BACKGROUND
phylogenetic relationships within and between animal species often depend on the markers studied, as different genes might have different modes of transmission and different histories  <cit> .
in addition, hybridization can result in discordant phylogenies between markers.
increasing evidence points to a contribution of reticulate evolution to the speciation process  <cit> .
in this context, information on the phylogenies of different markers for closely related species and subspecies is important to the study of processes underlying speciation  <cit> .
the study of chamois  allows exploring the effect of historical and evolutionary events on diversification.
it is distributed over most of the medium to high altitude mountain ranges of southern eurasia .
at present, chamois populations are classified into two species, r. pyrenaica and r. rupicapra  <cit> , on the basis of morphological and behavioral characters: rupicapra pyrenaica  from southwestern europe, and r. rupicapra  from central and southeastern europe and western asia  <cit> .
analysis of genetic variation in a limited number of subspecies for allozyme loci  <cit> , minisatellites  <cit> , rflps of mitochondrial dna  <cit>  and the major histocompatibility complex  <cit>  provided some support for this classification.
however, the nominal species are paraphyletic for mtdna  <cit> .
the quaternary glacial ages probably had a major effect on the phylogeography and evolution of the genus rupicapra, as it did on other animals in eurasia  <cit> .
the rupicaprini are thought to have originated in asia during the miocene period and the sudden appearance of rupicapra fossils in europe during the middle pleistocene age has been interpreted as resulting from immigration from the east during a cold climatic phase  <cit> .
in contrast with the fossil record, the divergence between the main mtdna clades has been estimated around  <dig> - <dig> mya  <cit>  but this cannot be directly assumed to be the divergence time between species.
the mitochondrial phylogeny showed three main lineages, originating during the early pleistocene  <cit> .
nuclear microsatellite genotypes formed three clearly defined groups as well; however those groups did not exactly match the mitochondrial lineages but are closer to morphology and taxonomic classification.
the phylogeographic patterns suggest an evolutionary history with range contractions and expansions related to climatic oscillations during the quaternary period and reflect a major effect of the alpine barrier on west-east differentiation.
the contrasting phylogenies of mtdna and nuclear microsatellites for populations of chartreuse and the western alps indicated events of range overlap and hybridization among highly divergent lineages in the central area of the distribution.
both markers showed differentiation between all pairs of populations  <cit>  and a geographic signature in the distribution of variability, suggesting that differentiation occurred without major migrations.
to further elucidate the processes leading to the origin and diversification of rupicapra, we studied the y-chromosome.
the y-chromosome is paternally inherited and does not undergo recombination at meiosis, providing a marker to study male dispersal  <cit> .
the study has the dual purpose of studying the patrilineal phylogenetic position of chamois, compared with capra, ovis and ammotragus, and the male dispersal within the genus rupicapra.
we present the analysis of a sequence of a fragment of the sry gene promoter together with two male-specific microsatellites umn <dig> and srym <dig>  in a sample set of  <dig> males,  <dig> of r. pyrenaica and  <dig> from r. rupricapra, which covers the entire distribution range of chamois.
comparison of the geographic distribution of male-specific markers with mtdna lineages  and autosomal markers  allows us to follow the evolutionary history of rupicapra in the context of the climatic oscillations of the pleistocene age.
RESULTS
sry promoter sequences
we have amplified and sequenced  <dig> nucleotides corresponding to a fragment of the sry gene promoter from  <dig> male chamois,  <dig> of the subspecies r. pyrenaica  and  <dig> of r. rupicapra .
the alignment resulted in only two haplotypes, one in r. pyrenaica and the other in r. rupicapra.
these haplotypes differ only in one nucleotide , which is a in the haplotype pyrenaica and g in rupicapra.
to investigate the evolutionary history of the y-chromosome of rupicapra, the two haplotypes were aligned with the sequences of other bovidae available in the genbank, ammotragus lervia, capra hircus, ovis aries and bos taurus .
in addition, two individual ammotragus lervia have been sequenced in our laboratory and both had identical sequence with a deletion of  <dig> nucleotides with respect to the rest of bovidae.
the aligned dataset contains  <dig> nucleotides  with  <dig> variable sites of which  <dig> are fixed and  <dig> are variable among caprinae.
the phylogenetic relationships were studied using neighbor-joining, maximum likelihood, maximum parsimony, or bayesian approaches under different models of nucleotide substitution, either the simple model of jukes-cantor or the substitution model that describes better the substitution pattern of the dataset, a tamura 3-parameter model  <cit>  with non-uniformity of evolutionary rate among sites .
the three parameters were nucleotide frequencies  <dig>  for a and t,  <dig>  for c and g, ts/tv ratio:  <dig>  and rate heterogeneity:  <dig> .
for the construction of the bayesian tree, the model of nucleotide substitution was hki+g  and the parameters were obtained by the program beast itself.
there were  <dig> parsimony-informative sites.
model-free parsimony analysis performed with mega led to three equally parsimonious trees with a total length of  <dig> steps.
the different methods of tree construction all led to topologies with two main well supported nodes , one grouping ovis with the published sequences of ammotragus and the other grouping rupicapra, capra and the sequence of ammotragus obtained in this work.
the relationships within this second group varied, depending on the method used for tree construction, and they were poorly supported.
