BACKGROUND
the bovine genome project  has provided researchers with an excellent resource for bovine phylogenetic studies.
genomic resources from b. taurus should have some transferability among a number of closely related bovids due to the likely divergence ages of the bovidae  <cit> .
to date several studies have suggested that primers designed in one species have a relatively high rate of pcr success in other families from the suborder ruminantia  <cit> .
therefore, sequencing exonic regions should have a high rate of success within the bovini tribe even from primers designed from the b. taurus genome.
bovinae subfamily relationships and phylogenetic reconstruction
the bovidae family consists of some of the most culturally and economically important species on the planet.
of the major representatives of the family bovidae arguably the most important would be the subfamily bovinae, which is divided into three main tribes ; the first two representatives of the bovinae, the tribes tragelaphini and boselaphini, comprise spiral, four-horned and much of the large ox-like antelope  <cit> .
representatives of these tribes are often hunted for meat and hide, and in the case of eland have been occasionally used to work in harness.
the bovini tribe comprises all of the major domestic bovine species, including a number of wild species, some of which are endangered or threatened with extinction  <cit> .
the first divergence within the bovini occurred between 5– <dig> million years ago  with the splitting of the buffalo or the subtribe bubalina  from the nonbuffalo or the subtribe bovina   <cit> .
these two subtribes consistently resolve themselves as dichotomous groups, and show no evidence of producing viable hybrid offspring  <cit> .
recently molecular methods have estimated the bovina bubalina split at approximately  <dig> mya  <cit> .
however, the earliest bovini fossils found south of the himalayas in india and pakistan have been dated at <  <dig> mya  <cit> .
phenotypic and stable carbon and oxygen isotope analysis suggest that these bovini representatives were behaviourally and ecologically intermediate to modern bovini and their progenitors the bosephalini  <cit> .
these early examples of the bovini appeared to become more adapted to drier more open habitats and were only beginning to develop affinities with grazing open grasslands and obligate drinking patterns  <cit> .
in addition early bovini fossils do not exist in nearby regions like afghanistan and iran suggesting that these early bovini were restricted to the open forests of india and pakistan until <  <dig> mya when the earliest bovini fossils begin to appear in africa, asia and europe, respectively.
this expansion coincides with a changing climate and expansion of c <dig> dominated grasslands  <cit> .
hence estimates of the bovinae bubalina split >  <dig> mya are most likely overestimates and a more realistic estimate is <  <dig> mya.
within the bovina subtribe, divergence of the remaining species also occurred recently, with a very sudden radiation in the early pleistocene ~ <dig> mya and as a result speciation has not been complete, with many members of the tribe producing viable hybrid offspring or infertile offspring in bulls that may have fertility restored by repeated backcrossing  <cit> .
the dramatic range expansion of domestic cattle has resulted in an increased threat of introgression by domestic cattle dna into the genomes of many wild populations, either intentionally or accidentally  <cit> , all of which may confuse phylogenetic relationships.
another confounding factor when reconstructing phylogenies may arise if a rapidly evolving clade's most recent common ancestor  was highly polymorphic, as this may result in the random assortment of genetic variation in different lineages and aberrant modes of inheritance for certain polymorphisms.
this phenomenon is known as lineage sorting, and it has been known to cause random associations among species, which may further confuse derived taxonomic relationships  <cit> .
recently phylogenetic reconstruction using mitochondrial lineages has led to clearer bovina phylogenies  <cit> .
however, because mitochondrial dna is maternally inherited, relationships driven by male mediated introgression may be missed.
this finding was confirmed with y-chromosomal phylogenies identifying close relationships between wisent and north american bison, while mitochondrial phylogenies identify an association between bos taurus and wisent  <cit> .
more recently the kouprey and banteng were shown to be both valid species despite the introgression of the kouprey mitochondrial genome in cambodian banteng  <cit> .
thus, some improvements have been made regarding the phylogenetic relationships of the bovinae subtribe, but there is still concern over the accuracy of many trees due to low confidence values and contrasting results from different data and phylogenetic methods.
this is especially evident when considering the position of the wisent in the bovine phylogeny and the time of divergence for these species.
despite concerns over the level of variation within nuclear gene sequences at the species level in the bovini  <cit> , by examining a number of nuclear genes  we should identify enough genetic variation to differentiate between species, populations or breeds.
also by examining variation within genes and exons in closely related species, one can analyse haplotypes for similarity among species over short  and large distances .
the presence of identical, long haplotypes in different species may be evidence of recent introgression as recombination is likely to break up linked haplotypes after a number of generations.
alternatively, associations generated by the random assortment of polymorphic alleles should not create similar patterns of linked haplotypes over any significant distance.
therefore, lineage sorting and introgression can be differentiated by the analysis of haplotype similarity and sequence variation in genomic dna between species.
the presence of alleles undergoing lineage sorting in the coding regions of the genome are of interest as their presence is most likely due to polymorphisms in a common ancestor that were sorted in a fashion that is inconsistent with the inferred phylogeny.
in some cases these polymorphisms can persist for generations, even past the lifespan of a species.
the fact that many of these polymorphisms do not appear to be linked over any large distances suggests they have been independently assorted and that they are ancient in origin and therefore not introduced by recent introgression.
if overdominance, selection or demographic factors, like the fixation of slightly deleterious alleles in small populations, have been responsible for the assortment of these ancient polymorphisms they must have an effect on phenotype and should therefore show high dn/ds ratios.
therefore, examining estimates of dn/ds at these sites should help infer whether they are neutral or nearly neutral and if so, whether they are useful for determining the neutral rate of mutation in the bovini.
in this paper we attempt to reconstruct phylogenies between  <dig> extant representatives of the bovinae subfamily.
we examined  <dig> autosomal gene sequences, from  <dig> different genes, sampled over  <dig> bovine chromosomes.
neighbour joining trees and the neutral substitution rate per site at synonymous  and noncoding  regions are presented for all autosomal sequences in an effort to identify relationships and divergence times between species, and test for evidence of introgression or lineage sorting.
