Genetically solving a zoological mystery: was the kouprey (Bos sauveli) a feral hybrid?

A famous zoological discovery of the 20th century was that of the kouprey Bos sauveli , a medium-sized ox inhabiting Cambodian forests. The kouprey was suspiciously intermediate between banteng oxen and domestic zebu cattle in its structure. Mitochondrial DNA sequences of mainland banteng are compared here with a published kouprey sequence, and the comparison demonstrates a close relationship. Either the kouprey derives partly from banteng or (less likely) these particular banteng acquired kouprey DNA via recent genetic introgression. The kouprey may have been a feral hybrid form, a descendant of domestic oxen, rather than a natural species.     

What is the taxonomic status of the Cambodian banteng and does it have close genetic links with the kouprey?

In a recent paper, Galbreath, Mordacq & Weiler (2006) concluded that the kouprey Bos sauveli was not a natural species, but rather a feral animal derived from hybridization between banteng Bos javanicus and zebu Bos taurus indicus . Here, we analyze two mitochondrial genes (cytochrome b and subunit II of the cytochrome c oxidase) for all the seven species of the subtribe Bovina, including new sequences for several specimens of banteng, zebu and gaur of Cambodia. Our analyses indicate that mitochondrial sequences of Cambodian banteng are divergent from those of Javan banteng (mean difference =4.27%), but similar to those of kouprey (1.25%). We propose two conflicting hypotheses to interpret these results: (1) the Cambodian and Indonesian banteng belong to two distinct species, and the kouprey derived from Cambodian banteng; (2) all subspecies of banteng belong to Bo. javanicus , but the mitochondrial genome of kouprey was transferred by natural hybridization into the ancestor of Cambodian banteng. Morphological, ecological and ethological characteristics of banteng and kouprey rather support the second hypothesis. However, we need to sequence nuclear markers, and to analyze banteng from Lao, Myanmar, Thailand and Vietnam, to give a definitive conclusion on the taxonomic status of banteng and kouprey.     

Molecular phylogeny of the tribe Bovini (Bovidae, Bovinae) and the taxonomic status of the Kouprey, Bos sauveli Urbain 1937

The kouprey is a very rare bovid species of the Indochinese peninsula, and no living specimen has been described for a long time, suggesting that it is possibly extinct. Its systematic position within the tribe Bovini remains confused since the analyses of morphological characters have led to several conflicting hypotheses. Some authors have also suggested that it could be a hybrid species produced by the crossing of the banteng with gaur, zebu, or water buffalo. Here we performed a molecular phylogeny of the tribe Bovini to determine the taxonomic status of the kouprey. DNA was extracted from the holotype specimen preserved in the MNHN collections. Phylogenetic analyses were carried out on a matrix including all the taxonomic diversity described in the tribe Bovini, and 2065 nucleotide characters, representing three different markers, i.e., the promotor of the lactoferrin and two mitochondrial genes (cytochrome b and subunit II of the cytochrome c oxidase). The results show that the kouprey belongs to the subtribe Bovina, and that three different clades can be evidenced into this group: the first includes the domestic ox, zebu, and European bison; the second incorporates the yak and American bison; and the third contains the kouprey, banteng and gaur. All hypotheses involving hybridization for the origin of the kouprey can be rejected, confirming that it is a real wild species. Molecular datings and biogeographic inferences suggest that the kouprey diverged from banteng and gaur during the Plio-Pleistocene of Asia. In addition, several molecular signatures were detected in the cytochrome b gene, permitting a molecular identification of the kouprey. We propose a conservation project based on a molecular taxonomy approach for tracking the kouprey in Indochina in order to determine whether some populations still survive in the wild.     

Phylogeny of bovine species based on AFLP fingerprinting

The Bovini species comprise both domestic and wild cattle species. Published phylogenies of this tribe based on mitochondrial DNA contain anomalies, while nuclear sequences show only low variation. We have used amplified fragment length polymorphism (AFLP) fingerprinting in order to detect variation in loci distributed over the nuclear genome. Computer-assisted scoring of electrophoretic fingerprinting patterns yielded 361 markers, which provided sufficient redundancy to suppress stochastic effects of intraspecies polymorphisms and length homoplasies (comigration of non-homologous fragments). Tree reconstructions reveal three clusters: African buffalo with water buffalo, ox with zebu, and bison with wisent. Similarity values suggest a clustering of gaur and banteng, but bifurcating clustering algorithms did not assign consistent positions to these species and yak. We propose that because of shared polymorphisms and reticulations, tree topologies are only partially adequate to represent the phylogeny of the Bovini. Principal-coordinate analysis positions zebu between a gaur/banteng cluster and taurine cattle. This correlates with the region of origin of these species and suggests that genomic distances between the cattle species have been influenced by genetic exchange between neighbouring ancestral populations.     

