BACKGROUND
the domestic goat , which counts a worldwide population of more than  <dig> ,000 specimens and about  <dig> breeds described , is among the “big five” livestock species defined by the food and agriculture organization   <cit>  and is an invaluable source of milk, meat, skin and fiber for poor small holders and shepherds in developing countries and marginal areas  <cit> .
on the basis of bone morphological changes associated with progressive taming  <cit> , zooarchaeology suggests that goat domestication began about  <dig>  years  ago in an area stretching from the high euphrates valleys in southeastern anatolia  to the zagros mountains in central iran  <cit>  and located within the natural distribution of the wild ancestor species, the bezoar capra aegagrus  <cit> .
this event is now considered as the final outcome of a gradual change from hunting to management of wild captive animals  <cit> .
due to their high rusticity and adaptability to harsh environments, goats represented a key resource during the neolithic agricultural revolution and for the human migration waves that spread neolithic culture out of the fertile crescent  <cit> .
previous studies of mitochondrial control-region haplotypes described six highly divergent haplogroups  in domestic goats: a, b, c  <cit> , d  <cit> , f  <cit>  and g  <cit> .
an additional haplogroup, named e, has been described by joshi et al.
<cit>  on the basis of two highly divergent control-region haplotypes.
this was recognized as a sub-clade of haplogroup a when compared to a larger dataset  <cit> .
the reported weak geographical structuring of goat mitochondrial variability was often interpreted as a consequence of the frequent transportation of goats along terrestrial and maritime routes of migration and commerce, probably during the early domestication phases  <cit> .
a subsequent comparison with wild stocks confirmed the presence of all “domestic” haplogroups in the current c. aegagrus populations, which could result from early translocations of animals and/or feralization before the worldwide spread of goats  <cit> .
the haplogroup a is largely predominant  among domestic goats, but rare  in the bezoar and never observed in the iranian zagros mountains.
the probable origin of haplogroup a occurred in eastern anatolia, where it is still present among wild populations, and its presence in eastern iran probably is the result of a subsequent feralization of domestic goats.
the most frequent haplogroup in wild populations is c  detected in most of the bezoar distribution area and more common in southern zagros/central iranian plateau.
the evidence that c control-region haplotypes from pakistan are the farthest from the domestic-related ones  <cit>  disproved the indus valley domestication hypothesis suggested by archaeological remains from mehrgarh   <cit> .
haplogroup f is still found in wild populations , but it is very rare in domestic goats , as it was identified only in three sicilian samples  <cit> .
the other haplogroups were found only in iranian  or in both northern iranian and eastern anatolian bezoars .
it has been proposed that these haplotypes might have entered the domestic goat gene pool either during the early spread of domestic goats, or due to small-scale domestication events.
these findings indicate that the process of goat domestication occurred not only in eastern anatolia, as marked by haplogroup a and supported by zooarchaeological data  <cit> , but possibly also in central iran .
this additional easternmost domestication event has been marked by haplogroup c, although it led only to a small contribution detectable in the mitochondrial gene pool of current domestic goats  and no archaeological substantiations  <cit> .
mtdna haplotypes belonging to haplogroups a and c have been found in ancient goat samples retrieved from an early neolithic site in southern france  <cit> .
the two haplogroups occurred with almost the same frequency among the analyzed bones , suggesting that domestic goat populations were already characterized by the mtdna variants a and c  <cit>  during the first colonization waves that brought neolithic farmers into the mediterranean area about  <dig>  ka ago  <cit> .
haplogroup divergence times calculated on different control-region datasets, usually by employing different calibration points, span from  <dig> to 940 ka , thus largely predating the domestication events .
these data suggest that many sub-haplogroups were already present among the bezoar populations and, therefore, that many lineages were included in the domesticated stocks.
previous studies on livestock species showed that phylogenies based on short mitochondrial sequences can be heavily affected by the confounding effects of homoplasies  and mitochondrial pseudogenes , which blur the real extent of lineage divergences/similarities.
the available goat sequencing data are usually restricted to a few hundreds of control-region base pairs , spanning from np  <dig> to np  <dig>  moreover, several complete goat mitochondrial genomes deposited in genbank are probably chimeric or affected by numts   <cit> , including the previously adopted mtdna goat reference sequence nc_ <dig>  <cit> .
in order to overcome these drawbacks, two recent papers have already extended the analysis to the mtdna coding genes, but either failed to explore the complete mitochondrial molecule  <cit>  or focused only on a new mtdna haplotype  <cit> .
we present the first extensive survey of the entire mitogenome variability based on  <dig> novel complete sequences from domestic goats  and wild relatives .
this analysis allowed us to accurately define those  haplogroups involved in the domestication process, and to provide haplogroup coalescence estimates falling at the interface between epipaleolithic and early neolithic periods and expansion times falling into the neolithic.
