Molecular phylogenetic inference of the howler monkey radiation (Primates: Alouatta).

Howler monkeys (Alouatta), comprising between nine and 14 species and ranging from southern Mexico to northern Argentina, are the most widely distributed platyrrhines. Previous phylogenetic studies of howlers have used chromosomal and morphological characters and a limited number of molecular markers; however, branching patterns conflict between studies or remain unresolved. We performed a new phylogenetic analysis of Alouatta using both concatenated and coalescent-based species tree approaches based on 14 unlinked non-coding intergenic nuclear regions. Our taxon sampling included five of the seven South American species (Alouatta caraya, Alouatta belzebul, Alouatta guariba, Alouatta seniculus, Alouatta sara) and the two recognized species from Mesoamerica (Alouatta pigra, Alouatta palliata). Similarly to previous studies, our phylogenies supported a Mesoamerican clade and a South American clade. For the South American howlers, both methods recovered the Atlantic Forest endemic A. guariba as sister to all remaining South American species, albeit with moderate support. Moreover, we found no support for the previously proposed sister relationship between A. guariba and A. belzebul. For the first time, a clade composed of A. sara and A. caraya was identified. The relationships among the other South American howlers, however, were not fully supported. Our estimates for divergence times within Alouatta are generally older compared to estimates in earlier studies. However, they conform to recent studies proposing a Miocene age for the Isthmus of Panama and for the uplift of the northern Andes. Our results also point to an early genetic isolation of A. guariba in the Atlantic Forest, in agreement with the hypothesis of biotic exchange across South American rain forests in the Miocene. Collectively, these findings contribute to a better understanding of the diversification processes among howler monkey species; however, they also suggest that further comprehension of the evolutionary history of the Alouatta radiation will rely on broadened taxonomic, geographic, and genomic sampling.

Comparing the selective landscape of TLR7 and TLR8 across primates reveals unique sites under positive selection in Alouatta.

Among primates, susceptibility to yellow fever (YFV), a single-stranded (ss) RNA virus, ranges from complete resistance to high susceptibility. Howler monkeys (genus Alouatta) are the most susceptible to YFV. In order to identify Alouatta-specific genetic factors that may be responsible for their susceptibility, we collected skin samples from howler monkey museum specimens of the species A. caraya and A. guariba clamitans. We compared the rate of nonsynonymous to synonymous (dN/dS) changes of Toll-like receptor (TLR) 7 and TLR8, the two genes responsible for detecting all ssRNA viruses, across the Primate order. Overall, we found that the TLR7 gene is under stronger purifying selection in howler monkeys compared to other New World and Old World primates, but TLR8 is under the same selective pressure. When we evaluated dN/dS at each codon, we found six codons under positive selection in Alouatta TLR8 and two codons under positive selection in TLR7. The changes in TLR7 are unique to A. guariba clamitans and are found in functionally important regions likely to affect detection of ssRNA viruses by TLR7/TLR8, as well as downstream signaling. These amino acid differences in A. guariba clamitans may play a role in YFV susceptibility. These results have implications for identifying genetic factors affecting YFV susceptibility in primates.

From Mexico to Michigan and back: An international collaboration investigating primate behavior, ecology, and evolution from multiple perspectives.

Evolutionary research benefits form the integration of laboratory and field components to determine factors and processes that affect the evolutionary trajectories of species. Our shared interest in understanding hybridization with genetic admixture as a process that may impact social, behavioral, and ecological features of primates, brought us together in a collaborative project aimed at addressing how vocal variation in two species of howler monkeys in Mexico affects and is affected by hybridization. To achieve this goal, we joined our academic expertise in studying primate genetics, ecology, and behavior under different natural and experimental conditions. We took advantage of decades of experience studying and handing wild howler monkeys for translocation projects to safely sample and study wild populations for this project. Here, we describe the history of our collaboration highlighting how our different perspectives, academic realities, and individual strengths built the foundation for our successful collaboration. We also share our perspectives on how this collaboration opened up new academic venues, broadened our individual perspectives on the integration of different research approaches to address a complex topic, and allowed us to recognize the strength of international collaboration.

Duplication and parallel evolution of the pancreatic ribonuclease gene (RNASE1) in folivorous non-colobine primates, the howler monkeys (Alouatta spp.).

