Pygathrix Research Articles

Characterization of the Complete Mitochondrial Genome of the Central Highland Grey-Shanked Douc Langur (Pygathrix cinerea), a Critically Endangered Species Endemic to Vietnam (Mammalia: Primates).

The grey-shanked douc langur (Pygathrix cinerea) is a recently described, critically endangered primate, endemic to Vietnam. In this study, we describe the Central Highland species' complete mitochondrial genome (mitogenome-mtDNA). It is a circular molecule with a length of 16,541 base pairs (bp). The genome consists of 37 genes, consistent with those found in most other vertebrates, including 13 protein coding genes, 22 transfer RNAs, and two ribosomal RNAs. A comparison with the mitogenomes of more than 50 primates showed that the mitogenome of Vietnamese Central Highland Pygathrix cinerea has a conservative gene order. We identified 43 nucleotide differences when comparing this genome with a previously published mitogenome of Pygathrix cinerea. It is evident that there are distinct differences between the Pygathrix cinerea we are currently studying and other Pygathrix cinerea specimens. These differences are unlikely to be solely the result of sequencing errors, as the mitogenomes were generated using high-quality methods. The genetic divergence observed between the two Pygathrix cinerea mitogenomes implies the potential existence of at least two distinct lineages or forms of this primate species within its native range in Vietnam.

ULTRASOUND-GUIDED PERCUTANEOUS TREATMENT OF HEPATIC HYDATIDOSIS IN TWO RED-SHANKED DOUC LANGURS (PYGATHRIX NEMAEUS).

Hydatid disease caused by Echinococcus ortleppi is a major cause of morbidity and mortality for critically endangered captive langurs in northern Vietnam. The most common sites for hydatid cyst development are the liver and the lungs. Chemotherapy trials with albendazole alone or in combination with praziquantel in these langurs had varying results and in most cases were ineffective. Ultrasound-guided percutaneous aspiration of cystic fluid followed by instillation and re-aspiration of a scolicidal agent (PAIR technique), has gained importance in the treatment of active hepatic cysts in humans and in many cases is preferred over surgical and endoscopic approaches. The PAIR technique was used in two red-shanked douc langurs (Pygathrix nemaeus) for the treatment of unilocular active hepatic cysts. The technique was found to be fairly easy to perform, safe, and effective and should be a useful treatment tool for hepatic hydatidosis in langurs and other nonhuman primate species.

RETROSPECTIVE REVIEW OF UROLITHIASIS-RELATED MORBIDITY AND MORTALITY IN ASIAN COLOBINE MONKEYS.

Obstructive and incidental urolithiasis cases were evaluated in 5 Asian colobine monkey species (n= 21 individuals) that included 12 silvery langurs (Trachypithecus cristatus), 6 spectacled langurs (Trachypithecus obscurus), 1 Javan langur (Trachypithecus auratus auratus), 1 François' langur (Trachypithecus francoisi), and 1 red-shanked douc langur (Pygathrix nemaeus) from eight zoologic institutions. All institutions that responded were Association of Zoos and Aquariums, European Association of Zoos and Aquaria, or World Association of Zoos and Aquariums accredited. Males were more commonly represented in the total number of cases (86%), and all cases of obstructive urolithiasis occurred in males. The most common clinical signs observed in obstructive cases included stranguria (58.8%), lethargy (41.2%), anorexia (29.4%), depression (17.6%), and penile manipulation (11.8%). Clinicopathologic abnormalities revealed azotemia (76.5%), anemia (35.3%), and hyperkalemia (23.5%). Eleven of the 21 cases included urinalysis results, and crystalluria was reported in all 11. Obstructive cases were more commonly managed surgically, with medical management following. Instances of individual obstruction ranged from 0 (incidental finding) to 18, with a median of 8 (mean of 4.3) recurrent obstructions. A total of 39 urolith analyses were available from 17 of 21 cases, with calcium carbonate being the most common type isolated (37 of 39, 94.9%). Calcium oxalate was observed in the remaining 5.1% of cases (2 of 39). No cases exhibited a consistent match of crystalluria and urolith type. Death or euthanasia secondary to obstructive urolithiasis occurred in 52.4% of cases. Urinary obstruction secondary to urolithiasis appears to be a relevant cause of morbidity and mortality in Asian colobine species, and further study into etiology and preventive medicine should be undertaken.