RESULTS
the phylogeny of goat mitochondrial genomes
an initial collection of  <dig> mtdna samples from c. aegagrus  and c. hircus  was preliminarily characterized through control-region sequencing .
this dataset, including  <dig> previously published samples  <cit> , was used to build a haplotype network .
overall, the network evidenced a high number of homoplasies and crosslinks, but it was useful to select  <dig> samples  for complete sequencing  using the criterion of including the widest possible range of mtdna variation.
the selected mitogenomes belonged to all known haplogroups, with few notable exceptions: i) the very rare haplogroup f, identified so far only in three domestic goats from sicily  <cit> , was not represented within our initial dataset of domestic goats; ii) the single control-region haplotype a in our c. aegagrus samples was re-classified as c by preliminary sequencing of some informative coding-region segments, which encompass diagnostic single nucleotide polymorphism  markers of a  and c ; iii) lastly, the few g control-region haplotypes were obtained from degraded dna molecules, which allowed only partial coding-region sequencing that most likely confirmed the g affiliation .
the  <dig> novel mitogenomes were compared with the revised goat reference sequence .
various measures of molecular diversity were evaluated on the final dataset of  <dig> mitogenomes, which included  <dig> different haplotypes.
after excluding ambiguous sites and indels , we identified a total number of  <dig> variant sites :  <dig> in the coding region  and  <dig> in the d-loop .
overall we observed an average number of  <dig>  ±  <dig>  nucleotide differences between two randomly chosen sequences.
figure s <dig> illustrates the distributions of nucleotide diversity  and total number of substitutions  along the mitogenome .
as expected, the highest diversity was observed around the hvs-i segment , with a peak of π =  <dig> .
the latter value is higher than those previously reported in horses  <cit> .
within the coding region, the highest number of variant sites was found in protein-coding genes , mostly synonymous mutations.
these data are consistent with previous studies on human and horse mitochondrial genomes .table  <dig> distribution and recurrence of mutations in the  <dig> goat mtdna sequences
length in base pairsa
alengths of protein-coding genes were readjusted and extended by considering the overlapping segments.
regarding the trna loci, the overlapping portions were counted only once
a parsimony approach was applied to infer evolutionary relationships from the final dataset of  <dig> complete mitogenomes.
eventually, only coding-region substitutions were included in the tree  because of the extraordinary control-region variability  and high indels’ instability.
the obtained topology confirmed all previously known control-region branches , but also revealed many different sub-branches, particularly within the major branch a. seven novel sub-branches, named a <dig> to a <dig>  were identified , all marked by coding-region transitions.
in order to convert mutational distances into time over the entire mitogenome, the goat mtdna sequences were compared with a published complete mitogenome  from a comisana sheep   <cit> , used as an outgroup.
diverse maximum likelihood  and bayesian analyses were employed, as described in materials and methods.
initially, an ml tree based on synonymous mutations alone was estimated .
in fact, contrary to the number of potential factors causing time-dependent rates  <cit> , synonymous mutations are virtually neutral and not subject to the effect of purifying selection, even though saturation might still be an issue with long time frames.
using codeml and the mammalian mtdna genetic code, we calibrated the synonymous molecular clock at  <dig>  × 10− <dig> substitutions per year  or  <dig> substitution every 3397 years, after verifying the clock model hypothesis .
this mutation rate was also used to convert into time the rho estimates based on synonymous mutations .
the deepest node corresponds to the single ancestral goat mitogenome , from which all modern goat mtdna sequences derive, and was dated ~460 ka.
the f bezoar mitogenome radiates first in the tree, then a major split  separates haplogroup c  from the remaining mtdna haplotypes.
a subsequent branching separates two sister clades  both dated about 50 ka.
haplogroup b encompasses four samples, including grs and one bezoar.
the remaining wild samples belong to haplogroup d together with two domestic goats from kyrgyzstan ; affiliation to haplogroup d was also confirmed by considering recently published partial coding sequences  <cit> .
the other bezoar mitogenomes, within haplogroups b and c, are ancestral to their most closely related domestic clusters b <dig> and c1a, dated  <dig>  and  <dig>  ka, respectively.
these estimates are very similar to those of the a  and g  clusters, which include only c. hircus mitogenomes.
in summary, all domestic clusters  originated between  <dig> and 9 ka ago, as confirmed by both ml and rho statistics estimates .fig.
<dig> schematic phylogeny of complete mtdnas from modern domestic and wild goats.
the tree encompasses  <dig> sequences and was rooted by using a published sheep  sequence .
