Incomplete lineage sorting is common in extant gibbon genera.

We sequenced reduced representation libraries by means of Illumina technology to generate over 1.5 Mb of orthologous sequence from a representative of each of the four extant gibbon genera (Nomascus, Hylobates, Symphalangus, and Hoolock). We used these data to assess the evolutionary relationships between the genera by evaluating the likelihoods of all possible bifurcating trees involving the four taxa. Our analyses provide weak support for a tree with Nomascus and Hylobates as sister taxa and with Hoolock and Symphalangus as sister taxa, though bootstrap resampling suggests that other phylogenetic scenarios are also possible. This uncertainty is due to short internal branch lengths and extensive incomplete lineage sorting across taxa. The true phylogenetic relationships among gibbon genera will likely require a more extensive whole-genome sequence analysis.

Phylogenetic separation in limb use in captive gibbons (Hylobatidae): a comparison across the primate order.

Although there have been few studies of self-scratching in primates, some have reported distinct differences in whether hands or feet are used, and these variations seem to reflect the evolutionary history of the Order. Monkeys and prosimians use both hands and feet to self-scratch while African great apes use hands almost exclusively. Gibbons represent an evolutionary divergence between monkeys and great apes and incidental observations at the Gibbon Conservation Center pointed to a difference in self-scratching among the four extant gibbon genera (Hoolock, Nomascus, Symphalangus, and Hylobates). To validate and further explore these preliminary observations, we collected systematic data on self-scratching from 32 gibbons, including nine species and all four genera. To supplement gibbon data, we also collected self-scratching information from 18 great apes (four species), five prosimians (two species), 26 New World Monkeys (nine species) and 20 Old World Monkeys (seven species). All monkeys and some prosimians used both hands and feet to self-scratch, whereas one prosimian species used only feet. All African great apes used hands exclusively (orangutans were an exception displaying occasional foot-use). This appears to represent a fundamental difference between monkeys and great apes in limb use. Interestingly, there was a clear difference in self-scratching between the four gibbon genera. Hylobates and Symphalangus self-scratched only with hands (like all African great apes), while Hoolock and Nomascus self-scratched with both hands and feet (like monkeys and prosimians). This difference in gibbon behavior may reflect the evolutionary history of gibbons as Hoolock and Nomascus are thought to have evolved before both Hylobates and Symphalangus. What evolutionary pressures led to this divergent pattern is currently opaque; however, this shift in limb preference may result from niche separation across the order facilitating differences in the behavioral repertoire associated with hind and forelimbs.

An Alu-based phylogeny of gibbons (hylobatidae).

Gibbons (Hylobatidae) are small, arboreal apes indigenous to Southeast Asia that diverged from other apes ∼15-18 Ma. Extant lineages radiated rapidly 6-10 Ma and are organized into four genera (Hylobates, Hoolock, Symphalangus, and Nomascus) consisting of 12-19 species. The use of short interspersed elements (SINEs) as phylogenetic markers has seen recent popularity due to several desirable characteristics: the ancestral state of a locus is known to be the absence of an element, rare potentially homoplasious events are relatively easy to resolve, and samples can be quickly and inexpensively genotyped. During radiation of primates, one particular family of SINEs, the Alu family, has proliferated in primate genomes. Nomascus leucogenys (northern white-cheeked gibbon) sequences were analyzed for repetitive content with RepeatMasker using a custom library. The sequences containing Alu elements identified as members of a gibbon-specific subfamily were then compared with orthologous positions in other primate genomes. A primate phylogenetic panel consisting of 18 primate species, including 13 gibbon species representing all four extant genera, was assayed for all loci, and a total of 125 gibbon-specific Alu insertions were identified. The resulting amplification patterns were used to generate a phylogenetic tree. We demonstrate significant support for Symphalangus as the most basal lineage within the family. Our findings also place Nomascus as a derived lineage, sister to Hoolock, with the Nomascus-Hoolock clade sister to Hylobates. Further, our analysis groups N. leucogenys and Nomascus siki as sister taxa to the exclusion of the other Nomascus species assayed. This study represents the first use of SINEs to determine the genus level phylogenetic relationships within the family Hylobatidae. These relationships have been resolved with robust support at most internal nodes, demonstrating the utility of SINE-based phylogenetic analysis. We postulate that hybridization and rapid radiation may have contributed to the complex and contradictory findings of the previous studies. Our findings will aid in the conservation of these threatened primates and inform future studies of the biogeographical history and distribution of modern gibbon species.