Group Composition and Social Structure of Red-Shanked Doucs (Pygathrix nemaeus) at Son Tra Nature Reserve, Vietnam

Multilevel societies, consisting of multiple one-male multi-female units, are relatively rare among primates, but are more widespread in the odd-nosed colobines than other taxa. Multilevel societies are found particularly in snub-nosed monkeys (Rhinopithecus)and have been debated in studies of proboscis monkeys (Proboscis). While it has been suggested that douc langurs (Pygathrix) may also form multilevel societies, the limited data available make the details of their social organization unclear. We aimed to establish a more comprehensive picture of the social organization of red-shanked doucs (Pygathrix nemaeus) and to address the question of whether this species forms multilevel societies, specifically collections of multiple distinct one-male units hereafter termed “bands.” We collected 259 h of behavioral data at Son Tra Nature Reserve in Vietnam from February 2010 to May 2011. The mean band size was approximately 18 individuals. Bands were comprised of approximately 2.7 units, and each unit contained approximately 6.5 individuals. Units had an average sex ratio of 1.0:1.6. We observed fission and fusion behaviors which were not correlated with phenological or weather measures. Activity budget data showed that fission and fusion behaviors between units were positively correlated with activity. Both vocalizations and vigilance increased when units engaged in fission. Based on this evidence, P. nemaeus at Son Tra Nature Reserve appear to engage in daily fission-fusion activity which does not vary between seasons. Additionally, our data suggest that these primates may form multilevel societies made up of distinct units. However, future data including proximity pattern analyses are necessary for confirmation.

Hand preference for a bimanual coordinated task in captive hatinh langurs (Trachypithecus hatinhensis) and grey-shanked douc langurs (Pygathrix cinerea)

Right-handedness in humans reflects the functional brain specialisation of the left hemisphere. To better understand the origins of this population-level tendency, it is crucial to understand manual lateralisation in other non-human primate species. The aim of this article is to present a first approach to the hand preference of two primates from Vietnam, the endangered hatinh langur (Trachypithecus hatinhensis) and the critically endangered grey-shanked douc langur (Pygathrix cinerea). Eighteen individuals from each species (N = 36) were evaluated by means of the bimanual coordinated tube task and their responses were recorded in terms of manual events and bouts. Our results showed that subjects presented strong individual-level preferences but not lateralisation at the group-level. No sex differences were detected within species. The index finger was used in all of the extractions during this bimanual task, alone (86 %) or in combination with other fingers (14 %). In addition, hatinh langurs exhibited a greater strength of hand preferences than grey-shanked douc langurs, pointing to a possible higher manual specialisation during the leaf-eating process. These findings help to broaden our scarce knowledge of manual laterality in Asian colobine monkeys and confirm the bimanual tube task as a sensitive measure for assessing manual laterality in non-human primates.

Unsupervised Acoustic Analysis of the Vocal Repertoire of the Gray-Shanked Douc Langur (Pygathrix cinerea)

Unsupervised analyses of nonhuman vocal repertoires are important not only because they allow decoding species-specific displays, but also to understand the evolution of communication signals, which need objective quantitative investigations. We recorded more than 1000 vocalizations emitted by captive gray-shanked douc langur (Pygathrix cinerea). Putative vocal categories were first identified by their overall acoustic structure, considering duration and spectral properties. We processed each call using an automatic pairwise Dynamic Time Warping comparison, limiting the use of a priori assumptions. We then performed a cluster analysis that indicated the presence of 25 clusters, which we then labeled to seven vocal types according to spectrographic information.