“a.g.m.” indicates the reconstructed ancestral goat mitogenome.
the topology was inferred by a maximum parsimony approach, while maximum likelihood  time divergences based on synonymous substitutions are shown below the branches.
the right inset shows the complete a branch rooted in d. further details are given in additional file 1: table s <dig> and figure s2
a.g.m.
b
athe entire genome was partitioned into six datasets: 1st, 2nd and 3rd positions of the codons, rnas, hvs-i and the remainder of the control region
bancestral goat mitogenome
ca 95 % c.i.
for the age of d <dig> is  <dig> to ln/n in units of 4120 years
a bayesian skyline plot  analysis was carried out to assess population expansions.
the overall bsp points to a steep increase of the female effective population size about 12–10 ka ago .
this analysis was performed on the complete molecule after establishing six different partitions .
the same partitions were also considered to perform ml analyses on the entire mitogenome.
the final outcomes were an overall mutational rate of  <dig>  × 10− <dig> substitutions per nucleotide per year  on the entire molecule and  <dig>  × 10− <dig> substitutions per nucleotide per year  on the coding region alone.
both analyses revealed that the molecular clock could not be rejected  when employing the complex gtr/rev model.
a clear sign of purifying selection is apparent when calculating the non-synonymous/synonymous ratio, i.e.
=  <dig>  .
as expected, this ratio is significantly lower  in the deep portion of the tree and the comparison among domestic branches reveals slightly significant differences  ; in particular the ɷ ratio of a is much higher than previously reported   <cit> .
similarly, age values of younger clades are much higher when considering the entire panel of mutations rather than those based on synonymous changes only , probably because the effect of purifying selection is incomplete and all the negatively selectable characters are still included  <cit> .fig.
<dig> bayesian skyline plot showing the goat population size trend with a generation time of  <dig>  years  <cit> .
the y axis indicates the effective number of females.
the thick solid line is the median estimate and the grey shading shows the 95 % highest posterior density limits.
the time axis is limited to 100 ka, beyond that time the curve remains linear
fig.
<dig> comparison of ml and rho-statistics ages relative to the ml synonymous estimates.
see table  <dig> for further details
geographic distributions of goat mtdna haplogroups
an analysis of the geographic distribution of goat haplogroups in eurasia and africa , based on the control-region data currently available in literature or deposited in genbank, confirms an overwhelming predominance of haplogroup a  among domestic goats all over the world .
the second most frequent haplogroup is b , more common in asia and southern africa, but also present in europe.
haplogroup g  is restricted to north-central africa and asia, while the presence of haplogroups c and d is limited to eurasia with an average frequency of  <dig>  % and  <dig>  %, respectively.
finally, haplogroup f was identified only in three sicilian domestic samples.
the bezoar samples analyzed so far are mainly from the middle east  and south asia , where they are mostly characterized by the occurrence of haplogroups c  and f .
c. aegagrus is also represented by haplogroups d , a , b  and g  in the middle east.
wild goats are also found on some mediterranean islands , even though the widely accepted opinion is that they derive from the feralization of very early domestic animals  <cit> .
finally, the few ancient mtdna haplotypes  were found in goat remains excavated in central/east asia , the near east  and europe .
the presence of a and c in the mediterranean area since ancient times could be due to the neolithic spread attested by the first appearance of cardial pottery in the eastern adriatic since  <dig>  ka ago .fig.
<dig> spatial frequency distributions of goat mtdna haplogroups in different geographic areas based on different datasets: modern breeds  a
; wild goats  b; and ancient goat remains c. see additional file  <dig>  for more information
discussion and
CONCLUSIONS
domestication of c. aegagrus
wild goats were widely distributed throughout southwestern eurasia during the middle paleolithic .
in an attempt to reconstruct past demographic histories based on phylogenetic inferences, the tree in fig.
<dig> suggests that a drastic bottleneck occurred during the late eurasian saalian glacial  , which is compatible with the age of the major macro-haplogroup a’b’c’d’g.
during the following riss-würm interglacial relapse , the a’b’d’g lineages evolved and expanded independently from the c-derived populations.
many lineages did not survive the following würm glacial period  and the drastic climatic changes during the last glacial maximum , but some of them, corresponding to the principal goat haplogroups, were preserved in the near east refuge areas  <cit> , survived the severe drop in temperature known as the younger dryas   <cit> , and provided the necessary substrate of variability for later goat domestication and management in an area from the zagros mountains to southeastern anatolia, as testified by the abundance of goat remains in neolithic sites from that regions  <cit> .
the main goat haplogroups were identified through control-region analyses following an approach similar to that adopted for other domesticated species, such as sheep .
however, in the case of both horses and bovines, the mtdna haplogroup classification received a major makeover from the exploration of the entire mitogenome variability .
a common feature of all livestock phylogenies is that the control-region molecular clocks turned out to be very inadequate for an accurate dating of mitochondrial lineage divergences.
when mtdna protein-coding genes were considered  <cit> , goat haplogroups were dated to the middle paleolithic, thus suggesting that multiple related lineages were domesticated ~11 ka ago in an area spanning from southeastern anatolia in turkey to zagros mountains in iran, by incorporating pre-existing variation .
this domestication center, further confirmed by zooarchaeological data  <cit> , left a clear signature in the mtdna gene pool of current domestic breeds, as attested by the large predominance of a haplotypes.
our analyses allowed us to assess the coding-region variability within this clade  and to date for the first time this major goat haplogroup to the epipaleolithic period; a few a sub-haplogroups were also phylogenetically defined and dated to the early neolithic.
the implication is that more than 90 % of current domestic goats might descend from a single foundress, represented by the internal node a in our phylogeny.
obviously, the absence of a bezoars in our dataset is an issue, as well as the long-term calibration point, which might represent an underestimate of the true sheep/goat divergence.
thus, by also considering the alternative hypothesis of many founder lineages within haplogroup a independently proposed by naderi et al.