Centromere remodeling in Hoolock leuconedys (Hylobatidae) by a new transposable element unique to the gibbons.

Gibbons (Hylobatidae) shared a common ancestor with the other hominoids only 15-18 million years ago. Nevertheless, gibbons show very distinctive features that include heavily rearranged chromosomes. Previous observations indicate that this phenomenon may be linked to the attenuated epigenetic repression of transposable elements (TEs) in gibbon species. Here we describe the massive expansion of a repeat in almost all the centromeres of the eastern hoolock gibbon (Hoolock leuconedys). We discovered that this repeat is a new composite TE originating from the combination of portions of three other elements (L1ME5, AluSz6, and SVA_A) and thus named it LAVA. We determined that this repeat is found in all the gibbons but does not occur in other hominoids. Detailed investigation of 46 different LAVA elements revealed that the majority of them have target site duplications (TSDs) and a poly-A tail, suggesting that they have been retrotransposing in the gibbon genome. Although we did not find a direct correlation between the emergence of LAVA elements and human-gibbon synteny breakpoints, this new composite transposable element is another mark of the great plasticity of the gibbon genome. Moreover, the centromeric expansion of LAVA insertions in the hoolock closely resembles the massive centromeric expansion of the KERV-1 retroelement reported for wallaby (marsupial) interspecific hybrids. The similarity between the two phenomena is consistent with the hypothesis that evolution of the gibbons is characterized by defects in epigenetic repression of TEs, perhaps triggered by interspecific hybridization.

Anatomical Contributions to Hylobatid Taxonomy and Adaptation.

Compared with the great apes, the small-bodied hylobatids were treated historically as a relatively uniform group with 2 genera, Hylobates and the larger-bodied Symphalangus. Four genera are now recognized, each with a different chromosome number: Hoolock (hoolock) (38), Hylobates (44), Nomascus (crested gibbon) (52), and Symphalangus (siamang) (50). Previous morphological studies based on relative bone lengths, e.g., intermembral indices; molar tooth sizes; and body masses did not distinguish the 4 genera from each other. We applied quantitative anatomical methods to test the hypothesis that each genus can be differentiated from the others using the relative distribution of body mass to the forelimbs and hind limbs. Based on dissections of 13 hylobatids from captive facilities, our findings demonstrate that each of the 4 genera has a distinct pattern of body mass distribution. For example, the adult Hoolock has limb proportions of nearly equal mass, a pattern that differentiates it from species in the genus Hylobates, e.g., H. lar (lar gibbon), H. moloch (Javan gibbon), H. pileatus (pileated gibbon), Nomascus, and Symphalangus. Hylobates is distinct in having heavy hind limbs. Although Symphalangus has been treated as a scaled up version of Hylobates, its forelimb exceeds its hind limb mass, an unusual primate pattern otherwise found only in orangutans. This research provides new information on whole body anatomy and adds to the genetic, ecological, and behavioral evidence for clarifying the taxonomy of the hylobatids. The research also underscores the important contribution of studies on rare species in captivity.

Patterns of genetic variation within and between Gibbon species.

Gibbons are small, arboreal, highly endangered apes that are understudied compared with other hominoids. At present, there are four recognized genera and approximately 17 species, all likely to have diverged from each other within the last 5-6 My. Although the gibbon phylogeny has been investigated using various approaches (i.e., vocalization, morphology, mitochondrial DNA, karyotype, etc.), the precise taxonomic relationships are still highly debated. Here, we present the first survey of nuclear sequence variation within and between gibbon species with the goal of estimating basic population genetic parameters. We gathered ~60 kb of sequence data from a panel of 19 gibbons representing nine species and all four genera. We observe high levels of nucleotide diversity within species, indicative of large historical population sizes. In addition, we find low levels of genetic differentiation between species within a genus comparable to what has been estimated for human populations. This is likely due to ongoing or episodic gene flow between species, and we estimate a migration rate between Nomascus leucogenys and N. gabriellae of roughly one migrant every two generations. Together, our findings suggest that gibbons have had a complex demographic history involving hybridization or mixing between diverged populations.

A sex difference in effect of prior experience on object-mediated problem-solving in gibbons.

