native horses of the iberian peninsula have played an important role in the development of current american horse breeds . approximately 10,000 years ago , north and south american equus species became extinct for several reasons ( grooves and ryder , 2000 ) . horses returned to the americas during the second trip of christopher columbus to the island of hispaniola in 1493 ( present - day dominican republic and haiti ) ( laguna - sanz , 1991 ; rodero et al . consequently , the first horses brought to america by columbus were not carefully selected , as revealed by complaints from christopher columbus to the catholic kings about the quality of these animals ( cabrera , 1945 ; sponenberg , 1992 ) . it is likely that the original horses were replaced before their departure to america by peasant stock breeds called jacas  located in sevilla ( rodero et al . , 1992 ; sponenberg , 1992 ) . in the years following the expeditions by columbus , conquering the new world became more important and the quality of the horses brought to america improved . as a result , horse breeds brought to america during this period varied in quality and origin , although most came from the southern iberian peninsula ( cabrera , 1945 ; rodero et al . traditionally , iberian horses have been classified into two groups : celtic ponies , in reference to breeds distributed throughout the north atlantic iberian region , and iberian horses located throughout the southern iberian peninsula ( aparicio , 1944 ; jordana et al . , 1995 ; several studies have postulated that iberian celtic ponies share a common ancestry with british ponies , whereas iberian horses share a common ancestry with the barb horse ( garcia - dory , 1980 ; sotillo and serrano , 1985 ; lvarez llana , 1995 ; martinez et al . the high degree of genetic diversity among iberian horse breeds suggests that the iberian peninsula served as a refugium for wild horses and has contributed considerably to local domestic stock , as well as to the domestic horse breeds of central europe and the united kingdom ( warmuth et al . , 2011 ) . one route was established by the expeditions of alonso de ojeda and diego de nicuesia in 1509 ( diaz , 1988 ) and went through panama via the gulf of uraba to nueva granada (= new granada ; present - day colombia ) . the second route was established by spanish expeditions through coro ( present - day venezuela ) to the eastern plains region known as llanos orientales  in nueva granada , in the current departments of arauca , casanare and meta ( montoya b , 1988 , undergraduate final work , universidad de los llanos , villavicencio , colombia ) . the horses that arrived via the first route are known today as the colombian paso fino horse breed and are found throughout most of colombia . the marshy areas from which the paso fino breed came favored the development of a typical gait very suitable for agriculture or riding . through years of selection , the colombian paso fino breed has been aptly divided into four lineages based on their natural gait : the colombian paso fino , colombian trocha , colombian trocha - gallop and colombian trot - gallop ( fedequinas , 2006 ) . horses that arrived in colombia via the second route gave rise to the colombian creole cattle horse breed ( known as criollo de vaquera ) . this breed occurs in three geographic regions , including the departments of arauca , casanare and meta , which make up part of the  llanos orientales  near the colombian - venezuelan border . traditionally , these horses have been used in agriculture and have been kept isolated in a state of semi - freedom in herds formed by mares , colts and stallions , without much selective pressure ( sandoval rfa and torres mti , 1996 , undergraduate final work , universidad de los llanos , villavicencio , colombia ) . the mitochondrial dna control region has been widely used to understand the origin and diversification of domestic horses ( vil et al . , 2001 ; mirol et al . , 2002 ; luis et al . , 2006 ; kavar and dov , 2008 ; warmuth et al . , 2011 ) . previous studies have discovered high levels of mitochondrial dna variability within and among horse breeds , without a clear geographical pattern of distribution ( hill et al . , 2002 ) . recent studies have confirmed the iberian peninsula as a geographic maternal and genetic refugium , a conclusion supported by the finding that several pre - domestic iberian maternal lineages survive in modern horses of iberian descent ( warmuth et al . , 2011 ) . ( 2002 ) , is well represented in both southern iberian and new world breeds , thus highlighting the importance of iberian breeds during re - population of the new world by horses ( royo et al . , 2005 ; luis et al . , 2006 ) . despite the clear connection between horses found in the iberian peninsula and horses found in the americas today , the influence of the iberian horse gene pool on colombian creole horses has not yet been analyzed . the aim of this study was to analyze the two