Functional morphology of the douc langur (Pygathrix spp.) scapula.

Most colobine monkeys primarily move through their arboreal environment quadrupedally. Douc langurs (Pygathrix spp.), however, are regularly observed to use suspensory behaviors at the Endangered Primate Rescue Center (EPRC) in Northern Vietnam. Previous work has linked variation in scapular morphology to different modes of primate arboreal locomotion. Here we investigate whether the shape of the Pygathrix scapula resembles obligate brachiators (gibbons) or obligate arboreal quadrupeds (other cercopithecoids). Using a MicroScribe G2X 3D digitizer, the positions of 17 landmarks were recorded on 15 different species of nonhuman primates (n = 100) from three categories of locomotor behavior: brachiator, arboreal quadruped, and unknown (Pygathrix). All analyses were conducted in the R package geomorph. A Procrustes analysis uniformly scaled the shape data and placed specimens into the same morphospace. A Principal Component Analysis was used to examine scapular shape and a Procrustes ANOVA was conducted to test for shape difference in the scapulae. A pairwise analysis was used to compare the means of the locomotor categories and identify any statistically significant differences. A phylogenetically controlled Procrustes ANOVA was also conducted using a phylogeny from 10kTrees. Results show Pygathrix scapular morphology is significantly different from both arboreal colobine quadrupeds (p < 0.01) and hylobatid brachiators (p < 0.01). It does, however, share some features with each including a long vertebral border, like other cercopithecoids, and a more laterally projecting acromion process, like the hylobatids. The principal difference segregating Pygathrix from both the arboreal quadrupeds and the brachiators is the more medially placed superior angle. These nuanced morphological characteristics associated with suspensory behaviors may be useful for inferring suspensory locomotion in the primate fossil record.

A list of big mammals recorded in Quangtri province and their conservation significance

Mammal fauna in Quangtri province was poorly studied. Most of surveys were conducted during period from 2002 to 2007 and concentrated in DaKrong nature reserve (NR) and Bac Huonghoa NR. Totally, 54 species of big mammals belonging to 20 families and 7 orders has been inventoried, of which 49 species with confirmed record and 6 species with temporary record. Number of species recorded for DaKrong PA is 49 species, for Bac Huonghoa PA is 47 species and Legendary Hochiminh trail PA is 25 species. Out of 54 species recorded, 37 species are of high conservation concern, including 30 species enlisted in Red Data Book of Vietnam (2000), 26 species in 2004 IUCN Red List of Threatened Species, 36 species in Governmental Decree 32/2006/ND-CP and 6 species endemic to Indochina. Forest ecosystems of Quangtri province are especially important for conservation of Sao la (Pseudoryx nghetinhensis), white-cheeked gibbon (Nomascus leucogenys), red-shanked douc langur (Pygathrix nemaeus), serow (Naemorhedus sumatraensis), Indochinese striped rabbit (Nesolagus timminsi) and (Trachypithecus laotum hatinhensis).

Seasonality of group size, feeding, and breeding in wild red‐shanked douc langurs (Lao PDR)

In Asian colobines, small one-male groups (OMG) seem to predominate alongside all-male groups (AMG), while larger multimale groups (MMG) are rare, but are reported for Hanuman langurs and red-shanked douc langurs. Recently, however, it has been speculated that the genus Pygathrix could have multilevel societies based on (1) a theoretical extension of the multilevel societies found in Rhinopithecus to all odd-nosed colobines and (2) first data for black-shanked douc langurs. This assumes bands composed of small OMG with a skewed adult sex ratio. Band size may vary with seasonal food availability resulting in smaller bands when feeding competition is increased. To investigate the social organization of red-shanked douc langurs and potential seasonal influences, we observed 2 unhabituated groups at Hin Namno National Protected Area, Lao PDR from March 2007 to August 2008 for 803 hr. We recorded births and performed group counts and scan sampling of feeding behavior. Most births (79% of N = 15) occurred from June-September, indicating a 4-month peak conception season from November to February. Group size averaged 24.5 individuals (range 17-45) with 2.45 adult males (range 1-4). Although the smaller group remained at a stable size (about 18 individuals), the larger group reduced from about 45 to 25 individuals during the 7-months long lean season, when less than 50% of the feeding time was spent on fruits. This suggests feeding competition as a potential cause of seasonal variation in group size. With 1.9 females per male the skew in adult sex ratio was much lower compared with Rhinopithecus, indicating MMG rather than multilevel societies. However, data on the spacing and interaction patterns between recognized individuals need to be collected and analyzed before the social organization can be determined. Detailed ecological data are furthermore required to investigate the basis for the seasonal changes in group size found.

