ (H16498; Marklund et al. 1994),

which amplify a 466-bp region situated between the

tRNAThr (position 15397; Xu & Arnason 1994) and the

central domain of the D-loop (position 15863; Xu & Arna-

son 1994). The 50 ll reaction mix contained approximately

100 ng of total horse DNA, 0.5 lM of each primer, 0.1 mM

of dNTPs and 2 U of Taq polymerase (Gibco BRL, Rockville,

MD, USA) in 20 mM Tris–HCl (pH 8.4), 50 mM KCl and

2 mM MgCl2, under mineral oil. The PCR consisted of a first

denaturation step at 96 �C for 2 min followed by 35 cycles

of 1 min at 94 �C, 30 s at 55 �C and 1 min at 72 �C, with

an elongation step of 5 min at 72 �C in the last cycle. The

size of the products was estimated by 1.5% agarose gel

electrophoresis with pBR322 MspI Digest as size marker.

Argentinean Creole horses, Peruvian Paso from Argen-

tina and Arabian (Argentina) were pre-selected before

sequencing by PCR–SSCP analysis, to include a range of

genetically distinct individuals. Fifteen microliters of each

PCR product was added to 40 ll of dye LIS (10% sucrose,

0.01% bromophenol blue and 0.01% xylene cyanol FF;

Maruya et al. 1996). The samples were then heated at

96 �C for 10 min, cooled on ice for at least 5 min and

loaded onto a 10% polyacrylamide gel (49 : 1 acryla-

mide : bisacrylamide). Electrophoresis was carried out at

4 �C, 200 V in 0.5 · Tris-Borate-EDTA (TBE) buffer for

18 h. The gels were subsequently fixed in 5% ethanol,

stained with 0.2% AgNO3 and revealed with 2% CaCO3.

Cloning and sequencing of the PCR products

The PCR products were purified using QIAquick columns

(QIAGEN, Hilden, Germany) and cloned into dT-tailed

pGEM-T easy vector (Promega, Madison, WI, USA) follow-

ing manufacturer’s recommendations. The DNA sequencing

was performed with an Applied Biosystems 377 automated

sequencer (BioResource Centre, Cornell University, Ithaca,

NY, USA) using T7 and M13 universal primers. At least two

independent clones were sequenced from each individual

and the unique substitutions were confirmed by sequencing

clones from a second PCR product.

Data analysis

Sequences of the D-loop were aligned using CLUSTAL-V

multiple alignment software (Higgins et al. 1992). Sites

representing a gap in any of the aligned sequences were

excluded from the analysis, and distances between D-loop

sequences were estimated using both the absolute number

of nucleotide differences and the Kimura two-parameter

distance (Kimura 1980) calculated on the basis of an equal


MtDNA variation in South American and Spanish horses 357

substitution rate per site. Phylogenies were constructed

using maximum parsimony with the PAUP 4.0 software

(Swofford 1997) and the NEIGHBOR program incorporated

in the PHYLIP package (Felsenstein 1991). For this analysis

sequences belonging to other horse breeds from GenBank

(http://www.ncbi.nlm.nih.gov/GenBank) were incorpor-

ated. In all analyses the sequence of Equus asinus (Xu &

Arnason 1996) was used as outgroup. The statistical con-

fidence of each node in the consensus trees was estimated by

1000 bootstrap resampling of the data. Analysis of mo-

lecular variance (AMOVA) and pairwise FST distances were

calculated using Arlequin (Schneider et al. 2000).

