BACKGROUND
gibbons, family hylobatidae, are small arboreal apes, which inhabit tropical and subtropical rainforests of southeast asia and adjacent regions .
together with humans and great apes, they belong to the primate superfamily hominoidea  <cit> .
among hominoids, gibbons were the first to branch off and they display a set of morphological and behavioural characteristics distinctly different from great apes and humans  <cit> .
most prominent in this respect is the predominantly monogamous life style, their territorial calls, and the typical brachiating locomotion  <cit> .
due to their extensive karyotypic diversity  <cit> , gibbons provide an excellent model organism to study chromosomal rearrangements and, hence, to better understand human diseases caused by such alterations.
although in several aspects unique among primates and with up to  <dig> species the most diverse group of apes, gibbons are still in the shadow of great apes in respect of scientific studies, conservation efforts and public awareness.
however, many gibbon species are on the brink of extinction and most of them are classified as "endangered" or even "critically endangered"  <cit> .
with approximately  <dig> individuals left in its native habitat, the hainan gibbon  is the rarest primate in the world  <cit> .
responsible for this critical situation is habitat loss and hunting, which both have seriously reduced gibbon populations throughout their range  <cit> .
hence, much more attention has to be drawn on the gibbons' situation and extensive conservation actions are urgently required to save them from extinction  <cit> .
while gibbons are widely considered to form a monophyletic clade, there is no consensus about the phylogeny and taxonomy within the family.
although various studies based on morphology, behaviour, vocalisation, protein electrophoresis, karyotyping and dna sequencing were conducted  <cit> , neither a congruent phylogeny nor a consistent taxonomic classification was obtained.
moreover, incomplete taxon sampling as well as misidentified specimens resulted in only fragmentary or even false conclusions.
accordingly, the classification of gibbon taxa at various taxonomic levels as well as their phylogenetic relationships remain disputed and a consensus is far from being available.
for example, in early studies, small apes were divided into two genera, with one  including the siamang, and the other  all the remaining species  <cit> .
later on, the family was split into four major clades, which were recognized as subgenera  <cit>  and eventually as genera  <cit> .
this division is now widely accepted and takes into account the fact that species within each of the four major clades share a number of characteristics, most importantly a distinctive diploid chromosome number: hoolock , hylobates , symphalangus  and nomascus   <cit> .
similarly, the number of species and subspecies is a matter of debate as well.
while symphalangus is consistently regarded as monotypic, the two hoolock subspecies were recently elevated to species  <cit> .
in nomascus originally only one species was recognized  <cit> , but in current classifications four to six species were suggested  <cit> .
in contrast, the genus hylobates already comprised at least four species in early classifications  <cit> , but recent studies proposed six or seven species  <cit> .
due to this incongruence we follow the most recent classification of the iucn red list  <cit>  with a total of  <dig> gibbon species .
*further research required
in the present study, we analyse the complete mitochondrial cytochrome b  gene from  <dig> individuals, which represent all gibbon genera and species, and most subspecies.
based on our data, we are able to 1) provide the most complete phylogeny of gibbons on all taxonomic levels, 2) estimate divergence times between lineages, 3) establish a reliable classification, 4) elucidate gibbon phylogeography, and 5) provide a tool for the species identification of gibbon individuals.
RESULTS
from all  <dig> gibbons, we successfully generated sequences of the complete mitochondrial cytb gene .
a contamination of our dataset with nuclear pseudogenes  can be regarded as minimal, because no multiple amplifications of different copies were detected by direct sequencing.
all sequences were correctly transcribed, and identical sequences were obtained for the same individual in cases where different material types were available.
moreover, no inconsistent positions were detected in alignments, which were assembled from overlapping sequences.
cross-contamination between individuals can be excluded as well, since all negative controls revealed no amplifications and randomly repeated pcrs for the same individual produced identical sequences.
among the  <dig> individual gibbons studied, no identical haplotypes were detected.
the cytb alignment comprising solely gibbons was characterized by  <dig> variable sites, of which  <dig> were parsimony-informative.
in the complete alignment, which additionally contained great ape, human and hamadryas baboon representatives, we observed  <dig> variable sites, of which  <dig> were parsimony-informative.
phylogenetic tree reconstructions based on maximum-parsimony , neighbor-joining , maximum-likelihood  and bayesian algorithms revealed various strongly supported clades, which corresponded to genera, species and subspecies .
all algorithms led to identical tree topologies, although several branching patterns gained only weak support.
according to our reconstructions, hominoids diverged into a clade consisting of gibbons, and another with great apes and human.
among the latter, pongo split off first, followed by gorilla, before finally pan and homo diverged.
within gibbons, a basal position of nomascus and a sister grouping of hylobates and hoolock was indicated, but support for this branching pattern was relatively low .