In foregut-fermenting mammals (e.g., colobine monkeys, artiodactyl ruminants) the enzymes pancreatic ribonuclease (RNASE1) and lysozyme C (LYZ), originally involved in immune defense, have evolved new digestive functions. Howler monkeys are folivorous non-colobine primates that lack the multi-chambered stomachs of colobines and instead digest leaves using fermentation in the caeco-colic region. We present data on the RNASE1 and LYZ genes of four species of howler monkey (Alouatta spp.). We find that howler monkey LYZ is conserved and does not share the substitutions found in colobine and cow sequences, whereas RNASE1 was duplicated in the common ancestor of A. palliata, A. seniculus, A. sara, and A. pigra. While the parent gene (RNASE1) is conserved, the daughter gene (RNASE1B) has multiple amino acid substitutions that are parallel to those found in RNASE1B genes of colobines. The duplicated RNase in Alouatta has biochemical changes similar to those in colobines, suggesting a novel, possibly digestive function. These findings suggest that pancreatic ribonuclease has, in parallel, evolved a new role for digesting the products of microbial fermentation in both foregut- and hindgut-fermenting folivorous primates. This may be a vital digestive enzyme adaptation allowing howler monkeys to survive on leaves during periods of low fruit availability.

Phylogeography of the Mantled Howler Monkey (Alouatta palliata; Atelidae, Primates) across Its Geographical Range by Means of Mitochondrial Genetic Analyses and New Insights about the Phylogeny of Alouatta.

We analyzed 156 specimens of diverse howler monkey taxa (Alouatta; Atelidae, Primates) for different mitochondrial genes (5,567 base pairs), with special emphasis on A. palliata and related taxa. Our results showed no relevant differences among individuals of different putative taxa, A. p. palliata, A. p. aequatorialis, A. coibensis coibensis, and A. c. trabeata. We found no spatial differences in genetic structure of A. p. palliata throughout Costa Rica, Nicaragua, and Honduras. A. p. mexicana (genetic distance: 1.6-2.1%) was the most differentiated taxon within A. palliata. Therefore, we postulate the existence of only 2 clearly defined subspecies within A. palliata (A. p. palliata and A. p. mexicana). A. palliata and A. pigra (traditionally considered a subspecies of A. palliata) are 2 clearly differentiated species as was demonstrated by Cortés-Ortiz and colleagues in 2003, with a temporal split between the 2 species around 3.6-3.7 million years ago (MYA). Our results with the Median Joining Network procedure showed that the ancestors of the cis-Andean Alouatta gave rise to the ancestors of the trans-Andean Alouatta around 6.0-6.9 MYA. As Cortés-Ortiz et al. showed, A. sara and A. macconnelli are differentiable species from A. seniculus, although the first 2 taxa were traditionally considered subspecies of A. seniculus. Our findings agree with the possibility that the ancestor of A. sara gave rise to the ancestor of A. pigra in northern South America. In turn, the ancestor of A. pigra originated the ancestor of A. palliata. Two of our results strongly support the hypothesis that the South American A. palliata (the putative A. p. aequatorialis) was the original population of this species; it has high genetic diversity and no evidence of population expansion. The Central America A. palliata is the derived population. It has low genetic diversity and there is clear evidence of population expansion. However, A. palliata and A. pigra probably migrated into Central America by 2 different routes: the Isthmus of Panama (A. palliata) and Caribbean island arch (A. pigra). Finally, the red howler monkeys from the island of Trinidad in the Caribbean Sea were not A. macconnelli (= A. s. stramineus) as Groves maintained in his influential 2001 publication on primate taxonomy. This taxon is more related to A. s. seniculus, although it formed a monophyletic clade. Future molecular and karyotypic studies will show if the Trinidad red howler monkeys should be considered as an extension of the Venezuelan taxon, A. arctoidea, as a subspecies of A. seniculus(A. s. seniculus), or, in the case of extensive chromosomal rearrangements, even a new species.

Does home range use explain the relationship between group size and parasitism? A test with two sympatric species of howler monkeys.

Group size is related to parasite infections in primates. This relationship probably reflects the fact that group size is associated with body contact between group members and with contact with contaminated items in the environment. The latter is highly associated with range use. In the present study we hypothesized that if infection by directly transmitted parasites (DTP) is mainly determined by the exposure of individuals to parasites that accumulate in the environment, and group size correlates negatively with the intensity of home range use, then smaller groups should be more infected by DTP. Additionally, groups that share a higher proportion of their home range with other groups should be more infected. To test our hypothesis we observed and collected fecal samples of two groups of Alouatta palliata (large group size) and two groups of A. pigra (small group size) that live sympatrically in a forest fragment located in Macuspana (Mexico). Group size was positively correlated with range area size and negatively correlated with the intensity of home range use. Range use variables were not related to either DTP prevalence or load. However, there were significant differences in DTP loads between groups, which were positively correlated with group size. Our results suggest that the intensity of home range use is a poor predictor of DTP infection parameters in groups with marked differences in size. Therefore, it is possible that the individual or combined effects of other ecological (e.g., microclimate), social (e.g., contact rate), or physiological (e.g., immune function) factors are more important in the dynamics of DTP in free-ranging primates.