Airborne Mycobacterium avium infection in a group of red‐shanked douc langurs (Pygathrix nemaeus nemaeus)

This report describes an airborne Mycobacterium avium (MA)-infection in two red-shanked douc langurs (Pygathrix nemaeus nemaeus) from Cologne zoo. The two individuals and their tissues were investigated clinically (including x-rays), in pathology, in pathohistology, in classical bacteriology and by polymerase chain reaction (PCR). Clinically, one individual displayed emaciation and a positive reaction in an intrapalpebral testing for M. bovis/MA. The other individual was without any symptoms and did not show any reaction in the intrapalpebral test. In x-ray photos of the lungs, calcified nodules were detected. In pathology, calcified and necrotic nodules were observed within the lungs and the bronchial lymph nodes. In pathohistology, both classical tuberculous granulomas, and few acid fast rods were seen in Ziehl-Neelsen-stain. However, classical bacteriology could not demonstrate mycobacteria. In PCR, MA-infection could be confirmed in one individual using the bronchial lymph nodes. It was an airborne infection; however, the definite source of infection in these cases remained unclear. Animals in contact to the langurs (house sparrows and mice) as well as water used in the building are the most promising candidates. The risk for a zoonotic transmission in these cases has been calculated to be low.

Distribution and Population Densities of Diurnal Primates in the Karst Forests of Phong Nha – Ke Bang National Park, Quang Binh Province, Central Vietnam

We studied the status and distribution of the diurnal primates in the Phong Nha – Ke Bang National Park (PNKB NP) from April to August 2007. In the past, researchers reported 9 primate species and subspecies for the karst forests of PNKB NP, which constitutes the most important protected area for the endangered Hatinh langur (Trachypithecus hatinhensis) in Vietnam. All 9 primate taxa are threatened due to hunting pressure and habitat loss. We applied line transect sampling in 4 areas. During a total of 117 transect inspections along 12 different transect routes, we confirmed 5 primate taxa and the analysis of population densities resulted in 2143 (±467) Hatinh langurs, 1316 (±871) red-shanked douc langurs (Pygathrix nemaeus), 930 (±489) stump-tailed macaques (Macaca arctoides), 986 (±883) eastern Assamese macaques (M. a. assamensis), and 18 (±18) southern white-cheeked crested gibbons (Nomascus siki) in the whole PNKB NP, which covers an area of ca. 85,000 ha. We could not detect the 2 nocturnal lorises, Bengal slow lorises (Nycticebus bengalensis) and pygmy slow lorises (N. pygmaeus), as well as rhesus macaques (Macaca mulatta) and northern pig-tail macaques (M. leonina). The distribution of the primates predominantly depended on human impact. We could not recognize a correlation between habitat constitution and abundance of primates. The population density estimates showed a much higher density of the Hatinh langur than previously assumed. Thus the importance of the PNKB NP for the conservation of this endangered langur increased significantly.