similarly, with the exception of a strongly supported h. agilis + h. albibarbis clade, also the relationships among the species of hylobates were not well resolved.
however, at least species monophylies were clearly confirmed, though a common origin of h. agilis was only weakly supported.
the relationships among the subspecies of h. muelleri and h. lar were less resolved.
in hoolock, the two species h. hoolock and h. leuconedys clearly segregated into two distinct clades.
within nomascus, relationships among species were completely resolved, suggesting a n. hainanus + n. nasutus clade as sister lineage to the remaining species.
among them, n. concolor branched off first, followed by the divergence of n. gabriellae and n. leucogenys/n.
siki.
the monophyly of n. leucogenys was significantly supported, but evidence for a common origin of n. siki individuals was not obtained.
within n. concolor, specimens identified as n. concolor lu formed a distinct clade, while the remaining subspecies clustered together without further subdivision.
however, support for a reciprocal monophyly of both clades was relatively low.
*means and 95% credibility intervals  are given for  <dig> nodes .
nodes used as calibrations are labelled with a "c", all others with an "n".
mrca denotes the most recent common ancestor.
c <dig> not shown in figure  <dig>  **support values as obtained from mp, nj, ml and bayesian reconstructions, respectively.
based on divergence age estimates, gibbons separated from great apes and humans  <dig>  million years ago  .
within hominids, pongo branched off first , followed by gorilla , before finally homo and pan diverged from each other .
the differentiation of pongo and pan into species occurred  <dig>  and  <dig>  mya, respectively.
in an initial radiation, gibbons diverged within a relative short time period of only  <dig>  million years  into four genera.
within hylobates, most species diverged from each other between  <dig>  and  <dig>  mya.
the only exception was the separation of h. albibarbis from h. agilis  <dig>  mya, which was in the time frame of subspecies splits within h. muelleri .
differentiation of h. lar into subspecies occurred even later .
the two hoolock species diverged  <dig>  mya from each other.
in nomascus, differentiation into species took place over a longer time period, lasting from  <dig>  until  <dig>  mya.
the most recent species divergence within nomascus occurred between n. siki and n. leucogenys , which was in a similar range as the separation of n. concolor lu from the other n. concolor subspecies .
discussion
by analysing all species and most subspecies, the present study provides the most complete view into the evolutionary history of the gibbon family.
however, as in earlier molecular studies on gibbons  <cit> , relationships on various taxonomic levels are less resolved and partially contradict earlier findings.
while the herein depicted branching pattern among genera is identical with that found in earlier studies using also cytb  <cit>  or d-loop  <cit>  sequences, it differs from another cytb-based study  <cit>  in placing nomascus and not symphalangus as most basal genus.
studies based on mitochondrial nd3-nd <dig> sequences  <cit>  or chromosomal rearrangements  <cit>  suggest hoolock as most ancestral lineage, and nomascus together with either hylobates  <cit>  or symphalangus  <cit>  as the most recently diverged genera.
for hylobates, our data indicate a basal position of h. klossii, and a further division into a clade consisting of h. lar, h. muelleri, h. agilis and h. albibarbis, and another one with h. moloch and h. pileatus.
various branching patterns among hylobates species are proposed  <cit> , which all differ from our one, but respective support values are similarly low as in our study.
in contrast, the relationships found among species of the genus nomascus are well resolved and identical with that suggested by  <cit> .
according to our and earlier data, relationships among gibbon genera and hylobates species remain disputed, which most likely can be explained by the separation of respective lineages within relative short time periods.
this becomes even more obvious when considering estimated divergence ages, which fall into four temporal windows.
in the first, between ~ <dig>  and ~ <dig>  mya, the four gibbon genera originated.
in a second radiation, between ~ <dig>  and ~ <dig>  mya, hylobates split into various species, and in a third burst, between ~ <dig>  and ~ <dig>  mya, h. muelleri, the h. agilis + h. albibarbis clade and hoolock further differentiated.
finally, in a fourth radiation, between ~ <dig>  and ~ <dig>  mya, h. lar diverged into subspecies.
in contrast, speciation in nomascus was a continuous process, lasting from  <dig>  until  <dig>  mya.
taxonomic implications
our data show that mitochondrial dna  provides a powerful tool for the identification and taxonomic classification of gibbons, because taxa form strongly supported monophyletic clades, or at least appear to form distinct lineages in those cases where only one individual per taxon was tested.
moreover, most differentiation events fall into four temporal periods, which allow a hierarchical ranking as proposed by goodman et al.