Has the kouprey (Bos sauveli Urbain, 1937) been domesticated in Cambodia?

The kouprey (Bos sauveli Urbain, 1937) is a very rare bovid species of Cambodia, which may be extinct in the wild, as no living specimen has been observed for a long time. Here, we describe a complete taxidermy mount, which presents astonishing morphological similarities with the kouprey. The animal was mounted in 1871 at the National Museum of Natural History in Paris, where it was referenced as No. 1871-576. It was deposited at the Natural History Museum of Bourges, France, in 1931, where it is still conserved today. To clarify the taxonomic status of the specimen of Bourges, DNA was extracted from a piece of bone taken on the mandible, and two different fragments of the mitochondrial cytochrome b gene were independently amplified and sequenced. The phylogenetic analyses show that the specimen of Bourges is robustly associated with the holotype of the kouprey, and that both are related to other wild species of Bos found in Indochina, i.e., banteng (B. javanicus) and gaur (B. frontalis). Because of doubts for sexing the animal, we applied a molecular test based on the PCR amplification of a DNA fragment specific to the Y chromosome. The results indicate that the specimen of Bourges is a male. The comparisons with male kouprey previously described in the literature reveal important differences concerning the body size, general coloration and horns. As these differences involve phenotypic traits that are strongly selected in case of domestication, we suggest that the specimen of Bourges was a domestic ox. This implies therefore that the kouprey may have been domesticated in Cambodia, and that several extant local races may be directly related to the kouprey.     

On the Origin of Indonesian Cattle

Background

Two bovine species contribute to the Indonesian livestock, zebu (Bos indicus) and banteng (Bos javanicus), respectively. Although male hybrid offspring of these species is not fertile, Indonesian cattle breeds are supposed to be of mixed species origin. However, this has not been documented and is so far only supported by preliminary molecular analysis.

Methods and Findings

Analysis of mitochondrial, Y-chromosomal and microsatellite DNA showed a banteng introgression of 10–16% in Indonesian zebu breeds. East-Javanese Madura and Galekan cattle have higher levels of autosomal banteng introgression (20–30%) and combine a zebu paternal lineage with a predominant (Madura) or even complete (Galekan) maternal banteng origin. Two Madura bulls carried taurine Y-chromosomal haplotypes, presumably of French Limousin origin. In contrast, we did not find evidence for zebu introgression in five populations of the Bali cattle, a domestic form of the banteng.

Conclusions

Because of their unique species composition Indonesian cattle represent a valuable genetic resource, which potentially may also be exploited in other tropical regions.     

Diversity and phylogeny of mitochondrial DNA isolated from mithun Bos frontalis located in Bhutan

We sequenced the 16S rRNA gene in mitochondrial DNA to characterize mithun located in Bhutan and to increase our understanding of its origin. We compared mithun with yak, European cattle, Bhutanese zebu and Indian zebu. Sequencing revealed low nucleotide diversity within the mithun population and their phylogenetic proximity to gaur. A close relationship between Bhutanese mithun and gaur was confirmed by an additional comparison with wild gaur specimens from three locations in Bhutan. Direct domestication of mithun from gaur was supported, while maternal contribution from the cattle lineage during domestication was not supported.     

Maternal and paternal lineages in cross-breeding bovine species. Has wisent a hybrid origin?

The tribe Bovini comprises cattle and cattle-like species. Reconstructions of their phylogeny have so far been incomplete and have yielded conflicting conclusions about the relationship of American bison and wisent (European bison). We have compared the sequences of three mitochondrial and two Y-chromosomal DNA segments. Mitochondrial DNA indicates that four distinct maternal lineages diverged after an early split-off of the buffalo species, leading to (1) taurine cattle and zebu, (2) wisent, (3) American bison and yak, and (4) banteng, gaur, and gayal, respectively. At a higher level, lineages (1) and (2) and lineages (3) and (4) are probably associated. In contrast, Y-chromosomal sequences indicate a close association of American and European bison, which is in agreement with their morphological similarity, complete fertility of hybrid offspring, and amplified fragment length polymorphism (AFLP) fingerprints of nuclear DNA. One explanation for the anomalous divergence of the mitochondrial DNA from the two bison species is lineage sorting, which implies that two distinct mitochondrial lineages coexisted in the bison-yak branch until the recent divergence of American bison and wisent. Alternatively, the wisent may have emerged by species hybridization initiated by introgression of bison bulls in another ancestral species. This "transpatric" mode of species formation would be consistent with the recent appearance of the wisent in the fossil record without clearly identifiable ancestors.     