Results

Single strand conformation polymorphism (SSCP)

A total of 91 horses were examined for SSCP, 45 belonging to

the Argentinean Creole breed, 16 Arabian and 30 Peruvian

Paso. The PCR products were of the same length, approxi-

mately 460 base pairs, which is in agreement with the pub-

lished horse sequences (Ishida et al. 1994). Heteroplasmy

was not detected in any of the horses examined. The SSCP

analysis revealed 14 variants (Table 1), which were consis-

tently obtained in different runs and also in different SSCP

conditions. As it can be seen from Table 1, Argentinean

Creole and Peruvian Paso horses have five distinct SSCP

patterns, and Arabian horses seven patterns. Arabian and

Peruvian Paso present six and three diagnostic patterns,

respectively (i.e. patterns not present in any other breed),

while there are only two SSCP variants found exclusively in

Argentine Creole horses. Three SSCP variants were shared

amongst the breeds studied, two of them between Argentin-

ean Creole and Peruvian Paso, and the other between

Argentinean Creole and Arabian. The most common variants

in each of the breeds analysed are unique to that specific breed.

Sequence variation

A total of 33 individuals from the Argentinean Creole,

Peruvian Paso and Arabian breeds were sequenced, cor-

responding to the 14 SSCP variants found. At least one

representative of each breed was sequenced for each one of

the variants. When the resulting sequences were not

identical for any particular SSCP pattern, all the individ-

uals showing that particular variant were sequenced.

Based on the analysis of 381 nucleotides between positions

15447 and 15827, 20 haplotypes were found. The 24

South American horses sequenced (ARC and PPA) showed

11 haplotypes. The number of haplotypes seems low

compared with other breeds analysed [13 haplotypes in 16

maternal lines of Lipizzan horses (Kavar et al. 1999), 27

haplotypes in 34 Arabian maternal lines (Bowling et al.

2000)]. Although this result could be indicating a bottle-

neck during the establishment of the New World breeds,

more information is needed to assess this phenomenon.

Most haplotypes were restricted to a particular breed, but

two of them where shared between Argentinean Creole

and Peruvian Paso horses from Argentina. Three of the

SSCP variants showed two different haplotypes each, with

the extreme case of four different haplotypes showing an

indistinguishable SSCP pattern (variant 3), differing in up

to 15 nucleotides.

Thirteen more individuals were sequenced from the

Spanish breeds Asturcon, Losino, Potoka, Mallorquina,

Menorquina, Spanish Pure breed or Andalusian and

Arabian. All but one of these breeds generated new haplotypes.

The only exception corresponded to one Andalusian animal

(AND1), which shared its haplotype with Argentinean

Creole horses (ARC1). This haplotype was the commonest

found in Argentinean horses, corresponding to 26 animals

showing SSCP variant 2, from which six horses were

sequenced. They were not randomly selected, but rather

were chosen by pedigree and also to represent three different

breeders in the country.

Analysis of the sequences showed 61 polymorphic posi-

tions, representing 16% of the total sequence obtained. The

mean divergence between sequences was 2.8% (range 0.3–

5.5%). All mutations detected corresponded to transitions,

with one position (15534) representing an insertion/dele-

tion of a single base pair located in the stretch of six

cytosines.

Number of animals showing each SSCP variant

Breed n nV 1 2 3 4 5 6 7 8 9 10 11 12 13 14

ARC 45 5 2 26 10 1 6 – – – – – – – – –

PPA 30 5 5 – – – 6 – – – – – 12 6 1 –

AR 16 7 – – 1 – – 1 3 6 3 1 – – – 1

ARC, Argentinean Creole; PPA, Peruvian Paso from Argentina; AR, Arabian, n ¼ number of

individuals; nV ¼ number of SSCP variants.

Table 1 Number and type of SSCP variants

detected in each breed.


Mirol et al.358

Phylogenetic relationships

Table 2 shows a summary of nucleotide diversities (p) and

pairwise genetic distances among individuals within and

between breeds. An AMOVA analysis showed substantial

subdivision between breeds (FST ¼ 0. 1815, P < 0.0001),

but with a large fraction of the variation (81.85%) found

within populations. Furthermore, variation within breeds

(x ¼ 0.0270, SE ¼ 0.0035) was as high as variation

between breeds (x ¼ 0.0269, SE ¼ 0.0019).