<cit> , though the threshold for the recognition of a certain taxonomic unit whether genus, species, or subspecies remains disputed.
hence, to provide a more reliable classification, we compare divergence ages among gibbon lineages with those among other asian primates and hominids.
accordingly and concordant with recent classifications  <cit> , the four major gibbon lineages are proposed as distinct genera , since they split from each other in a similar time range as did colobine genera  or african great apes and human  <cit> .
most species of hylobates and nomascus emerged in or around the second radiation, which is on the same time scale as species splits within pongo and pan, and the separation of species groups within macaca  <cit>  and trachypithecus  <cit> .
thus, taxa originating in this time period should be recognized as distinct species , and might be even classified as species groups.
further differentiation events among gibbons occurred in the third time period, which is in a similar window as several speciation events within macaques  <cit> .
accordingly, h. leuconedys and h. albibarbis should be separated from h. hoolock and h. agilis on species level, respectively, and the three subspecies of h. muelleri could be considered for elevation to species level.
moreover, h. agilis is divided into two clades, which refer to individuals identified by pelage coloration as h. agilis agilis and h. agilis unko.
however, in a recent work based on a larger number of individuals a reciprocal monophyly of both lineages is doubted  <cit> , and, hence, we provisionally recognize h. agilis as monotypic.
for h. lar, only a few unambiguously identified specimens were available for our study, but these represent at least four of the five recognized subspecies, while the identity of the putative h. lar yunnanensis individual remains uncertain.
based on our data, h. lar subspecies form distinct lineages, which diverged relative recently.
we provisionally accept all five subspecies, though ongoing studies might reject some or all of them.
for n. concolor, our data indicate a separation of n. concolor lu from the remaining subspecies, which form a clade without further subdivision into taxa.
hence and concordant with monda et al.
<cit>  and roos et al.
<cit> , we provisionally classify n. concolor furvogaster and n. concolor jingdongensis as synonyms of n. concolor concolor, while we feel n. concolor lu is a separate subspecies.
we further separate n. gabriellae from n. siki/n.
leucogenys on species level, while it is questionable whether the latter two should be recognized as species or subspecies.
our study reveals a split between both taxa just  <dig>  mya, which is in a similar range as the subspecies differentiation within h. lar or n. concolor.
hence, a separation of both taxa only on subspecies level would be indicated.
however, both taxa show slight differences in vocalisation and facial colouration  <cit> , and carbone et al.
<cit>  found a chromosomal inversion unique to n. leucogenys.
accordingly, we follow here the current view and recognize n. leucogenys and n. siki as distinct species.
in summary, we recognize four gibbon genera with  <dig> species and seven subspecies .
biogeographic implications
multiple radiations in the evolutionary history of gibbons suggest a complicated biogeographic pattern leading to the current distribution of gibbon taxa.
since gibbons are arboreal  <cit> , radiations most likely were correlated with expanding forest habitats.
in fact, the complete range of gibbons experienced complex geographical and environmental changes during the last ten million years.
notably, in the late miocene as well as in the plio- and pleistocene, a series of dramatic climatic changes influenced the geography and vegetation in the region, leading to shifts in the extension and distribution of different habitat types  <cit> .
in particular, periods of maximum glaciation might have reduced rainforest cover, resulting in the appearance of more open and deciduous vegetation types in many parts of the region .
moreover, due to the alternately falling and rising sea water levels during the several glacial and interglacial periods  <cit> , connections and separations of landmasses were common, and repeated migration between islands and today's mainland was possible  <cit> .
by combining the available information, we develop the following dispersal scenario for gibbons, which is in general agreement with that proposed by chatterjee  <cit> , harrison et al.
<cit> , and jablonski and chaplin  <cit> , but which differs substantially from them in some aspects.
accordingly, gibbons most likely originated on the asian mainland, because all four gibbon genera occur there.
specifically, the hengduan mountains in the border region of today's burma, india and china might have been a possible diversification hotspot  <cit> .
in the region, all the larger southeast asian rivers  rise, which are all well known as barriers for arboreal primates  <cit> .
although these rivers changed their courses several times, their upper reaches in the hengduan mountains exist at least since the early miocene  <cit> .
recently, the hengduan mountains were also proposed as a region of differentiation for colobine monkeys, and, most interestingly, respective splitting events occurred on a similar time scale as in gibbons .
in fact, in the late miocene, widely distributed rain forest habitats promoted range extension for arboreal primates  <cit> .
accordingly, in the late miocene, nomascus invaded the region east of the mekong, hoolock entered the region west of the salween, and hylobates and symphalangus migrated into the area in-between and later on into sundaland.
hylobates successfully colonized large parts of sundaland, but also survived on the asian mainland.
shortly after its arrival in sundaland in the pliocene, populations on the asian mainland, the malay peninsula, sumatra, borneo, java and the mentawai archipelago became isolated.
at the same time, various species groups of the genera macaca and trachypithecus diverged  <cit> , indicating dramatic environmental changes.
in fact, this time period was characterized by global warming and sea levels similar to today  <cit> , which prevented migration between landmasses and, thus, promoted speciation due to vicariance.
whether symphalangus experienced a similar range expansion in sundaland like hylobates, remains questionable.
