![Geographical distribution of gibbons based on [2,5,23,41]. Dotted and solid lines indicate country borders and major rivers, respectively. Historical distribution of N. hainanus and N. nasutus is hatched.](https://www.researchgate.net/publication/41941560/figure/fig1/AS:216485705326593@1428625622300/Geographical-distribution-of-gibbons-based-on-2-5-23-41-Dotted-and-solid-lines_Q320.jpg)
RESEARC H ARTIC LE Open Access
Mitochondrial evidence for multiple radiations in
the evolutionary history of small apes
Van Ngoc Thinh
1*
, Alan R Mootnick
2
, Thomas Geissmann
3
, Ming Li
4
, Thomas Ziegler
5
, Muhammad Agil
6
,
Pierre Moisson
7
, Tilo Nadler
8
, Lutz Walter
1,9
, Christian Roos
1,9*
Abstract
Background: 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 16 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 85 indi viduals representing all gibbon species, including most subspecies.
Results: 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 ~6.7 and ~8.3
mya, the four gibbon genera diverged from each other. In the second (~3.0 - ~3.9 mya) and in the third period
(~1.3 - ~1.8 mya), Hylobates and Hoolock differentiated. Finally, between ~0.5 and ~1.1 mya, Hylobates lar diverged
into subspecies. In contrast, differe ntiation of Nomascus in to species and subspecies was a continuous and
prolonged process lasting from ~4.2 until ~0.4 mya.
Conclusions: 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.
Background
Gibbons, family Hylobatidae, are small arboreal apes,
which inhabit tropical and subtropical rainforests of
Southeast Asia and adjacent regions (Figure 1). Together
with humans and great apes, they belong to the primate
superfamily Hominoidea [1-4]. Among homino ids, gib-
bonswerethefirsttobranchoffandtheydisplayaset
of morphological and behavioural characteristics dis-
tinctly different from great apes and humans [1,5,6].
Most prominent in this respect is the predominantly
monogamous life style, their territorial calls, and the
typical brachiating locomotion [1,4-7]. Due to their
extensive karyotypic diversity [8-11], gibbons provide an
excellent model organism to study chromosomal rear-
rangements and, hence, to better understand human dis-
eases caused by such alterations.
Although in several aspects unique among primates
andwithupto16speciesthemostdiversegroupof
apes, gibbons are s till 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” [12].
With approximately 20 individuals left in its native habi-
tat, the Hainan gibbon ( Nomascus hainanus) is the rar-
est primate in the world [6,13,14]. Responsible for this
critical situation is habitat loss and hunting, which both
* Correspondence: vanthinhngoc@yahoo.com; croos@dpz.eu
1
Primate Genetics Laboratory, German Primate Center, Kellnerweg 4, 37077
Göttingen, Germany
Thinh et al. BMC Evolutionary Biology 2010, 10:74
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© 2010 Thinh et al; licensee BioMed Central Ltd. This is an Open Access articl e distributed und er the terms of the Creative Commons
Attribution License (http://creative commons.org/licenses/by/2.0), which permits unrestri cted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Figure 1 Geographical distribution of gibbons based on [2,5,23,41]. Dotted and solid lines indicate country borders and major rivers,
respectively. Historical distribution of N. hainanus and N. nasutus is hatched.
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have seriously reduced gibbon populations throughout
their range [15,16]. Hence, much more attention has to
be drawn on the gibbons’ situation and extensive con-
servation actions are urgently required to save them
from extinction [16].
While gibbons are widely considered to form a mono-
phyletic clade, there is no consensus about the phylo-
gen y and taxonomy within the family. Although vari ous
studies based on morphology, behaviour, vocalisation,
protein electrophoresis, karyotyping and DNA sequen-
cing w ere conducted [3-5,7,17-35], neither a congruent
phylogeny nor a consistent taxonomic classification was
obtained. Moreover, incomplete taxon s ampling as well
as misidentified specimens resulted in only fragmentary
or even false conclusions. Acco rdingly, 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 (Symphalangus) including the
siamang, and the other (Hylobates) all the remaining spe-
cies [17,36]. Later on, the family was split into four major
clades, which were re cognized as subgenera [2,5,21] and
eventually as genera [4,16,29,37,38]. This division is now
widely accepted and takes into account the fact that spe-
cies within each of the four major clades share a number
of characteristics, most importantly a distinctive diploid
chromosome number: Hoolock (2n = 38), Hylobates
(2n = 44), Symphalangus (2n = 50) and Nomascus
(2n = 52) [8]. Similarly, the number of s pecies and sub-
species is a matter of debate as well. While Symphalangus
is consistently regarded as monotypic, the two Hoolock
subspecies were recently elevated to species [38]. In
Nomascus originally only one species was recognized
[17,18,20,39], but in current classifications four to six
species were suggested [2,4,12,16,34]. In contrast, the
genus Hylobates already comprised at least four species
in early c lassifications [17,39], but recent studies pro-
posed six or seven species [2,4,16]. Due to this incongru-
ence we follow the most recent classification of the
IUCN Re d List [12] with a total of 16 gibbon species
(Table 1).