all the  <dig> different bovidae sequences present, like pyrenaica, a in site  <dig> in our alignment, suggesting that this is the ancestral haplotype.
the estimated divergence time of the two rupicapra haplotypes from the sry promoter sequence was  <dig> kya .
the mean substitution rate per nucleotide calculated with tracer from the mcmc samples was  <dig> e- <dig> ±  <dig> e- <dig> per million years.
y-chromosome microsatellites
out of  <dig> microsatellite markers tested, only umn <dig> and srym <dig> produced male-specific products.
amplification from  <dig> males,  <dig> of r. pyrenaica and  <dig> from r. rupricapra, revealed two different length fragments for umn <dig> and seven for srym <dig> .
for each microsatellite, fragments within each length variant were further analyzed by cloning and sequencing, and the architecture was compared with their homologous loci in other bovidae.
the umn <dig> repeated motif was found to be n differing from the repeat n reported in bos taurus.
rupicapra pyrenaica presented two alleles,  <dig> and  <dig>  differing in one repetition, and r. rupicapra was monomorphic, with only the  <dig> allele.
the microsatellite srym <dig> lacks the pentanucleotide  and the dinucleotide  motifs common in sheep  <cit>  and instead presents a trinucleotide m and a mononucleotide n motifs.
rupicapra pyrenaica haplotypes were man and r. rupicapra haplotypes were mn, these two structures were reported in ammotragus lerviae  and ovis aries breed balami , respectively.
combinations of variation in number of repeats in both motifs in r. rupicapra resulted in homoplasy, where pcr products with the same size had different sequence architecture.
the trinucleotide motif, m, was polymorphic in the species r. rupicapra but not in r. pyrenaica while the mononucleotide motif was polymorphic in both subspecies .
the abbreviated name of the subspecies are: par, parva; pyr, pyrenaica; orn, ornata; cat, cartusiana; rupw, rupicapraw; rupc, rupicaprac; rupe, rupicaprae; tat, tatrica; cap, carpatica; bal, balcanica; asi, asiatica; and cau, caucasica.
number of samples sequenced for srym <dig> are given in parenthesis.
network of y haplotypes
altogether, ten haplotypes could be differentiated in the rupicapra genus .
total y-chromosome haplotype diversity was  <dig>  with on average one distinct haplotype over  <dig>  individuals .
three private haplotypes were found in west chamois r. pyrenaica, giving a haplotypic diversity of  <dig> % and the other seven haplotypes were private of the east chamois r. rupicapra with a diversity of  <dig> %.
the network of haplotypes  revealed two haplogroups that concur with the taxonomy of chamois.
these two clades are separated by two nucleotide substitutions  and a mean distance of  <dig>  microsatellite repeats.
the connections between haplotypes show a strong geographic signal with links always between neighboring populations.
the same network is obtained whether or not the nucleotide substitutions are included.
discussion
analysis of chromosome y presented a pattern of variation different from the one obtained from either mitochondrial or biparental nuclear dna, both on diversity as well as on the spread and geographic boundaries of dispersion.
however, the east/west phylogeographic signal in the distribution of haplotypes is present again and once more the suture zone places in the alps.
it is remarkable that, contrary to mtdna and autosomal microsatellites that formed three clades , the variation for the y-chromosome conforms to the two species currently accepted, rupicapra pyrenaica and r. rupicapra.
this could explain the concurrence of coat patterns, cranial morphometry and several courtship behavioral patterns in iberian and apennine chamois  <cit>  that remained unexplained from the study of mtdna and nuclear microsatellites  <cit> .
phylogenetic relationships between chamois and other caprini
the phylogeny of sry promoter shows an association between rupicapra, capra and the ammotragus sequence obtained in this work .
this association concurs with the relationships revealed from the study of the complete mitochondrial genome  <cit> .
previously, meadows and kijas  <cit>  reported a very close relation between the sry promoter of ammotragus  and ovis, but we found  <dig> differences  between this previously reported sequence and the new sequences produced in our laboratory .
this large difference is not expected between two individuals of the same species.
in contrast, the reported sequence of ammotragus presents only  <dig> differences  with ovis canadensis.
our sequence has been obtained from good quality samples  from two specimens  with identical results.
so, we think that the sequence reported by meadows and kijas could be contaminated with dna of ovis canadensis.
an alternative interpretation to take into account is the polyphyly of ammotragus.
the affinities of ammotragus with either capra or ovis have been widely discussed in the literature as it exhibits a particular combination of goat-like and sheep-like characters  <cit> .
the structure of the microsatellite srym <dig> of west chamois is identical to ammotragus and different to most ovis, but the african breed balami of o. aries shares the repeat structure with ammotragus and rupicapra.
this observation had lead meadows and kijas  <cit>  to hypothesize the possible gene flow from barbary to domestic sheep.
the observed similarities between ammotragus lerviae and the genus rupicapra and the apparent spread of male chamois south to north reopen the question of the possible position of ammotragus as an ancestor of the caprinae  <cit> .
it can also be noted that rupicapra and ammotragus have similar karyotype with  <dig> chromosomes  <cit> .
additional studies of y-chromosome phylogenies of caprinae could offer very important information to clarify this issue.
patrilineal phylogeography of chamois
when comparing the phylogenetic trees based on mtdna or the sequences of the sry promoter , a clear difference emerges.