RESULTS
sequencing
in total  <dig> amplicons from  <dig> genes were sequenced across  <dig> representatives of the bovinae subfamily, including members of the bovini and tragelaphini tribes, which we used to infer the sequence from the most recent common ancestor .
from the  <dig> amplicons  <dig> amplified in at least one sample.
the relative proportions from the amplicons that successfully amplified in each species were particularly high, ranging from  <dig>  in eland to  <dig>  in gaur .
surprisingly some of the wild relatives showed better amplification  than different breeds of b. taurus .
all sequences were submitted to genbank and their primers and accession numbers are also available in additional file  <dig>
genetic distances, divergence times and phylogenetic reconstruction
both di and ds are generally considered neutral substitutions and are often used for discriminating between phylogenetic lineages.
table  <dig> shows pairwise comparisons for di and ds between  <dig> bovinae representatives, lower and upper diagonal respectively.
the minimum di is within breeds of indian river buffalo, followed by breeds of domestic cow.
the largest di is between species like eland and the domestic cow .
similarly, the minimum ds is found between breeds, and the maximum ds is between eland and other members of the bovidae, such as bison, gaur, mithan, and domestic cow, respectively .
interestingly, ds is often greater than di.
subramanian and kumar  <cit>  suggest that an elevation in gc content at synonymous sites may be responsible for this phenomenon.
species:
species:
no significant differences were detected between overall base frequencies  for any member of the bovini tribe, and substitution rates between ancestral and eland sequences with all members of the bovini appear to be largely the same.
a small bias was detected for transition  to transversion  substitutions, with a ratio of ti/tv =  <dig>  .
therefore, kimura's two parameter model should be appropriate for phylogenetic reconstruction.
however, the relatively small genetic distances detected  suggest that the p-distances calculated in table  <dig> should also be accurate despite the transition bias  <cit> .
aberrant sites
to detect if recent gene flow or ancient polymorphisms were responsible for differences between our and other recently published phylogenies, we analysed all of the segregating sites from figure  <dig> for the presence of anomalies.
we reconstructed a number of phylogenetic trees to identify the tree that minimises the number of aberrant sites, which we defined as any site that must have arisen due to a double mutation or lineage sorting of an ancient polymorphism .
we identified  <dig> aberrant sites, of which  <dig> were not tree specific and were aberrant in all trees leaving  <dig> informative sites.
from the  <dig> non-tree specific aberrant sites  <dig> ancient sites are segregating within both the bovina and bubalina lineages.
a legend to all aberrant sites, their position and the relative age  is provided in additional file  <dig>
to examine these aberrant sites in more detail we have analysed a number of phylogenetic trees that have been suggested due to the poor bootstrapping confidence in figure  <dig>  and trees presented in recent work by verkaar et al and hassanin and ropiquet  <cit> .
the phylogenetic tree that generates the smallest number of anomalies  places yak off the lineage leading to domestic cattle and bison off the lineage leading to the indochinese cattle , ); however, similar numbers  are also seen for the tree that places bison and yak as a lineage of the indochinese clade ,  indochinese cattle)), which is only worse by one aberrant site.
therefore, some evidence exists for the trees recently proposed by hassanin and ropiquet  <cit>  and verkaar et al.
<cit> .
however, it appears that a number of aberrant sites are generating an association between yak and domestic cattle, which has confounded the phylogenetic reconstruction.
removing all aberrant sites from the analysis leaves fewer substitutions to accurately estimate genetic distances between members of the bovina subtribe and the tree is less certain .
introgression and ancient polymorphism
to identify the source of the aberrant substitutions within the bovini tribe, we compared all variable sites in the alignment to find sites shared with b. taurus at normal and aberrant positions .
we examined these positions within gaur, mithan, banteng, bison and yak for similarities to domestic cattle over extended haplotypes resolved from aberrant and all variable sites.
we found that yak shares the largest number of alleles in common with domestic cattle for all variable and aberrant sites .
perhaps the most interesting finding is the high number of identical haplotypes yak and domestic cattle share over haplotypes resolved from the aberrant sites per amplicon .
when compared to haplotypes resolved from the all variable sites, yak and domestic cattle share the largest number in common.
however, there are relatively few haplotypes that are similar for any of the species  and domestic cattle, and this is probably a function of the species specific mutations that have accumulated per amplicon since divergence from the mrca.
and the number of substitutions similar to b. taurus over haplotypes generated from all variable and from aberrant sites for each amplicon.
gene_amplicon
nrip1_e <dig>
nrip1_e <dig>
pit1_e1
pit1_e3
pit1_e5
pit1_e6
itgbp5_e3
itgbp5_e5
itgbp5_e6
itgbp5_e10
5ht1f_e1
igfbp2_e1
igfbp5_e2
igfbp5_e3
hfabp_e2
hfabp_e3
lacs3_e1
lacs3_e3
lacs3_e4
lacs3_e6
5ht6_e <dig>
gmeb1_e1
gmeb1_e3
gmeb1_e4
gmeb1_e5
gmeb1_e6
gmeb1_e7
gmeb1_e8
egf_e1
era_e3
era_e4
era_e5
era_e6
era_e7
pabpc1_e2
pabpc1_e3
pabpc1_e5
pabpc1_e9
mfge8_e2
mfge8_e3
mfge8_e5
*number of exons represented as e where e = exon and n = number, therefore, pit1_e <dig> is exon  <dig> of pit <dig>  exons that extend over the largest sequencing read, which is typically  <dig> bp are represented as e where j equals order of read from 5-prime to 3-prime.
therefore nrip_e <dig>  and nrip_e <dig>  represent two subsequent reads of the l
the number of aberrant alleles making each haplotype are given in brackets as the .
table displays haplotype blocks shared with hereford and  <dig> members of the bovinae for exons within the each gene and for genes on the same chromosome.
to determine if the high proportion of haplotypes, resolved from aberrant sites, shared between yak and domestic cattle may be evidence of recent introgression, we looked for extended haplotypes that are shared between species.
thus, the extent of linkage between neighbouring amplicons and genes on the same chromosome was examined.
table  <dig> shows the b. taurus haplotype per amplicon from all aberrant sites and the species that share the same haplotype.
a number of haplotypes are shared with all species.
many of the haplotypes that are based on one aberrant site  may be similar by chance and therefore may not be very informative.
however, by extending haplotypes to adjacent exons and genes more power to detect recent hybridisation should be available.
but as haplotypes are extended to closely linked amplicons or neighbouring genes on the same chromosome this similarity rapidly disappears .
in a further attempt to identify recent introgression, sequence diversity estimates were examined between neighbouring amplicons and genes.
figure  <dig> shows plots of sequence divergence at noncoding sites  for hereford vs yak , hereford vs holstein  and yak vs gaur .
a similar pattern is seen to that described in table  <dig>  a number of amplicons show similar amounts of sequence divergence between hereford vs yak as is seen between hereford vs holstein .
however, genetic diversity at closely linked amplicons remains low between breeds of b. taurus for closely spaced genes on chromosome  <dig>  and chromosome  <dig> , which suggests that these animals have recently shared a common ancestor.
in contrast, comparisons for domestic cattle vs yak, and yak vs gaur  rarely show low sequence diversity over similar linked regions, and thus, genetic associations are most likely not the result of recent introgression.
an examination of the river and swamp buffalo samples from southeast asia  has identified large segments of chromosome  <dig> and  <dig> that are identical.
thus bubb has a closer relationship to swamp buffalo  than to other members of river buffalo that originate from india, which may suggest some level of introgression in these two samples as large portions of chromosomes  <dig> and  <dig> in bubb appear to be of swamp buffalo origin .
classifying aberrant sites: age and role of selection
a number of aberrant sites were identified that were inconsistent with any of the phylogenetic trees suggested from either molecular or morphological characteristics.
in total  <dig> aberrant sites occurred in coding regions, of which  <dig> are ka and  <dig> are ks from  <dig>  synonymous and  <dig>  nonsynonymous sites, which resulted in a dn/ds ratio of  <dig> .