In the present study, we analyse the complete mito-
chondri al cytochrome b (cytb) gene from 85 individuals,
which represent all gibbon genera and species, and most
subspecies. Based on our da ta, we are able to 1) provide
Table 1 Common names, IUCN classification and proposed classification of gibbons.
Common name IUCN classification [12] Proposed classification
Kloss’ s gibbon Hylobates klossii Hylobates klossii
Eastern Müller’s Bornean gibbon Hylobates muelleri muelleri Hylobates muelleri*
Northern Müller’s Bornean gibbon Hylobates muelleri funereus Hylobates funereus*
Abbott’s Müller’s Bornean gibbon Hylobates muelleri abbotti Hylobates abbotti*
Agile gibbon Hylobates agilis Hylobates agilis*
Bornean white-bearded gibbon Hylobates albibarbis Hylobates albibarbis
Malayan lar gibbon Hylobates lar lar Hylobates lar lar*
Sumatran lar gibbon Hylobates lar vestitus Hylobates lar vestitus*
Mainland lar gibbon Hylobates lar entelloides Hylobates lar entelloides*
Carpenter’s lar gibbon Hylobates lar carpenteri Hylobates lar carpenteri*
Yunnan lar gibbon Hylobates lar yunnanensis Hylobates lar yunnanensis*
Silvery Javan gibbon Hylobates moloch Hylobates moloch*
Pileated gibbon Hylobates pileatus Hylobates pileatus
Western hoolock gibbon Hoolock hoolock Hoolock hoolock
Eastern hoolock gibbon Hoolock leuconedys Hoolock leuconedys
Siamang Symphalangus syndactylus Symphalangus syndactylus*
Hainan gibbon Nomascus hainanus Nomascus hainanus
Cao-vit crested gibbon Nomascus nasutus Nomascus nasutus
Black crested gibbon Nomascus concolor concolor Nomascus concolor concolor*
West Yunnan black crested gibbon Nomascus concolor furvogaster Nomascus concolor concolor*
Central Yunnan black crested gibbon Nomascus concolor jingdongensis Nomascus concolor concolor*
Laotian black crested gibbon Nomascus concolor lu Nomascus concolor lu*
Northern white-cheeked gibbon Nomascus leucogenys Nomascus leucogenys*
Southern white-cheeked gibbon Nomascus siki Nomascus siki*
Red-cheeked gibbon Nomascus gabriellae Nomascus gabriellae
16 species, 12 subspecies 18 species, 7 subspecies
*further research required
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the most complete phylogeny of gibbons on all taxo-
nomic 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 85 gibbons, we successfully generated
sequences of the comple te mitochondrial cytb gene
(1,140 bp). A contamination of our dataset with nuclear
pseudogenes (numts) 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
mater ial types were available. Moreover, no inconsistent
positions were detected in alignments, which were
assembled from overlapping sequences. Cross-contami-
nation 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 85 individual gibbons studied, no identical
haplotypes were detected. The cytb alignment compris-
ing solely gibbons was characterized by 429 variable
sites, of which 374 were parsimony-informative. In the
complete alignment, which additionally contained great
ape, human and hamadryas b aboon representative s, we
observed 565 variable sites, of which 462 were parsi-
mony-informative.
Phylogenetic tree reconstructions based on maximum-
parsimony (MP), neighbor-joining (NJ), maximum-likeli-
hood (ML) and Bayesian algorithms revealed various
strongly supported clades, which corresponded to gen-
era, species and subspecies (Figure 2). All algorithms led
to identical tree topologies, although several branching
patterns gained only weak support. According to our
reconstructions, hominoids diverged into a clade con-
sisting of gibbons, and another with great apes and
human. Among the latter, Pongo split off first, followed
by Gorilla,beforefinallyPan and Homo diverged.