all the rupicapra belong to one unique clade for the sry promoter while three, well differentiated, clades formed for mtdna.
the observed number of substitutions per nucleotide between the pairs of species ovis-rupicapra, capra-rupicapra and ovis-capra for the sequences of mtdna in our former study  <cit>  were  <dig> ,  <dig>  and  <dig>  respectively to be compared with  <dig> ,  <dig>  and  <dig>  substitutions per nucleotide respectively for the sry promoter sequence.
the distance between pairs of species for mtdna is about two or three times that of the sry promoter, consistent with observations in other mammals including humans  <cit> .
the level of differentiation among y-chromosomes in chamois is remarkably low.
the haplogroups y-rpyr and y-rrup within the rupicapra genus differ by one single nucleotide, leading to an estimated average number of substitutions per nucleotide of  <dig>  that is  <dig>  times lower than the average distance between the three clades of mtdna .
the time of divergence between the sry haplotypes estimated from the phylogenetic tree places the split  <dig> kya, in the middle of the pleistocene.
thus, all modern chamois seem to descend of one very young male lineage.
the low diversity in the number of microsatellite repeats, both between species and within species, compared with the y-specific evolutionary mutation rate of  <dig>  × 10- <dig> mutations per generation  <cit> , gives further support to this interpretation.
thereafter, our data suggest that the divergence of y-chromosomal variants took place well after the divergence of mtdna lineages  <cit> , in a period compatible with the sudden appearance of rupicapra fossils in europe  <cit> .
the examination of the y-chromosome network of haplotypes offers insight into the patrilineal history of dispersion.
there is an association between the network of haplotypes and geography  with a west-east cline of ancestral to derived chromosomes and the signature of the alpine barrier.
attending to the snp in the sry promoter it can be noted that the haplogroup y-rpyr presents the ancestral plesiomorph state , shared by all the other species in the phylogeny, while the haplogroup y-rrup has the derived state g .
regarding the snp in the microsatellite srym <dig>  y-rpyr presents the nucleotide a like ammotragus, hence it could be assumed to represent the ancestral state, while y-rrup presents t .
from here we propose that one of the haplotypes of the y-pyr group represents the ancestral state.
the pattern of microsatellite variation within each haplogroup also supports this interpretation as explained below.
first, let us recall that the mutation profiles of microsatellites depend on the size of the repeat motif, the rate of mutation of pentanucleotides is about half of that of trinucleotides, and mononucleotide repeats present a very high mutation rate  <cit> .
our data on pentanucleotide, trinucleotide and mononucleotide difference between r. pyrenatica and r. rupicapra in number of repeats  concur with this observation.
hence, microsatellites with longer motifs retain a stronger phylogenetic signal than shorter ones.
it has been shown that in microsatellites with few repetitions, mutation is biased towards increase in number of repeats  <cit> .
attending to the network of haplotypes, it can be seen that rpyr <dig>  sampled from r. p. pyrenaica and r. p. parva, presents the lowest number of repeats for all, the penta, the tri and the mononucleotide motifs, this is probably the closest to the basal haplotype.
the number of repeats of the pentanucleotide microsatellite umn <dig> is variable only in western chamois, the allele  <dig>  with one additional repeat is found in r. p. pyrenaica and r. p. parva.
the number of trinucleotide repeats and mononucleotide repeats of srym <dig> increases west to east.
the mononucleotide motif seems to have suffered mutations forth and back in the eastmost populations.
we conclude that the patrilineal dispersion of rupicapra was south-west to north-east.
the cline of ancestral to derived chromosomes could be originated by serial funding events, probably related to the glacial-interglacial cycles of the quaternary.
male dispersion could start from the iberian peninsula or from italy  to the east and the two haplogroups differentiate one to each side of the alps.
the lower number of repeats points to haplotype rpyr <dig> present in iberia as the closest to the ancestor.
alternatively, the haplotype rpyr <dig> in the center of the network and differing from rpyr <dig> solely in the mononucleotide could be also a good candidate.
differences between markers in patterns of variation and the evolutionary history of chamois
the discordant patterns of mtdna and autosomal microsatellites that have been described in a previous study  <cit>  indicated the occurrence of hybridization among divergent lineages of chamois in the central area of the distribution.
chromosome y data add complexity to this mosaic; the geographical sorting of variation for the different kind of marker is summarized in figure  <dig>  the three kinds of markers have specific evolutionary characteristics that need to be taken into account to explain differing variation patterns  <cit> .
these differences are on  mutation rates,  selection,  effective population size and  dispersal.
being the y a male-specific chromosome, and given that the mutation rate is higher in males than in females  <cit> , we would expect higher divergence for y-chromosome than for autosomal microsatellites; our data are contrary to this expectation.
positive selection could act on the whole y-chromosome and, given that the y-chromosome does not recombine, it will be selected as a block in a selective sweep with the consequence of homogenization  <cit> .
in addition, the lack of recombination must be responsible for the effect known as muller's ratchet, the random accumulation of deleterious mutations that cannot be removed by recombination  <cit> .
selection against these mutations further reduces variability.
selection has been proposed as the main factor to explain low levels of genetic variability in the y-chromosome  <cit>  and must contribute to the reduced variability among y-chromosomes in chamois.