from the  <dig> aberrant sites  <dig> appear to be polymorphic since the common ancestor with buffalo, while the remaining  <dig> would have been polymorphic in the common ancestor of the bos and bison genera.
of the  <dig> ancient polymorphisms  <dig> are ka and  <dig> are ks.
therefore these  <dig> ancient polymorphisms were maintained for millions of years.
they must have been polymorphic in the mrca to the bovina and bubalina subtribes some 5– <dig> mya and appear to have persisted to at least the divergence of the bovina subtribe around  <dig> mya.
the small values estimated for the dn/ds ratios calculated suggest that these polymorphisms may have been neutral.
discussion
sequencing
a high proportion of successful amplification was achieved for all species within the bovinae subfamily from primers designed using the bos taurus genome despite divergence times of 8– <dig> my .
the high rate of amplification between species should facilitate further population genetic studies in the bovini and possibly other ruminant subfamilies.
bovine phylogeny reconstruction, mutation and substitution
within the bovini, difficulties resolving interspecific relationships arose when comparing pairwise differences between species for di and ds with those deduced from the neighbour joining algorithm.
in general the phylogenetic reconstruction created using kimura's two parameter method and the neighbour joining algorithm agreed with the p-distances calculated for di and ds in table  <dig>  with strong separation between bubalina and bovina subtribes and strong support for separation of bubalus and syncerus genera, confirming previous studies from molecular data  <cit> .
within the bubalus there appears to be a separation between swamp and river buffalo and a possible separation within river buffalo that may be due to due to geographic patterns and/or introgression of swamp and river buffalo.
the phylogenetic tree we identified does not agree with some of the individual genetic distances within the bovina.
for instance, the genetic distance between yak and bison for di and ds both imply that the species are most likely a monophyletic group that could either be grouped with the b. taurus lineage or the lineage leading to the cattle from indochina  but the tree places them as separate divergences from the lineage leading to b. taurus.
this disparity may be the result of more complicated methods of deducing genetic distance, like the kimura two parameter model, inflating variances over simpler methods, such as the p-distances, thereby increasing the chance of topographical errors  <cit> .
low bootstrapping levels may be the result of low genetic differentiation between these three lineages and the presence of aberrant polymorphisms, confusing phylogenetic reconstruction.
examining a number of manually inferred trees that attempt to minimise the number of aberrant polymorphisms identified a tree that has bison as a divergence from the gaur/mithan/banteng lineage and yak as a divergence of the b. taurus clade.
but this relationship does not seem logical considering the close relationship identified between bison and yak for ds and di distances.
therefore, the large number of aberrant sites that yak shares with hereford may confuse the phylogenetic reconstruction, as the neighbour joining algorithm deals with these aberrant polymorphisms in the same manner as all other polymorphisms.
removing aberrant polymorphisms from the analysis identified a phylogenetic relationship in which all members of the bovina appear to have diverged roughly at the same time in a star like phylogeny.
however, bootstrap values were even lower than those deduced from the entire dataset, highlighting the difficulty of reconstructing phylogenies from a group of species that have undergone rapid genetic isolation.
of course, low bootstrap values are expected for a star-like phylogeny.
all trees show a close association between members of the bovina subtribe, with all major lineages diverging very rapidly some 1– <dig> million years ago, which appears to be associated with climatic oscillations during the late pleistocene  <cit> .
all trees show that bison and yak are single or multiple divergences from the clade leading to b. taurus, which disagrees with previous work by verkaar et al  <cit>  and hassanin and ropiquet  <cit>  that place bison and yak as a single divergence from the indochinese cattle.
more recently hassanin and ropiquet  <cit>  show bison and yak as a single divergence from the lineage leading to b. taurus.
however, these studies were typically based only on one or two thousand nucleotide characters or from a small number of genetic markers and therefore, each tree typically had low bootstrapping values or resulted in variations to this tree when differing methods or maternally, paternally or bi-parentally inherited data are used, suggesting that some of these relationships are still contentious.
the phylogenetic reconstruction presented above is based on over  <dig>  bi-parentally inherited nucleotide characters and should have more power to resolve some of the more confusing intergenic relationships within the bovini.
the association identified between bison, yak and b. taurus may also explain the similarity seen between wisent and b. taurus.
although, no wisent sample was represented in our study, verkaar et al  <cit>  and hassanin and ropiquet  <cit>  have recently hypothesised that as a result of repeated male mitigated introgression of american bison populations into eurasian cattle like populations lead to a new species , which comprises the phenotype, autosomal genes and y-chromosome of american bison and the mitochondrial genome from the maternal ancestor   <cit> .
thus, the constant hybridisation by the progenitors of bison and b. taurus may have had a large impact on the genomes of both species.
surprisingly, one of the bubalus bubalis  samples was more closely related to the bubalis carabenensis  than to other river buffalo.
this relationship was puzzling, as previous studies have found that the two subspecies are genetically distinct, have different chromosome numbers and their divergence time has been estimated tens of thousands to over one million years  <cit> .
however, despite the obvious phenotypic and genetic differences, the two subspecies are known to hybridise, especially in south east asia where a hybrid zone for buffalo may exist  <cit> .
also in some instances animals can show the phenotype of the swamp buffalo but have the chromosomal and genetic make up of the river buffalo, as is the case for animals in sri lanka  <cit> .
therefore, the phylogenetic reconstruction may have been confused due to an analysis of a hybrid river buffalo  that has large regions of chromosome  <dig> and  <dig> that apparently originated from swamp buffalo.
alternatively, the phenotypic and karyoptypic plasticity shown within water buffalo may indicate that genetic differences between river and swamp buffalo could all be within species polymorphism.
aberrant sites, introgression and ancient polymorphism
one of the most astonishing features of the phylogenetic reconstruction was the identification of a large number of aberrant sites that did not fit any known phylogenetic reconstructions.
a number of explanations may account for these phylogenetic aberrations, such as double mutations, introgressive hybridisation and lineage sorting.
the probability of aberrant sites as a result of double mutations can be calculated as follows:
which is the probability of no mutation in one lineage with mutation rate μ over t generations.
therefore, the probability of mutation in one lineage is
thus the probability that the same site mutates in two different lineages is
and the expected number of double mutations across n sites is
the probability that both mutations result in the same base is approximately 1/ <dig>  hence the expected number of double mutations in two lineages is
therefore, even if t is set to  <dig> million years as a conservative estimate for the divergence of b. taurus and b. bubalis and n =  <dig>  then for equation  <dig> one would expect around  <dig> bases to have undergone a double mutation resulting in the same derived allele from the ~ <dig>  bases sequenced in each lineage since the divergence of b. taurus and b. bubalus from a common ancestor some  <dig> mya.
the time of divergence is somewhat less within the bovini but there are more than  <dig> lineages.
however, the likelihood of a mutation affecting the exact same base ~ <dig> times in our dataset is highly improbable, so in general, most abnormalities must be evidence of lineage sorting or introgressive hybridisation and not double mutations.