Understanding the functionally relevant properties of objects is likely facilitated by learning with a critical role for past experience. However, current evidence is conflicting regarding the effect of prior object exposure on acquisition of object manipulation skills. This may be due to the influence of life history variables on the capacity to benefit from such experience. This study assessed effect of task-relevant object exposure on object-mediated problem-solving in 22 gibbons using a raking-in task. Despite not using tools habitually, 14 gibbons spontaneously used a rake to obtain a reward. Having prior experience with the rake in an unrewarded context did not improve learning efficiency in males. However, females benefitted significantly from the opportunity to interact with the rake before testing, with reduced latencies to solution compared to those with no previous exposure. These results reflect potential sex differences in approach to novelty that moderate the possible benefits of prior experience. Due to their relatively high energetic requirements, reproductively active females may be highly motivated to explore potential resources; however, increased investment in developing offspring could make them more guarded in their investigations. Previous exposure that allows females to learn of an object's neutrality can offset this cautious exploration.

A comparative study of crested gibbons (Nomascus).

Crested gibbons (Nomascus) are in the rarest genus of the family Hylobatidae, with the Hainan gibbon (Nomascus hainanus) being the rarest primate in the world. In the past, the number of species in this genus has been at the center of much controversy, in part, because their color changes during immaturity as well as other factors, such as physical similarities in genitalia, creating difficulties in accurately determining the sex of individuals. Furthermore, owing to their rarity, illusiveness, and the rough terrain that comprises their native habitat, Nomascus is one of the least studied Hylobatidae. This article represents the most comprehensive dissemination of visual characteristics of the genus Nomascus to assist in the accurate identification of captive and wild crested gibbons. Through differences in pelage color, skeletal anatomy, dentition, vocalizations, behavior, distribution, and genetic studies, we are able to determine more accurately whether or not a subspecies should be elevated to species level. From the current data, there are six species and one subspecies in the genus Nomascus. However, reports of a recently identified light-cheeked gibbon (Nomascus sp.) in northeast Cambodia, Central Vietnam, and South Lao PDR, will add additional taxa to this genus.

Mitochondrial evidence for multiple radiations in the evolutionary history of small apes.

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 individuals 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 approximately 6.7 and approximately 8.3 mya, the four gibbon genera diverged from each other. In the second (approximately 3.0 - approximately 3.9 mya) and in the third period (approximately 1.3 - approximately 1.8 mya), Hylobates and Hoolock differentiated. Finally, between approximately 0.5 and approximately 1.1 mya, Hylobates lar diverged into subspecies. In contrast, differentiation of Nomascus into species and subspecies was a continuous and prolonged process lasting from approximately 4.2 until approximately 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.

A small, variable, and irregular killer cell Ig-like receptor locus accompanies the absence of MHC-C and MHC-G in gibbons.

The killer cell Ig-like receptors (KIRs) of NK cells recognize MHC class I ligands and function in placental reproduction and immune defense against pathogens. During the evolution of monkeys, great apes, and humans, an ancestral KIR3DL gene expanded to become a diverse and rapidly evolving gene family of four KIR lineages. Characterizing the KIR locus are three framework regions, defining two intervals of variable gene content. By analysis of four KIR haplotypes from two species of gibbon, we find that the smaller apes do not conform to these rules. Although diverse and irregular in structure, the gibbon haplotypes are unusually small, containing only two to five functional genes. Comparison with the predicted ancestral hominoid KIR haplotype indicates that modern gibbon KIR haplotypes were formed by a series of deletion events, which created new hybrid genes as well as eliminating ancestral genes. Of the three framework regions, only KIR3DL3 (lineage V), defining the 5' end of the KIR locus, is present and intact on all gibbon KIR haplotypes. KIR2DL4 (lineage I) defining the central framework region has been a major target for elimination or inactivation, correlating with the absence of its putative ligand, MHC-G, in gibbons. Similarly, the MHC-C-driven expansion of lineage III KIR genes in great apes has not occurred in gibbons because they lack MHC-C. Our results indicate that the selective forces shaping the size and organization of the gibbon KIR locus differed from those acting upon the KIR of other hominoid species.

Evolutionary breakpoints in the gibbon suggest association between cytosine methylation and karyotype evolution.