principal horse populations in colombia , the colombian paso fino and the colombian creole cattle horse , in order to clarify their maternal genetic origin and the current matrilineages in both breeds . in total , 116 blood samples were collected from unrelated animals representing the colombian paso fino ( cpf ) , the colombian creole cattle horse ( from the casanare ( cvc ) and meta ( cvm ) populations ) and the following five iberian horse breeds : asturcon ( ast ) , caballo gallego ( cga ) , jaca navarra ( jna ) , losino ( los ) and pura raza espaola ( pre ) ( table 1 ) . dna was extracted using a qiagen dneasy blood & tissue kit ( qiagen , germany ) according to the manufacturer s instructions . a 364-bp fragment of the mtdna d - loop that included positions 1546915832 ( xu and arnason , 1994 ) was sequenced . the primer sequences were designed using the primer 0.5 software ( gcg software package , university of wisconsin , usa ) . the primers used were d - loopf ( 5-ccaccatcaacacccaaagc-3 ) and d - loopr ( 5-gccctgaagaaagaaccagat-3 ) . the amplified fragments were purified using a purelink pcr purification kit ( invitrogen ) , according to the manufacturer s instructions . sequencing was done using an abi prism 310 genetic analyzer ( applied biosystems ) and the sequences were aligned using clustalx ( thompson et al . , 1997 ) . the mean number of pairwise differences ( mnpd ) , nucleotide diversity and fst distances were obtained using mega 4.1 ( tamura et al . , 2007 ) and arlequin 3.11 ( excoffier et al . , 2005 ) softwares . partitioning of the total genetic variance into components based on intergroup ( colombian and spanish ) , interbreed and inter - individual differences was done using analysis of molecular variance ( amova ) implemented in arlequin 3.11 ( excoffier et al . , the sequencing of mtdna from the 116 blood samples of colombian and spanish horse breeds revealed 51 haplotypes defined by 69 polymorphisms ( 52 transitions and 17 transversions ) distributed among 63 distinct sites . five sites contained transitions and transversions and one site had two unique transversions ( figure s1 ) . for each breed , we identified between six and 15 haplotypes . the colombian creole cattle horse from casanare ( cvc ) ( 15 ) and asturcon ( ast ) ( 10 ) showed the highest number of haplotypes among the colombian creole horse populations and spanish breeds , respectively ( table 1 ) . the cvc and ast populations also had a higher frequency of haplotypes exclusive of the other breeds studied ( 60% and 70% , respectively ) . seven haplotypes were common to spanish and colombian horse breeds ( haplotypes 2 , 3 , 8 , 13 , 15 , 16 and 21 ) . only 10 haplotypes were found in more than one breed and 41 haplotypes were unique to a single breed ( table 1 ) . the cvc ( 9 ) and jaca navarra ( jna ) ( 7 ) populations had a higher number of exclusive haplotypes among colombian and spanish breeds , respectively . haplotypes 2 ( 24% ) and 3 ( 10% ) had a total frequency of 34% , whereas the frequencies of all other individual haplotypes varied from 0.9% to 5% of the total haplotypes ( table 1 ) . thirty - eight haplotypes coincided with jansen s haplogroup classification ( jansen et al . , 2002 ) and were assigned to nine previously defined haplogroups ( a1 , a3 , a5 , a6 , c1 , c2 , d1 , d3 and f1 ) ( table 1 ) ; the remaining 13 haplotypes were classified as new haplotypes ( table 1 ) . the most frequently identified haplotypes in the colombian and spanish breeds were type d1 ( 47% ) , a3 ( 10% ) and c2 ( 9% ) . haplotype d1 was considered to be representative of iberian and north african breeds and was found in every breed analyzed except jaca navarra ( table 2 ) . the skeleton network revealed four main clusters that corresponded to the four haplogroups ( a , c , d , and f ) described by the classification system of jansen et al . the haplotypes belonging to haplogroup d showed a star - like skeleton in which the center of the star corresponded to the haplotype with the highest frequency ( 2 ) surrounded by a number of peripheral haplotypes of lower frequency . most of the different horse breeds were represented in all four haplogroups , the exceptions being pura raza espaola , which was absent in haplogroup c , and losino and jaca navarra , which were absent in haplogroup f. the haplotype diversity , nucleotide diversity and mean number of pairwise differences were similar for the colombian and iberian breeds ( table 3 ) . however , the colombian paso fino breed had lower nucleotide diversity than the other colombian and spanish breeds . haplotype diversity ranged from 0.97 in pura raza espaola to 1 in the gallego breed . among colombian breeds , the colombian creole cattle horse from meta had the highest value ( 0.92 ) whereas the colombian paso fino breed had the lowest nucleotide diversity ( 0.0114 ) and the lowest mean number of pairwise differences ( 4.2 ) the average fst distance between any two breeds ranged from just