a similar pattern was found in ursidae where y-chromosome genes have fewer substitutions than expected in external branches  <cit> .
the chromosome y and mtdna both have effective population size of one quarter of that of an autosome because they are haploid and transmitted by one sex, but the effective population size of y-chromosome must be lower because the variance in number of descendants is larger in males than in females, especially in a polygynous species.
hence, the y-chromosome is more prone to genetic drift effects.
however, genetic drift cannot be invoked as the main cause for the y-chromosome phylogeography because in such case no such strong geographical signal would be expected.
differences in male and female dispersal can be related to different patterns of variation for patrilineal, matrilineal and biparental markers.
recent migration of males could be responsible of the low y-chromosome diversity observed.
taking into account the above considerations, together with the assumed asiatic origin of rupicapra and the similarity of the y-chromosome of rupicapra and ammotragus, we propose two speculative hypotheses to explain the observed results: a) the rupicapra genus has its origin in southern europe and spread west to east.
the differences in divergence between mtdna and y-chromosome are due to the particular evolutionary characteristics of the y-chromosome, or b) males from an ancestral population highly differentiated from a second ancestral taxa present in europe entered from the south, hybridizing and spreading westwards.
under the first hypothesis, the origin of the genus rupicapra needs to be placed in south europe from where it spread eastwards.
this is contrary to the general belief that chamois, as caprines in general, originated in asia  <cit>  but, following ropiquet and hassanin  <cit> , this hypothesis is not really supported by the fossil record.
these authors propose that caprines originated in the mediterranean islands during the miocene and from here may have invaded africa, europe and asia after the tortonian salinity crisis  or after the messinian salinity crisis .
the similarity among y-chromosomes under this hypothesis must be attributed to male dispersal and female phylopatry, together with selective sweeps and purifying selection that acted over the chromosome as a whole.
the second hypothesis is the hybridization between two ancestral caprinae in the beginning of the evolutionary history of the genus rupicapra.
the hybridization between highly divergent lineages at speciation of rupicapra had been previously hypothesized after the observation of a pseudogene of cytochrome b from a highly divergent lineage in the nucleus of present day chamois  <cit> .
an ancestral caprinae related with ammotragus could have reached the south mediterranean, most probably the iberian peninsula, and hybridize with the ancestral rupicapra female.
the dispersal of species across the strait of gibraltar during pleistocene has been proposed for other vertebrates  <cit>  and has been related to the substantially lower sea level associated with major pleistocene glaciations  <cit> .
new male y-chromosome lineages would have dispersed during the pleistocene, replacing older ones.
hybridization, after secondary contact among related taxa, has been inferred in the evolution of the genus capra  <cit>  as well as in the evolution of some species of bos  <cit>  and has been recently observed between native and introduced species of cervus in scotland  <cit> .
it can be added that hybrids from ammotragus male and capra female have been artificially obtained  <cit> .
many recent studies have shown contrasting phylogenies for different kinds of markers that lead to the view of hybridization as an important mechanism in the evolution of animal species  <cit> .
our results on y-chromosome reconcile the sudden occurrence of rupicapra in the middle-late pleistocene with the existence of very old mtdna lineages.
in addition, the proposed migration of chamois west to east can explain the reported more conservative features of r. pyrenaica  <cit> .
the alternative hypotheses presented here can be tested in future research that includes the comparative study of chromosome y in a broader representation of caprinae and additional nuclear sequences of the different populations of rupicapra to search for the signature of possible hybridizations.
CONCLUSIONS
the y-chromosome is highly informative to follow the dispersal of populations of caprinae.
the phylogenetic analysis of the sry gene promoter shows an association between rupicapra, capra and ammotragus, not in agreement with a previous report, which grouped ammotragus with ovis.
the structure of the srym <dig> microsatellite of rupicapra is equal to ammotragus and the african breed balami of o. aries.
the patrilineal relationships of caprine deserve a reinvestigation.
within rupicapra, the diversity of y-chromosomes is very low.
the combined data define  <dig> y-chromosome haplotypes forming a west-to-east geographical cline of ancestral to derived alleles.
haplotypes form two haplogroups, which concur with taxonomic classification, instead of the three clades formed for mtdna and biparental microsatellites.
we propose that, contrary to the presumed asian origin, the paternal lineage of chamois originated in the mediterranean, most probably in the iberian peninsula, and dispersed eastwards through serial funding events during the glacial-interglacial cycles of the quaternary.
the differences in patterns of variation among y-chromosome, mtdna and biparental microsatellites reflect the evolutionary characteristics of the different markers, as well as the effects of sex-biased dispersal and species phylogeography.