<cit>  and nomura et al.
<cit> , we might suggest that at least seven of them have been identified in our dataset as represented by the ancestral mitogenomes of sub-haplogroups a1–a <dig>
moreover, in some instances, our phylogenetic reconstruction clearly shows that other domestic clusters are nested within wild branches and each of them descends from a unique wild haplotype.
therefore, excluding the rare but possible occurrence of stochastic backcrossing between wild and domestic animals, the first domestication process probably involved only four additional female goat populations corresponding to one founder haplotype for each of the other domestic lineages, i.e.
b <dig>  d <dig>  c1a and g,.
we cannot exclude that, when analyzing further c. aegagrus samples at the maximum level of resolution, several additional sub-groups could be identified within each haplogroup, as previously discussed for the a lineages.
likewise, the possibility that some of the mitochondrial haplotypes belonging to lineages b <dig>  d <dig> and c1a and presently found in domestic animals might derive from introgression of wild lineages cannot be completely ruled out.
yet, events of introgression from wild animals into domestic herds are incidental and usually male-mediated  <cit> , and they were not identified so far even when analyzing a larger dataset of wild and domestic control-region sequences  <cit> .
we have also verified that all available control-region haplotypes  belonging to haplogroups b, c and d could be specifically assigned either to wild or domestic branches as expected from individual phenotypes , without finding any notable exceptions.
the bayesian analysis of our goat sequences also shows that, after domestication, the early domestic populations soon experienced a drastic demographic expansion about 12–10 ka ago .
however, since our tree includes samples from a very wide geographical area, we cannot exclude that additional and perhaps more recent signals of local demographic expansions could be detected by analyzing larger and more geographically structured datasets, particularly along the domestication routes.
a more fascinating scenario could also be envisioned for haplogroup c since this haplogroup might represent a marker of a concomitant secondary domestication, as already suggested by control-region analyses  <cit> .
to support this scenario, our age estimate for c1a is compatible with the first domestication wave and, more importantly, the c clade represents an independent branch in our tree, which is separated from the a’b’d’g cluster by over one hundred thousand years, thus suggesting a genetically distinct population origin.
the prevalence of haplogroup a: a close similarity between cattle and goat domestication
phylogenetic analyses based on entire mitogenomes revealed that, similarly to horses and taurine cattle, the ancient goat populations might have passed through a severe bottleneck during the late saalian glacial maximum with a single sequence  from that period as the common ancestor of most goat mitogenomes.
however, the recent goat evolutionary history suggests more similarities with bos primigenius domestication .
present-day cattle and goat mitogenomes belong to very few haplogroups, with most of them  falling within a single branch , whose sequence coalescence time corresponds to a very similar time estimate of about 11–13 ka.
as in the case of the cattle t <dig> branch, this study and other published data support a neolithic origin for all goat a mitogenomes  from a middle eastern bezoar population.
at least four other minor lineages  directly related to bezoar ancestors  were domesticated.
however, unlike the bovine minor clades p and q that suggest a possible european domestication or introgression, the geographical distribution of the low-frequency goat clusters indicates that secondary domestication foci were located in the same area between anatolian turkey and iran.
this leaves open the possibility of a more eastward iranian center involving the furthest phylogenetically-related haplotype c <dig>  moreover, the new coding-region diagnostic mutational motifs defined in the present study  could be employed to revise phylogenetic relationships between modern breeds and ancient remains with the overall objective of testing the proposed scenario of secondary early domestication processes that took place before the occurrence of morphological modifications, and evaluating whether selection differentially affected mitogenome variants during the development of economically important breeds.