also a high proportion  of aberrant sites were identified as sites that were deemed to be polymorphic in bubalina and bovina subtribes.
members of these two subtribes separated 5– <dig> mya and no hybridisation has ever been documented between any members of these two subtribes  <cit> .
therefore, these aberrant sites cannot have arisen due to recent hybridisation events.
if 40% of the aberrant sites are due to polymorphisms that existed in the common ancestor at bubalina and bovina, it seems likely that other polymorphisms in the common ancestor of bovina will also have contributed to the other 60% of aberrant sites.
however, recent introgression might have also contributed, as the incomplete speciation of the tribe has resulted in many viable hybrid offspring from various crossings  <cit> .
the presence of identical haplotypes between bovina representatives and domestic cattle may also be a result of ancient geneflow between animals before reproductive isolation was achieved, but in essence this hypothesis would virtually be identical to the presence of ancestral polymorphisms.
another possibility for this finding may also be explained by hybridisation between another closely related species.
a previous study from the labs of university station in texas also found an anomalous association between yak and taurine cattle, due to an animal descending from a b. indicus cow  <cit> .
the fact that a bos indicus sample was not included in the data set, meant tests could not determine if some of the aberrant sites in our sample were due to gene flow between b. grunniens and b. indicus and this will need to be investigated further.
the high degree of sequence similarity detected between yak and domestic cattle at all variable sites in the alignment, aberrant sites and over extended haplotypes suggests that hybridisation may have also occurred between these two species .
yak-taurine hybrids are reported in china and mongolia where their altitudinal ranges intersect  <cit> .
however, no evidence of linked haplotypes at aberrant sites between neighbouring genes or on very small scales between exons within the same gene was detected .
genes were selected from tightly linked regions of the b. taurus genome and the majority of amplicons within genes were only separated by 1– <dig> kilobases.
no information on the genetic distance between genes has been collected but, the small physical distances between amplicons on the same gene suggest the recombination rate between many of these amplicons would be far below 1% per generation.
the fact that all animals rarely share haplotypes with b. taurus between neighbouring amplicons and genes suggests that there is no evidence of linked haplotypes.
this finding is also reflected in plots of sequence diversity between yak and domestic cattle with low sequence diversity detected for some amplicons but consistently larger estimates found for neighbouring amplicons from the same gene .
thus recent introgression has not played a large role in the presence of these aberrant sites within the bovina and they may be due to homologous polymorphisms that existed in the mrca that have undergone lineage sorting in subsequent generations.
however, the lack of information on the genetic ancestry for all of our animals may require some caution when interpreting some of our results.
ancestral polymorphisms that have undergone lineage sorting in the bovini would have required an ancestral species with an extremely large population size that was very polymorphic.
palaeontological evidence generally agrees with this assumption, with the earliest fossil for the bovini tribe found in asia ~ <dig> mya, with evidence of populations existing solely in pakistan and india, south of the himalayas.
it was not until about 6– <dig> mya that the himalayas began increasing in size and areas of desert were becoming seasonally humid facilitating wide dispersal of bovini ancestors  <cit> .
thus the  <dig> aberrant sites identified in the bubalina and bovina subtribes most likely originate in this once large restricted and panmictic population.
the fact that each allele is found in the bubalina and bovina suggest that the polymorphisms must have remained segregating in both groups for what would appear to be ~ <dig> million years.
the remaining aberrant sites appear to have arisen in the independent ancestors of the bovina, which suggests that the respective population sizes for these populations must have also been very large.
these sites may have persisted for at least  <dig> million years, which is the approximate divergence time for the major bovina lineages from molecular and palaeontological data  <cit> .
the finding that some polymorphisms in the bovini have been segregating for so long is completely novel.
these ancestral polymorphisms have existed for very long periods and been maintained as neutral or overdominant polymorphisms.
the low dn/ds among these polymorphisms suggest that they are neutral because it is unlikely the synonymous polymorphisms show overdominance.
knowledge of polymorphisms segregating in humans and other primates has been known for some time with examples of polymorphisms in abo, mn and lewis blood groups  <cit> , although it was not known whether these polymorphisms were the result of the same genetic variations.
recently, work on the mhc has identified a number of polymorphisms that are trans-specific and have existed in humans and other primates for millions of years  <cit> .
however, the majority of these genes are involved with disease resistance and the maintenance of polymorphism has been hypothesised to exist due to balancing selection.
to our knowledge this is one of the only examples where polymorphisms at the same alleles have been maintained for such long periods outside of the mhc or involving genes that are responsible for disease resistance.
the difficulties in phylogenetic reconstruction caused by lineage sorting in closely related species have previously been reported for neutral genetic markers and mitochondrial control regions  <cit> .
because the majority of phylogenetic studies typically only involve a few genetic markers or concentrate on specific regions of the mitochondrial genome the exact rate of alleles undergoing lineage sorting is unknown, especially for nuclear genes, but the maintenance of polymorphic sites beyond the lifespan of a species is expected to be rare  <cit> .
therefore, the presence of alleles undergoing lineage sorting in coding regions of the bovine genome should be extremely scarce.
however, a relatively large number of these aberrant sites were detected within genomic coding regions of the bovini tribe.
these substitutions most likely would have been neutral or nearly neutral in the ancestral population from which they were derived, and thus the probability of their loss and fixation in future lineages is governed by the neutral theory of molecular evolution  <cit> .
by sequencing a large number of close relatives researchers should be able to identify a high number of these aberrant sites and infer the dn/ds rate among neutral mutations.
CONCLUSIONS
in conclusion, a partial phylogenetic reconstruction of the bovinae subfamily has identified three distinct lineages that have arisen after the bovini split from the bosephalaphini and the tragelaphini tribes.
within the bovini tribe two subtribes the bubalina and bovina were resolved, which we estimate diverged approximately 5– <dig> mya.
about 2– <dig> mya the bovina subtribe was still a single widespread species.
it then appears that the bovina split into  <dig> major groups: domestic cattle, bison/yak and the indochinese cattle or gaur/mithan and banteng and this occurred very rapidly.
polymorphisms in the ancestral species continued to segregate for long periods of time, which has lead to some alleles undergoing lineage sorting.
the lineage sorting we have detected appears to have confused the algorithms and tree drawing programs used for this analysis, and the apparent star like phylogeny will most likely continue to confuse phylogenetic reconstructions.
the majority of these sites appear to be neutral and must have arose in a polymorphic ancestor and continued to segregate within species for millions of years.
the age and maintenance of these ancestral polymorphisms in coding regions may be useful for studying the neutral rate of evolution and could also help explain the discrepancies between effective population size and levels of polymorphism in domesticated cattle.