today the genus appears only on sumatra and the malay peninsula, and fossil data provide only evidence for its historical occurrence on java and sumatra  <cit> .
in the early pleistocene, further differentiation in hylobates occurred on borneo and sumatra, and in hoolock on the mainland which is on a similar time scale when macaque species diverged  <cit> , and which might has been triggered by the shrinking of forest habitats due to cold phases .
notably, h. albibarbis is mitochondrially closer related to sumatran h. agilis than to the other bornean gibbons, and acoustic, morphological and chromosomal data suggest an intermediate position  <cit> .
accordingly, h. albibarbis might be the product of an ancient hybridization event, in which proto-h. agilis invaded borneo during sea level lowstands  <cit> , and successfully reproduced with proto-h. muelleri.
as we find mtdna of proto-h. agilis in h. albibarbis, female introgression is the most likely hybridization scenario, which is in agreement with recent findings, that gibbon females disperse over longer distances than males  <cit> .
finally, in a last range expansion in the early to middle pleistocene, h. lar colonized, starting from its sumatran refuge, the malaysian peninsular and mainland southeast asia .
in contrast to the biogeographic pattern found in hylobates and to the scenario proposed by chatterjee  <cit> , for nomascus not a radiation but a successive migration from north to south over a long time period becomes evident.
based on our data, nomascus originated in the border region of vietnam and china in the early pliocene and it took to the early pleistocene until the genus reached the southern extend of its current distribution in southern vietnam and cambodia.
conservation implications
all gibbon species are on the brink of extinction and, with the exception of h. leuconedys , are classified as "endangered" or even "critically endangered"  <cit> .
with approximately  <dig> individuals left in its native habitat, the hainan gibbon  is the rarest primate in the world  <cit> , and the situation for its closest relative, the cao-vit crested gibbon  with approximately  <dig> individuals left  <cit> , as well as for other gibbon species, the situation is alarming.
reasons for the decline of gibbons are manifold, but habitat loss due to forest clearance for agricultural use, oil palm or rubber plantations, gold mining, or charcoal and timber production, as well as illegal hunting for food and sport, and the trade for pets or medicine are major threats to wild gibbon populations  <cit> .
to save gibbons from extinction, urgent actions are required to prevent ongoing habitat destruction and hunting, and to build up a viable gene pool in captivity for later release purposes.
specifically, to prevent or at least reduce hunting, hunting hotspots have to be identified.
therefore, it is crucial to confirm the taxon identity and if possible the geographical origin of confiscated gibbons or their remains.
similarly, to avoid artificial hybrids, only gibbons with clear taxon identity should be considered for reproduction in zoos or rescue centres.
finally, if captive gibbons are reintroduced into the wild, it has to be ascertained that these gibbons are pure individuals and of the same taxon as those, which naturally occur in the area they are to be released.
an accurate taxonomic identification of gibbons based on vocal data or pelage colouration is sometimes complicated  <cit> .
in this respect, mtdna analysis might be a promising tool.
as shown in our study, gibbon taxa can be diagnosed through mtdna, and, hence, a secure identification can easily be obtained.
yet since mtdna is only maternally inherited, possible hybrids will not be detected in such analysis, so that additional markers should be studied as well.
CONCLUSIONS
due to a nearly complete taxon sampling, the present study provides the most comprehensive insights into the evolutionary and biogeographic history of the hylobatid family.
based on estimated divergence ages and unresolved relationships among gibbon taxa on various levels, several radiation-like splitting events are indicated, which suggest a complex biogeographic history of gibbons.
presumably, most of these differentiation events occurred in wave-like range expansions in sundaland and the asian mainland followed by vicariance effects, most likely caused by alternately shrinking and expanding rain forest habitats and by repeated separations and connections of landmasses.
in contrast, in the region east of the mekong river gibbons underwent a successive north-to-south migration.
our study also shows that mtdna provides a solid platform for the taxonomic classification of gibbons and that mtdna can be successfully applied to accurately identify the species affiliation of gibbon individuals, which is urgently required for conservation purposes.
however, to completely understand the phylogeny and phylogeography of gibbons, to identify hybrids in captivity, or to trace possible ancient hybridization events as it might be indicated for h. albibarbis, further studies including extended mitochondrial as well as autosomal, x and y chromosomal sequence data, are necessary.

