Introduction

Possessing multiple ancient features of their earliest forms originated from the auroch (Bos taurus primigenius), and distinguished by its extensive management¹, the Lidia bovine is a primitive breed whose roots can be traced back to approximately 250 yr ago in the Iberian Peninsula in order to satisfy a demand of cattle destined to participate in popular spectacles. At present, shows involving cattle are found in geographical areas comprising mainly the southwest region of Europe (Italy, France, Spain and Portugal) and along the American continent involving approximately 14 countries². These kinds of spectacles have their origins in the early Mediterranean civilizations, where bovines of untamed behavior, lacking of docile temperament, participated in ceremonies and rituals as an assigned symbol of the nature´s strength³. After, in the 13^th Century those practices evolved into social events called tauromachies or “tauromaquias”, a term that makes reference of a cultural and subjective representation of all types of games involving cattle and not as a single term for identifying one single practice (since sometimes the term is associated exclusively with the Spanish bullfight or “corrida”). To date, tauromaquias assemble a social and semantic construction, are an important livestock economic source and reinforce local and regional identities of the countries where are still found^2,4. Diversity in orography and climate along with historical factors and traditions, led place to the development of different variants of bovine populations. There all were selected based upon behavioral performance of aggressiveness: the Andalusian and Navarro-Aragonese that in Spain gave rise to the original Lidia breed population, in Portugal the Lidia Portuguese breed and in France the Landaise and Camargue’s cattle populations⁵.

The specialization and intensification of animal husbandry did not take place until ~250 yr ago with the emergence of many specialized breeds during the industrial revolution. In Spain, to become breeder of this type of cattle provide more status to the members of aristocracy and gentry, who in search of improving the behavioral skills of their “aggressive” bovines developed a documented breeding system, giving rise to the original Lidia breed population^4,6. Moreover, these breeders concerned about raising bovines that could be distinguished for performing different type of behavior (sometimes demanded for the different type of festivities) established closed family trees that prompted to a fragmentation of the racial group into small lineages⁷.

In America, specifically in Mexico, bovines with these behavioral characteristics were imported during the colonial period (after the conquest of the Aztec empire in 1521) to take part in the festivities that were inherited as traditions of the Spanish colonizers². The Lidia breed specialization began between 1908 and 1912 when mainly two families of breeders (Llaguno and González) imported a reduced number of Spanish Lidia bovines. Each family kept different breeding strategies, the Llaguno family followed a closed breeding scheme reproducing the new imported animals among them, and the González family reproduced the imported animals with local Mexican bovines selected for aggressiveness⁸.

Mexican Lidia census suffered dramatic losses during the post-revolution period, which lasted ten years (1910-1920). After those years, breeders recovered their Lidia production opting for raise cattle that derived either from the Llaguno or González families. In recent years, during 1996 and 1997, some Mexican breeders imported close to 1,000 Spanish Lidia bovines before closing borders of importations from Spain⁹. To date, this recent refreshment suggests a strong impact in the genetic structure of the herds belonging from the breeders that took part in those importations. But still, the major part of the Mexican Lidia population derives from the elder Llaguno and González families⁸. Despite both Mexican and Spanish Lidia populations are demographically well stablished, their low effective population size places them at risk of extinction⁷.

Previous studies on the Spanish Lidia population found a genetic uniqueness in the breed, which is given by a high genetic differentiation between lineages⁶. Moreover, Eusebi et al¹⁰ studied the genetic diversity of the Mexican Lidia population and its divergence from the Spanish Lidia population and found high genetic differentiation among them. However, both studies have been conducted by using neutral autosomal microsatellites, and recently, the availability of SNP panels allow the investigation of livestock genetic diversity and genetic structure at higher level of resolution, hard to reach with other types of markers.

In this study, a subset of 573 SNPs with low gametic disequilibrium were selected from the 50K medium density genotyping array (Illumina Inc., San Diego, CA) to assess the genetic diversity and structure of the Mexican and Spanish Lidia populations and thereafter analyze the relationships among these two populations, in order to explore the degree of admixture among them.