 phylogenetic relationships within and between animal species often depend on the markers studied, as different genes might have different modes of transmission and different histories. in addition, hybridization can result in discordant phylogenies between markers. increasing evidence points to a contribution of reticulate evolution to the speciation process. in this context, information on the phylogenies of different markers for closely related species and subspecies is important to the study of processes underlying speciation. the study of chamois allows exploring the effect of historical and evolutionary events on diversification. it is distributed over most of the medium to high altitude mountain ranges of southern eurasia. at present, chamois populations are classified into two species, r. pyrenaica and r. rupicapra, on the basis of morphological and behavioral characters: rupicapra pyrenaica from southwestern europe, and r. rupicapra from central and southeastern europe and western asia. analysis of genetic variation in a limited number of subspecies for allozyme loci, minisatellites, rflps of mitochondrial dna and the major histocompatibility complex provided some support for this classification. however, the nominal species are paraphyletic for mtdna. the quaternary glacial ages probably had a major effect on the phylogeography and evolution of the genus rupicapra, as it did on other animals in eurasia. the rupicaprini are thought to have originated in asia during the miocene period and the sudden appearance of rupicapra fossils in europe during the middle pleistocene age has been interpreted as resulting from immigration from the east during a cold climatic phase. in contrast with the fossil record, the divergence between the main mtdna clades has been estimated around - mya but this cannot be directly assumed to be the divergence time between species. the mitochondrial phylogeny showed three main lineages, originating during the early pleistocene. nuclear microsatellite genotypes formed three clearly defined groups as well; however those groups did not exactly match the mitochondrial lineages but are closer to morphology and taxonomic classification. the phylogeographic patterns suggest an evolutionary history with range contractions and expansions related to climatic oscillations during the quaternary period and reflect a major effect of the alpine barrier on west-east differentiation. the contrasting phylogenies of mtdna and nuclear microsatellites for populations of chartreuse and the western alps indicated events of range overlap and hybridization among highly divergent lineages in the central area of the distribution. both markers showed differentiation between all pairs of populations and a geographic signature in the distribution of variability, suggesting that differentiation occurred without major migrations. to further elucidate the processes leading to the origin and diversification of rupicapra, we studied the y-chromosome. the y-chromosome is paternally inherited and does not undergo recombination at meiosis, providing a marker to study male dispersal. the study has the dual purpose of studying the patrilineal phylogenetic position of chamois, compared with capra, ovis and ammotragus, and the male dispersal within the genus rupicapra. we present the analysis of a sequence of a fragment of the sry gene promoter together with two male-specific microsatellites umn and srym in a sample set of males, of r. pyrenaica and from r. rupricapra, which covers the entire distribution range of chamois. comparison of the geographic distribution of male-specific markers with mtdna lineages and autosomal markers allows us to follow the evolutionary history of rupicapra in the context of the climatic oscillations of the pleistocene age.  sry promoter sequences we have amplified and sequenced nucleotides corresponding to a fragment of the sry gene promoter from male chamois, of the subspecies r. pyrenaica and of r. rupicapra. the alignment resulted in only two haplotypes, one in r. pyrenaica and the other in r. rupicapra. these haplotypes differ only in one nucleotide, which is a in the haplotype pyrenaica and g in rupicapra. to investigate the evolutionary history of the y-chromosome of rupicapra, the two haplotypes were aligned with the sequences of other bovidae available in the genbank, ammotragus lervia, capra hircus, ovis aries and bos taurus. in addition, two individual ammotragus lervia have been sequenced in our laboratory and both had identical sequence with a deletion of nucleotides with respect to the rest of bovidae. the aligned dataset contains nucleotides with variable sites of which are fixed and are variable among caprinae. the phylogenetic relationships were studied using neighbor-joining, maximum likelihood, maximum parsimony, or bayesian approaches under different models of nucleotide substitution, either the simple model of jukes-cantor or the substitution model that describes better the substitution pattern of the dataset, a tamura 3-parameter model with non-uniformity of evolutionary rate among sites. the three parameters were nucleotide frequencies for a and t, for c and g, ts/tv ratio: and rate heterogeneity:. for the construction of the bayesian tree, the model of nucleotide substitution was hki+g and the parameters were obtained by the program beast itself. there were parsimony-informative sites. model-free parsimony analysis performed with mega led to three equally parsimonious trees with a total length of steps. the different methods of tree construction all led to topologies with two main well supported nodes, one grouping ovis with the published sequences of ammotragus and the other grouping rupicapra, capra and the sequence of ammotragus obtained in this work. the relationships within this second group varied, depending on the method used for tree construction, and they were poorly supported. all the different bovidae sequences present, like pyrenaica, a in site in our alignment, suggesting that this is the ancestral haplotype. the estimated divergence time of the two rupicapra haplotypes from the sry promoter sequence was kya. the mean substitution rate per nucleotide calculated with tracer from the mcmc samples was e- ± e- per million years. y-chromosome microsatellites out of microsatellite markers tested, only umn and srym produced male-specific products. amplification from males, of r. pyrenaica and from r. rupricapra, revealed two different length fragments for umn and seven for srym. for each microsatellite, fragments within each length variant were further analyzed by cloning and sequencing, and the architecture was compared with their homologous loci in other bovidae. the umn repeated motif was found to be n differing from the repeat n reported in bos taurus. rupicapra pyrenaica presented two alleles, and differing in one repetition, and r. rupicapra was monomorphic, with