 gibbons, family hylobatidae, are small arboreal apes, which inhabit tropical and subtropical rainforests of southeast asia and adjacent regions. together with humans and great apes, they belong to the primate superfamily hominoidea. among hominoids, gibbons were the first to branch off and they display a set of morphological and behavioural characteristics distinctly different from great apes and humans. most prominent in this respect is the predominantly monogamous life style, their territorial calls, and the typical brachiating locomotion. due to their extensive karyotypic diversity, gibbons provide an excellent model organism to study chromosomal rearrangements and, hence, to better understand human diseases caused by such alterations. although in several aspects unique among primates and with up to species the most diverse group of apes, gibbons are still in the shadow of great apes in respect of scientific studies, conservation efforts and public awareness. however, many gibbon species are on the brink of extinction and most of them are classified as "endangered" or even "critically endangered". with approximately individuals left in its native habitat, the hainan gibbon is the rarest primate in the world. responsible for this critical situation is habitat loss and hunting, which both have seriously reduced gibbon populations throughout their range. hence, much more attention has to be drawn on the gibbons' situation and extensive conservation actions are urgently required to save them from extinction. while gibbons are widely considered to form a monophyletic clade, there is no consensus about the phylogeny and taxonomy within the family. although various studies based on morphology, behaviour, vocalisation, protein electrophoresis, karyotyping and dna sequencing were conducted, neither a congruent phylogeny nor a consistent taxonomic classification was obtained. moreover, incomplete taxon sampling as well as misidentified specimens resulted in only fragmentary or even false conclusions. accordingly, the classification of gibbon taxa at various taxonomic levels as well as their phylogenetic relationships remain disputed and a consensus is far from being available. for example, in early studies, small apes were divided into two genera, with one including the siamang, and the other all the remaining species. later on, the family was split into four major clades, which were recognized as subgenera and eventually as genera. this division is now widely accepted and takes into account the fact that species within each of the four major clades share a number of characteristics, most importantly a distinctive diploid chromosome number: hoolock, hylobates, symphalangus and nomascus. similarly, the number of species and subspecies is a matter of debate as well. while symphalangus is consistently regarded as monotypic, the two hoolock subspecies were recently elevated to species. in nomascus originally only one species was recognized, but in current classifications four to six species were suggested. in contrast, the genus hylobates already comprised at least four species in early classifications, but recent studies proposed six or seven species. due to this incongruence we follow the most recent classification of the iucn red list with a total of gibbon species. *further research required in the present study, we analyse the complete mitochondrial cytochrome b gene from individuals, which represent all gibbon genera and species, and most subspecies. based on our data, we are able to 1) provide the most complete phylogeny of gibbons on all taxonomic levels, 2) estimate divergence times between lineages, 3) establish a reliable classification, 4) elucidate gibbon phylogeography, and 5) provide a tool for the species identification of gibbon individuals.  from all gibbons, we successfully generated sequences of the complete mitochondrial cytb gene. a contamination of our dataset with nuclear pseudogenes can be regarded as minimal, because no multiple amplifications of different copies were detected by direct sequencing. all sequences were correctly transcribed, and identical sequences were obtained for the same individual in cases where different material types were available. moreover, no inconsistent positions were detected in alignments, which were assembled from overlapping sequences. cross-contamination between individuals can be excluded as well, since all negative controls revealed no amplifications and randomly repeated pcrs for the same individual produced identical sequences. among the individual gibbons studied, no identical haplotypes were detected. the cytb alignment comprising solely gibbons was characterized by variable sites, of which were parsimony-informative. in the complete alignment, which additionally contained great ape, human and hamadryas baboon representatives, we observed variable sites, of which were parsimony-informative. phylogenetic tree reconstructions based on maximum-parsimony, neighbor-joining, maximum-likelihood and bayesian algorithms revealed various strongly supported clades, which corresponded to genera, species and subspecies. all algorithms led to identical tree topologies, although several branching patterns gained only weak support. according to our reconstructions, hominoids diverged into a clade consisting of gibbons, and another with great apes and human. among the latter, pongo split off first, followed by gorilla, before finally pan and homo diverged. within gibbons, a basal position of nomascus and a sister grouping of hylobates and hoolock was indicated, but support for this branching pattern was relatively low. similarly, with the exception of a strongly supported h. agilis + h. albibarbis clade, also the relationships among the species of hylobates were not well resolved. however, at least species monophylies were clearly confirmed, though a common origin of h. agilis was only weakly supported. the relationships among the subspecies of h. muelleri and h. lar were less resolved. in hoolock, the two species h. hoolock and h. leuconedys clearly segregated into two distinct clades. within nomascus, relationships among species were completely resolved, suggesting a n. hainanus + n. nasutus clade as sister lineage to the remaining species. among them, n. concolor branched off first, followed by the divergence of n. gabriellae and n. leucogenys/n. siki. the monophyly