When and how did Bos indicus introgress into Mongolian cattle?

The Mongolian cattle are one of the most widespread breeds with strictly Bos taurus morphological features in northern China. In our current study, we presented a diversity of mitochondrial DNA (mtDNA) D-loop region and Y chromosome SNP markers in 25 male and 8 female samples of Mongolian cattle from the Xinjiang Uygur autonomous region in Western China, and detected 21 B. taurus and four Bos indicus (zebu) mtDNA haplotypes. Among four B. indicus mtDNA haplotypes, two haplotypes belonged to I1 haplogroup and the remaining two haplotypes belonged to I2 haplogroup. In contrast, all 25 male Mongolian cattle samples revealed B. taurus Y chromosome haplotype and no B. indicus haplotypes were found. Historical and archeological records indicate that B. taurus was introduced to Xinjiang during the second millennium BC and B. indicus appeared in this region by the second century AD. The two types of cattle coexisted for many centuries in Xinjiang, as depicted in clay and wooden figurines unearthed in the Astana cemetery in Turfan (3rd–8th century AD). Multiple lines of evidence suggest that the earliest B. indicus introgression in the Mongolian cattle may have occurred during the 2nd–7th centuries AD through the Silk Road around the Xinjiang region. This conclusion differs from the previous hypothesis that zebu introgression to Mongolian cattle happened during the Mongol Empire era in the 13th century.     

The Origins of Domesticated Cattle

The origin of domestic cattle has perplexed archaeologists for more than a century. Researchers have proposed various theories, which offer alternative spatial and chronological models for the origin and spread of domesticated cattle. One point of discussion is whether domestic cattle had a single or multiple origins; however, most authorities considered that the first steps towards cattle domestication were taken in southwest Asia and that domesticated cattle entered Europe with pastoralists migrating from this region. Domesticated taurine cattle were thought to have entered Africa in successive waves from southwest Asia, while zebu cattle migrated into Africa at a later date from Arabia and the Indian subcontinent. Analysis of mitochondrial DNA (mtDNA) shows that taurine and zebu cattle divergence before the Holocene and were probably domesticated independently. Recent mtDNA sequence data shows that African and European taurine cattle were probably domesticated independently, but that there was a process of genetic introgression between taurine and zebu cattle in Africa. Ancient DNA studies over the last 10 years suggest that Northern European aurochsens apparently contributed little or nothing to domestic cattle while Southern European aurochsens apparently made a significant input. However, Middle Eastern aurochsen, unfortunately not typed yet, are expected to be to be very similar to European breeds as well, both because archeological data suggest that the major center of domestication for European Bos taurus breeds was the Fertile Crescent (9), and also because a mtDNA sequence from a Syrian specimen dated at 8,000–9,000 years ago shows a typical European haplotype found both in modern breeds and the Italian aurochsen. Evidence seems to suggest that small to moderate levels of local gene flow from wild Bos primigenius females in selected breeds were either accepted or may be reinforced by Neolithic breeders.     

Paternally inherited markers in bovine hybrid populations

The genetic integrity of crossfertile bovine- or cattle-like species may be endangered by species hybridization. Previously, amplified fragment length polymorphism, satellite fragment length polymorphism and microsatellite assays have been used to analyze the species composition of nuclear DNA in taurine cattle, zebu, banteng and bison populations, while mitochondrial DNA reveals the origin of the maternal lineages. Here, we describe species-specific markers of the paternally transmitted Y-chromosome for the direct detection of male-mediated introgression. Convenient PCR-restriction fragment length polymorphism and competitive PCR assays are shown to differentiate the Y-chromosomes of taurine cattle, American bison and European bison, and to detect the banteng origin of Indonesian Madura and Bali cattle bulls.     