Diet of Pygathrix nigripes in Southern Vietnam

Black-shanked douc langurs (Pygathrix nigripes) are Southeast Asian colobines about which primatologists know very little, but they are classed as endangered because of population decline due to habitat loss. Two preliminary studies have shown that this monkey is primarily folivorous, but there are few details of plant selection or seasonal changes in diet. We set out to observe douc langurs directly in the wild to quantify the diet during wet and dry seasons. We confirmed that the species relies on foliage throughout the year, but it includes significant proportions of fruit and flowers in its diet when those items are available. The douc langurs selected various parts from 152 species of plants in 2 national parks, but there did not appear to be favorite species that were heavily selected over others. In both the wet and dry seasons, the black-shanked douc langurs ate mostly leaves, but the diversity of plants consumed increased and the proportion of fruit eaten almost doubled in the wet season. There were diurnal shifts in food selection, but we found no evidence that the douc langurs were foraging strategically to maximize their protein or energy intake.

The Time is Now: Survival of the Douc Langurs of Son Tra, Vietnam

Red-shanked douc langur (Pygathrix nemaeus) groups were located in the Son Tra Nature Reserve, Da Nang, Vietnam, and two preliminary censuses were undertaken in December 2006 and April 2007. Several teams located and counted Douc Langur groups by direct observation. Large numbers of groups with multiple members were located at various sites in the nature reserve. These findings are highly significant because reports over the last several years have recorded low numbers of Douc groups, and some have predicted an entire loss of doucs in Son Tra Nature Reserve. The numbers of Douc groups and individuals is also very significant since they may represent at least 60% of all of the living red shank Douc Langurs in Vietnam.

Molecular phylogenetic affinities of the simakobu monkey ( Simias concolor)

The simakobu (Simias concolor), or pig-tailed snubnosed langur, is one of four endemic primate species on the Mentawai Islands oV the west coast of Sumatra, Indonesia, and is currently considered a monotypic genus (BrandonJones et al., 2004; Miller, 1903). This species, like many other colobines, is a specialized folivore found in tropical rainforests. The simakobu monkey is a member of the “odd-nosed” group of Asian colobines, an informal grouping that also includes the proboscis monkey of Borneo (Nasalis larvatus), the snub-nosed monkeys of China and Vietnam (Rhinopithecus spp.), and the douc langurs of Vietnam, Laos, and Cambodia (Pygathrix spp.). There is no agreement, however, on the relationships among these genera, nor whether they even form a natural taxonomic or phylogenetic group (Delson, 1975; Groves, 1970, 2001; Jablonski, 1998; though see Sterner et al., in press). Geographically, the simakobu is found closest to the proboscis monkey, and some morphological data have indicated that these species are sister taxa. Both Simias and Nasalis exhibit narrow, long-faced skulls; long narrow nasal bones; and similar hair patterns (Groves, 1970). These two species appear to share more traits than do Rhinopithecus and Pygathrix, and some researchers have suggested that Simias should be subsumed into the genus Nasalis (Delson, 1975; Groves, 1970), perhaps as a subgenus (Delson, 1975); Groves (2001; Brandon-Jones, et al., 2004) appears to have retreated from the close linkage of these genera, though is not necessarily opposed to it (Groves, pers. comm.).

Mitochondrial data support an odd-nosed colobine clade

To obtain a more complete understanding of the evolutionary history of the leaf-eating monkeys we have examined the mitochondrial genome sequence of two African and six Asian colobines. Although taxonomists have proposed grouping the “odd-nosed” colobines (proboscis monkey, douc langur, and the snub-nosed monkey) together, phylogenetic support for such a clade has not been tested using molecular data. Phylogenetic analyses using parsimony, maximum likelihood, and Bayesian methods support a monophyletic clade of odd-nosed colobines consisting of Nasalis, Pygathrix, and Rhinopithecus, with tentative support for Nasalis occupying a basal position within this clade. The African and Asian colobine lineages are inferred to have diverged by 10.8 million years ago (mya or Ma). Within the Asian colobines the odd-nosed clade began to diversify by 6.7 Ma. These results augment our understanding of colobine evolution, particularly the nature and timing of the colobine expansion into Asia. This phylogenetic information will aid those developing conservation strategies for these highly endangered, diverse, and unique primates.