Gibbon species have accumulated an unusually high number of chromosomal changes since diverging from the common hominoid ancestor 15-18 million years ago. The cause of this increased rate of chromosomal rearrangements is not known, nor is it known if genome architecture has a role. To address this question, we analyzed sequences spanning 57 breaks of synteny between northern white-cheeked gibbons (Nomascus l. leucogenys) and humans. We find that the breakpoint regions are enriched in segmental duplications and repeats, with Alu elements being the most abundant. Alus located near the gibbon breakpoints (<150 bp) have a higher CpG content than other Alus. Bisulphite allelic sequencing reveals that these gibbon Alus have a lower average density of methylated cytosine that their human orthologues. The finding of higher CpG content and lower average CpG methylation suggests that the gibbon Alu elements are epigenetically distinct from their human orthologues. The association between undermethylation and chromosomal rearrangement in gibbons suggests a correlation between epigenetic state and structural genome variation in evolution.

A chromosomal inversion unique to the northern white-cheeked gibbon.

The gibbon family belongs to the superfamily Hominoidea and includes 15 species divided into four genera. Each genus possesses a distinct karyotype with chromosome numbers varying from 38 to 52. This diversity is the result of numerous chromosomal changes that have accumulated during the evolution of the gibbon lineage, a quite unique feature in comparison with other hominoids and most of the other primates. Some gibbon species and subspecies rank among the most endangered primates in the world. Breeding programs can be extremely challenging and hybridization plays an important role within the factors responsible for the decline of captive gibbons. With less than 500 individuals left in the wild, the northern white-cheeked gibbon (Nomascus leucogenys leucogenys, NLE) is the most endangered primate in a successful captive breeding program. We present here the analysis of an inversion that we show being specific for the northern white-cheeked gibbon and can be used as one of the criteria to distinguish this subspecies from other gibbon taxa. The availability of the sequence spanning for one of the breakpoints of the inversion allows detecting it by a simple PCR test also on low quality DNA. Our results demonstrate the important role of genomics in providing tools for conservation efforts.

Primate TNF promoters reveal markers of phylogeny and evolution of innate immunity.

BACKGROUND: Tumor necrosis factor (TNF) is a critical cytokine in the immune response whose transcriptional activation is controlled by a proximal promoter region that is highly conserved in mammals and, in particular, primates. Specific single nucleotide polymorphisms (SNPs) upstream of the proximal human TNF promoter have been identified, which are markers of human ancestry. METHODOLOGY/PRINCIPAL FINDINGS: Using a comparative genomics approach we show that certain fixed genetic differences in the TNF promoter serve as markers of primate speciation. We also demonstrate that distinct alleles of most human TNF promoter SNPs are identical to fixed nucleotides in primate TNF promoters. Furthermore, we identify fixed genetic differences within the proximal TNF promoters of Asian apes that do not occur in African ape or human TNF promoters. Strikingly, protein-DNA binding assays and gene reporter assays comparing these Asian ape TNF promoters to African ape and human TNF promoters demonstrate that, unlike the fixed differences that we define that are associated with primate phylogeny, these Asian ape-specific fixed differences impair transcription factor binding at an Sp1 site and decrease TNF transcription induced by bacterial stimulation of macrophages. CONCLUSIONS/SIGNIFICANCE: Here, we have presented the broadest interspecies comparison of a regulatory region of an innate immune response gene to date. We have characterized nucleotide positions in Asian ape TNF promoters that underlie functional changes in cell type- and stimulus-specific activation of the TNF gene. We have also identified ancestral TNF promoter nucleotide states in the primate lineage that correspond to human SNP alleles. These findings may reflect evolution of Asian and African apes under a distinct set of infectious disease pressures involving the innate immune response and TNF.

Molecular refinement of gibbon genome rearrangements.

The gibbon karyotype is known to be extensively rearranged when compared to the human and to the ancestral primate karyotype. By combining a bioinformatics (paired-end sequence analysis) approach and a molecular cytogenetics approach, we have refined the synteny block arrangement of the white-cheeked gibbon (Nomascus leucogenys, NLE) with respect to the human genome. We provide the first detailed clone framework map of the gibbon genome and refine the location of 86 evolutionary breakpoints to <1 Mb resolution. An additional 12 breakpoints, mapping primarily to centromeric and telomeric regions, were mapped to approximately 5 Mb resolution. Our combined FISH and BES analysis indicates that we have effectively subcloned 49 of these breakpoints within NLE gibbon BAC clones, mapped to a median resolution of 79.7 kb. Interestingly, many of the intervals associated with translocations were gene-rich, including some genes associated with normal skeletal development. Comparisons of NLE breakpoints with those of other gibbon species reveal variability in the position, suggesting that chromosomal rearrangement has been a longstanding property of this particular ape lineage. Our data emphasize the synergistic effect of combining computational genomics and cytogenetics and provide a framework for ultimate sequence and assembly of the gibbon genome.

