hDomestication and Phylogeography of Taurine and Zebu Cattle ( BOS tuum and Bos indicus )

Genetic variation at 20 microsatellite loci was surveyed to determine the evolutionary relationships and molecular biogeography of 20 different cattle populations from Africa, Europe and Asia. Phylogenetic reconstruction and multivariate analysis highlighted a marked distinction between humpless (taurine) and humped (zebu) cattle, providing strong support for a separate origin for domesticated zebu cattle. A molecular clock calculation using bison (Bison sp.) as an outgroup gave an estimated divergence time between the two subspecies of 610,000-850,000 years. Substantial differences in the distribution of alleles at 10 of these loci were observed between zebu and taurine cattle. These markers subsequently proved very useful for investigations of gene flow and admixture in African populations. When these data were considered in conjunction with previous mitochondrial and Y chromosomal studies, a distinctive male-mediated pattern of zebu genetic introgression was revealed. The introgression of zebu-specific alleles in African cattle afforded a high resolution perspective on the hybrid nature of African cattle populations and also suggested that certain West African populations of valuable disease-tolerant taurine cattle are under threat of genetic absorption by migrating zebu herds. M ICROSATELLITES or simple tandem repeat (STR) genetic markers have become the mainstay of genetic linkage mapping efforts. Recently, they have also been used to address questions concerning the genetic diversity and evolutionary history of various organisms. In particular, the genetic origins of human populations have been usefully explored using microsatellite polymorphisms ( BOWCOCK et al. 1994; DEKA et al. 1995; GOLDSTEIN et al. 1995a). We have also previously used microsatellite polymorphisms to investigate genetic variation among European breeds of cattle ( MACHUGH et al. 1994). Microsatellite analysis of genetic diversity provides two distinct levels of information. In addition to allele frequency differences among populations, it also prcvides information about the cladistic relationships between alleles and groups of alleles on the basis of differences in allelic repeat length. Novel methods have therefore been developed to investigate the mutational dynamics of microsatellites and their application to population genetic problems. The stepwise mutation model (SMM) , in which microsatellite allele length is treated as an incremental quantitative character, has been an important component of these new analytical methods ( SHRTVER et al. 1993, 1995; GOLDSTEIN et al. 1995b). Domesticated cattle are the major component of pasCorresponding authw: David MacHugh, Genetics Department, Trinity College, Dublin 2, Ireland. E-mail: [email protected] toral economies throughout the world. Through milk, they provide the bulk of the animal protein consumed by many human societies, and contribute other important commodities including meat, hides, traction and dung. The biological systematics and evolutionary relationships of cattle have always been highly contentious. In particular, the relationship between the two main types of cattle, humped zebu (Bos indicus) and humpless taurine (Bos t a u m s ) , has been an active area of research with various hypotheses having been proposed to account for the morphological and genetic differences observed between the two subspecies. One school of thought asserts that domesticated cattle were first developed from a single wild ancestor, the aurochs (Bos pimigenius primigenius) during the early Neolithic phase of the agricultural communities in the Near East (circa 8000-9000 BP ) . Bos indicus populations are then thought to have been produced at a later date through breeding and selection from Bos t a u m s cattle ( EPSTEIN 1971; EPSTEIN and MASON 1984; PAYNE 1991 ) . The alternative viewpoint contends that domesticated zebu populations were developed independently by a separate group of early pastoralists and that the candidate domestication centers were the Neolithic societies of Baluchistan in present-day Pakistan. The southern Asian subspecies of aurochs (Bos primigenius namadicus) would then be the most likely progenitor of domesticated zebu cattle. This interpretation is sup ported by recent archaeological and genetic evidence (MEADOW 1993; LOFTUS et al. 1994a; BRADLEY et al. Genetics 146 1071-1086 (July, 1997) 1072 D. E. MacHugh et al. 1996). In particular, through analysis of mitochondrial DNA (mtDNA) sequence variation in extant cattle populations, Loftus et al. (1994a) were able to demonstrate that taurine and zebu cattle probably diverged at least 210,000 years ago, well outside the range required for human-mediated development of zebu cattle from taurine progenitors. The history and biogeography of cattle populations in Africa represent a complex interaction of ecological, genetic and anthropological factors. The original indigenous cattle of Africa are universally considered to have been exclusively taurine. These populations are thought to have arisen from migrations of early pastoralists from the Near East ( EPSTEIN 1971; PAYNE 1991 ) . Recent archaeological evidence has, however, questioned this viewpoint, suggesting that the African aurochs ( Bos primigenius opisthonomus) may have been domesticated independently somewhere on the African continent ( WENDOW and SCHILD 1994). Although, zebu cattle are thought to have been first introduced into Africa about 4000 years ago, they only started to become widespread about 700 AD with the Arabic migrations into North and East Africa. At present, Africa represents