of n. leucogenys was significantly supported, but evidence for a common origin of n. siki individuals was not obtained. within n. concolor, specimens identified as n. concolor lu formed a distinct clade, while the remaining subspecies clustered together without further subdivision. however, support for a reciprocal monophyly of both clades was relatively low. *means and 95% credibility intervals are given for nodes. nodes used as calibrations are labelled with a "c", all others with an "n". mrca denotes the most recent common ancestor. c not shown in figure **support values as obtained from mp, nj, ml and bayesian reconstructions, respectively. based on divergence age estimates, gibbons separated from great apes and humans million years ago. within hominids, pongo branched off first, followed by gorilla, before finally homo and pan diverged from each other. the differentiation of pongo and pan into species occurred and mya, respectively. in an initial radiation, gibbons diverged within a relative short time period of only million years into four genera. within hylobates, most species diverged from each other between and mya. the only exception was the separation of h. albibarbis from h. agilis mya, which was in the time frame of subspecies splits within h. muelleri. differentiation of h. lar into subspecies occurred even later. the two hoolock species diverged mya from each other. in nomascus, differentiation into species took place over a longer time period, lasting from until mya. the most recent species divergence within nomascus occurred between n. siki and n. leucogenys, which was in a similar range as the separation of n. concolor lu from the other n. concolor subspecies. discussion by analysing all species and most subspecies, the present study provides the most complete view into the evolutionary history of the gibbon family. however, as in earlier molecular studies on gibbons, relationships on various taxonomic levels are less resolved and partially contradict earlier findings. while the herein depicted branching pattern among genera is identical with that found in earlier studies using also cytb or d-loop sequences, it differs from another cytb-based study in placing nomascus and not symphalangus as most basal genus. studies based on mitochondrial nd3-nd sequences or chromosomal rearrangements suggest hoolock as most ancestral lineage, and nomascus together with either hylobates or symphalangus as the most recently diverged genera. for hylobates, our data indicate a basal position of h. klossii, and a further division into a clade consisting of h. lar, h. muelleri, h. agilis and h. albibarbis, and another one with h. moloch and h. pileatus. various branching patterns among hylobates species are proposed, which all differ from our one, but respective support values are similarly low as in our study. in contrast, the relationships found among species of the genus nomascus are well resolved and identical with that suggested by. according to our and earlier data, relationships among gibbon genera and hylobates species remain disputed, which most likely can be explained by the separation of respective lineages within relative short time periods. this becomes even more obvious when considering estimated divergence ages, which fall into four temporal windows. in the first, between ~ and ~ mya, the four gibbon genera originated. in a second radiation, between ~ and ~ mya, hylobates split into various species, and in a third burst, between ~ and ~ mya, h. muelleri, the h. agilis + h. albibarbis clade and hoolock further differentiated. finally, in a fourth radiation, between ~ and ~ mya, h. lar diverged into subspecies. in contrast, speciation in nomascus was a continuous process, lasting from until mya. taxonomic implications our data show that mitochondrial dna provides a powerful tool for the identification and taxonomic classification of gibbons, because taxa form strongly supported monophyletic clades, or at least appear to form distinct lineages in those cases where only one individual per taxon was tested. moreover, most differentiation events fall into four temporal periods, which allow a hierarchical ranking as proposed by goodman et al., though the threshold for the recognition of a certain taxonomic unit whether genus, species, or subspecies remains disputed. hence, to provide a more reliable classification, we compare divergence ages among gibbon lineages with those among other asian primates and hominids. accordingly and concordant with recent classifications, the four major gibbon lineages are proposed as distinct genera, since they split from each other in a similar time range as did colobine genera or african great apes and human. most species of hylobates and nomascus emerged in or around the second radiation, which is on the same time scale as species splits within pongo and pan, and the separation of species groups within macaca and trachypithecus. thus, taxa originating in this time period should be recognized as distinct species, and might be even classified as species groups. further differentiation events among gibbons occurred in the third time period, which is in a similar window as several speciation events within macaques. accordingly, h. leuconedys and h. albibarbis should be separated from h. hoolock and h. agilis on species level, respectively, and the three subspecies of h. muelleri could be considered for elevation to species level. moreover, h. agilis is divided into two clades, which refer to individuals identified by pelage coloration as h. agilis agilis and h. agilis unko. however, in a recent work based on a larger number of individuals a reciprocal monophyly of both lineages is doubted, and, hence, we provisionally recognize h. agilis as monotypic. for h. lar, only a few unambiguously identified specimens were available for our study, but these represent at least four of the five recognized subspecies, while the identity of the putative h. lar yunnanensis individual remains uncertain. based on our data, h. lar subspecies form distinct lineages, which diverged relative recently. we provisionally accept all five subspecies, though ongoing studies might reject some or all of them. for n. concolor, our data indicate a separation of n. concolor lu from the remaining subspecies, which form a clade without further subdivision into taxa. hence and concordant with monda et