Learning the ropes: The ontogeny of locomotion in red-shanked douc (Pygathrix nemaeus), Delacour's (Trachypithecus delacouri), and Hatinh langurs (Trachypithecus hatinhensis) I. positional behavior

Recent studies at the Endangered Primate Rescue Center (EPRC) in the Socialist Republic of Vietnam's Cuc Phuong National Park by Byron et al. ([2002] Am. J. Phys. Anthropol. [Suppl.] 34:51) and Covert and Byron ([2002] Caring for Primates) revealed unexpected locomotor and postural behaviors in the red-shanked douc langur (Pygathrix nemaeus). This paper further elucidates the question of red-shanked douc suspensory behavior, and provides initial positional behavior data for two other rare Asian colobines, by comparing the ontogenetic positional behavior of red-shanked douc langurs, Delacour's langurs (Trachypithecus delacouri), and Hatinh langurs (Trachypithecus hatinhensis) at the EPRC. Two hundred and seven hours of positional behavior data were collected, with approximately equal amounts of data on each species, and equal amounts on adults and those less than 18 months in age. All young animals were more active than adults, used a wider repertoire of locomotor behaviors, and expressed suspensory behaviors more frequently than did the adults. Young animals also "invented" one new locomotor and two new postural behaviors. These differences are due to both play and explorative behavior, as well as to the youths' changing musculoskeletal systems. The number of positional behaviors utilized by the adults of these species is quite similar to one another (23-32), as is that utilized by the young (51-56). Douc langurs in both age categories used suspensory behaviors more frequently than did Delacour's and Hatinh langurs. Because the uniformity of enclosures offers a control, the results of this study generate hypotheses regarding adaptive radiations and niche partitioning in wild populations.

Ovarian Cycle and Effect of Social Changes on Adrenal and Ovarian Function in Pygathrix nemaeus

To assist captive breeding of the endangered red-shanked douc langur (Pygathrix nemaeus), basic knowledge on female reproductive physiology is important. We aimed 1) to characterize the pattern of fecal estrogens and progestogens during the ovarian cycle and 2) to use the information to provide reliable data on ovarian cycle characteristics. Moreover, we examined the potential impact of a change in group composition on ovarian activity and adrenal function, the latter being measured by fecal glucocorticoid excretion. We collected fecal samples during 2 mo from 5 adult females of 3 groups under undisturbed conditions and from the same 5 adult females and 2 juvenile females and 2 adult males during 3 mo during which there were changes in housing and group composition. During the undisturbed period, measurement of fecal 5-P-3OH, a major progesterone metabolite, and total estrogens revealed in all adult females a cyclic pattern, from which we documented a cycle length of 26.4 ± 1.8 days, comprising a follicular phase of 13.1 ± 2.9 days and a luteal phase of 14.8 ± 1.2 days. In contrast, during the period of social changes, 3 of the 5 females showed clear signs of cycle irregularity as indicated by extended follicular phases, abbreviated luteal phases and/or anovulation. Moreover, the period of social changes was associated with an overall significant elevation in fecal glucocorticoid excretion. Our data provide the first detailed information on the endocrine characterization of the ovarian cycle in Pygathrix nemaeus and suggest that social changes have the potential to impair ovarian function, likely as a result of increased activation of the HPA-axis due to stressful situations. However, because of relatively small sample size, particularly concerning the latter finding, more data are needed to confirm these results.