Ocorrência de Amblyomma aureolatum (Pallas, 1772) e A. ovale (Kock, 1844) (Acari: Ixodidae) parasitando Alouatta clamitans Cabrera, 1940 (Primates: Atelidae) na região norte do estado de Santa Catarina / Ocurrence of Amblyomma aureolatum (Pallas, 1772) and A. ovale (Kock, 1844) (Acari: Ixodidae) parasitizing Alouatta clamitans Cabrera, 1940 (Primates: Atelidae) in the north of Santa Catarina state, Brazil

The authors confirm the presence of one female and two males of Amblyomma aureolatum and five females and one male of A. ovale parasitizing southern brown howler monkeys in the municipalities of Indaial, Blumenau, Garuva, and Jaraguá do Sul, Santa Catarina State, Brazil.

Coalescent analysis of mtDNA indicates Pleistocene divergence among three species of howler monkey (Alouatta spp.) and population subdivision within the Atlantic Coastal Forest species, A. guariba.

We have used coalescent analysis of mtDNA cytochrome b (cyt b) sequences to estimate times of divergence of three species of Alouatta--A. caraya, A. belzebul, and A. guariba--which are in close geographic proximity. A. caraya is inferred to have diverged from the A. guariba/A. belzebul clade approximately 3.83 million years ago (MYA), with the later pair diverging approximately 1.55 MYA. These dates are much more recent than previous dates based on molecular-clock methods. In addition, analyses of new sequences from the Atlantic Coastal Forest species A. guariba indicate the presence of two distinct haplogroups corresponding to northern and southern populations with both haplogroups occurring in sympatry within Sao Paulo state. The time of divergence of these two haplogroups is estimated to be 1.2 MYA and so follows quite closely after the divergence of A. guariba and A. belzebul. These more recent dates point to the importance of Pleistocene environmental events as important factors in the diversification of A. belzebul and A. guariba. We discuss the diversification of the three Alouatta species in the context of recent models of climatic change and with regard to recent molecular phylogeographic analyses of other animal groups distributed in Brazil.

Anotaciones de la ecología alimentaria de monos aulladores negros en un fragmento con condiciones de hacinamiento (Balancán, Tabasco, México) / Anotações sobre a ecologia alimentar de bugios num fragmento superlotado (Balancán, Tabasco, México) / Annotations on the feeding ecology of black howler monkeys in an overcrowded fragment in Balancán, Tabasco, México

Se describió el comportamiento de alimentación del mono aullador negro centroamericano (Alouatta pigra) en un fragmento de menos de una hectárea de selva húmeda perennifolia rodeada de pastizales de uso ganadero, árboles dispersos y huertos de mango en la región de Balancán (Tabasco, México). Objetivo: reportar la dieta y actividad del mono aullador negro en condiciones de hacinamiento durante el mes más lluvioso en Balancán, Tabasco, México. Materiales y métodos: las observaciones conductuales (81 h) se hicieron mediante la metodología animal focal de una tropa de A. pigra constituida por tres individuos (un macho adulto, un hembra adulta y un infante). Resultados: del tiempo total de observación, la alimentación representó el 15,97%, el descanso el 78% y la locomoción el 4,57%. En el área de estudio se registraron 14 especies de plantas, de las cuales 10 fueron consumidas por los monos aulladores. El 55% del tiempo de alimentación fue invertido al consumo de frutos, 27% a las hojas, 17% al tallo y 1% a los retoños. La especie de árbol más consumida fue Spondias mombin, con el 61% del tiempo invertido. Conclusión: al igual que otros estudios, se confirmó la tendencia alimentaria folívoro-frugívora del género Alouatta. Se provee información sobre la flexibilidad que tienen los monos aulladores negros para vivir y mantenerse en condiciones de extrema perturbación donde su hábitat es de 0,1 hectáreas con pocas especies de árboles para utilizar, lo que además obliga a los individuos a hacer uso del suelo para trasladarse. Palabras clave: Alouatta pigra, Dieta, Patrón de actividad, Frugivoría, Hábitat fragmentado...