 the domestic goat, which counts a worldwide population of more than,000 specimens and about breeds described, is among the “big five” livestock species defined by the food and agriculture organization and is an invaluable source of milk, meat, skin and fiber for poor small holders and shepherds in developing countries and marginal areas. on the basis of bone morphological changes associated with progressive taming, zooarchaeology suggests that goat domestication began about years ago in an area stretching from the high euphrates valleys in southeastern anatolia to the zagros mountains in central iran and located within the natural distribution of the wild ancestor species, the bezoar capra aegagrus. this event is now considered as the final outcome of a gradual change from hunting to management of wild captive animals. due to their high rusticity and adaptability to harsh environments, goats represented a key resource during the neolithic agricultural revolution and for the human migration waves that spread neolithic culture out of the fertile crescent. previous studies of mitochondrial control-region haplotypes described six highly divergent haplogroups in domestic goats: a, b, c, d, f and g. an additional haplogroup, named e, has been described by joshi et al. on the basis of two highly divergent control-region haplotypes. this was recognized as a sub-clade of haplogroup a when compared to a larger dataset. the reported weak geographical structuring of goat mitochondrial variability was often interpreted as a consequence of the frequent transportation of goats along terrestrial and maritime routes of migration and commerce, probably during the early domestication phases. a subsequent comparison with wild stocks confirmed the presence of all “domestic” haplogroups in the current c. aegagrus populations, which could result from early translocations of animals and/or feralization before the worldwide spread of goats. the haplogroup a is largely predominant among domestic goats, but rare in the bezoar and never observed in the iranian zagros mountains. the probable origin of haplogroup a occurred in eastern anatolia, where it is still present among wild populations, and its presence in eastern iran probably is the result of a subsequent feralization of domestic goats. the most frequent haplogroup in wild populations is c detected in most of the bezoar distribution area and more common in southern zagros/central iranian plateau. the evidence that c control-region haplotypes from pakistan are the farthest from the domestic-related ones disproved the indus valley domestication hypothesis suggested by archaeological remains from mehrgarh. haplogroup f is still found in wild populations, but it is very rare in domestic goats, as it was identified only in three sicilian samples. the other haplogroups were found only in iranian or in both northern iranian and eastern anatolian bezoars. it has been proposed that these haplotypes might have entered the domestic goat gene pool either during the early spread of domestic goats, or due to small-scale domestication events. these findings indicate that the process of goat domestication occurred not only in eastern anatolia, as marked by haplogroup a and supported by zooarchaeological data, but possibly also in central iran. this additional easternmost domestication event has been marked by haplogroup c, although it led only to a small contribution detectable in the mitochondrial gene pool of current domestic goats and no archaeological substantiations. mtdna haplotypes belonging to haplogroups a and c have been found in ancient goat samples retrieved from an early neolithic site in southern france. the two haplogroups occurred with almost the same frequency among the analyzed bones, suggesting that domestic goat populations were already characterized by the mtdna variants a and c during the first colonization waves that brought neolithic farmers into the mediterranean area about  ka ago. haplogroup divergence times calculated on different control-region datasets, usually by employing different calibration points, span from to 940 ka, thus largely predating the domestication events. these data suggest that many sub-haplogroups were already present among the bezoar populations and, therefore, that many lineages were included in the domesticated stocks. previous studies on livestock species showed that phylogenies based on short mitochondrial sequences can be heavily affected by the confounding effects of homoplasies and mitochondrial pseudogenes, which blur the real extent of lineage divergences/similarities. the available goat sequencing data are usually restricted to a few hundreds of control-region base pairs, spanning from np to np moreover, several complete goat mitochondrial genomes deposited in genbank are probably chimeric or affected by numts, including the previously adopted mtdna goat reference sequence nc_. in order to overcome these drawbacks, two recent papers have already extended the analysis to the mtdna coding genes, but either failed to explore the complete mitochondrial molecule or focused only on a new mtdna haplotype. we present the first extensive survey of the entire mitogenome variability based on novel complete sequences from domestic goats and wild relatives. this analysis allowed us to accurately define those haplogroups involved in the domestication process, and to provide haplogroup coalescence estimates falling at the interface between epipaleolithic and early neolithic periods and expansion times falling into the neolithic.  the phylogeny of goat mitochondrial genomes an initial collection of mtdna samples from c. aegagrus and c. hircus was preliminarily characterized through control-region sequencing. this dataset, including previously published samples, was used to build a haplotype network. overall, the network evidenced a high number of homoplasies and crosslinks, but it was useful to select samples for complete sequencing using the criterion of including the widest possible range of mtdna variation. the selected mitogenomes belonged to all known haplogroups, with few notable exceptions: i) the very rare haplogroup f, identified so far only in three domestic goats from sicily, was not represented within our initial dataset of domestic goats; ii) the single control-region haplotype a in our c. aegagrus samples was re-classified as c by preliminary sequencing of some informative coding-region segments, which encompass diagnostic single nucleotide polymorphism markers of a and c; iii) lastly, the few g control-region haplotypes were obtained from degraded dna molecules, which allowed only partial coding-region sequencing that most likely confirmed the g affiliation. the novel mitogenomes were compared with the revised goat reference sequence. various measures of molecular diversity were evaluated on the final dataset of mitogenomes, which included different haplotypes. after excluding ambiguous sites and indels, we identified a total number of variant sites: in the coding region and in the d-loop. overall we observed an average number of  ±  nucleotide differences between two randomly chosen sequences. figure s illustrates the distributions of nucleotide diversity and total number of substitutions along the mitogenome. as expected, the highest