Material and methods

Blood samples of 468 Lidia bovines were collected: 119 belonging to the Mexican population and 349 to the Spanish population. Classification of the Spanish lineages was given according to Cañón et al⁶ and, for the Mexican Lidia population the samples arise from 20 breeders studied independently but classified into the family that they belong to (González or Llaguno), according to standards set by the by the Mexican Lidia Breeders Association⁹. More information is available in Table 1.

Animals were randomly chosen according to their origin, and qualified veterinarians collected the samples during routine practices in the framework of official programs aimed at applying preventive medicine. Blood samples were maintained in Magic Buffer® DNA solution¹¹ until DNA extraction by standard phenol/chloroform methods¹². Genotypes were obtained with the Illumina 50k BeadChip (Illumina Inc., San Diego, CA) and SNP quality was analyzed with the Genome Studio software (Illumina). Thereupon, by using the PLINK software¹³ the dataset of SNPs was filtered according to the following excluding criteria: SNPs located on sexual chromosomes; individuals with >20% missing genotypes; SNPs with a minimum allele frequency <0.01; markers that did not match Hardy-Weinberg equilibrium expectations (P<10^-6); and a restricted linkage disequilibrium criterion of r²<0.01; thus assuring low gametic disequilibrium rate among markers. Finally, the information derived from 573 SNPs spanning across all the bovine autosomal chromosomes, were selected.

Statistical estimates of genetic diversity were performed followed by a multifactorial correspondence analysis estimated to quantify genetic diversity; these analyses were carried out with the GENETIX v.4.0.5 software¹⁴. The proportion of mixed ancestries among populations was inferred with STRUCTURE v.2.1. software¹⁵ which uses a hierarchical Bayesian model to infer a population structure from multilocus genotypes and assign each individual into that supposed population, assuming that each individual may have mixed ancestry from different underlying populations. The figurative number of populations or genetic clusters (K) ranged from 2 to 4 with six replicate chains for each value of K. The runs sharing maximum likelihood pattern were selected to be displayed in a graphic constructed with the DISTRUCT v.1.1. software¹⁶.

Results

Population relationships and clustering

The Bayesian approach implemented in STRUCTURE software¹⁵ was used to analyse the clustering and genetic relationship among both Mexican and Spanish populations, acronyms are stated as defined in Table 1, displaying names of the breeders and their belonging family of the Mexican population, and names of the lineages of the Spanish population. The contribution of the assumed ancestral populations is graphically presented in Figure 1, with K populations going from 2 to 4.

The acronyms are as defined in Table 1 and each acronym encloses the number of breeders belonging to each lineage.

Figura 1 Analysis of the genetic structure of the Mexican breeders and the Spanish lineages, the plot shows common genetic ancestors, or model based population assignments (K), for values going from from k=2 (upper) to k=4 (lower) 

In the Mexican population, from k=2 to k=4 a single ancestral population is observed in most of the breeders of the Llaguno family (Gar, Igu, Boq, Mat, Mon, Zar, Riv, Rey, Bar, Teo, Xaj and Mat), with a clear separation between González and Llaguno families. Mixed contributions with some of the Spanish lineages (Alb, Sal and Col) are observed in all of the González breeders (Sec, Cas and Har) and some breeders from Llaguno family (Tor, Jos, Fer, Crl and Enc) when k=4.

In the Spanish population when k=2 most of the lineages belong to a same single ancestral population with some mixed contributions observed in Alb, Sal and Col lineages. Then when k=4 three different ancestral groups or clusters are differentiated: one conformed by Alb, Sal and Col lineages, a second cluster conformed by Cua, Con, Veg, Miu, Vil, Sie Mur, Gam, Ana, Maz, Fel, Pab and Ara and a third cluster conformed of Dom, Ata, Ant, Cor, Crm, Bal and Urc.

In general, among Spanish and Mexican populations, both showed different genetic ancestral origin with an exception of mixed contributions in the Mexican breeders of the González family and Tor, Jos, Fer, Crl, and Enc breeders from the Llaguno family with the Spanish lineages of Alb, Sal and Col.