al. and roos et al., we provisionally classify n. concolor furvogaster and n. concolor jingdongensis as synonyms of n. concolor concolor, while we feel n. concolor lu is a separate subspecies. we further separate n. gabriellae from n. siki/n. leucogenys on species level, while it is questionable whether the latter two should be recognized as species or subspecies. our study reveals a split between both taxa just mya, which is in a similar range as the subspecies differentiation within h. lar or n. concolor. hence, a separation of both taxa only on subspecies level would be indicated. however, both taxa show slight differences in vocalisation and facial colouration, and carbone et al. found a chromosomal inversion unique to n. leucogenys. accordingly, we follow here the current view and recognize n. leucogenys and n. siki as distinct species. in summary, we recognize four gibbon genera with species and seven subspecies. biogeographic implications multiple radiations in the evolutionary history of gibbons suggest a complicated biogeographic pattern leading to the current distribution of gibbon taxa. since gibbons are arboreal, radiations most likely were correlated with expanding forest habitats. in fact, the complete range of gibbons experienced complex geographical and environmental changes during the last ten million years. notably, in the late miocene as well as in the plio- and pleistocene, a series of dramatic climatic changes influenced the geography and vegetation in the region, leading to shifts in the extension and distribution of different habitat types. in particular, periods of maximum glaciation might have reduced rainforest cover, resulting in the appearance of more open and deciduous vegetation types in many parts of the region. moreover, due to the alternately falling and rising sea water levels during the several glacial and interglacial periods, connections and separations of landmasses were common, and repeated migration between islands and today's mainland was possible. by combining the available information, we develop the following dispersal scenario for gibbons, which is in general agreement with that proposed by chatterjee, harrison et al., and jablonski and chaplin, but which differs substantially from them in some aspects. accordingly, gibbons most likely originated on the asian mainland, because all four gibbon genera occur there. specifically, the hengduan mountains in the border region of today's burma, india and china might have been a possible diversification hotspot. in the region, all the larger southeast asian rivers rise, which are all well known as barriers for arboreal primates. although these rivers changed their courses several times, their upper reaches in the hengduan mountains exist at least since the early miocene. recently, the hengduan mountains were also proposed as a region of differentiation for colobine monkeys, and, most interestingly, respective splitting events occurred on a similar time scale as in gibbons. in fact, in the late miocene, widely distributed rain forest habitats promoted range extension for arboreal primates. accordingly, in the late miocene, nomascus invaded the region east of the mekong, hoolock entered the region west of the salween, and hylobates and symphalangus migrated into the area in-between and later on into sundaland. hylobates successfully colonized large parts of sundaland, but also survived on the asian mainland. shortly after its arrival in sundaland in the pliocene, populations on the asian mainland, the malay peninsula, sumatra, borneo, java and the mentawai archipelago became isolated. at the same time, various species groups of the genera macaca and trachypithecus diverged, indicating dramatic environmental changes. in fact, this time period was characterized by global warming and sea levels similar to today, which prevented migration between landmasses and, thus, promoted speciation due to vicariance. whether symphalangus experienced a similar range expansion in sundaland like hylobates, remains questionable. today the genus appears only on sumatra and the malay peninsula, and fossil data provide only evidence for its historical occurrence on java and sumatra. in the early pleistocene, further differentiation in hylobates occurred on borneo and sumatra, and in hoolock on the mainland which is on a similar time scale when macaque species diverged, and which might has been triggered by the shrinking of forest habitats due to cold phases. notably, h. albibarbis is mitochondrially closer related to sumatran h. agilis than to the other bornean gibbons, and acoustic, morphological and chromosomal data suggest an intermediate position. accordingly, h. albibarbis might be the product of an ancient hybridization event, in which proto-h. agilis invaded borneo during sea level lowstands, and successfully reproduced with proto-h. muelleri. as we find mtdna of proto-h. agilis in h. albibarbis, female introgression is the most likely hybridization scenario, which is in agreement with recent findings, that gibbon females disperse over longer distances than males. finally, in a last range expansion in the early to middle pleistocene, h. lar colonized, starting from its sumatran refuge, the malaysian peninsular and mainland southeast asia. in contrast to the biogeographic pattern found in hylobates and to the scenario proposed by chatterjee, for nomascus not a radiation but a successive migration from north to south over a long time period becomes evident. based on our data, nomascus originated in the border region of vietnam and china in the early pliocene and it took to the early pleistocene until the genus reached the southern extend of its current distribution in southern vietnam and cambodia. conservation implications all gibbon species are on the brink of extinction and, with the exception of h. leuconedys, are classified as "endangered" or even "critically endangered". with approximately individuals left in its native habitat, the hainan gibbon is the rarest primate in the world, and the situation for its closest relative, the cao-vit crested gibbon with approximately individuals left, as well as for other gibbon species, the situation is alarming. reasons for the decline of gibbons are manifold, but habitat loss due to forest clearance for agricultural use, oil palm or rubber plantations, gold mining, or charcoal and timber