Diet Versus Phylogeny: a Comparison of Gut Microbiota in Captive Colobine Monkey Species.

Both diet and host phylogeny shape the gut microbial community, and separating out the effects of these variables can be challenging. In this study, high-throughput sequencing was used to evaluate the impact of diet and phylogeny on the gut microbiota of nine colobine monkey species (N = 64 individuals). Colobines are leaf-eating monkeys that fare poorly in captivity-often exhibiting gastrointestinal (GI) problems. This study included eight Asian colobines (Rhinopithecus brelichi, Rhinopithecus roxellana, Rhinopithecus bieti, Pygathrix nemaeus, Nasalis larvatus, Trachypithecus francoisi, Trachypithecus auratus, and Trachypithecus vetulus) and one African colobine (Colobus guereza). Monkeys were housed at five different captive institutes: Panxi Wildlife Rescue Center (Guizhou, China), Beijing Zoo, Beijing Zoo Breeding Center, Singapore Zoo, and Singapore Zoo Primate Conservation Breeding Center. Captive diets varied widely between institutions, but within an institution, all colobine monkey species were fed nearly identical or identical diets. In addition, four monkey species were present at multiple captive institutes. This allowed us to parse the effects of diet and phylogeny in these captive colobines. Gut microbial communities clustered weakly by host species and strongly by diet, and overall, colobine phylogenetic relationships were not reflected in gut microbiota analyses. Core microbiota analyses also identified several key taxa-including microbes within the Ruminococcaceae and Lachnospiraceae families-that were shared by over 90% of the monkeys in this study. Microbial species within these families include many butyrate producers that are important for GI health. These results highlight the importance of diet in captive colobines.

Parallel adaptive origins of digestive RNases in Asian and African leaf monkeys.

Similar morphological or physiological changes occurring in multiple evolutionary lineages are not uncommon. Such parallel changes are believed to be adaptive, because a complex character is unlikely to originate more than once by chance. However, the occurrence of adaptive parallel amino acid substitutions is debated. Here I propose four requirements for establishing adaptive parallel evolution at the protein sequence level and use these criteria to demonstrate such a case. I report that the gene encoding pancreatic ribonuclease was duplicated independently in Asian and African leaf-eating monkeys. Statistical analyses of DNA sequences, functional assays of reconstructed ancestral proteins and site-directed mutagenesis show that the new genes acquired enhanced digestive efficiencies through parallel amino acid replacements driven by darwinian selection. They also lost a non-digestive function independently, under a relaxed selective constraint. These results demonstrate that despite the overall stochasticity, even molecular evolution has a certain degree of repeatability and predictability under the pressures of natural selection.

Changes in fecal estrogen levels and sexual behavior in captive sichuan snub-nosed monkeys (Rhinopithecus roxellana) following a male replacement.

Rhinopithecus roxellana are generally seasonal breeders, although copulation can occur throughout the year. Previous studies suggest that estradiol modulates female sexual behavior during the mating season. However, the effects of social context on estrogen levels and behavior have not been fully explored. We studied the relationship between sexual behaviors and fecal estrogens in a group of captive R. roxellana during a period of social instability. We collected behavioral data for six months and collected fecal samples at 2-3-day intervals for four months spanning the mating and nonmating seasons, and analyzed fecal estrogen levels via RIA. Females showed clear cyclic solicitation and copulation peaks in the mating season, which corresponded with sharp peaks in fecal estrogens. During the nonmating season, solicitation rates, copulation rates, and fecal estrogens were generally low. However, one nonpregnant female displayed a sharp peak in solicitations, copulations, and estrogens during the nonmating season 10-14 days after a male replacement. Our results provide preliminary evidence that social and behavioral changes affect estrogen levels in R. roxellana.

Is Malaysia's banded langur, Presbytis femoralis femoralis, actually Presbytis neglectus neglectus? Taxonomic revision with new insights on the radiation history of the Presbytis species group in Southeast Asia.

The disjunct distribution of Presbytis femoralis subspecies across Sumatra (P. f. percura), southern (P. f. femoralis) and northern (P. f. robinsoni) Peninsular Malaysia marks the unique vicariance events in the Sunda Shelf. However, the taxonomic positions and evolutionary history of P. f. femoralis are unresolved after decades of research. To elucidate this evolutionary history, we analyzed 501 base pairs of the mitochondrial HVSI gene from 25 individuals representing Malaysia's banded langur, with the addition of 29 sequences of Asian Presbytis from Genbank. Our results revealed closer affinity of P. f. femoralis to P. m. mitrata and P. m. sumatrana while maintaining the monophyletic state of P. f. femoralis as compared to P. f. robinsoni. Two central theses were inferred from the results; (1) P. f. femoralis does not belong in the same species classification as P. f. robinsoni, and (2) P. f. femoralis is the basal lineage of the Presbytis in Peninsular Malaysia. Proving the first hypothesis through genetic analysis, we reassigned P. f. femoralis of Malaysia to Presbytis neglectus (Schlegel's banded langur) (Schlegel in Revue Methodique, Museum d'Histoire Naturelle des Pays-Bas 7:1, 1876) following the International Code of Zoological Nomenclature (article 23.3). The ancestors of P. neglectus are hypothesized to have reached southern Peninsular Malaysia during the Pleistocene and survived in refugium along the western coast. Consequently, they radiated upward, forming P. f. robinsoni and P. siamensis resulting in the highly allopatric distribution in Peninsular Malaysia. This study has successfully resolved the taxonomic position of P. neglectus in Peninsular Malaysia while providing an alternative biogeographic theory for the Asian Presbytis.

Effect of diet on the feces quality in javan langur (Trachypithecus auratus auratus).

A high intake of easily fermentable carbohydrates and a low intake of fiber material are generally regarded as major factors affecting the health of captive langurs. The effect on fecal consistency of excluding fruits and vegetables from the diet was evaluated in Javan langurs (Trachypithecus auratus auratus). Cross-over trials were carried out at Rotterdam Zoo and at the Apenheul Zoo, The Netherlands. During the first and third dietary period, the langurs were fed their usual diet, which contained fruits, vegetables, langur pellets, and browse. During the second period, the vegetables and fruits were excluded from the diet and the diet essentially consisted of pellets and browse. Feces consistency was scored using a fecal score chart developed for langurs. During the second feeding period the feces consistency improved significantly in animals at both zoos. Across all trials, a firmer feces consistency was correlated with an increase in dietary cell wall (measured as neutral detergent fiber) and a decrease in dietary water. It is suggested that the combined decrease in the intake of soluble sugars, the increase of fiber intake, and a lower amount of dietary water in the diet resulted in more solid stools. The results indicate that a dietary neutral detergent fiber content of approximately 46% in dry matter will result in a feces consistency indicative of undisturbed gut function.

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.