Evolution and domestication of the Bovini species

Domestication of the Bovini species (taurine cattle, zebu, yak, river buffalo and swamp buffalo) since the early Holocene (ca. 10 000 BCE) has contributed significantly to the development of human civilization. In this study, we review recent literature on the origin and phylogeny, domestication and dispersal of the three major Bos species – taurine cattle, zebu and yak – and their genetic interactions. The global dispersion of taurine and zebu cattle was accompanied by population bottlenecks, which resulted in a marked phylogeographic differentiation of the mitochondrial and Y-chromosomal DNA. The high diversity of European breeds has been shaped through isolation-by-distance, different production objectives, breed formation and the expansion of popular breeds. The overlapping and broad ranges of taurine and zebu cattle led to hybridization with each other and with other bovine species. For instance, Chinese gayal carries zebu mitochondrial DNA; several Indonesian zebu descend from zebu bull × banteng cow crossings; Tibetan cattle and yak have exchanged gene variants; and about 5% of the American bison contain taurine mtDNA. Analysis at the genomic level indicates that introgression may have played a role in environmental adaptation.     

Low genetic diversity in the bottlenecked population of endangered non-native banteng in northern Australia

Undomesticated (wild) banteng are endangered in their native habitats in Southeast Asia. A potential conservation resource for the species is a large, wild population in Garig Gunak Barlu National Park in northern Australia, descended from 20 individuals that were released from a failed British outpost in 1849. Because of the founding bottleneck, we determined the level of genetic diversity in four subpopulations in the national park using 12 microsatellite loci, and compared this to the genetic diversity of domesticated Asian Bali cattle, wild banteng and other cattle species. We also compared the loss of genetic diversity using plausible genetic data coupled to a stochastic Leslie matrix model constructed from existing demographic data. The 53 Australian banteng sampled had average microsatellite heterozygosity (HE) of 28% compared to 67% for outbred Bos taurus and domesticated Bos javanicus populations. The Australian banteng inbreeding coefficient (F) of 0.58 is high compared to other endangered artiodactyl populations. The 95% confidence bounds for measured heterozygosity overlapped with those predicted from our stochastic Leslie matrix population model. Collectively, these results show that Australian banteng have suffered a loss of genetic diversity and are highly inbred because of the initial population bottleneck and subsequent small population sizes. We conclude that the Australian population is an important hedge against the complete loss of wild banteng, and it can augment threatened populations of banteng in their native range. This study indicates the genetic value of small populations of endangered artiodactyls established ex situ.     

A bubaline-derived satellite DNA probe uncovers generic affinities of gaur with other bovids

DNA typing using genome derived cloned probes may be conducted for ascertaining genetic affinities of closely related species. We analysed gaurBos gaurus, cattleBos indicus, buffaloBubalus bubalis, sheepOvis aries and goatCapra hircus DNA using buffalo derived cloned probe pDS5 carrying an array ofBamHI satellite fraction of 1378 base residues to uncover its genomic organization. Zoo-blot analysis showed that pDS5 does not cross hybridize with non-bovid animals and surprisingly with female gaur genomic DNA. The presence of pDS5 sequences in the gaur males suggests their possible location on the Y chromosome. Genotyping of pDS5 withBamHI enzyme detected mostly monomorphic bands in the bubaline samples and polymorphic ones in cattle and gaur giving rise to clad specific pattern. Similar typing withRsaI enzyme also revealed clad specific band pattern detecting more number of bands in buffalo and fewer in sheep, goat and gaur samples. Copy number variation was found to be prominent in cattle and gaur withRsaI typing. Our data based on matched band profiles (MBP) suggest that gaur is genetically closer to cattle than buffalo contradicting the age-old notion held by some that gaur is a wild buffalo. The pDS5 clone has a potential for estimating the generic and genetic relationship amongst closely related bovid species.     

On the origin of cattle: How aurochs became cattle and colonized the world

Since their domestication in the Neolithic, cattle have belonged to our cultural heritage. The reconstruction of their history is an active field of research 1 that contributes to our understanding of human history. Archeological data are now supplemented by analyses of modern and ancient samples of cattle with DNA markers of maternal, paternal, or autosomal inheritance. The most recent genetic data suggest that maternal lineages of taurine cattle originated in the Fertile Crescent with a possible contribution of South‐European wild cattle populations, while zebu cattle originate from the Indus Valley. Subsequently, cattle accompanied human migrations, which led to the dispersal of domestic cattle of taurine, indicine, or mixed origin over Asia, Africa, Europe, and the New World. This has resulted in their adaptation to different environments and considerable variation in appearance and performance. More recently, rational management of breeding led to international movements of sires, which again changed the global patterns of genetic diversity.     