under 3% to 12% ( table 3 ) . the colombian creole horse populations had values at the lower end of this range ( 2.74.9% ) whereas the spanish breeds had higher values ranging from 6.2% to 11.5% . amova of the various haplotypes revealed no significant genetic differences among the spanish and colombian breeds ( fct = 1% ) ( table 4 ) . however , this lack of genetic differentiation between groups was not reflected in the differences among breeds within groups or among breeds between different groups ( fst = 5% and fsc = 5% ) . several studies have confirmed the importance of iberian horses in the repopulation of the new world with equus species ( mirol et al . , 2002 ; luis et al . , 2006 ) . however , this is the first genetic analysis of the two principal colombian equine breeds ( colombian creole cattle horse or criollo de vaquera and colombian paso fino ) . haplotypes of haplogroup d were found more often than any other haplotype in the colombian and iberian horse breeds analyzed . haplogroup d is representative of the ancestral horse populations of the iberian peninsula , thus supporting historical records of iberian ancestry in the development of colombian horse breeds ( miretti et al . , 2004 ; primo , 2004 ; luis et al . , 2006 ) . haplogroup a has previously been found in the marismeo breed , which is an ancient semi - feral population that occurs in doana natural park in spain ( royo et al . , 2005 ) . we have identified haplotype a in some of the colombian horse breeds analyzed , including the sevillan jacas breed , which is closely related to the extant marismeo horse breed , thus explaining the origin of haplogroup a in colombian horse breeds ( rodero et al . , 1992 ; sponenberg , 1992 ) . this finding confirms historical documents that report the presence of the sevillan jacas breed among the horses brought to america . the identification of haplogroup c in the colombian horse breeds studied here most likely reflects the authorization to import celtic horses to nueva granada , which included present - day panama and colombia ( luis et al . , 2006 ) . haplogroup f was not identified in the colombian horse breeds , but the closely - related haplotypes 11 and 19 were found in the colombian creole cattle horse ( criollo de vaquera ) breed . haplogroup f , which is found in middle eastern breeds , could be related to the berber phenotype found in colombian breeds . further research in this area should shed light on the corresponding haplotype - phenotype relationships . the proportion of the total variance explained by the genetic differences among iberian and colombian horse breeds was low ( 1% and data not shown ) as a consequence of the iberian origin of the colombian breeds . nevertheless , the variance explained by the differences between breeds was higher ( 6% ) because of reproductive isolation after the colonization event and because of genetic drift . the reduced diversity of new world horse breeds when compared with iberian breeds is consistent with an iberian origin . previous studies that included south american horse breeds support this theory ( luis et al . , 2006 ) . as expected , the colombian paso fino breed had a lower degree of genetic diversity than iberian breeds . in contrast , the colombian creole cattle horse breed showed a similar degree of genetic diversity to that of the iberian breeds analyzed . the colombian paso fino breed is considered to have undergone selective breeding because of its characteristic gait , which renders it very suitable for riding . conversely , the colombian creole cattle horse breed has been kept in relative isolation with very little selective pressure . the different selection processes applied to colombian breeds could explain the high genetic diversity found in the colombian creole cattle horse relative to the colombian paso fino horse . our network analysis of the mtdna d - loop sequences discovered 51 haplotypes that could be classified into four main clusters that formed the four principal haplogroups ( a , c , d and f ) . the heterogeneity of the marismeo breed , in which haplogroup a has previously been identified , could explain the high number of unidentified haplotypes ( six of 13 ) that aligned within this haplogroup . in conclusion , four principal matrilineages have been found in the two principal colombian horse breeds . haplogroup d , which is representative of the ancestral iberian horse population , was discovered most often in colombian breeds , thus supporting the ancestral iberian origin of colombian horses . the higher degree of genetic diversity found in the colombian creole cattle horse when compared with either the colombian paso fino or other south american horse breeds is a consequence of significantly different selective pressures in both populations . the following online material is available for this article : figure s1 - variable sites in a 364-bp fragment of the mitochondrial dna d - loop of colombian and spanish horse breeds .