Finally, in the correspondence analysis (Figure 2) a genetic discrimination between the Mexican and Spanish populations can be seen, with some exceptions like Sec and Tor breeders from the Mexican population who are placed closer to the Spanish Lineages than to the Mexican breeders. Furthermore, the Spanish Col, Sal and Alb: lineages are situated closer to the Mexican breeders than to the rest of the Spanish lineages.

Figura 2 Correspondence analysis of the Spanish and Mexican Lidia breed populations 

Discussion

High gene diversity values were found in both the Spanish (0.48) and the Mexican populations (0.46). This value obtained in the Mexican population is remarkable, since lower gene diversity values were expected to obtain considering that, most of the current Mexican population arose from a few individuals of the Spanish Lidia population. On the contrary, similar diversity values were observed in both populations, so it is reasonable to consider certain degree of introgression with local Creole cattle populations of diverse origin during the establishment of the Mexican Lidia breed population.

Moreover, significant F _IS (P<0.01) values were observed in both populations which means a subdivision within each, higher (0.21) in the Spanish than in the Mexican population (0.06). This subdivision in lineages or breeders results in the preservation of more genetic variance¹⁷, but a faster loss of genetic diversity within sub-population can be expected. Additionally, a loss of diversity due to population bottlenecks and founder effects result in increased inbreeding, resulting that the preservation of heterozygosity in the whole population is at the expense of a progressive poor genetic health within each sub-population.

Genetic diversity analysis revealed significantly higher genetic distances (P<0.05) in the Spanish population compared to the genetic distances of the Mexican population, with whole population F _ST values of 0.18 and 0.10 respectively (Table 3). Similar results were observed by Eusebi et al¹⁰ with data obtained with microsatellite markers. In the Mexican population the lower genetic distances among breeders means higher animal exchangeability, a common practice in Mexico and less usual in Spain, where higher genetic distances between lineages were obtained, thus explained by higher genetic isolation among lineages.

Furthermore, genetic structure analysis revealed in both, Correspondence and Bayesian clustering analysis a clear separation among families (González and Llaguno) of the Mexican population and in the Spanish population three clusters are observed at k=4. The cluster with Albaserrada (Alb), Saltillo (Sal) and Santa Coloma (Col) is placed closer (correspondence analysis) and share genetic structure with the Mexican González family and some Llaguno breeders (Tor, Crl, Jos and Enc), leaving clearly differentiated the remaining Llaguno breeders. This similarity of Spanish Alb, Sal and Col lineages with the above mentioned González family and the few Llaguno breeders is not surprising, given the fact that those breeders were involved in the imports of 1996 and 1997, introducing mainly animals from Santa Coloma (Col) and in lesser extent Saltillo (Sal) and Vega Villar (Veg)⁸. But it is worth to note the proximity of Albaserrada (Alb) lineage to the Mexican population, since Albaserrada herds have been raised under strict closed breeding schemes from 1912 onwards¹⁸. This genetic closeness is explained by two similar historical and genetical phenomenons’ as Albaserrada lineage derive from Saltillo and Santa Coloma lineages⁶ and in parallel, those similar Mexican breeders constructed their herds by mating animals from the same lineages as ancestors.

A deeper analysis of the Mexican population structure revealed that anthropogenic barriers are well documented drivers of the genetic differentiation observed among breeders (e.g., the clear genetic division observed between the González and Llaguno families). Both families where located respectively in the North and south central regions of Mexico and became much like hegemony of Lidia cattle, being in charge to supply Lidia cattle to emerging farmers in their regions. In addition, both families’ bovines did not mix each other⁸, confirming the different genetic origin among them.

Conclusions and implications

Isolation along with a small founder population size shaped by a classic bottleneck effect can explain the differentiation of the Llaguno Family of the Mexican population from the Spanish Lineages of which it arose. To all this, a possible introgression of Creole Cattle populations located at the north and south central regions of Mexico¹⁹ could explain this gain of diversity. A trace-back analysis of the extant cattle populations in those regions could be footprints in the way to explain the major ancestors of the Mexican Llaguno family.