diversity was observed around the hvs-i segment, with a peak of π = . the latter value is higher than those previously reported in horses. within the coding region, the highest number of variant sites was found in protein-coding genes, mostly synonymous mutations. these data are consistent with previous studies on human and horse mitochondrial genomes.table distribution and recurrence of mutations in the goat mtdna sequences length in base pairsa alengths of protein-coding genes were readjusted and extended by considering the overlapping segments. regarding the trna loci, the overlapping portions were counted only once a parsimony approach was applied to infer evolutionary relationships from the final dataset of complete mitogenomes. eventually, only coding-region substitutions were included in the tree because of the extraordinary control-region variability and high indels’ instability. the obtained topology confirmed all previously known control-region branches, but also revealed many different sub-branches, particularly within the major branch a. seven novel sub-branches, named a to a were identified, all marked by coding-region transitions. in order to convert mutational distances into time over the entire mitogenome, the goat mtdna sequences were compared with a published complete mitogenome from a comisana sheep, used as an outgroup. diverse maximum likelihood and bayesian analyses were employed, as described in materials and methods. initially, an ml tree based on synonymous mutations alone was estimated. in fact, contrary to the number of potential factors causing time-dependent rates, synonymous mutations are virtually neutral and not subject to the effect of purifying selection, even though saturation might still be an issue with long time frames. using codeml and the mammalian mtdna genetic code, we calibrated the synonymous molecular clock at  × 10− substitutions per year or substitution every 3397 years, after verifying the clock model hypothesis. this mutation rate was also used to convert into time the rho estimates based on synonymous mutations. the deepest node corresponds to the single ancestral goat mitogenome, from which all modern goat mtdna sequences derive, and was dated ~460 ka. the f bezoar mitogenome radiates first in the tree, then a major split separates haplogroup c from the remaining mtdna haplotypes. a subsequent branching separates two sister clades both dated about 50 ka. haplogroup b encompasses four samples, including grs and one bezoar. the remaining wild samples belong to haplogroup d together with two domestic goats from kyrgyzstan; affiliation to haplogroup d was also confirmed by considering recently published partial coding sequences. the other bezoar mitogenomes, within haplogroups b and c, are ancestral to their most closely related domestic clusters b and c1a, dated and  ka, respectively. these estimates are very similar to those of the a and g clusters, which include only c. hircus mitogenomes. in summary, all domestic clusters originated between and 9 ka ago, as confirmed by both ml and rho statistics estimates.fig. schematic phylogeny of complete mtdnas from modern domestic and wild goats. the tree encompasses sequences and was rooted by using a published sheep sequence. “a.g.m.” indicates the reconstructed ancestral goat mitogenome. the topology was inferred by a maximum parsimony approach, while maximum likelihood time divergences based on synonymous substitutions are shown below the branches. the right inset shows the complete a branch rooted in d. further details are given in additional file 1: table s and figure s2 a.g.m. b athe entire genome was partitioned into six datasets: 1st, 2nd and 3rd positions of the codons, rnas, hvs-i and the remainder of the control region bancestral goat mitogenome ca 95 % c.i. for the age of d is to ln/n in units of 4120 years a bayesian skyline plot analysis was carried out to assess population expansions. the overall bsp points to a steep increase of the female effective population size about 12–10 ka ago. this analysis was performed on the complete molecule after establishing six different partitions. the same partitions were also considered to perform ml analyses on the entire mitogenome. the final outcomes were an overall mutational rate of  × 10− substitutions per nucleotide per year on the entire molecule and  × 10− substitutions per nucleotide per year on the coding region alone. both analyses revealed that the molecular clock could not be rejected when employing the complex gtr/rev model. a clear sign of purifying selection is apparent when calculating the non-synonymous/synonymous ratio, i.e.  = . as expected, this ratio is significantly lower in the deep portion of the tree and the comparison among domestic branches reveals slightly significant differences; in particular the ɷ ratio of a is much higher than previously reported. similarly, age values of younger clades are much higher when considering the entire panel of mutations rather than those based on synonymous changes only, probably because the effect of purifying selection is incomplete and all the negatively selectable characters are still included.fig. bayesian skyline plot showing the goat population size trend with a generation time of  years. the y axis indicates the effective number of females. the thick solid line is the median estimate and the grey shading shows the 95 % highest posterior density limits. the time axis is limited to 100 ka, beyond that time the curve remains linear fig. comparison of ml and rho-statistics ages relative to the ml synonymous estimates. see table  for further details geographic distributions of goat mtdna haplogroups an analysis of the geographic distribution of goat haplogroups in eurasia and africa, based on the control-region data currently available in literature or deposited in genbank, confirms an overwhelming predominance of haplogroup a among domestic goats all over the world. the second most frequent haplogroup is b, more common in asia and southern africa, but also present in europe. haplogroup g is restricted to north-central africa and asia, while the presence of haplogroups c and d is limited to eurasia with an average frequency of  % and  %, respectively. finally, haplogroup f was identified only in three sicilian domestic samples. the bezoar samples analyzed so far are mainly from the middle east and south asia, where they are mostly characterized by the occurrence of haplogroups c and f. c. aegagrus is also represented by haplogroups d, a, b and g in the middle east. wild goats are also found on some mediterranean islands, even though the widely accepted opinion is that they derive from the feralization of very early domestic animals. finally, the few ancient mtdna haplotypes were found in goat remains excavated in central/east asia, the near east and europe. the presence of a and c in the mediterranean area since ancient times could be due to the neolithic spread attested by the first appearance of cardial pottery in the eastern adriatic since  ka ago.fig. spatial frequency distributions of goat mtdna haplogroups in different geographic areas based on different datasets: modern breeds a; wild goats b; and ancient goat remains c. see additional file for more information discussion and  domestication