Annotations on the feeding ecology of black howler monkeys in an overcrowded fragment in Balancán, Tabasco, México. We described the feeding behavior of Central-American black howler monkey (Alouatta pigra) in a fragment of less than one hectare of humid evergreen forest surrounded by grassland for livestock use, isolated trees and mango orchards, in the region of Balancán (Tabasco, México). Objective: To record the diet and activity of black howler monkeys in crowded conditions during the wettest month in Balancán (Tabasco, México). Methods and methods: The behavioral observations (81 h) were made using the focal animal methodology with an A. pigra troop made up of three individuals (1 adult male, 1 adult female and 1 infant). Results: Feeding time represented 15.97%, rest 78% and locomotion 4.57% of total time. In the study area we recorded 14 plant species, with 10 being consumed by howler monkeys. 55% of feeding time was devoted to the consumption of fruits, 27% to leaves, 17% to stems, and 1% to sprouts. Spondias mombin was the tree species most consumed, with 61% of the time invested on it. Conclusion: Like other studies, we confirmed the frugivore-folivore feeding tendency of the genus Alouatta. We provide information on the black howler monkey flexibility to live and maintain themselves in extreme perturbation conditions, where their habitat is 0.1 ha with few tree species to use, forcing individuals to move around the ground...

Anotações sobre a ecologia alimentar de bugios num fragmento superlotado (Balancán, Tabasco, México). Descreve-se o comportamento alimentar do bugio centro-americano (Alouatta pigra) num fragmento menor que 1 ha de floresta úmida perenifólia rodeada por pastos para uso do gado, árvores dispersas e pomares de manga na região de Balacán (Tabasco, México). Objetivo: Registrar a dieta e atividade do bugio em condições de superlotação durante o mês mais chuvoso em Balacán, Tabasco, México. Materiais e métodos: As observações de comportamento (81 h) se fizeram por abordagem animal focal de um grupo de A. pigra constituído por três indivíduos (um macho adulto, uma fêmea adulta e um infante). Resultados: Do tempo total de observação, a alimentação representou 15,97%, o descanso 78% e a locomoção 4,57%. Na área de estudo se registraram 14 espécies de plantas das quais 10 foram consumidas pelos bugios. O 55% do tempo de alimentação foi gasto para o consumo de frutas, 27% às folhas, 17% ao caule e 1% aos brotos. A espécie de árvores mais consumida foi Spondias mombin, com 61% do tempo investido. Conclusão: Assim como outros estudos confirma-se a tendência alimentaria folívora-frugívora do gênero Alouatta. Fornece-se informação sobre a flexibilidade que têm os bugios para viver e ficar em condições de extrema perturbação, onde seu habitat é de 0,1 ha com poucas espécies de árvores para seu uso. Forçando, além disso, aos indivíduos a usar o terreno para se deslocar...

The Y chromosome of the Atelidae family (Platyrrhini): study by chromosome microdissection.

In order to study the intergeneric variability of the Y chromosome, we describe the hybridization of the Y chromosome of Brachytelesarachnoides, obtained by microdissection, to metaphases of Atelesbelzebuthmarginatus, Lagothrixlagothricha, and Alouatta male specimens. Brachytelesarachnoides (Atelinae) has 62 chromosomes and a very small Y chromosome. Our results showed that the Brachytelesarachnoides Y chromosome probe hybridized to Lagothrixlagothricha metaphases yielding one hybridization signal on only the tiny Y chromosome, and when hybridized with Atelesbelzebuthmarginatus metaphases it yielded one hybridization signal on two thirds of the small acrocentric Y chromosome. However, no hybridization signal was observed in Alouatta metaphases (subfamily Alouattinae), a closely related genus in the Atelidae family. Furthermore, our data support a close phylogenetic relationship among Brachyteles, Ateles, and Lagothrix and their placement in the Atelinae subfamily, but exclude Alouatta from this group indicating its placement as basal to this group.

Hybridization in large-bodied New World primates.