Within gibbons, a basal position of Nomascus and a sis-
ter grouping of Hylobates and Hoolock was indicated,
but support for this branching patte rn was relatively low
(Table 2). Similarly, with the exception of a strongly
supported H. agilis + H. albibarbis clade, also the rela-
tionships among the species of Hylobates were not well
resolved. However, a t least spe cies 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. leucone-
dys clearly segregated into two distinct clades. Within
Nomascus, rel ationships among species were completely
resolved, suggesting a N. hainanus + N. nasutus clade as
sister lineage t o the remaining species. Among them, N.
concolor branched off first, followed by the divergence
of N. gabr iellae and N. leucogenys/N. siki.Themono-
phyly of N. leucogenys was significantly supported, but
evidence for a common origin of N. siki individuals was
not obtaine d. Within N. concolor, specimens identified
as N. concolor lu formed a distinct c lade, while the
remaining sub species clustered tog ether without furth er
subdivision. However, support for a reciprocal mono-
phyly of both clades was relatively low.
Based on divergence age estimates, gibbons separated
from great apes and humans 16.26 million years ago
(mya) (for 95% credibility intervals see Table 2). Withi n
hominids, Pongo branched off first (13.83 mya), followed
by Gorilla (8.90 mya), before finally Homo and Pan
diverged from each other (6.56 mya). The differentiation
of Pongo and Pan into species occurred 4.12 and 2.74
mya, respectively. In an initial radiation, gibbons
diverged within a relative short time pe riod of only 1.65
million years (6.69-8.34 mya) into four genera. Within
Hylobates, most species diverged from each other
between 3.02 and 3.90 mya. The only exception was the
separation of H. albibarbis from H. a gilis
1.56 mya,
which was in the time frame of subspecies splits within
H. muelleri (1.42-1.78 mya). Differentiation of H. lar
into subspecies occurred even later (0.52-1.05 mya). The
two Hoolock species diverged 1.42 mya from ea ch other.
In Nomascus, differentiation into species took place over
a longer time peri od, lasting from 4.24 until 0.55 mya.
The most recent species divergence within Nomascus
occurred be tween N. siki and N. leucogenys (0.55 mya),
which was in a similar range as the separation of N. con-
color lu from the other N. concolor subspecies (0.43
mya).
Discussion
By analysing all species and most subspecies, the present
study provides the most complete view into the evolu-
tionary history of the gibbon family. However, as in ear-
lier molecular studies on gibbons [26-35], relationships
on various taxonomic levels are less resolved and par-
tially contradict earlier findings. While the herein
depicted branching pattern among genera is identical
with that found in earlier studies using also cytb [32] or
D-loop [29] sequences, it differs from another cytb-
based study [28] in placing Nomascus and not Symp ha-
langus as most basal genus. Studies based on mitocho n-
drial ND3-ND4 sequences [31] or chro mosomal
rearrangements [8] sug gest Hoolock as most ancestral
lineage, and Nomascus together with either Hylobates
[31] or Symphalangus [8] as the most recently diverged
genera. For Hylobates, our data indicate a basal position
of H. klossii, and a further division into a clade
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Figure 2 Ultrametric tree showing phylogenetic relationships and estimat ed divergence ages among studied gibbon individuals
based on complete mitochondrial cytb sequence data. For individual codes see Additional File 1. Circles indicate bootstrap or posterior
probability values (filled circles: >90%, >0.95, open circles: <70%, <0.80). Nodes of interest are arbitrarily numbered (N1-N45). C2 and C3 refer to
two of the three nodes used for calibration (C1 not shown). Light green bars indicate the four radiations. A geological time scale is given below.
Full details of age estimates and node supports are presented in Table 2.
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Table 2 Support values and Bayesian divergence date estimates (in mya)*.