of c. aegagrus wild goats were widely distributed throughout southwestern eurasia during the middle paleolithic. in an attempt to reconstruct past demographic histories based on phylogenetic inferences, the tree in fig.  suggests that a drastic bottleneck occurred during the late eurasian saalian glacial, which is compatible with the age of the major macro-haplogroup a’b’c’d’g. during the following riss-würm interglacial relapse, the a’b’d’g lineages evolved and expanded independently from the c-derived populations. many lineages did not survive the following würm glacial period and the drastic climatic changes during the last glacial maximum, but some of them, corresponding to the principal goat haplogroups, were preserved in the near east refuge areas, survived the severe drop in temperature known as the younger dryas, and provided the necessary substrate of variability for later goat domestication and management in an area from the zagros mountains to southeastern anatolia, as testified by the abundance of goat remains in neolithic sites from that regions. the main goat haplogroups were identified through control-region analyses following an approach similar to that adopted for other domesticated species, such as sheep. however, in the case of both horses and bovines, the mtdna haplogroup classification received a major makeover from the exploration of the entire mitogenome variability. a common feature of all livestock phylogenies is that the control-region molecular clocks turned out to be very inadequate for an accurate dating of mitochondrial lineage divergences. when mtdna protein-coding genes were considered, goat haplogroups were dated to the middle paleolithic, thus suggesting that multiple related lineages were domesticated ~11 ka ago in an area spanning from southeastern anatolia in turkey to zagros mountains in iran, by incorporating pre-existing variation. this domestication center, further confirmed by zooarchaeological data, left a clear signature in the mtdna gene pool of current domestic breeds, as attested by the large predominance of a haplotypes. our analyses allowed us to assess the coding-region variability within this clade and to date for the first time this major goat haplogroup to the epipaleolithic period; a few a sub-haplogroups were also phylogenetically defined and dated to the early neolithic. the implication is that more than 90 % of current domestic goats might descend from a single foundress, represented by the internal node a in our phylogeny. obviously, the absence of a bezoars in our dataset is an issue, as well as the long-term calibration point, which might represent an underestimate of the true sheep/goat divergence. thus, by also considering the alternative hypothesis of many founder lineages within haplogroup a independently proposed by naderi et al. and nomura et al., we might suggest that at least seven of them have been identified in our dataset as represented by the ancestral mitogenomes of sub-haplogroups a1–a moreover, in some instances, our phylogenetic reconstruction clearly shows that other domestic clusters are nested within wild branches and each of them descends from a unique wild haplotype. therefore, excluding the rare but possible occurrence of stochastic backcrossing between wild and domestic animals, the first domestication process probably involved only four additional female goat populations corresponding to one founder haplotype for each of the other domestic lineages, i.e. b d c1a and g,. we cannot exclude that, when analyzing further c. aegagrus samples at the maximum level of resolution, several additional sub-groups could be identified within each haplogroup, as previously discussed for the a lineages. likewise, the possibility that some of the mitochondrial haplotypes belonging to lineages b d and c1a and presently found in domestic animals might derive from introgression of wild lineages cannot be completely ruled out. yet, events of introgression from wild animals into domestic herds are incidental and usually male-mediated, and they were not identified so far even when analyzing a larger dataset of wild and domestic control-region sequences. we have also verified that all available control-region haplotypes belonging to haplogroups b, c and d could be specifically assigned either to wild or domestic branches as expected from individual phenotypes, without finding any notable exceptions. the bayesian analysis of our goat sequences also shows that, after domestication, the early domestic populations soon experienced a drastic demographic expansion about 12–10 ka ago. however, since our tree includes samples from a very wide geographical area, we cannot exclude that additional and perhaps more recent signals of local demographic expansions could be detected by analyzing larger and more geographically structured datasets, particularly along the domestication routes. a more fascinating scenario could also be envisioned for haplogroup c since this haplogroup might represent a marker of a concomitant secondary domestication, as already suggested by control-region analyses. to support this scenario, our age estimate for c1a is compatible with the first domestication wave and, more importantly, the c clade represents an independent branch in our tree, which is separated from the a’b’d’g cluster by over one hundred thousand years, thus suggesting a genetically distinct population origin. the prevalence of haplogroup a: a close similarity between cattle and goat domestication phylogenetic analyses based on entire mitogenomes revealed that, similarly to horses and taurine cattle, the ancient goat populations might have passed through a severe bottleneck during the late saalian glacial maximum with a single sequence from that period as the common ancestor of most goat mitogenomes. however, the recent goat evolutionary history suggests more similarities with bos primigenius domestication. present-day cattle and goat mitogenomes belong to very few haplogroups, with most of them falling within a single branch, whose sequence coalescence time corresponds to a very similar time estimate of about 11–13 ka. as in the case of the cattle t branch, this study and other published data support a neolithic origin for all goat a mitogenomes from a middle eastern bezoar population. at least four other minor lineages directly related to bezoar ancestors were domesticated. however, unlike the bovine minor clades p and q that suggest a possible european domestication or introgression, the geographical distribution of the low-frequency goat clusters indicates that secondary domestication foci were located in the same area between anatolian turkey and iran. this leaves open the possibility of a more eastward iranian center involving the furthest phylogenetically-related haplotype c moreover, the new coding-region diagnostic mutational motifs defined in the present study could be employed to revise phylogenetic relationships between modern breeds and ancient remains with the overall objective of testing the proposed scenario of secondary early domestication processes that took place before the occurrence of morphological modifications, and evaluating whether selection differentially affected mitogenome variants during the development of economically important breeds. 