 the bovine genome project has provided researchers with an excellent resource for bovine phylogenetic studies. genomic resources from b. taurus should have some transferability among a number of closely related bovids due to the likely divergence ages of the bovidae. to date several studies have suggested that primers designed in one species have a relatively high rate of pcr success in other families from the suborder ruminantia. therefore, sequencing exonic regions should have a high rate of success within the bovini tribe even from primers designed from the b. taurus genome. bovinae subfamily relationships and phylogenetic reconstruction the bovidae family consists of some of the most culturally and economically important species on the planet. of the major representatives of the family bovidae arguably the most important would be the subfamily bovinae, which is divided into three main tribes; the first two representatives of the bovinae, the tribes tragelaphini and boselaphini, comprise spiral, four-horned and much of the large ox-like antelope. representatives of these tribes are often hunted for meat and hide, and in the case of eland have been occasionally used to work in harness. the bovini tribe comprises all of the major domestic bovine species, including a number of wild species, some of which are endangered or threatened with extinction. the first divergence within the bovini occurred between 5– million years ago with the splitting of the buffalo or the subtribe bubalina from the nonbuffalo or the subtribe bovina. these two subtribes consistently resolve themselves as dichotomous groups, and show no evidence of producing viable hybrid offspring. recently molecular methods have estimated the bovina bubalina split at approximately mya. however, the earliest bovini fossils found south of the himalayas in india and pakistan have been dated at < mya. phenotypic and stable carbon and oxygen isotope analysis suggest that these bovini representatives were behaviourally and ecologically intermediate to modern bovini and their progenitors the bosephalini. these early examples of the bovini appeared to become more adapted to drier more open habitats and were only beginning to develop affinities with grazing open grasslands and obligate drinking patterns. in addition early bovini fossils do not exist in nearby regions like afghanistan and iran suggesting that these early bovini were restricted to the open forests of india and pakistan until < mya when the earliest bovini fossils begin to appear in africa, asia and europe, respectively. this expansion coincides with a changing climate and expansion of c dominated grasslands. hence estimates of the bovinae bubalina split > mya are most likely overestimates and a more realistic estimate is < mya. within the bovina subtribe, divergence of the remaining species also occurred recently, with a very sudden radiation in the early pleistocene ~ mya and as a result speciation has not been complete, with many members of the tribe producing viable hybrid offspring or infertile offspring in bulls that may have fertility restored by repeated backcrossing. the dramatic range expansion of domestic cattle has resulted in an increased threat of introgression by domestic cattle dna into the genomes of many wild populations, either intentionally or accidentally, all of which may confuse phylogenetic relationships. another confounding factor when reconstructing phylogenies may arise if a rapidly evolving clade's most recent common ancestor was highly polymorphic, as this may result in the random assortment of genetic variation in different lineages and aberrant modes of inheritance for certain polymorphisms. this phenomenon is known as lineage sorting, and it has been known to cause random associations among species, which may further confuse derived taxonomic relationships. recently phylogenetic reconstruction using mitochondrial lineages has led to clearer bovina phylogenies. however, because mitochondrial dna is maternally inherited, relationships driven by male mediated introgression may be missed. this finding was confirmed with y-chromosomal phylogenies identifying close relationships between wisent and north american bison, while mitochondrial phylogenies identify an association between bos taurus and wisent. more recently the kouprey and banteng were shown to be both valid species despite the introgression of the kouprey mitochondrial genome in cambodian banteng. thus, some improvements have been made regarding the phylogenetic relationships of the bovinae subtribe, but there is still concern over the accuracy of many trees due to low confidence values and contrasting results from different data and phylogenetic methods. this is especially evident when considering the position of the wisent in the bovine phylogeny and the time of divergence for these species. despite concerns over the level of variation within nuclear gene sequences at the species level in the bovini, by examining a number of nuclear genes we should identify enough genetic variation to differentiate between species, populations or breeds. also by examining variation within genes and exons in closely related species, one can analyse haplotypes for similarity among species over short and large distances. the presence of identical, long haplotypes in different species may be evidence of recent introgression as recombination is likely to break up linked haplotypes after a number of generations. alternatively, associations generated by the random assortment of polymorphic alleles should not create similar patterns of linked haplotypes over any significant distance. therefore, lineage sorting and introgression can be differentiated by the analysis of haplotype similarity and sequence variation in genomic dna between species. the presence of alleles undergoing lineage sorting in the coding regions of the genome are of interest as their presence is most likely due to polymorphisms in a common ancestor that were sorted in a fashion that is inconsistent with the inferred phylogeny. in some cases these polymorphisms can persist for generations, even past the lifespan of a species. the fact that many of these polymorphisms do not appear to be linked over any large distances suggests they have been independently assorted and that they are ancient in origin and therefore not introduced by recent introgression. if overdominance, selection or demographic factors, like the fixation of slightly deleterious alleles in small populations, have been responsible for the assortment of these ancient polymorphisms they must have an effect on phenotype and should therefore show high dn/ds ratios. therefore, examining estimates of dn/ds at these sites should help infer whether they are neutral or nearly neutral and if so, whether they are useful for determining the neutral rate of mutation in the bovini. in this paper we attempt to reconstruct phylogenies between extant representatives of the bovinae subfamily. we examined autosomal gene sequences, from different genes, sampled over bovine chromosomes. neighbour joining trees and the neutral substitution rate per site at synonymous and noncoding regions are presented for all autosomal sequences in an effort to identify relationships and divergence times between species, and test for evidence of introgression or lineage sorting.  sequencing in total amplicons from genes were sequenced across representatives of the bovinae subfamily, including members of the bovini and tragelaphini tribes, which we used to infer the sequence from the most recent common ancestor. from the amplicons amplified in at least one sample. the relative proportions from the amplicons that successfully amplified in each species were particularly high, ranging from in eland to in gaur. surprisingly some of the wild relatives showed better amplification than different breeds of b. taurus. all sequences were submitted to genbank and their primers and accession numbers are also available in additional file genetic distances, divergence times and phylogenetic reconstruction both di and ds are generally considered neutral substitutions and are often used for discriminating between phylogenetic lineages. table shows pairwise comparisons for di and ds between bovinae representatives, lower and upper diagonal respectively. the minimum di is within breeds of indian river buffalo, followed by breeds of domestic cow. the largest di is between species like eland and the domestic cow. similarly, the minimum ds is found between breeds, and the maximum ds is between eland and other members of the bovidae, such as bison, gaur, mithan, and domestic cow, respectively. interestingly, ds is often greater than di. subramanian and kumar suggest that an elevation in gc content at synonymous sites may be responsible for this phenomenon. species: species: no significant differences were detected between overall base frequencies for any member of the bovini tribe, and substitution rates between ancestral and eland sequences with all members of the bovini appear to be largely the same. a small bias was detected for transition to transversion substitutions, with a ratio of ti/tv =. therefore, kimura's two parameter model should be appropriate for phylogenetic reconstruction. however, the relatively small genetic distances detected suggest that the p-distances calculated in table should also be accurate despite the transition bias. aberrant sites to detect if recent gene flow or ancient polymorphisms were responsible for differences between our and other recently published phylogenies, we analysed all of the segregating sites from figure for the presence of anomalies. we reconstructed a number of phylogenetic trees to identify the tree that minimises the number of aberrant sites, which we defined as any site that must have arisen due to a double mutation or lineage sorting of an ancient polymorphism. we identified aberrant sites, of which were not tree specific and were aberrant in all trees leaving informative sites. from the non-tree specific aberrant sites ancient sites are segregating within both the bovina and bubalina lineages. a legend to all aberrant sites, their position and the relative age is provided in additional file to examine these aberrant sites in more detail we have analysed a number of phylogenetic trees that have been suggested due to the poor bootstrapping confidence in figure and trees presented in recent work by verkaar et al and hassanin and ropiquet. the phylogenetic tree that generates the smallest number of anomalies places yak off the lineage leading to domestic cattle and bison off the lineage leading to the indochinese cattle, ); however, similar numbers are also seen for the tree that places bison and yak as a lineage of the indochinese clade, indochinese cattle)), which is only worse by one aberrant site. therefore, some evidence exists for the trees recently proposed by hassanin and ropiquet and verkaar et al.. however, it appears that a number of aberrant sites are generating an association between yak and domestic cattle, which has confounded the phylogenetic reconstruction. removing all aberrant sites from the analysis leaves fewer substitutions to accurately estimate genetic distances between members of the bovina subtribe and the tree is less certain. introgression and ancient polymorphism to identify the source of the aberrant substitutions within the bovini tribe, we compared all variable sites in the alignment to find sites shared with b. taurus at normal and aberrant positions. we examined these positions within gaur, mithan, banteng, bison and yak for similarities to domestic cattle over extended haplotypes resolved from aberrant and all variable sites. we found that yak shares the largest number of alleles in common with domestic cattle for all variable and aberrant sites. perhaps the most interesting finding is the high number of identical haplotypes yak and domestic cattle share over haplotypes resolved from the aberrant sites per amplicon. when compared to haplotypes resolved from the all variable sites, yak and domestic cattle share the largest number in common. however, there are relatively few haplotypes that are similar for any of the species and domestic cattle, and this is probably a function of the species specific mutations that have accumulated per amplicon since divergence from the mrca. and the number of substitutions similar to b. taurus over haplotypes generated from all variable and from aberrant sites for each amplicon. gene_amplicon nrip1_e nrip1_e pit1_e1 pit1_e3 pit1_e5 pit1_e6 itgbp5_e3 itgbp5_e5 itgbp5_e6 itgbp5_e10 5ht1f_e1 igfbp2_e1 igfbp5_e2 igfbp5_e3 hfabp_e2 hfabp_e3 lacs3_e1 lacs3_e3 lacs3_e4 lacs3_e6 5ht6_e gmeb1_e1 gmeb1_e3 gmeb1_e4 gmeb1_e5 gmeb1_e6 gmeb1_e7 gmeb1_e8 egf_e1 era_e3 era_e4 era_e5 era_e6 era_e7 pabpc1_e2 pabpc1_e3 pabpc1_e5 pabpc1_e9 mfge8_e2 mfge8_e3 mfge8_e5 *number of exons represented as e where e = exon and n = number, therefore, pit1_e is exon of pit exons that extend over the largest sequencing read, which is typically bp are represented as e where j equals order of read from 5-prime to 3-prime. therefore nrip_e and nrip_e represent two subsequent reads of the l the number of aberrant alleles making each haplotype are given in brackets as the. table displays haplotype blocks shared with hereford and members of the bovinae for exons within the each gene and for genes on the same chromosome. to determine if the high proportion of haplotypes, resolved from aberrant sites, shared between yak and domestic cattle may be evidence of recent introgression, we looked for extended haplotypes that are shared between species. thus, the extent of linkage between neighbouring amplicons and genes on the same chromosome was examined. table shows the b. taurus haplotype per amplicon from all aberrant sites and the species that share the same haplotype. a number of haplotypes are shared with all species. many of the haplotypes that are based on one aberrant site may be similar by chance and therefore may not be very informative. however, by extending haplotypes to adjacent exons and genes more power to detect recent hybridisation should be available. but as haplotypes are extended to closely linked amplicons or neighbouring genes on the same chromosome this similarity rapidly disappears. in a further attempt to identify recent introgression, sequence diversity estimates were examined between neighbouring amplicons and genes. figure shows plots of sequence divergence at noncoding sites for hereford vs yak, hereford vs holstein and yak vs gaur. a similar pattern is seen to that described in table a number of amplicons show similar amounts of sequence divergence between hereford vs yak as is seen between hereford vs holstein. however, genetic diversity at closely linked amplicons remains low between breeds of b. taurus for closely spaced genes on chromosome and chromosome, which suggests that these animals have recently shared a common ancestor. in contrast, comparisons for domestic cattle vs yak, and yak vs gaur rarely show low sequence diversity over similar linked regions, and thus, genetic associations are most likely not the result of recent introgression. an examination of the river and swamp buffalo samples from southeast asia has identified large segments of chromosome and that are identical. thus bubb has a closer relationship to swamp buffalo than to other members of river buffalo that originate from india, which may suggest some level of introgression in these two samples as large portions of chromosomes and in bubb appear to be of swamp buffalo origin. classifying aberrant sites: age and role of selection a number of aberrant sites were identified that were inconsistent with any of the phylogenetic trees suggested from either molecular or morphological characteristics. in total aberrant sites occurred in coding regions, of which are ka and are ks from synonymous and nonsynonymous sites, which resulted in a dn/ds ratio of. from the aberrant sites appear to be polymorphic since the common ancestor with buffalo, while the remaining would have been polymorphic in the common ancestor of the bos and bison genera. of the ancient polymorphisms are ka and are ks. therefore these ancient polymorphisms were maintained for millions of years. they must have been polymorphic in the mrca to the bovina and bubalina subtribes some 5– mya and appear to have persisted to at least the divergence of the bovina subtribe around mya. the small values estimated for the dn/ds ratios calculated suggest that these polymorphisms may have been neutral. discussion sequencing a high proportion of successful amplification was achieved for all species within the bovinae subfamily from primers designed using the bos taurus genome despite divergence times of 8– my. the high rate of amplification between species should facilitate further population genetic studies in the bovini and possibly other ruminant subfamilies. bovine phylogeny reconstruction, mutation and substitution within the bovini, difficulties resolving interspecific relationships arose when comparing pairwise differences between species for di and ds with those deduced from the neighbour joining algorithm. in general the phylogenetic reconstruction created using kimura's two parameter method and the neighbour joining algorithm agreed with the p-distances calculated for di and ds in table with strong separation between bubalina and bovina subtribes and strong support for separation of bubalus and syncerus genera, confirming previous studies from molecular data. within the bubalus there appears to be a separation between swamp and river buffalo and a possible separation within river buffalo that may be due to due to geographic patterns and/or introgression of swamp and river buffalo. the phylogenetic tree we identified does not agree with some of the individual genetic distances within the bovina. for instance, the genetic distance between yak and bison for di and ds both imply that the species are most likely a monophyletic group that could either be grouped with the b. taurus lineage or the lineage leading to the cattle from indochina but the tree places them as separate divergences from the lineage leading to b. taurus. this disparity may be the result of more complicated methods of deducing genetic distance, like