Node Support values** Divergence Mean (95% CI)
C1 Papio - Hominoidea 24.04 (22.01-26.08)
N1 Hylobatidae - Hominidae 16.26 (14.69-18.16)
C2 96/92/92/0.99 Pongo - Gorilla/Pan/Homo 13.83 (13.28-14.41)
N2 91/93/98/1.0 Gorilla - Pan/Homo 8.90 (7.58-10.22)
C3 97/96/91/1.0 Pan - Homo 6.56 (6.01-7.08)
N3 100/91/97/0.99 Pan troglodytes - P. paniscus 2.74 (2.03-3.51)
N4 100/98/96/0.99 Pongo pygmaeus - P. abelii 4.12 (3.14-5.13)
N5 100/100/100/1.0 Nomascus - Symphalangus/Hoolock/Hylobates 8.34 (7.14-9.68)
N6 56/69/67/0.78 Symphalangus - Hoolock/Hylobates 7.22 (5.99-8.44)
N7 65/54/54/0.71 Hoolock - Hylobates 6.69 (5.56-7.88)
N8 100/93/94/0.99 Hylobates klossii - H. pileatus/H. moloch/H. agilis/H. albibarbis/H. lar/H. muelleri 3.91 (3.25-4.59)
N9 <50/68/<50/<0.50 H. pileatus/H. moloch - H. agilis/H. albibarbis/H. lar/H. muelleri 3.65 (3.05-4.25)
N10 <50/<50/<50/0.62 H. muelleri - H. agilis/H. albibarbis/H. lar 3.40 (2.81-3.99)
N11 <50/53/<50/0.69 H. agilis/H. albibarbis - H. lar 3.02 (2.43-3.60)
N12 100/99/100/1.0 H. agilis - H. albibarbis 1.56 (1.19-1.98)
N13 <50/52/<50/<0.50 H. pileatus - H. moloch 3.29 (2.64-3.97)
N14 96/96/98/1.0 H. muelleri funereus - H. m. abbotti/H. m. muelleri 1.78 (1.33-2.25)
N15 56/57/<50/<0.50 H. muelleri abbotti - H. m. muelleri 1.42 (1.02-1.81)
N16 63/<50/67/0.79 H. agilis agilis - H. a. unko 1.30 (0.95-1.68)
N17 100/100/99/1.0 H. lar vestitus - H. l. lar/H. l. entelloides/H. l. carpenteri/H. l. yunnanensis 1.05 (0.75-1.35)
N18 <50/<50/50/0.76 H. l. lar - H. entelloides/H. l. carpenteri/H. l. yunnanensis 0.86 (0.60-1.13)
N19 <50/63/65/0.79 H. l. entelloides - H. l. carpenteri/H. l. yunnanensis 0.62 (0.41-0.83)
N20 <50/66/66/0.78 H. l. carpenteri - H. l. yunnanensis 0.52 (0.32-0.71)
N21 100/100/99/1.0 MRCA H. klossii 0.53 (0.29-0.81)
N22 99/96/97/1.0 MRCA H. muelleri muelleri 0.62 (0.38-0.88)
N23 100/100/100/1.0 MRCA H. albibarbis 0.44 (0.22-0.68)
N24 100/100/100/1.0 MRCA H. agilis unko 0.13 (0.02-0.25)
N25 99/96/94/1.0 MRCA H. agilis agilis 0.61 (0.36-0.89)
N26 95/98/92/1.0 MRCA H. lar carpenteri 0.17 (0.05-0.28)
N27 96/94/96/1.0 MRCA H. lar entelloides 0.18 (0.07-0.31)
N28 100/100/94/1.0 MRCA H. pileatus 0.41 (0.21-0.64)
N29 100/100/100/1.0 MRCA H. moloch 0.56 (0.30-0.84)
N30 100/100/100/1.0 Hoolock hoolock - H. leuconedys 1.42 (0.97-1.90)
N31 99/95/93/0.96 MRCA H. leuconedys 0.51 (0.28-0.80)
N32 100/100/100/1.0 MRCA
H. hoolock 0.07 (0.00-0.17)
N33 100/99/99/1.0 MRCA Symphalangus syndactylus 0.83 (0.51-1.18)
N34 100/100/99/1.0 Nomascus hainanus/N. nasutus - N. concolor/N. gabriellae/N. leucogenys/N. siki 4.24 (3.46-5.06)
N35 91/92/92/0.99 N. hainanus - N. nasutus 3.25 (2.49-3.99)
N36 94/91/96/1.0 N. concolor - N. gabriellae/N. leucogenys/N. siki 2.83 (2.21-3.50)
N37 96/92/98/1.0 N. gabriellae - N. leucogenys/N. siki 1.74 (1.28-2.22)
N38 100/99/93/1.0 N. leucogenys - N. siki 0.55 (0.35-0.77)
N39 100/100/100/1.0 N. concolor lu - N. c. concolor/N. c. furvogaster/N. c. jingdongensis 0.43 (0.25-0.63)
N40 100/100/99/1.0 MRCA N. nasutus 0.23 (0.08-0.39)
N41 <50/<50/67/0.75 MRCA N. concolor lu 0.19 (0.05-0.35)
N42 59/<50/<50/<0.50 MRCA N. concolor concolor/N. c. furvogaster/N. jingdongensis 0.32 (0.19-0.48)
N43 100/100/98/1.0 MRCA N. gabriellae 0.39 (0.21-0.57)
N44 92/91/98/1.0 MRCA N. leucogenys 0.33 (0.18-0.47)
N45 <50/<50/<50/0.58 MRCA N. siki 0.38 (0.18-0.55)
*Means and 95% credibility intervals (CI) are given for 48 nodes (see also Figure 2). Nodes used as calibrations are labelled with a “C”, all others with an “N”.