a mosaic of cattle morphologies with zebu cattle and intermediate forms, often referred to as "sanga," predominating over most of the continent ( PAYNE 1970; EPSTEIN 1971). It has previously been suggested that the genetic signature of zebu gene flow in Africa may represent an unusual pattern of malemediated introgression ( BRADLEY et aZ. 1994; LOETUS et al. 1994a; BRADLEY et al. 1996). Taurine populations predominate only in subtropical regions of West Africa where an inherited resistance to trypanosomiasis ( trypanotolerance) allows them to inhabit areas infested with tsetse flies ( Glossina sp.) . Zebu cattle possess no innate resistance to trypanosomiasis and have only started to penetrate these regions with the assistance of veterinary prophylaxis and the destruction of the tsetse habitat through widespread deforestation ( LHOSTE 1991 ) . These recent migrations of zebu cattle pose a serious threat to the genetic integrity of valuable trypanotolerant populations of taurine cattle in the southern areas of West and Central Africa. To clarify the genetic relationships among the major groups of cattle and to characterize the extent and pattern of zebu genetic introgression in African populations, we have screened 20 different microsatellite loci in a large number of cattle representing 20 distinct populations from Africa, Europe and Asia. The patterns of genetic variation observed within and among populations should provide a large body of data to test hypotheses concerning the genetic affinities and origins of domesticated cattle, the dynamics of zebu genetic introgression in Africa and should also provide a method to assess the genetic integrity of threatened taurine populations in West Africa. MATERIALS AND METHODS Sample collection and DNA extraction: DNA samples were collected from 728 individual cattle representing 20 different populations originating from Africa, Asia and Europe. Samples were collected during a series of field sampling missions from artificial insemination ( A I ) stations, agricultural research institutions and village herds. Every attempt was made to ensure each population sample was representative. A strict, distributed sampling strategy was employed and herd book and breeding records were consulted while farmers and breeders were questioned about the origins and familial relationships of individual animals. The populations, their sample sizes and other relevant information are detailed in Table 1. In addition, a small number of samples were taken from three related species. These were American bison (Bison bison; n = 2 ) , European bison (Bison bonasus; n = 2) and Banteng ( Bibos bantmg; n = 2 ) . DNA was extracted from blood samples using standard procedures ( SAMBROOK et al. 1989 ) . Semen straws were used as a DNA source for some of the European breeds and these were processed according to a protocol described by ANDERSON et al. (1986). Microsatellite genotype analysis: A battery of 36 randomly chosen microsatellite loci were used from the array of publicdomain markers available in the bovine genome mapping literature. These microsatellite markers were variants of the (CA), class and were characterized and developed using standard methods. Eventually, 20 of the 36 markers that amplified robustly and repeatedly were used for full screens of the total sample panel. These 20 microsatellites are detailed in Table 2 with associated reaction conditions. PCR amplifications of individual loci were performed as follows. Reactions were performed using 96-well microtitre plates with 5-10 ng template DNA in 11 p1 reaction volumes using 0.5 unit of Taq polymerase with reaction buffer comprising 50 mM KCl; 10 mM TrisHCI pH 9.0; 1 .O-2.5 mM MgC12 (see Table 2) ; 1 % Triton X100; 200 p~ dATP, dGTP, dTTP; 10 p~ dCTP; 0.3 PM of each primer was added, as was 0.5 pCi [(Y-"~P] dCTP. A 10 pl oil overlay was then added and amplifications were performed in a Hybaid OmniGene thermal cycler using a 4 min denaturation step at 94", followed by 35 cycles of 45 sec at 93", 45 sec at 54-63' (see Table 2 ) , 45 sec at 72" and a final extension step at 72" for 4 min. Samples were then mixed with 10 PI formamide solution. After heat treatment at 93", 3p1 aliquots of these mixtures were then loaded on 6% denaturing polyacrylamide sequencing gels. M13mp18 plasmid DNA was used to provide sequence ladders for initial allele size calibration. Samples with appropriate genotypes were then selected to provide unambiguous length standards for each microsatellite. The allelic data from each autoradiogram were entered manually into a computer database. Data for each gel was then printed out and double-checked against the original autoradiogram. Basic data analysis: Allele frequencies were determined by direct counting. Allele frequency distributions for the 20 microsatellite loci are available via anonymous file-transfer at the following ftp address: acer.gen.tcd.ie/pub/cow-microsat/. As discussed below, 10 out of the 20 microsatellites displayed alleles that were present at high frequency in Asian zebu breeds, intermediate frequencies in African zebu populations, low frequencies in African taurine populations and that were either absent or at low frequencies in European breeds. As an illustration of this phenomenon, Figure 1 shows the allelic distributions for two of these loci in pooled populations samples. These private ( NEEI. 1973) or zebu-diagnostic alleles were used to evaluate gene flow and genetic admixture in African individuals and populations (see below). Observed heterozygosity and unbiased estimates of gene diversity (expected heterozygosity) with associated standard errors were 1073 Microsatellite Variation in Cattle