Unexpected locomotor behaviour: brachiation by an Old World monkey (Pygathrix nemaeus) from Vietnam

AbstractMore than 70 h of positional behaviour data were collected on the red‐shanked douc langur Pygathrix nemaeus, Delacour's langur Trachypithecus delacouri, Hatinh langur Trachypithecus laotum, and the white‐cheeked crested gibbon Hylobates leucogenys in January and February of 2001 at the Endangered Primate Rescue Center of Cuc Phuong National Park, Ninh Binh Province, Vietnam. Equal amounts of instantaneous and continuous bout data were collected for each of the four species. All animals were housed in enclosures of 10×5×3.5 m with similar substrates, and lived in small, species‐appropriate social groups. The positional behaviour of white‐cheeked crested gibbons was as expected with &gt;80% of locomotor bouts and 60% of postures being suspensory in nature. The positional behaviour of the Delacour's and Hatinh langurs was similar to that reported in the literature for other Asian colobines with &gt;90% of the locomotor bouts being quadrupedal walking, running, and bounding on tops of arboreal supports. Postures were dominated by sitting in and sitting out with forelimb suspension associated with &lt;10% of these behaviours. The positional behaviour of the red‐shanked douc langur is intermediate in nature between the Delacour's and Hatinh langur and the white‐cheeked crested gibbon. Locomotion is nearly evenly divided between arm‐swinging (46%) and quadrupedal walking and running bouts (54%). Forelimb suspensory postures were three times more frequent for the red‐shanked douc langur than they were for the Delacour's langur.

Parallel functional changes in the digestive RNases of ruminants and colobines by divergent amino acid substitutions.

A morphological or physiological trait may appear multiple times in evolution. At the molecular level, similar protein functions may emerge independently in different lineages. Whether these parallel functional changes are due to parallel amino acid substitutions has been a subject of debate. Here, I address this question using digestive ribonucleases (RNases) of two groups of foregut-fermenting mammals: ruminant artiodactyls and colobine monkeys. The RNase1 gene was duplicated twice in ancestral ruminants at least 40 MYA, and it was also duplicated in the douc langur, an Asian colobine, approximately 4 MYA. After duplication, similar functional changes occurred in the ruminant and monkey enzymes. Interestingly, five amino acid substitutions in ruminant RNases that are known to affect its catalytic activity against double-stranded (ds) RNA did not occur in the monkey enzyme. Rather, a similar functional change in the monkey was caused by a different set of nine substitutions. Site-directed mutagenesis was used to make three of the five ruminant-specific substitutions in the monkey enzyme. Functional assays of these mutants showed that one of the three substitutions has a similar effect in monkeys, the second has a stronger effect, and the third has an opposite effect. These results suggest that (1) an evolutionary problem can have multiple solutions, (2) the same amino acid substitution may have opposite functional effects in homologous proteins, (3) the stochastic processes of mutation and drift play an important role even at functionally important sites, and (4) protein sequences may diverge even when their functions converge.

Pseudogenization of the tumor-growth promoter angiogenin in a leaf-eating monkey

Physiological functions of human genes may be studied by gene-knockout experiments in model organisms such as the mouse. This strategy relies on the existence of one-to-one gene orthology between the human and mouse. When lineage-specific gene duplication occurs and paralogous genes share a certain degree of functional redundancy, knockout mice may not provide accurate functional information on human genes. Angiogenin is a small protein that stimulates blood-vessel growth and promotes tumor development. Humans and related primates only have one angiogenin gene, while mice have three paralogous genes. This makes it difficult to generate angiogenin-knockout mice and even more difficult to interpret the genotype-phenotype relation from such animals should they be generated. We here show that in the douc langur ( Pygathrix nemaeus), an Asian leaf-eating colobine monkey, the single-copy angiogenin gene has a one-nucleotide deletion in the sixth codon of the mature peptide, generating a premature stop codon. This nucleotide deletion is found in five unrelated individuals sequenced, and therefore is likely to have been fixed in the species. Five colobine species that are closely related to the douc langur have intact angiogenin genes, suggesting that the pseudogenization event was recent and unique to the douc langur lineage. This natural knockout experiment suggests that primate angiogenin is dispensable even in the wild. Further physiological studies of douc langurs may offer additional information on the role of this cancer-related gene in normal physiology of primates, including humans. Our findings also provide a strong case for the importance of evolutionary analysis in biomedical studies of gene functions.