MRCA denotes the most recent common ancestor. C1 not shown in Figure 2. **Support values as obtained from MP, NJ, ML and Bayesian reconstructions,
respectively.
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consisting of H. lar, H. muelleri, H. agilis and H. albi-
barbis, and another one with H. moloch and H. pil eatus.
Various branching pattern s 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 spe-
cies of the genus Nomascus are well resolved and identi-
cal 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 esti-
mated 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,theH. agilis + H. albibarbis clade and
Hoolock further differentiated. Finally, in a fourth radia-
tion, 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.
Taxonomic implications
Our data show that mitochondrial DNA (mtDNA) pro-
vides a powerful tool for the identificat ion and taxo-
nomic 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 peri-
ods, which allow a hierarchical ranking as proposed b y
Goodman et al. [40], though the threshold for the recog-
nition of a certain taxonomic unit whether genus, spe-
cies, or subspecies remains dispute d. 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
areproposedasdistinctgenera(Table1),sincethey
split from each other in a similar time range as did colo-
bine 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 Afri-
can great apes and human [40,42]. Most species of Hylo-
bates an d Nomascus emergedinoraroundthesecond
radiation, which is on the same time scale as species
splits within Pongo and Pan, and the separation of spe-
cies groups within Macaca [44,45] and Trachy pithecus
[46]. Thus, taxa originating in this t ime 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 g roups. Further d if-
ferentiati on events among gibbons occurred in the third
time period, which is in a similar window as several spe-
ciation events within macaques [ 44,45]. Acc ordingly,
H. leuconedys and H. albib arbis should be separated
from H. hoolock and H. agilis on species level, respec-
tively, 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 indivi-
duals 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 [47], and, hence, we provi-
sionally recognize H. agilis as m onotypic. 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 indiv idual remains
uncertain. Based on our data, H. lar subspecies form
distinct lineages, w hich diverged re lative recently. W e
provisionally accept all five subspecies, though ongoing
studies might reject some or all of them. For N. conco-
lor, 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
withMondaetal.[33]andRoosetal.[34],weprovi-
sionally 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. leucog enys
on species level, while it is questionable whether the lat-
ter 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 differ-
entiation within H. lar or N. concolor. Hence, a separa-
tion of both ta xa 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. [48] found a chromosomal inversion unique to N.
leucogenys. Accordingly, we follow here the current view
and recognize N. leucogenys and
N. siki as distinct spe-
cies. In summary, we recognize four gibbon genera with
18 species and seven subspecies (Table 1).
Biogeographic implications
Multiple radiations in the evolutionary history of gib-
bons suggest a complicated biogeographic pattern lead-
ing to the current distribution of gibbon taxa. Since
gibbons are arboreal [7,39], radiations most likely were
correlated with expanding forest habitats. In fac t, the
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complete range of gibbons experienced complex geogra-
phical 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 [58]]. Moreover, due to the alternately
falli ng and rising sea water levels during the se veral gla-
cial 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 gen-
eral agreement with that pr oposed by Chatterjee [32,68],
Harrison et al. [69], and Jablonski and Chaplin [70], but
which differs substantially from them in some aspects.