the current extensive use of the domestic goat is the result of its medium size and high adaptability as multiple breeds. domestic goat mitogenomes descended from a small number of founding haplotypes that underwent domestication after surviving the last glacial maximum in the near eastern refuges. we present the first extensive survey of goat mitogenomic variability based on complete sequences selected from an initial collection of samples that represent different breeds of c. hircus, as well as its wild sister species, bezoar from iran. a common standard by which to analyze maternally-inherited variability of livestock species is through complete sequencing of the entire mitogenome. zooarchaelogical data indicate that domestication first occurred in southeastern anatolia. more than 90 % of goats examined were in haplogroup a. these domestic lineages are predominantly nested within c. aegagrus branches, diverged concomitantly at the interface between the epipaleolithic and early neolithic periods, and underwent a dramatic expansion starting from ~12–10 ka ago. our phylogenetic analyses dated the most recent common ancestor of c. hircus to ~ years ago and identified five distinctive domestic haplogroups. the novel diagnostic mutational motifs defined here, which distinguish wild and domestic haplogroups, could be used to understand phylogenetic relationships among modern breeds and ancient remains and to evaluate whether selection differentially affected mitochondrial genome variants during the development of economically important breeds.
 the current extensive use of the domestic goat is the result of its medium size and high adaptability as multiple breeds. the extent to which its genetic variability was influenced by early domestication practices is largely unknown. a common standard by which to analyze maternally-inherited variability of livestock species is through complete sequencing of the entire mitogenome.  we present the first extensive survey of goat mitogenomic variability based on complete sequences selected from an initial collection of samples that represent different breeds of c. hircus, as well as its wild sister species, bezoar from iran. our phylogenetic analyses dated the most recent common ancestor of c. hircus to ~ years ago and identified five distinctive domestic haplogroups. more than 90 % of goats examined were in haplogroup a. these domestic lineages are predominantly nested within c. aegagrus branches, diverged concomitantly at the interface between the epipaleolithic and early neolithic periods, and underwent a dramatic expansion starting from ~12–10 ka ago.  domestic goat mitogenomes descended from a small number of founding haplotypes that underwent domestication after surviving the last glacial maximum in the near eastern refuges. all modern haplotypes a probably descended from a single female c. aegagrus. zooarchaelogical data indicate that domestication first occurred in southeastern anatolia. goats accompanying the first neolithic migration waves into the mediterranean were already characterized by two ancestral a and c variants. the ancient separation of the c branch suggests a genetically distinct population that could have been involved in a second event of domestication. the novel diagnostic mutational motifs defined here, which distinguish wild and domestic haplogroups, could be used to understand phylogenetic relationships among modern breeds and ancient remains and to evaluate whether selection differentially affected mitochondrial genome variants during the development of economically important breeds. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords goat mitochondrial genomemtdna haplogroupsdomesticationorigin of capra hircuscapra aegagrusmiur: progetti firb-futuro in ricerca 2008rbfr08u07machilli alessandro miur: progetti firb-futuro in ricerca 2012rbfr126b8iolivieri anna miur: prin-progetti ricerca di interesse nazionale 20122012ja4bty_002achilli alessandro miur: prin-progetti ricerca di interesse nazionale 20092009p2cnkk_005achilli alessandro miur: prin-progetti ricerca di interesse nazionale 20072007xrbaenajmone marsan paolo eu fp project nextgenkbbe-2009-1-1-03issue-copyright-statement© the author 2015 