production, as well as illegal hunting for food and sport, and the trade for pets or medicine are major threats to wild gibbon populations. to save gibbons from extinction, urgent actions are required to prevent ongoing habitat destruction and hunting, and to build up a viable gene pool in captivity for later release purposes. specifically, to prevent or at least reduce hunting, hunting hotspots have to be identified. therefore, it is crucial to confirm the taxon identity and if possible the geographical origin of confiscated gibbons or their remains. similarly, to avoid artificial hybrids, only gibbons with clear taxon identity should be considered for reproduction in zoos or rescue centres. finally, if captive gibbons are reintroduced into the wild, it has to be ascertained that these gibbons are pure individuals and of the same taxon as those, which naturally occur in the area they are to be released. an accurate taxonomic identification of gibbons based on vocal data or pelage colouration is sometimes complicated. in this respect, mtdna analysis might be a promising tool. as shown in our study, gibbon taxa can be diagnosed through mtdna, and, hence, a secure identification can easily be obtained. yet since mtdna is only maternally inherited, possible hybrids will not be detected in such analysis, so that additional markers should be studied as well.  due to a nearly complete taxon sampling, the present study provides the most comprehensive insights into the evolutionary and biogeographic history of the hylobatid family. based on estimated divergence ages and unresolved relationships among gibbon taxa on various levels, several radiation-like splitting events are indicated, which suggest a complex biogeographic history of gibbons. presumably, most of these differentiation events occurred in wave-like range expansions in sundaland and the asian mainland followed by vicariance effects, most likely caused by alternately shrinking and expanding rain forest habitats and by repeated separations and connections of landmasses. in contrast, in the region east of the mekong river gibbons underwent a successive north-to-south migration. our study also shows that mtdna provides a solid platform for the taxonomic classification of gibbons and that mtdna can be successfully applied to accurately identify the species affiliation of gibbon individuals, which is urgently required for conservation purposes. however, to completely understand the phylogeny and phylogeography of gibbons, to identify hybrids in captivity, or to trace possible ancient hybridization events as it might be indicated for h. albibarbis, further studies including extended mitochondrial as well as autosomal, x and y chromosomal sequence data, are necessary. 
with up to species, gibbons form the most diverse group of living hominoids, but the number of taxa, their phylogenetic relationships and their phylogeography is controversial. to further the discussion of these issues we analyzed the complete mitochondrial cytochrome b gene from individuals representing all gibbon species, including most subspecies. although relationships among gibbon taxa on various levels remain unresolved, the present study provides a more complete view of the evolutionary and biogeographic history of the hylobatid family, and a more solid genetic basis for the taxonomic classification of the surviving taxa. nevertheless, the new data permitted the estimation of divergence ages for all taxa for the first time and showed that most lineages emerged during four short time periods. gibbons or small apes inhabit tropical and subtropical rain forests in southeast asia and adjacent regions, and are, next to great apes, our closest living relatives. based on phylogenetic tree reconstructions, several monophyletic clades were detected, corresponding to genera, species and subspecies. the phylogenetic relationships among the four genera were also not well resolved. we also show that mtdna constitutes a useful marker for the accurate identification of individual gibbons, a tool which is urgently required to locate hunting hotspots and select individuals for captive breeding programs. in the first, between ~ and ~ mya, the four gibbon genera diverged from each other. a significantly supported branching pattern was obtained for members of the genus nomascus but not for the genus hylobates.
 gibbons or small apes inhabit tropical and subtropical rain forests in southeast asia and adjacent regions, and are, next to great apes, our closest living relatives. with up to species, gibbons form the most diverse group of living hominoids, but the number of taxa, their phylogenetic relationships and their phylogeography is controversial. to further the discussion of these issues we analyzed the complete mitochondrial cytochrome b gene from individuals representing all gibbon species, including most subspecies.  based on phylogenetic tree reconstructions, several monophyletic clades were detected, corresponding to genera, species and subspecies. a significantly supported branching pattern was obtained for members of the genus nomascus but not for the genus hylobates. the phylogenetic relationships among the four genera were also not well resolved. nevertheless, the new data permitted the estimation of divergence ages for all taxa for the first time and showed that most lineages emerged during four short time periods. in the first, between ~ and ~ mya, the four gibbon genera diverged from each other. in the second and in the third period, hylobates and hoolock differentiated. finally, between ~ and ~ mya, hylobates lar diverged into subspecies. in contrast, differentiation of nomascus into species and subspecies was a continuous and prolonged process lasting from ~ until ~ mya.  although relationships among gibbon taxa on various levels remain unresolved, the present study provides a more complete view of the evolutionary and biogeographic history of the hylobatid family, and a more solid genetic basis for the taxonomic classification of the surviving taxa. we also show that mtdna constitutes a useful marker for the accurate identification of individual gibbons, a tool which is urgently required to locate hunting hotspots and select individuals for captive breeding programs. further studies including nuclear sequence data are necessary to completely understand the phylogeny and phylogeography of gibbons. 