Acco rdingly, 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 diversifica tion hotspot [71,72]. In the
region, all the larger Southeast Asian rivers (Mekong,
Salween, Yangtze) rise, which are all well known as bar-
riers for arboreal primates [54]. Although these rivers
changed their courses several times, their upper reaches
in the H engduan mountains exist at least since the early
Miocene [73]. 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
[RoosC,ZinnerD,SchwarzC,NashSD,XingJ,Batzer
MA, Leendertz FH, Ziegler T, Perwitasari-Farajallah D,
Nadler T, Walter L, Osterholz M: Nuclear versus mito-
chondrial 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 Sunda-
land, 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 a rchipe lago became iso-
lated. At the same time, various species groups of the gen-
era 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 migra-
tion 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 [69]. In the early Pleistocene, further differentia-
tion 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 [[74], but see [58]]. 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 pro-
duct 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.Aswe
find mtDNA of proto-H. agil is 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 [76].
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 [70]].
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 orig i-
nated in the border region of Vi etnam and China in the
early Pliocene and it took to the early Pleistocene until
the genus reached t he 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 (Vulnerable), are
classified as “Endangered” or even “ Critically Endan-
gered” [12,16]. With approximately 20 individuals left in
its native habit at, the Hainan gibbon (N. hainanus)is
the rarest primate in the world [6,13,14], and the situa-
tion for its closest relative, the Cao-vit crested gibbon
(N. nasutu s) 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 fo rest 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
Thinh et al. BMC Evolutionary Biology 2010, 10:74
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or medicine are major threats to wild gibbon popula-
tions [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 identi-
fied. Therefore, it is crucial to confirm the taxon identity
and if possible the geographical origin of confiscated
gibbons or t heir remains. Similarly, to avoid artificial
hybrids, only gibbons with clear taxon identity should
be considered for reproduction in zoos or rescue cen-
tres. Finally, if captive gibbons are rei ntroduced into the
wild, it has to be ascertained that these gibbons are pure
individuals and of the same taxon as those, which natu-
rally occur in the area they are to be released.
An accurate taxonomic identification of gibbons based
on vocal data or pelage colouration is sometimes com-
plicated [4,5]. In this respect, mtDNA analysis might be
a promising to ol. 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. B ased on estimated divergence ages and unre-
solved relationships among gibbon taxa on various
levels, several radiation-like splitting events are indi-
cated, which suggest a complex biogeographic history of
gibbons. Presumably, most of these differentiation
events occurred in wave-like range expansions in Sunda-
land and the Asian mainland followe d by vicariance
effects, most likely caused by alternately shrinking and
expanding rain forest habitats an d by repeated separa-
tions 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 phylogeo-
graphy of gibbons, to identify hybrids in captivity, or to
trace possible ancient hybridization events as it might
be indicated for H. albibarbis, further s tudies i ncluding
extended mi tochondrial as well as autosomal, X and Y
chromosomal sequence data, are necessary.
Methods
Sample Collection
A total of 85 specimens representing all species and
most subspecies of hylobatids were included in our
study. Blood, tissue, faecal or hair samples were col-
lected during field surveys, in zoos or rescue centres, or
from museum specimens between 1995 and 2008 (Addi-
tional file 1). Blood and hair samples were taken during
routine health checks by veterinarians. Tissue samples
were obtained only from deceased animals. Taxon iden-
tity of individuals was ascertained by pelage coloration,
morphology and if possible by vocalization and geo-
graphic origin. With the e xception of some H. lar indi-
viduals for which subspecies identity could not be
traced, only clearly identified specimens were included
in our study. Fresh tissue or faecal samples were pre-
served in 80-90% ethanol and dry samples (tissue,
museum skins, hair samples) were pla ced in plastic bags
without any additive. Samples were stored at ambient
temperature for up to six months before further
processing.
Laboratory Methods
Total genomic DNA was extracted with the DNeasy Blood
& Tissue and QIAamp DNA Stool Mini kits from Qiagen.
When hair follicle cells were used, up to three hairs were
directly implemented into the PCR reaction. From high-
quality DNA, the complete mitochondrial cytb gene was
PCR-amplified in a single fragment with the p rimers
5’ -AATGATATGAAAAACCATCGTTGTA-3’ and
5’-TTCATTTCCGGCTTACAAGAC-3’ . For low-qualit y
DNA, extracted from faeces or museums material, two to
seven overlapping PCR products were amplified with pri-
mers constructed on the basis of sequences from conspe-
cifics (respective primers are available from the authors
upon request). For all amplifications, wax-mediated hot-
start PCRs were performed for 40 cycles, e ach with a
denaturation step at 92°C for 1 min, annealing at 60°C for
1 min, and extension at 72°C for 0.5-1.5 min, followed by
a final extension step at 72°C for 5 min. The results of the
PCR amplifications were checked on 1% agarose gels. Sub-
sequently, PCR products were cleaned with the Qiagen
Gel Extraction kit and sequenced on an ABI 3130xl
sequencer using the BigDye Cycle Sequencing kit.