only the allele. the microsatellite srym lacks the pentanucleotide and the dinucleotide motifs common in sheep and instead presents a trinucleotide m and a mononucleotide n motifs. rupicapra pyrenaica haplotypes were man and r. rupicapra haplotypes were mn, these two structures were reported in ammotragus lerviae and ovis aries breed balami, respectively. combinations of variation in number of repeats in both motifs in r. rupicapra resulted in homoplasy, where pcr products with the same size had different sequence architecture. the trinucleotide motif, m, was polymorphic in the species r. rupicapra but not in r. pyrenaica while the mononucleotide motif was polymorphic in both subspecies. the abbreviated name of the subspecies are: par, parva; pyr, pyrenaica; orn, ornata; cat, cartusiana; rupw, rupicapraw; rupc, rupicaprac; rupe, rupicaprae; tat, tatrica; cap, carpatica; bal, balcanica; asi, asiatica; and cau, caucasica. number of samples sequenced for srym are given in parenthesis. network of y haplotypes altogether, ten haplotypes could be differentiated in the rupicapra genus. total y-chromosome haplotype diversity was with on average one distinct haplotype over individuals. three private haplotypes were found in west chamois r. pyrenaica, giving a haplotypic diversity of % and the other seven haplotypes were private of the east chamois r. rupicapra with a diversity of %. the network of haplotypes revealed two haplogroups that concur with the taxonomy of chamois. these two clades are separated by two nucleotide substitutions and a mean distance of microsatellite repeats. the connections between haplotypes show a strong geographic signal with links always between neighboring populations. the same network is obtained whether or not the nucleotide substitutions are included. discussion analysis of chromosome y presented a pattern of variation different from the one obtained from either mitochondrial or biparental nuclear dna, both on diversity as well as on the spread and geographic boundaries of dispersion. however, the east/west phylogeographic signal in the distribution of haplotypes is present again and once more the suture zone places in the alps. it is remarkable that, contrary to mtdna and autosomal microsatellites that formed three clades, the variation for the y-chromosome conforms to the two species currently accepted, rupicapra pyrenaica and r. rupicapra. this could explain the concurrence of coat patterns, cranial morphometry and several courtship behavioral patterns in iberian and apennine chamois that remained unexplained from the study of mtdna and nuclear microsatellites. phylogenetic relationships between chamois and other caprini the phylogeny of sry promoter shows an association between rupicapra, capra and the ammotragus sequence obtained in this work. this association concurs with the relationships revealed from the study of the complete mitochondrial genome. previously, meadows and kijas reported a very close relation between the sry promoter of ammotragus and ovis, but we found differences between this previously reported sequence and the new sequences produced in our laboratory. this large difference is not expected between two individuals of the same species. in contrast, the reported sequence of ammotragus presents only differences with ovis canadensis. our sequence has been obtained from good quality samples from two specimens with identical results. so, we think that the sequence reported by meadows and kijas could be contaminated with dna of ovis canadensis. an alternative interpretation to take into account is the polyphyly of ammotragus. the affinities of ammotragus with either capra or ovis have been widely discussed in the literature as it exhibits a particular combination of goat-like and sheep-like characters. the structure of the microsatellite srym of west chamois is identical to ammotragus and different to most ovis, but the african breed balami of o. aries shares the repeat structure with ammotragus and rupicapra. this observation had lead meadows and kijas to hypothesize the possible gene flow from barbary to domestic sheep. the observed similarities between ammotragus lerviae and the genus rupicapra and the apparent spread of male chamois south to north reopen the question of the possible position of ammotragus as an ancestor of the caprinae. it can also be noted that rupicapra and ammotragus have similar karyotype with chromosomes. additional studies of y-chromosome phylogenies of caprinae could offer very important information to clarify this issue. patrilineal phylogeography of chamois when comparing the phylogenetic trees based on mtdna or the sequences of the sry promoter, a clear difference emerges. all the rupicapra belong to one unique clade for the sry promoter while three, well differentiated, clades formed for mtdna. the observed number of substitutions per nucleotide between the pairs of species ovis-rupicapra, capra-rupicapra and ovis-capra for the sequences of mtdna in our former study were, and respectively to be compared with, and substitutions per nucleotide respectively for the sry promoter sequence. the distance between pairs of species for mtdna is about two or three times that of the sry promoter, consistent with observations in other mammals including humans. the level of differentiation among y-chromosomes in chamois is remarkably low. the haplogroups y-rpyr and y-rrup within the rupicapra genus differ by one single nucleotide, leading to an estimated average number of substitutions per nucleotide of that is times lower than the average distance between the three clades of mtdna. the time of divergence between the sry haplotypes estimated from the phylogenetic tree places the split kya, in the middle of the pleistocene. thus, all modern chamois seem to descend of one very young male lineage. the low diversity in the number of microsatellite repeats, both between species and within species, compared with the y-specific evolutionary mutation rate of × 10- mutations per generation, gives further support to this interpretation. thereafter, our data suggest that the divergence of y-chromosomal variants took place well after the divergence of mtdna lineages, in a period compatible with the sudden appearance of rupicapra fossils in europe. the examination of the y-chromosome network of haplotypes offers insight into the patrilineal history of dispersion. there is an association between the network of haplotypes and geography with a west-east cline of ancestral to derived chromosomes and the signature of the alpine barrier. attending to the snp in the sry promoter it can be noted that the haplogroup y-rpyr presents the ancestral plesiomorph state, shared by all the other species in the phylogeny, while the haplogroup y-rrup has the derived state g. regarding the snp in the microsatellite srym y-rpyr presents the nucleotide a like ammotragus, hence it could be assumed