The data were first evaluated for phylogenetic informa-

tion. The distribution of 10 000 randomly generated trees

was left-skewed (g1 ¼ )0.6998, P < 0.01; Hillis & Huel-

senbeck 1992), indicating phylogenetic signal in the data

set. Maximum parsimony and neighbour-joining trees

showed similar patterns. A heuristic search resulted in three

equally parsimonious trees of length 123, 76 steps shorter

than the shortest randomly generated tree. The consensus

tree is shown in Fig. 1a. Two main clusters of haplotypes

can be recognized. The first one includes haplotypes from

Arabian (8 out of 11), Peruvian Paso (three out of six),

Argentinean Creole (two out of seven), and all Menorquina

and Potoka. The second group contains most of Argentin-

ean Creole (five out of seven), half of the Peruvian Paso

haplotypes, some Arabian (3 out of 11), Asturcon, one

Mallorquina and all Andalusian and Losino. The neighbour-

joining tree (Fig. 1b) differs in the clustering of AND3,

ARC2, ARB3, PPA4, LO1 and LO2, which in the distance

analysis appeared as a clade related to the first group of

haplotypes.

Although the results of a bootstrap analysis with 1000

replications showed significant probabilities for most of the

external nodes, only the second group of sequences resulted

in a significant bootstrap value (79%), which is mainly

because of shared substitutions at positions 15494, 15496,

15534, 15603 and 15649. The lack of support of most of

the nodes is not surprising given the previous bootstrap

values found in most of the phylogenetic analysis of horses

published to date, where only a few nodes of the recon-

structed phylogeny showed significant probabilities (Kavar

et al. 1999; Kim et al. 1999; Bowling et al. 2000).

Both maximum parsimony and neighbour-joining ana-

lysis showed no clear relationship between the South

American breeds (Argentinean Creole and Peruvian Paso)

and any of the Spanish breeds analysed, apart from the

decisive fact of Argentinean Creole and Andalusian horses

sharing one haplotype.

In order to compare the results obtained using mitoc-

hondrial DNA and previous results based on microsatellite

data (Canon et al. 2000), maximum parsimony and

neighbour-joining trees were also constructed for the subset

of Spanish horses (trees not shown). Parsimony and dis-

tance analysis resulted in identical trees, with bootstrap

values higher than 50% in most of the nodes. There was a

first cluster of sequences including ARSP, Potoka and

Menorquina breeds (bootstrap 51%). Each one of the three

breeds represented in this group was monophyletic with

bootstrap values higher than 90%. A second group consis-

ted of Losino and one Andalusian haplotype (bootstrap

78%), and a third group included AST, AND1 and MA2

(bootstrap 86%). As in the trees in Fig. 1, the MA1 haplo-

type did not cluster with any other sequence.

The results are quite different from the ones obtained

using microsatellites (Canon et al. 2000), where a clear

clustering of the Atlantic breeds (including Asturcon,

Potoka, and Losino) vs. a cluster of Mediterranean breeds

(Mallorquina and Menorquina) was obtained.

We also analyse the sequences reported in this work in a

wider context. Vila et al. (2001) have found that a

wide variety of mitochondrial haplotypes of horse breeds

clustered in seven different clades with low bootstrap sup-

port, indicating a high number of ancestral matrilines of

ancient origin. We selected 19 haplotypes (accession num-

bers: AF326677, AF326676, AF326678, AF356672,

Table 2 Nucleotide diversity (p) and mean number and range of nucleotide differences within and between breeds. References as in Table 1.

Intra-breed values are in bold.