Sequences were assembled with Geneiou s v4.6.1 [78] and
checked for their pote ntial to be correctly transcribed.
Gibbon haplotypes were deposited at GenBank and are
available under the accession numbers GU321245-
GU321329 (see also Additional file 1).
To prevent cross-species contaminations, laboratory
procedures followed described standards [46]. To exclude
contaminations of the dataset with numts, we mainly
used material in which nuclear DNA is highly degraded
Thinh et al. BMC Evolutionary Biology 2010, 10:74
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Page 9 of 13
(faeces, museum tissue) [79,80]. Moreover, the applied
primers are known to amplify solely th e mitochon drial
copy of the gene in hylobatids [34], and for cross-valida-
tion purposes, for some specimens, sequences were gen-
erated using different material types (blood, faeces).
Statistical Methods
For phylogenetic reconstructions, we expanded ou r data-
set with orthologous sequences from various hominids
(Homo,Pan,Gorilla,Pongo)andPapio hamadryas,
which was used as outgroup. Phylogenetic trees were
constructed with MP and NJ algorithms as implemented
in PAUP v4.0b10 [81] as well as with ML and Bayesian
algorithms, using the programs GARLI v0.951 [82] and
MrBayes v3.1.2 [ 83,84]. For MP anal ysis, all char acters
were treated as unordered and equally weighted through-
out. A heuristic search was performed with the maxi-
mumnumberoftreessetto100.ForNJandML
reconstructions, the optimal nucleotide substitution
model (GTR + Γ) was chosen using Akaike information
criterion (AIC) as implemented in MODELTEST v3.7
[85]. Relative support of internal nodes was performed by
bootstrap analyses with 10,000 (MP, NJ) or 500 replica-
tions (ML). In GARLI, only the model specificat ion set-
tings were adjusted according to the dataset, while all
other settings were left at their default value. ML major-
ity-rule consensus trees were calculated in PAUP. For
Bayesian reconstructions, the dataset was partitioned into
codon positions, each with its own substitution model.
We use d f our Markov Chain Monte Carlo (M CMC)
chains with the default temperature of 0.1. Four repeti-
tions were run for 10,000,000 generations with tree and
parameter sampling occurring every 100 generations.
The first 25% of samples were discarded as burnin, leav-
ing 75,001 trees per run. Posterior probabilities for each
split and a phylogram with mean branch lengths were
calculated from the posterior density of trees.
To estimate divergence times, a Bayesian MCMC
method, which employs a relaxed molecular clock
approach [86], as implemented in BEAST v1.4.8 [87],
was used. A relaxed lognormal model of lin eage varia-
tion and a Yule prior for branching rates was assume d.
The alignment was partitioned into codon positions,
and the substitution model, rate heterogeneity and base
frequencies were unlinked across codon positions. Opti-
mal nucleot ide substitution models were chosen using
AIC in MODELTEST.
For calibrations we used the fossil-based divergence
between Homo and Pan, which was dated at 6 - 7 mya
[88-90], the separation of Pongo from the Homo/Pan line-
age~14mya[91],andthedivergenceofhominoidsand
cercopithecoids ~23 mya [92,93]. Instead of hardbounded
calibration points, we used the published dates as a normal
distribution prior for the respective node. For the Homo -
Pan divergence, this translates into a normal distribution
with a mean of 6.5 mya and a standard deviation (SD ) of
0.5 mya, for the separation of Pongo from the Homo/Pan
clade into a mean of 14.0 mya and a SD of 1.0 mya, and
for the hominoid - cercopithecoid divergence into a mean
of 23 mya and a SD of 2 mya.