Genetic analysis of a wild-caught hybrid between non-sister Heliconius butterfly species

Interspecific hybridization occurs regularly in wild Heliconius butterflies, although hybrid individuals are usually very rare. However, hybridization generally occurs only between the most closely related species. We report a rare naturally occurring hybrid between non-sister species and carry out the first genetic analysis of such distant hybridization. Mitochondrial and nuclear genes indicate that the specimen is an F1 hybrid between a female Heliconius ethilla and a male Heliconius melpomene, originating from a group of 13 species estimated to have diverged over 2.5 Myr ago. The presence of such distant natural hybrids, together with evidence for backcrossing, suggests that gene flow across species boundaries can take place long after speciation. Adaptive genes such as those involved in wing coloration could thus be widely shared among members of this highly mimetic genus.     

Cattle demographic history modelled from autosomal sequence variation

The phylogeography of cattle genetic variants has been extensively described and has informed the history of domestication. However, there remains a dearth of demographic models inferred from such data. Here, we describe sequence diversity at 37 000 bp sampled from 17 genes in cattle from Africa, Europe and India. Clearly distinct population histories are suggested between Bos indicus and Bos taurus, with the former displaying higher diversity statistics. We compare the unfolded site frequency spectra in each to those simulated using a diffusion approximation method and build a best-fitting model of past demography. This implies an earlier, possibly glaciation-induced population bottleneck in B. taurus ancestry with a later, possibly domestication-associated demographic constriction in B. indicus. Strikingly, the modelled indicine history also requires a majority secondary admixture from the South Asian aurochs, indicating a complex, more diffuse domestication process. This perhaps involved multiple domestications and/or introgression from wild oxen to domestic herds; the latter is plausible from archaeological evidence of contemporaneous wild and domestic remains across different regions of South Asia.     

Weights of gaur (Bos gaurus) and banteng (Bos javanicus) killed by tigers in Thailand

The primary prey of tigers across much of South‐East Asia has been depleted, reducing the ability of already limited habitat to support tigers. To better understand the extent to which two of the largest prey species, gaur (Bos gaurus) and banteng (Bos javanicus), contribute to the tiger's diet, we estimated the average size of these species killed by tigers. This information is needed to more accurately calculate biomass of these species in the tiger's diet and to devise strategies to increase tiger carrying capacity where habitat is fragmented and limited in west‐central Thailand. We used temporally clumped locations of 24 satellite radio‐collared tigers to identify their kill sites and obtained mandibles from 82 gaur and 79 banteng. Kills were aged by teeth eruption sequence, sectioning the M1 molar and counting cementum annuli. Of all gaur killed, 45.2% were adults; of all banteng killed, 55.7% were adults. The average weight of banteng killed was 423.9 kg, which was similar to the 397.9 kg average weight for gaur. The mean weight of both prey species is 3.5–4.5 times greater than the predicted 1:1 preferred prey to predator ratio. In the absence of medium‐sized prey, killing these larger animals may be especially critical for female tigers provisioning nearly independent young when male offspring are already larger than the mother. This is the first study to present data on the average weights of gaur and banteng killed in South‐East Asia, and these results suggest that these are key prey species to target in tiger prey recovery efforts.     

Complete mitogenome reveals genetic divergence and phylogenetic relationships among Indian cattle (Bos indicus) breeds

Indigenous cattle of India belong to the species, Bos indicus and they possess various adaptability and production traits. However, little is known about the genetic diversity and origin of these breeds. To investigate the status, we sequenced and analyzed the whole mitochondrial DNA (mtDNA) of seven Indian cattle breeds. In total, 49 single-nucleotide variants (SNVs) were identified among the seven breeds analyzed. We observed a common synonymous SNV in the COII gene (m.7583G?>?A) of all the breeds studied. The phylogenetic analysis and genetic distance estimation showed the close genetic relationship among the Indian cattle breeds, whereas distinct genetic differences were observed between Bos indicus and Bos taurus cattle. Our results indicate a common ancestor for European Zwergzebu breed and South Indian cattle. The estimated divergence time demonstrated that the Bos indicus and Bos taurus cattle lineages diverged 0.92 million years ago. Our study also demonstrates that ancestors of present zebu breeds originated in South and North India separately ～30,000 to 20,000 years ago. In conclusion, the identified genetic variants and results of the phylogenetic analysis may provide baseline information to develop appropriate strategies for management and conservation of Indian cattle breeds.