to represent the ancestral state, while y-rrup presents t. from here we propose that one of the haplotypes of the y-pyr group represents the ancestral state. the pattern of microsatellite variation within each haplogroup also supports this interpretation as explained below. first, let us recall that the mutation profiles of microsatellites depend on the size of the repeat motif, the rate of mutation of pentanucleotides is about half of that of trinucleotides, and mononucleotide repeats present a very high mutation rate. our data on pentanucleotide, trinucleotide and mononucleotide difference between r. pyrenatica and r. rupicapra in number of repeats concur with this observation. hence, microsatellites with longer motifs retain a stronger phylogenetic signal than shorter ones. it has been shown that in microsatellites with few repetitions, mutation is biased towards increase in number of repeats. attending to the network of haplotypes, it can be seen that rpyr sampled from r. p. pyrenaica and r. p. parva, presents the lowest number of repeats for all, the penta, the tri and the mononucleotide motifs, this is probably the closest to the basal haplotype. the number of repeats of the pentanucleotide microsatellite umn is variable only in western chamois, the allele with one additional repeat is found in r. p. pyrenaica and r. p. parva. the number of trinucleotide repeats and mononucleotide repeats of srym increases west to east. the mononucleotide motif seems to have suffered mutations forth and back in the eastmost populations. we conclude that the patrilineal dispersion of rupicapra was south-west to north-east. the cline of ancestral to derived chromosomes could be originated by serial funding events, probably related to the glacial-interglacial cycles of the quaternary. male dispersion could start from the iberian peninsula or from italy to the east and the two haplogroups differentiate one to each side of the alps. the lower number of repeats points to haplotype rpyr present in iberia as the closest to the ancestor. alternatively, the haplotype rpyr in the center of the network and differing from rpyr solely in the mononucleotide could be also a good candidate. differences between markers in patterns of variation and the evolutionary history of chamois the discordant patterns of mtdna and autosomal microsatellites that have been described in a previous study indicated the occurrence of hybridization among divergent lineages of chamois in the central area of the distribution. chromosome y data add complexity to this mosaic; the geographical sorting of variation for the different kind of marker is summarized in figure the three kinds of markers have specific evolutionary characteristics that need to be taken into account to explain differing variation patterns. these differences are on mutation rates, selection, effective population size and dispersal. being the y a male-specific chromosome, and given that the mutation rate is higher in males than in females, we would expect higher divergence for y-chromosome than for autosomal microsatellites; our data are contrary to this expectation. positive selection could act on the whole y-chromosome and, given that the y-chromosome does not recombine, it will be selected as a block in a selective sweep with the consequence of homogenization. in addition, the lack of recombination must be responsible for the effect known as muller's ratchet, the random accumulation of deleterious mutations that cannot be removed by recombination. selection against these mutations further reduces variability. selection has been proposed as the main factor to explain low levels of genetic variability in the y-chromosome and must contribute to the reduced variability among y-chromosomes in chamois. a similar pattern was found in ursidae where y-chromosome genes have fewer substitutions than expected in external branches. the chromosome y and mtdna both have effective population size of one quarter of that of an autosome because they are haploid and transmitted by one sex, but the effective population size of y-chromosome must be lower because the variance in number of descendants is larger in males than in females, especially in a polygynous species. hence, the y-chromosome is more prone to genetic drift effects. however, genetic drift cannot be invoked as the main cause for the y-chromosome phylogeography because in such case no such strong geographical signal would be expected. differences in male and female dispersal can be related to different patterns of variation for patrilineal, matrilineal and biparental markers. recent migration of males could be responsible of the low y-chromosome diversity observed. taking into account the above considerations, together with the assumed asiatic origin of rupicapra and the similarity of the y-chromosome of rupicapra and ammotragus, we propose two speculative hypotheses to explain the observed results: a) the rupicapra genus has its origin in southern europe and spread west to east. the differences in divergence between mtdna and y-chromosome are due to the particular evolutionary characteristics of the y-chromosome, or b) males from an ancestral population highly differentiated from a second ancestral taxa present in europe entered from the south, hybridizing and spreading westwards. under the first hypothesis, the origin of the genus rupicapra needs to be placed in south europe from where it spread eastwards. this is contrary to the general belief that chamois, as caprines in general, originated in asia but, following ropiquet and hassanin, this hypothesis is not really supported by the fossil record. these authors propose that caprines originated in the mediterranean islands during the miocene and from here may have invaded africa, europe and asia after the tortonian salinity crisis or after the messinian salinity crisis. the similarity among y-chromosomes under this hypothesis must be attributed to male dispersal and female phylopatry, together with selective sweeps and purifying selection that acted over the chromosome as a whole. the second hypothesis is the hybridization between two ancestral caprinae in the beginning of the evolutionary history of the genus rupicapra. the hybridization between highly divergent lineages at speciation of rupicapra had been previously hypothesized after the observation of a pseudogene of cytochrome b from a highly divergent lineage in the nucleus of present day chamois. an ancestral caprinae related with ammotragus could have reached the south mediterranean, most probably the iberian peninsula, and hybridize with the ancestral rupicapra female. the dispersal of species across the strait of gibraltar during pleistocene has been proposed for other vertebrates and has been related to the substantially lower sea level associated with major pleistocene glaciations. new male y-chromosome lineages would have dispersed during