the kimura two parameter model, inflating variances over simpler methods, such as the p-distances, thereby increasing the chance of topographical errors. low bootstrapping levels may be the result of low genetic differentiation between these three lineages and the presence of aberrant polymorphisms, confusing phylogenetic reconstruction. examining a number of manually inferred trees that attempt to minimise the number of aberrant polymorphisms identified a tree that has bison as a divergence from the gaur/mithan/banteng lineage and yak as a divergence of the b. taurus clade. but this relationship does not seem logical considering the close relationship identified between bison and yak for ds and di distances. therefore, the large number of aberrant sites that yak shares with hereford may confuse the phylogenetic reconstruction, as the neighbour joining algorithm deals with these aberrant polymorphisms in the same manner as all other polymorphisms. removing aberrant polymorphisms from the analysis identified a phylogenetic relationship in which all members of the bovina appear to have diverged roughly at the same time in a star like phylogeny. however, bootstrap values were even lower than those deduced from the entire dataset, highlighting the difficulty of reconstructing phylogenies from a group of species that have undergone rapid genetic isolation. of course, low bootstrap values are expected for a star-like phylogeny. all trees show a close association between members of the bovina subtribe, with all major lineages diverging very rapidly some 1– million years ago, which appears to be associated with climatic oscillations during the late pleistocene. all trees show that bison and yak are single or multiple divergences from the clade leading to b. taurus, which disagrees with previous work by verkaar et al and hassanin and ropiquet that place bison and yak as a single divergence from the indochinese cattle. more recently hassanin and ropiquet show bison and yak as a single divergence from the lineage leading to b. taurus. however, these studies were typically based only on one or two thousand nucleotide characters or from a small number of genetic markers and therefore, each tree typically had low bootstrapping values or resulted in variations to this tree when differing methods or maternally, paternally or bi-parentally inherited data are used, suggesting that some of these relationships are still contentious. the phylogenetic reconstruction presented above is based on over bi-parentally inherited nucleotide characters and should have more power to resolve some of the more confusing intergenic relationships within the bovini. the association identified between bison, yak and b. taurus may also explain the similarity seen between wisent and b. taurus. although, no wisent sample was represented in our study, verkaar et al and hassanin and ropiquet have recently hypothesised that as a result of repeated male mitigated introgression of american bison populations into eurasian cattle like populations lead to a new species, which comprises the phenotype, autosomal genes and y-chromosome of american bison and the mitochondrial genome from the maternal ancestor. thus, the constant hybridisation by the progenitors of bison and b. taurus may have had a large impact on the genomes of both species. surprisingly, one of the bubalus bubalis samples was more closely related to the bubalis carabenensis than to other river buffalo. this relationship was puzzling, as previous studies have found that the two subspecies are genetically distinct, have different chromosome numbers and their divergence time has been estimated tens of thousands to over one million years. however, despite the obvious phenotypic and genetic differences, the two subspecies are known to hybridise, especially in south east asia where a hybrid zone for buffalo may exist. also in some instances animals can show the phenotype of the swamp buffalo but have the chromosomal and genetic make up of the river buffalo, as is the case for animals in sri lanka. therefore, the phylogenetic reconstruction may have been confused due to an analysis of a hybrid river buffalo that has large regions of chromosome and that apparently originated from swamp buffalo. alternatively, the phenotypic and karyoptypic plasticity shown within water buffalo may indicate that genetic differences between river and swamp buffalo could all be within species polymorphism. aberrant sites, introgression and ancient polymorphism one of the most astonishing features of the phylogenetic reconstruction was the identification of a large number of aberrant sites that did not fit any known phylogenetic reconstructions. a number of explanations may account for these phylogenetic aberrations, such as double mutations, introgressive hybridisation and lineage sorting. the probability of aberrant sites as a result of double mutations can be calculated as follows: which is the probability of no mutation in one lineage with mutation rate μ over t generations. therefore, the probability of mutation in one lineage is thus the probability that the same site mutates in two different lineages is and the expected number of double mutations across n sites is the probability that both mutations result in the same base is approximately 1/ hence the expected number of double mutations in two lineages is therefore, even if t is set to million years as a conservative estimate for the divergence of b. taurus and b. bubalis and n = then for equation one would expect around bases to have undergone a double mutation resulting in the same derived allele from the ~ bases sequenced in each lineage since the divergence of b. taurus and b. bubalus from a common ancestor some mya. the time of divergence is somewhat less within the bovini but there are more than lineages. however, the likelihood of a mutation affecting the exact same base ~ times in our dataset is highly improbable, so in general, most abnormalities must be evidence of lineage sorting or introgressive hybridisation and not double mutations. also a high proportion of aberrant sites were identified as sites that were deemed to be polymorphic in bubalina and bovina subtribes. members of these two subtribes separated 5– mya and no hybridisation has ever been documented between any members of these two subtribes. therefore, these aberrant sites cannot have arisen due to recent hybridisation events. if 40% of the aberrant sites are due to polymorphisms that existed in the common ancestor at bubalina and bovina, it seems likely that other polymorphisms in the common ancestor of bovina will also have contributed to the other 60% of aberrant sites. however, recent introgression might have also contributed, as the incomplete speciation of the tribe has resulted in many viable hybrid offspring from various crossings. the presence of identical haplotypes between bovina representatives and domestic cattle may also be a result of ancient geneflow between animals before reproductive isolation was achieved, but in essence this hypothesis would virtually be identical to the presence of ancestral polymorphisms. another possibility for this finding may also be explained by hybridisation between another closely related species. a previous study from the labs of university station in texas also found an anomalous association between yak and taurine cattle, due to an animal descending from a b. indicus cow. the fact that a bos indicus sample was not included in the data set, meant tests could not determine if some of the aberrant sites in our sample were due to gene flow between b. grunniens and b. indicus and this will need to be investigated further. the high degree of sequence similarity detected between yak and domestic cattle at all variable sites in the alignment, aberrant sites and over extended haplotypes suggests that hybridisation may have also occurred between these two species. yak-taurine hybrids are reported in china and mongolia where their altitudinal ranges intersect. however, no evidence of linked haplotypes at aberrant sites between neighbouring genes or on very small scales between exons within the same gene was detected. genes were selected from tightly linked regions of the b. taurus genome and the majority of amplicons within genes were only separated by 1– kilobases. no information on the genetic distance between genes has been collected but, the small physical distances between amplicons on the same gene suggest the recombination rate between many of these amplicons would be far below 1% per generation. the fact that all animals rarely share haplotypes with b. taurus between neighbouring amplicons and genes suggests that there is no evidence of linked haplotypes. this finding is also reflected in plots of sequence diversity between yak and domestic cattle with low sequence diversity detected for some amplicons but consistently larger estimates found for neighbouring amplicons from the same gene. thus recent introgression has not played a large role in the presence of these aberrant sites within the bovina and they may be due to homologous polymorphisms that existed in the mrca that have