ARC PPA AR AND AST LO PO MA ME

p 0.018 0.020 0.029 0.020 – 0.008 0.005 0.032 0.008

ARC 6.92 (1–13) 8.71 (0–15) 10.62 (3–19) 7.21 (0–11) 8.71 (4–13) 9.57 (7–13) 12.21 (6–18) 7.71 (1–13) 11.93 (9–16)

PPA 7.77 (1–15) 11 (4–20) 8.5 (1–14) 11 (5–14) 8.75 (6–13) 11.5 (5–19) 9.12 (3–14) 10.5 (5–17)

AR 11.15 (2–20) 10.82 (2–17) 13.73 (6–21) 10.73 (5–15) 11.19 (1–17) 11.27 (2–18) 11.04 (3–17)

AND 10 7 (4–10) 6.5 (2–12) 14 (13–15) 7.5 (2–10) 11.5 (10–13)

AST – 10 (8–12) 18 (17–19) 9 (6–12) 13.5 (12–15)

LO 4 12 (11–13) 9.5 (6–12) 10.5 (8–13)

PO 2 14 (11–17) 11.5 (9–14)

MA 12 12.5 (10–15)

ME 3



AF326674, AF326669, AF064628, AF014416, AF014410,

AF072996, AF14413, AF072990, AF072988, AF072992,

AF072987, AF014409, AF064632, AF072977, D23666)

in order to represent the seven clades described. A maxi-

mum parsimony and neighbour-joining analysis were con-

ducted with the total data set of 53 sequences. The

neighbour-joining tree is shown in Fig. 2. The structure of

the tree is the same as the one obtained in Vila et al. (2001).

The Spanish and South American haplotypes are dispersed

along the different clades.

Discussion

In a previous analysis based on five polymorphic protein

loci, Peral Garcıa et al. (1996) found a close relationship

between Andalusian, Barb, Argentinean Creole horses

and Peruvian Paso. Bowling (1994) has also suggested a

close association between Andalusian and Argentinean

Creole based on blood type markers, although in this case

the Nei’s distances among all breeds and feral popula-

tions examined were so similar that the results were not

conclusive.

Our results indicate that there is a close relationship

between Andalusian and Argentinean Creole horses. It is

very noteworthy that ARC1, the most common haplotype

found in the South American breed with 26 animals

showing SSCP variant 2, is identical to one of the haplo-

types found in the Andalusian horses examined. A longer

sequence of 468 bp (positions 15396–15862) was com-

pared between both haplotypes and proved to be identical.

(a)

Figure 1 (a) Consensus of the three most

parsimonious trees found in the sample of

South American and Spanish horses analysed.

Figures on the internodes are bootstrap prob-

abilities (in percentage) based on 1000 repli-

cations. (b) Neighbour-joining tree based on

the Kimura two-parameters distances. Boot-

strap values on the internodes. Haplotype

names as in the text.


Mirol et al.360

The mean number of nucleotide differences between both

breeds was 7.21 (range 0–11), the lowest of all interbreed

comparisons, and very similar to the mean number of

nucleotide differences within the Argentinean Creole horses

(6.92, range 1–13). The Nei’s genetic distances were higher

in all comparisons with Celtic and Arabian horses. With the

Peruvian Paso from Argentina, the mean number of nuc-

leotide differences was 8.71 (range 0–15). There were two

haplotypes shared between both breeds (CP1 and CP2). The

origin of the Peruvian Paso can be traced to the 16th

century, from Barb and Andalusian horses brought by

Spanish conquerors to Peru, so their close association with

the Argentinean breed is not surprising. Furthermore, the

Argentinean Peruvian Paso is a young breed, and so, even

when stallions were brought from Peruvian Paso in Peru,

the mares were frequently not pure Peruvian Paso, and

their mtDNA was probably of mixed ancestry. The mean

number of nucleotide differences between Peruvian Paso

and Andalusian is 8.50 (range 1–14), again the lowest of all

comparisons and similar to the comparison Argentinean

Creole–Peruvian Paso and the Peruvian Paso intrabreed

differences (7.78, range 1–15).