the pleistocene, replacing older ones. hybridization, after secondary contact among related taxa, has been inferred in the evolution of the genus capra as well as in the evolution of some species of bos and has been recently observed between native and introduced species of cervus in scotland. it can be added that hybrids from ammotragus male and capra female have been artificially obtained. many recent studies have shown contrasting phylogenies for different kinds of markers that lead to the view of hybridization as an important mechanism in the evolution of animal species. our results on y-chromosome reconcile the sudden occurrence of rupicapra in the middle-late pleistocene with the existence of very old mtdna lineages. in addition, the proposed migration of chamois west to east can explain the reported more conservative features of r. pyrenaica. the alternative hypotheses presented here can be tested in future research that includes the comparative study of chromosome y in a broader representation of caprinae and additional nuclear sequences of the different populations of rupicapra to search for the signature of possible hybridizations.  the y-chromosome is highly informative to follow the dispersal of populations of caprinae. the phylogenetic analysis of the sry gene promoter shows an association between rupicapra, capra and ammotragus, not in agreement with a previous report, which grouped ammotragus with ovis. the structure of the srym microsatellite of rupicapra is equal to ammotragus and the african breed balami of o. aries. the patrilineal relationships of caprine deserve a reinvestigation. within rupicapra, the diversity of y-chromosomes is very low. the combined data define y-chromosome haplotypes forming a west-to-east geographical cline of ancestral to derived alleles. haplotypes form two haplogroups, which concur with taxonomic classification, instead of the three clades formed for mtdna and biparental microsatellites. we propose that, contrary to the presumed asian origin, the paternal lineage of chamois originated in the mediterranean, most probably in the iberian peninsula, and dispersed eastwards through serial funding events during the glacial-interglacial cycles of the quaternary. the differences in patterns of variation among y-chromosome, mtdna and biparental microsatellites reflect the evolutionary characteristics of the different markers, as well as the effects of sex-biased dispersal and species phylogeography. 
the differences in patterns of variation among y-chromosome, mtdna and biparental microsatellites reflect the evolutionary characteristics of the different markers as well as the effects of sex-biased dispersal and species phylogeography. phylogenetic analysis of the alignment showed the clustering of rupicapra with capra and the ammotragus sequence obtained in this study, different from the previously reported sequence of ammotragus which groups with ovis. we analyzed the y-chromosome of males covering the distribution range of the rupicapra genus. the chamois, distributed over most of the medium to high altitude mountain ranges of southern eurasia, provides an excellent model for exploring the effects of historical and evolutionary events on diversification. within rupicapra, the combined data define y-chromosome haplotypes forming two haplogroups, which concur with taxonomic classification, instead of the three clades formed for mtdna and nuclear microsatellites. populations have been grouped into two species, rupicapra pyrenaica from southwestern europe and r. rupicapra from eastern europe. we sequenced a fragment of the sry gene promoter and characterized the male specific microsatellites umn and srym the sry promoter sequences of two samples of barbary sheep were also determined and compared with the sequences of bovidae available in the genbank. here we investigate the pattern of variation of the y-chromosome to obtain information on the patrilineal phylogenetic position of the genus rupicapra and on the male-specific dispersal of chamois across europe. the study of ancestral and derived characters in the y-chromosome suggests that, contrary to the presumed asian origin, the paternal lineage of chamois originated in the mediterranean, most probably in the iberian peninsula, and dispersed eastwards through serial funding events during the glacial-interglacial cycles of the quaternary. the variation shows a west-to-east geographical cline of ancestral to derived alleles.
 the chamois, distributed over most of the medium to high altitude mountain ranges of southern eurasia, provides an excellent model for exploring the effects of historical and evolutionary events on diversification. populations have been grouped into two species, rupicapra pyrenaica from southwestern europe and r. rupicapra from eastern europe. the study of matrilineal mitochondrial dna and biparentally inherited microsatellites showed that the two species are paraphyletic and indicated alternate events of population contraction and dispersal-hybridization in the diversification of chamois. here we investigate the pattern of variation of the y-chromosome to obtain information on the patrilineal phylogenetic position of the genus rupicapra and on the male-specific dispersal of chamois across europe.  we analyzed the y-chromosome of males covering the distribution range of the rupicapra genus. we sequenced a fragment of the sry gene promoter and characterized the male specific microsatellites umn and srym the sry promoter sequences of two samples of barbary sheep were also determined and compared with the sequences of bovidae available in the genbank. phylogenetic analysis of the alignment showed the clustering of rupicapra with capra and the ammotragus sequence obtained in this study, different from the previously reported sequence of ammotragus which groups with ovis. within rupicapra, the combined data define y-chromosome haplotypes forming two haplogroups, which concur with taxonomic classification, instead of the three clades formed for mtdna and nuclear microsatellites. the variation shows a west-to-east geographical cline of ancestral to derived alleles.  the phylogeny of the sry-promoter shows an association between rupicapra and capra. the position of ammotragus needs a reinvestigation. the study of ancestral and derived characters in the y-chromosome suggests that, contrary to the presumed asian origin, the paternal lineage of chamois originated in the mediterranean, most probably in the iberian peninsula, and dispersed eastwards through serial funding events during the glacial-interglacial cycles of the quaternary. the diversity of y-chromosomes in chamois is very low. the differences in patterns of variation among y-chromosome, mtdna and biparental microsatellites reflect the evolutionary characteristics of the different markers as well as the effects of sex-biased dispersal and species phylogeography. 