undergone lineage sorting in subsequent generations. however, the lack of information on the genetic ancestry for all of our animals may require some caution when interpreting some of our results. ancestral polymorphisms that have undergone lineage sorting in the bovini would have required an ancestral species with an extremely large population size that was very polymorphic. palaeontological evidence generally agrees with this assumption, with the earliest fossil for the bovini tribe found in asia ~ mya, with evidence of populations existing solely in pakistan and india, south of the himalayas. it was not until about 6– mya that the himalayas began increasing in size and areas of desert were becoming seasonally humid facilitating wide dispersal of bovini ancestors. thus the aberrant sites identified in the bubalina and bovina subtribes most likely originate in this once large restricted and panmictic population. the fact that each allele is found in the bubalina and bovina suggest that the polymorphisms must have remained segregating in both groups for what would appear to be ~ million years. the remaining aberrant sites appear to have arisen in the independent ancestors of the bovina, which suggests that the respective population sizes for these populations must have also been very large. these sites may have persisted for at least million years, which is the approximate divergence time for the major bovina lineages from molecular and palaeontological data. the finding that some polymorphisms in the bovini have been segregating for so long is completely novel. these ancestral polymorphisms have existed for very long periods and been maintained as neutral or overdominant polymorphisms. the low dn/ds among these polymorphisms suggest that they are neutral because it is unlikely the synonymous polymorphisms show overdominance. knowledge of polymorphisms segregating in humans and other primates has been known for some time with examples of polymorphisms in abo, mn and lewis blood groups, although it was not known whether these polymorphisms were the result of the same genetic variations. recently, work on the mhc has identified a number of polymorphisms that are trans-specific and have existed in humans and other primates for millions of years. however, the majority of these genes are involved with disease resistance and the maintenance of polymorphism has been hypothesised to exist due to balancing selection. to our knowledge this is one of the only examples where polymorphisms at the same alleles have been maintained for such long periods outside of the mhc or involving genes that are responsible for disease resistance. the difficulties in phylogenetic reconstruction caused by lineage sorting in closely related species have previously been reported for neutral genetic markers and mitochondrial control regions. because the majority of phylogenetic studies typically only involve a few genetic markers or concentrate on specific regions of the mitochondrial genome the exact rate of alleles undergoing lineage sorting is unknown, especially for nuclear genes, but the maintenance of polymorphic sites beyond the lifespan of a species is expected to be rare. therefore, the presence of alleles undergoing lineage sorting in coding regions of the bovine genome should be extremely scarce. however, a relatively large number of these aberrant sites were detected within genomic coding regions of the bovini tribe. these substitutions most likely would have been neutral or nearly neutral in the ancestral population from which they were derived, and thus the probability of their loss and fixation in future lineages is governed by the neutral theory of molecular evolution. by sequencing a large number of close relatives researchers should be able to identify a high number of these aberrant sites and infer the dn/ds rate among neutral mutations.  in conclusion, a partial phylogenetic reconstruction of the bovinae subfamily has identified three distinct lineages that have arisen after the bovini split from the bosephalaphini and the tragelaphini tribes. within the bovini tribe two subtribes the bubalina and bovina were resolved, which we estimate diverged approximately 5– mya. about 2– mya the bovina subtribe was still a single widespread species. it then appears that the bovina split into major groups: domestic cattle, bison/yak and the indochinese cattle or gaur/mithan and banteng and this occurred very rapidly. polymorphisms in the ancestral species continued to segregate for long periods of time, which has lead to some alleles undergoing lineage sorting. the lineage sorting we have detected appears to have confused the algorithms and tree drawing programs used for this analysis, and the apparent star like phylogeny will most likely continue to confuse phylogenetic reconstructions. the majority of these sites appear to be neutral and must have arose in a polymorphic ancestor and continued to segregate within species for millions of years. the age and maintenance of these ancestral polymorphisms in coding regions may be useful for studying the neutral rate of evolution and could also help explain the discrepancies between effective population size and levels of polymorphism in domesticated cattle. 
a partial phylogenetic reconstruction of extant members of the bovini tribe from complete or partially sequenced autosomal genes is presented. closer examination of these sites has identified that they are the result of ancient polymorphisms that have subsequently undergone lineage sorting in the bovini tribe, of which have remained polymorphic since bos and bison species last shared a common ancestor with bubalus between 5– million years ago. the presence of these sites could help account for the apparent contradiction between levels of polymorphism and effective population size in domesticated cattle. interestingly, a number of these aberrant sites are in coding sections of the genome and their identification may have important implications for studying the neutral rate of mutation at nonsynonymous sites. not surprisingly, a number of phylogenetic studies from molecular and morphological data have resulted in ambiguous trees and relationships amongst species, especially for yak and bison species. we identified distinct lineages after the bovini split from the boselaphini and tragelaphini tribes, which has lead to the buffalo clade and a more recent divergence leading to the banteng, gaur and mithan and domestic cattle clades. however, there was some ambiguity as to whether this was a divergence from the banteng/gaur/mithan or the domestic cattle clade. it appears that a number of sites with ambiguous inheritance have been maintained in subsequent populations by chance and that they have contributed to an association between yak and domestic cattle and an unreliable phylogenetic reconstruction for the bison/yak clade. uncertainty arises in our phylogenetic reconstructions because many species in the bovini diverged over a short period of time.
 the bovinae subfamily incorporates an array of antelope, buffalo and cattle species. all of the members of this subfamily have diverged recently. not surprisingly, a number of phylogenetic studies from molecular and morphological data have resulted in ambiguous trees and relationships amongst species, especially for yak and bison species. a partial phylogenetic reconstruction of extant members of the bovini tribe from complete or partially sequenced autosomal genes is presented.  we identified distinct lineages after the bovini split from the boselaphini and tragelaphini tribes, which has lead to the buffalo clade and a more recent divergence leading to the banteng, gaur and mithan and domestic cattle clades. a fourth lineage may also exist that leads to bison and yak. however, there was some ambiguity as to whether this was a divergence from the banteng/gaur/mithan or the domestic cattle clade. from an analysis of approximately sites that were amplified in all species sites were identified with ambiguous inheritance, in that all trees implied more than one mutation at the same site. closer examination of these sites has identified that they are the result of ancient polymorphisms that have subsequently undergone lineage sorting in the bovini tribe, of which have remained polymorphic since bos and bison species last shared a common ancestor with bubalus between 5– million years ago.  uncertainty arises in our phylogenetic reconstructions because many species in the bovini diverged over a short period of time. it appears that a number of sites with ambiguous inheritance have been maintained in subsequent populations by chance and that they have contributed to an association between yak and domestic cattle and an unreliable phylogenetic reconstruction for the bison/yak clade. interestingly, a number of these aberrant sites are in coding sections of the genome and their identification may have important implications for studying the neutral rate of mutation at nonsynonymous sites. the presence of these sites could help account for the apparent contradiction between levels of polymorphism and effective population size in domesticated cattle. 