Even when the distances between Argentinean Creole,

Peruvian Paso and Andalusian are low, haplotypes repre-

senting both Argentinean Creole and Peruvian Paso

appeared in the two main clades found in the parsimony

and neighbour-joining trees (Fig. 1a,b). This could be

interpreted as mixed ancestry or multiple origin of the South

American breeds, as was suggested when similar results

were obtained in studies of mtDNA of Lipizzan horses (Kavar

et al. 1999) and Cheju horses (Kim et al. 1999). An alter-

native explanation could be that the phylogenetic

(b)

Figure 1b Continued.



reconstruction is reflecting very ancient maternal lineages,

present in the Spanish ancestors and so in the South

American descendants. This hypothesis is in agreement

with the tree depicted in Fig. 2. Haplotypes of Argentinean

Creole horses appeared in four different clades (A, C, D and

F, nomenclature of Vila et al. 2001), and Argentinean

Peruvian Paso in three clades (A, C and D). Andalusian

horses are clustered in clades C and D, which also contain

Argentinean Creole and Peruvian Paso. Vila et al. (2001)

propose that modern horse sequences do not define mono-

phyletic groups with respect to wild progenitors, as would

be expected if they were founded from a limited wild stock.

They assume that the high diversity of matrilines observed

suggests the use of a large number of wild populations in the

origin of the domestic horse. In this context, the lack of

strongly supported phylogenetic relationships among the

breeds analysed in this work, indicates the retention of very

ancient mitochondrial diversity.

The phylogenetic pattern is rather different if microsat-

ellites are used as molecular markers. As the development of

distinct breeds usually follows a pattern of very restricted

selection based in a few males serving many females, indi-

viduals from the same breed are generally clustered together

in a phylogenetic tree when microsatellites are used (Vila

et al. 2001). In a previous work, Canon et al. (2000) studied

the genetic structure of Spanish Celtic horses, including

Asturcon, Losino, Potoka, Mallorquina and Menorquina,

using microsatellites. The genetic distances between breeds

Figure 2 Parsimony tree obtained when some

of the sequences reported in Vila et al. (2001)

were included in the analysis. Pleist: remains of

horses dated 12 000–28 000 years ago, Anc:

remains of horses dated 1000–2000 years ago

(Vila et al. 2001). Figures in the internodes

represent bootstrap values, and letters indicate

the different clades described by Vila et al. In

bold and underlined, sequences from the horse

breeds analysed in the present work.


Mirol et al.362

were in all cases higher than the ones we obtained here

using mtDNA (Table 2), and many of the breeds were

defined as monophyletic groups. They also found a clear

clustering among all Atlantic breeds (Asturcon, Losino,

Potoka), different from the clade containing the Mediterra-

nean breeds (Mallorquina and Menorquina). This tree is

different from the one based on mtDNA, where only two of

the Spanish breeds (Potoka and Menorquina) constitute

monophyletic groups, and there is no association between

Atlantic breeds on the one hand, and Mediterranean breeds

on the other. This difference is reflecting the maternally

dominated genetic flow between breeds and the male-biased

selection in the breeds development.

In conclusion, we provide the first mitochondrial char-

acterization of South American and Spanish horse breeds.

In addition, our results support the very ancient origin of

the matrilines in horses, from the perspective of modern

New World breeds.

Accession numbers

GenBank accession numbers for the sequences presented

here are AF465984 to AF466016.

Acknowledgements

This paper was supported by National Research Council and

Universidad Nacional de La Plata grants to PPG and FND,

and SECyT and Fundacion Antorchas grants to PMM. The

authors wish to thank Mariana Kienast for the collection of

the samples and Jeremy B. Searle and Chris Faulkes for very

useful comments.

References

Avise J.C. (1994) Molecular Markers, Natural History and Evolution.

Chapman & Hall, New York.

Bowling A.T. (1994) Population genetics of Great Basin feral

horses. Animal Genetics 25, 67–74.

Bowling A.T., Del Valle A. & Bowling M. (2000) A pedigree-based

study of mitochondrial D-loop sequence variation among Ara-

bian horses. Animal Genetics 31, 1–7.