A high-resolution map of synteny disruptions in gibbon and human genomes.

Gibbons are part of the same superfamily (Hominoidea) as humans and great apes, but their karyotype has diverged faster from the common hominoid ancestor. At least 24 major chromosome rearrangements are required to convert the presumed ancestral karyotype of gibbons into that of the hominoid ancestor. Up to 28 additional rearrangements distinguish the various living species from the common gibbon ancestor. Using the northern white-cheeked gibbon (2n = 52) (Nomascus leucogenys leucogenys) as a model, we created a high-resolution map of the homologous regions between the gibbon and human. The positions of 100 synteny breakpoints relative to the assembled human genome were determined at a resolution of about 200 kb. Interestingly, 46% of the gibbon-human synteny breakpoints occur in regions that correspond to segmental duplications in the human lineage, indicating a common source of plasticity leading to a different outcome in the two species. Additionally, the full sequences of 11 gibbon BACs spanning evolutionary breakpoints reveal either segmental duplications or interspersed repeats at the exact breakpoint locations. No specific sequence element appears to be common among independent rearrangements. We speculate that the extraordinarily high level of rearrangements seen in gibbons may be due to factors that increase the incidence of chromosome breakage or fixation of the derivative chromosomes in a homozygous state.

A whole-arm translocation (WAT8/9) separating Sumatran and Bornean agile gibbons, and its evolutionary features.

Gibbons, like orangutans, are a group of threatened Asian apes, so that genetic monitoring of each species or subspecies is a pressing need for conservation programmes. We conducted a project to take, as far as possible, samples of known origin from wild-born animals from Sumatra and Borneo (Central Kalimantan) for genetic monitoring of agile gibbons. As a result, we found a whole arm translocation between chromosomes 8 and 9 (WAT8/9) specific to Sumatran agile gibbons. Furthermore, population surveys suggested that the form with the WAT8/9 seems to be incompatible with an ancestral form, suggesting that the former might have extinguished the latter from Sumatran populations by competition. In any case, this translocation is a useful chromosomal marker for identifying Sumatran agile gibbons. Population genetic analyses with DNA showed that the molecular genetic distance between Sumatran and Bornean agile gibbons is the smallest, although the chromosomal difference is the largest. Thus, it is postulated that WAT8/9 occurred and fixed in a small population of Sumatra after migration and geographical isolation at the last glacial period, and afterwards dispersed rapidly to other populations in Sumatra as a result of the bottleneck effect and a chromosomal isolating mechanism.

Genetic mechanism and property of a whole-arm translocation (WAT) between chromosomes 8 and 9 of agile gibbons (Hylobates agilis).

C-banding analysis with 47 gibbons of the subgenus Hylobates (Hylobates) (44-chromosome gibbons) uncovered that the gibbons had a characteristic complicated C-banding pattern. The C-band pattern also revealed that a whole-arm translocation (WAT) between chromosomes 8 and 9 existed only in the species H. agilis (agile gibbon). Comprehensive consideration allows postulation that the translocation seemed to be restricted to two subspecies: H. agilis agilis (mountain agile gibbon) and H. agilis unko (lowland agile gibbon), found in Sumatra and part of the Malay Peninsula. Moreover, combined intensive analyses of C-banding and chromosome painting provided strong evidence for a plausible evolutionary pathway of chromosome differentiation of chromosomes 8 and 9. The C-banded morph 8M(t/c) seemed to be the primary type of chromosome 8 in the subgenus and to have altered into the three morphs through three pericentric inversions. The newest morph (8A(M/ci)) produced by the third inversion exchanged the long arm for chromosome 9, and subsequently constructed the WAT morphs of 8/9A(Mc/ct) and 9/8M(i/ci).

Taxonomic note: Bunopithecus: A genus-level taxon for the hoolock gibbon (Hylobates hoolock).

The recent discovery that the hoolock gibbon (Hylobates hoolock [Harlan, 1834]) has a karyotype distinct from all other hylobatids provides a new and strong motive for revising gibbon taxonomy and establishing hoolocks in a separate, higher taxon. Revising Groves's taxonomy of 1972, we propose that hoolock, along with the fossil species sericus, occupy a subgenus, Bunopithecus. With the newly added taxon, the genus Hylobates would thus contain four subgenera: Bunopithecus, Hylobates, Nomascus, and Symphalangus.