Since the estimation of phylogenetic relationships was
not the main aim of this analysis, for the calculation an a-
priori fixed tree topology as obtained from NJ reconstruc-
tions using the GTR + Γ model (Figure 2) was implemen-
ted. Four replicates were run for 10,000,000 generations
with tree and parameter sampling occurring every 100
generations. The adequacy of a 10% burnin and conver-
gence of all parameters were assessed by visual inspection
of the trace of the parameters across generations using
TRACER v1.4.1 [94]. Subsequently, the sampling distribu-
tions were combined (25% burnin) using the software Log-
Combiner v1.4.8, and a consensus chronogram with node
height distribution was generated and visualized with
TreeAnnotator v1.4.8 and FigTree v1.2.2 [95].
Additional file 1: Origin, material type, sample provider/collector and
GenBank accession numbers of studied gibbon specimens.
Click here for file
[ http://www.biomedcentral.com/content/supplementary/1471-2148-10-
74-S1.XLS ]
Acknowledgements
We are grateful to the following colleagues, zoos, and institutions for
providing permits or valuable gibbon materials: Claudia Barelli, Gareth
Goldthorpe, Andrew Kitchener, Nicolas Lormée, Annette Schrod, Chris
Smeenk, Berlin Zoo, Duisburg Zoo, Leipzig Zoo, Munich Zoo, Nuremberg
Zoo, Rostock Zoo, Schwerin Zoo, Wuppertal Zoo, Zurich Zoo, Besancon Zoo,
Plock Zoo, Banham Zoo, Bristol Zoo, Howletts Wild Animal Park, Paignton
Zoo, Twycross Zoo, Beijing Zoo, Dhaka Zoo, Jakarta Zoo, Taman Safari,
Singapore Zoo, Perth Zoo, Louisiana Purchase Gardens and Zoo, Bogor
Agricultural University, the Indonesian Institute for Science, Museum für
Naturkunde Berlin, National Museums Scotland Edinburgh, Natural History
Museum Leiden, National Museum of Natural History Washington, Institute
of Zoology of the Chinese Academy of Sciences Beijing, Bawangling
National Nature Reserve, Bokeo Nature Reserve, Cat Tien National Park,
Phong Nha-Ke Bang National Park and Khao Yai National Park. No
international or national rules and regulations have been violated during
sampling, and shipping. Many thanks also to Christiane Schwarz for her
excellent laboratory work, and to Colin Groves and two anonymous
reviewers for valuable comments on an earlier version of the manuscript.
This study was financially supported by the German Primate Center, and the
Biodiversitäts-Pakt of the Wissenschaftsgemeinschaft Gottfried-Wilhelm
Leibniz.
Author details
1
Primate Genetics Laboratory, German Primate Center, Kellnerweg 4, 37077
Göttingen, Germany.
2
Gibbon Conservation Center, PO Box 800249, Santa
Clarita, CA 91380, USA.
3
Anthropological Institute, University Zurich-Irchel,
Winterthurerstrasse 190, 8057 Zurich, Switzerland.
4
Laboratory of Animal
Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of
Sciences, 1 Beichen West Road, Chaoyang District, Beijing 100101, PR China.
5
Siberut Conservation Programme, Reproductive Biology Unit, German
Primate Center, Kellnerweg 4, 37077 Göttingen, Germany.
6
Department of
Clinic, Reproduction and Pathology, Faculty of Veterinary Medicine , Bogor
Agricultural University, Jl. Agatis, Kampus IPB Darmaga, 16680 Bogor,
Indonesia.
7
Parc Zoologique et Botanique de Mulhouse, 51, rue du Jardin
Thinh et al. BMC Evolutionary Biology 2010, 10:74
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Page 10 of 13
Zoologique, 68100 Mulhouse, France.
8
Frankfurt Zoological Society,
Endangered Primate Rescue Center, Cuc Phuong National Park, Nho Quan
District, Ninh Binh Province, Vietnam.
9
Gene Bank of Primates, German
Primate Center, Kellnerweg 4, 37077 Göttingen, Germany.
Authors’ contributions
VNT collected samples, did laboratory work, analysed the data, and wrote
the paper. ARM, TG, LM, TZ, MA, PM, and TN collected samples and wrote
the paper. LW analysed data, and wrote the paper. CR designed the study,
collected samples, did laboratory work, analysed data, and wrot e the paper.
All authors read and approved the final manuscript.
Received: 20 October 2009
Accepted: 12 March 2010 Published: 12 March 2010
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doi:10.1186/1471-2148-10-74
Cite this article as: Thinh et al.: Mitochondrial evidence for multiple
radiations in the evolutionary history of small apes. BMC Evolutionary
Biology 2010 10:74.
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