Canon J., Checa M.L., Carleos C., Vega Pla J.L. & Dunner S. (2000)

The genetic structure of Spanish Celtic horse breeds from mi-

crosatellite data. Animal Genetics 31, 39–48.

Felsenstein (1991) PHYLIP (Phylogeny Inference Package), Version

3.4. University of Washington, Seattle, WA.

Higgins D.G., Bleasby A.J. & Funchs R. (1992) CLUSTAL V:

improved software for multiple sequence alignment. Computer

Applications in the Biosciences 8, 189–91.

Hillis D.M. & Huelsenbeck J.P. (1992) Signal, noise and reliability in

molecular phylogenetic analysis. Journal of Heredity 83, 189–95.

Ishida N., Hasegawa T., Takeda K., Sakagami M., Onishi A.,

Inumaru S., Kamtsu M. & Mukoyama H. (1994) Polymorphic

sequence in the D-loop region of equine mitochondrial DNA.

Animal Genetics 25, 215–21.

Kavar T., Habe F., Brem G. & Dovc, P. (1999) Mitochondrial D-loop

sequence variation among the 16 maternal lines of the Lipizzan

horse breed. Animal Genetics 30, 423–30.

Kim K.-I., Yang Y.-H., Lee S.-S., Park C., Ma R., Bouzat J.L. & Lewin

H.A. (1999) Phylogenetic relationships of Cheju horses to other

horses breeds as determined by mtDNA D-loop sequence poly-

morphism. Animal Genetics 30, 102–8.

Kimura M. (1980) A simple method for estimating evolutionary

rate of base substitutions through comparative studies of nuc-

leotide sequences. Journal of Molecular Evolution 16, 111–20.

Marklund S., Chaudhary R., Marklund L., Sandberg K. & Andersson

L. (1994) Extensive mtDNA diversity in horses revealed by PCR–

SSCP analysis. Animal Genetics 26, 193–6.

Maruya E., Saji H. & Yokoyama S. (1996) PCR–LIS–SSCP (Low

ionic strenght stranded conformatioon polymorphism) – a simple

method for high resolution allele typing of HLA-DQB1, and DPB1.

Genome Research 6, 51–7.

Peral Garcıa P., Kienast M., Villegas E., Dıaz S. & Dulout F. (1996)

Estudio de relaciones geneticas entre razas equinas mediante el

analisis multivariado. Agro Sur 24 (1), 39–47.

Rodero, A., Delgado, J.V. & Rodero, E. (1992) Primitive Andalusian

livestock and their implications in the discovery of America.

Archivos de Zootecnia 41, 383–400.

Schneider S., Roessli D. & Excoffier L. (2000) Arlequin: A Software for

Population Genetics Data Analysis, Ver 2.000. Genetics and

Biometry Laboratory, Department of Anthropology, University of

Geneva, Geneva.

Swofford D.L. (1997) PAUP: Phylogenetic Analysis Using Parsimony.

Smithsonian Institution, Washington, DC.

Tudela J. (1987) El Legado de Espana a America. Ediciones Pegaso,

Madrid.

Vila C., Leonard J.A., Gotherstrom A., Marklund S., Sandberg K.,

Liden K., Wayne R.K. & Ellegren H. (2001) Widespread origins of

domestic horse lineages. Science 291, 474–7.

Xu X. & Arnason U. (1994) The complete mitochondrial DNA

sequence of the horse, Equus caballus: extensive heteropplasmy of

the control region. Gene 148, 357–62.

Xu X. & Arnason U. (1996) The complete mitochondrial DNA

(mtDNA) of the donkey and mtDNA comparison among four

closely related mammalian species-pairs. Journal of Molecular

Evolution 43, 438–46.



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Phylogenetic relationships of Argentinean Creole horses and other South American and Spanish breeds inferred from mitochondrial DNA sequences