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 [26–35], 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-ND4 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 [27, 31, 32, 35], 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 [30, 31, 33, 34].
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 ~6.7 and ~8.3 mya, the four gibbon genera originated. In a second radiation, between ~3.0 and ~3.9 mya, Hylobates split into various species, and in a third burst, between ~1.3 and ~1.8 mya, H. muelleri, the H. agilis + H. albibarbis clade and Hoolock further differentiated. Finally, in a fourth radiation, between ~0.5 and ~1.1 mya, H. lar diverged into subspecies. In contrast, speciation in Nomascus was a continuous process, lasting from 4.24 until 0.55 mya.
Our data show that mitochondrial DNA (mtDNA) 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 [4, 12, 16, 29, 34, 37, 38, 41], the four major gibbon lineages are proposed as distinct genera (Table 1), since they split from each other in a similar time range as did colobine genera [[42, 43], Roos C, Zinner D, Schwarz C, Nash SD, Xing J, Batzer MA, Leendertz FH, Ziegler T, Perwitasari-Farajallah D, Nadler T, Walter L, Osterholz M: Nuclear versus mitochondrial DNA: evidence for hybridization in colobine monkeys, submitted] or African great apes and human [40, 42]. 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 [44, 45] and Trachypithecus . Thus, taxa originating in this time period should be recognized as distinct species (H. moloch, H. pileatus, H. klossii, H. lar, H. muelleri, H. agilis/H. albibarbis, H. hoolock/H. leuconedys, N. nasutus, N. hainanus, N. concolor, N. gabriellae/N. leucogenys/N. siki), 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 [44, 45]. 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 0.55 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 [4, 5, 15], 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 18 species and seven subspecies (Table 1).
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 [7, 39], 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 [49–54]. 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 [[52–57], but see ]. Moreover, due to the alternately falling and rising sea water levels during the several glacial and interglacial periods [59–64], connections and separations of landmasses were common, and repeated migration between islands and today's mainland was possible [65–67].
By combining the available information, we develop the following dispersal scenario for gibbons, which is in general agreement with that proposed by Chatterjee [32, 68], 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 [71, 72]. In the region, all the larger Southeast Asian rivers (Mekong, Salween, Yangtze) 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 [Roos C, Zinner D, Schwarz C, Nash SD, Xing J, Batzer MA, Leendertz FH, Ziegler T, Perwitasari-Farajallah D, Nadler T, Walter L, Osterholz M: Nuclear versus mitochondrial DNA: evidence for hybridization in colobine monkeys, submitted]. In fact, in the late Miocene, widely distributed rain forest habitats promoted range extension for arboreal primates [50, 54]. 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 [44–46], indicating dramatic environmental changes. In fact, this time period was characterized by global warming and sea levels similar to today [54, 61–63], 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 [44, 45], and which might has been triggered by the shrinking of forest habitats due to cold phases [, but see ]. 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 [2, 5, 47, 75]. Accordingly, H. albibarbis might be the product of an ancient hybridization event, in which proto-H. agilis invaded Borneo during sea level lowstands [61–64], 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 [see also ].
In contrast to the biogeographic pattern found in Hylobates and to the scenario proposed by Chatterjee [32, 68], 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.
All gibbon species are on the brink of extinction and, with the exception of H. leuconedys (Vulnerable), are classified as "Endangered" or even "Critically Endangered" [12, 16]. With approximately 20 individuals left in its native habitat, the Hainan gibbon (N. hainanus) is the rarest primate in the world [6, 13, 14], and the situation for its closest relative, the Cao-vit crested gibbon (N. nasutus) with approximately 100 individuals left [12, 77], 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 [15, 16].
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 [4, 5]. 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.