Well-documented cases of natural hybridization among primates are not common. In New World primates, natural hybridization has been reported only for small-bodied species, but no genotypic data have ever been gathered that confirm these reports. Here we present genetic evidence of hybridization of two large-bodied species of neotropical primates that diverged approximately 3 MYA. We used species-diagnostic mitochondrial and microsatellite loci and the Y chromosome Sry gene to determine the hybrid status of 36 individuals collected from an area of sympatry in Tabasco, Mexico. Thirteen individuals were hybrids. We show that hybridization and subsequent backcrosses are directionally biased and that the only likely cross between parental species produces fertile hybrid females, but fails to produce viable or fertile males. This system can be used as a model to study gene interchange between primate species that have not achieved complete reproductive isolation.

Sympatry between Alouatta caraya and Alouatta clamitans and the rediscovery of free-ranging potential hybrids in Southern Brazil.

Records of sympatry between Alouatta caraya and A. clamitans are rare despite their extensive range overlap. An example of their current sympatry and the rediscovery of free-ranging potential hybrids of A. caraya and A. clamitans in the forests of the Upper Paraná River, Southern Brazil, are reported in this paper. Eight groups were observed in the study area: five monospecific groups of A. caraya, two of A. clamitans, and a group containing two adult males and two adult females of A. caraya and a sub-adult male and two adult females identified as Alouatta sp. The color of the last three individuals was a mosaic between the two species; this is consistent with previously described variations in museum specimens collected in the Paraná River in the 1940s that had been identified as potential hybrids. The results from this study emphasize the need for scientific studies in the region of the Ilha Grande National Park, one of the few regions in the Paraná River that currently harbors both howler species.

A phylogeny of howler monkeys (Cebidae: Alouatta) based on mitochondrial, chromosomal and morphological data

The current taxonomic status of the species and subspecies belonging to the genus Alouatta is addressed by combined phylogenetic analysis using morphological, kariotipyc and molecular data (mitochondrial genes cytocrome oxidase II and cytochrome B). Our result demonstrated that Alouatta palliata is the most basal taxon for the genus in concordance with previous studies, as well as showing the validity of the taxon Alouatta sara as a species. Also our analysis shows that the sex chromosome has evolved from a XY/XX system to a X1X2Y1Y2/X1X1X2X2 system within the genus, as well as an increase in the size and complexity of the hioideal bone.

Genetic evidence for the coexistence of pheromone perception and full trichromatic vision in howler monkeys.

Vertebrate pheromones are water-soluble chemicals perceived mainly by the vomeronasal organ (VNO) for intraspecific communications. Humans, apes, and Old World (OW) monkeys lack functional genes responsible for the pheromone signal transduction and are generally insensitive to vomeronasal pheromones. It has been hypothesized that the evolutionary deterioration of pheromone sensitivity occurred because pheromone communication became redundant after the emergence of full trichromatic color vision via the duplication of the X-chromosome-linked red/green opsin gene in the common ancestor of hominoids and OW monkeys. Interestingly, full trichromacy also evolved in the New World (NW) howler monkeys via an independent duplication of the same gene. Here we sequenced from three species of howler monkeys an essential component of the VNO pheromone transduction pathway, the gene encoding the ion channel TRP2. In contrast to those of hominoids and OW monkeys, the howler TRP2 sequences have none of the characteristics of pseudogenes. This and other observations indicate that howler monkeys have maintained both their systems of pheromone communication and full trichromatic vision, suggesting that the presence of full trichromacy alone does not lead to the loss of pheromone communication. We suggest that the ecological differences between OW and NW primates, particularly in habitat selection, may have also affected the evolution of pheromone perception.

A phylogeny of howler monkeys (Cebidae: Alouatta) based on mitochondrial, chromosomal and morphological data.

The current taxonomic status of the species and subspecies belonging to the genus Alouatta is addressed by combined phylogenetic analysis using morphological, kariotipyc and molecular data (mitochondrial genes cytocrome oxidase II and cytochrome B). Our result demonstrated that Alouatta palliata is the most basal taxon for the genus in concordance with previous studies, as well as showing the validity of the taxon Alouatta sara as a species. Also our analysis shows that the sex chromosome has evolved from a XY/XX system to a X1X2Y1Y2/X1X1X2X2 system within the genus, as well as an increase in the size and complexity of the hioideal bone.

Phylogeny of neotropical monkeys: the interplay of morphological, molecular, and parasitological data.

Separate independent hypotheses of the phylogenetic relationships among the Platyrrhini monkeys have been produced in a recent past, either based upon morphological or molecular data, but the results are generally conflicting and the phylogeny of the group still is debated. The high host specificity observed among primates and their oxyurid parasites allows to consider the result of a morphologically based cladistic analysis of the pinworms of the Platyrrhini as an estimate of the phylogeny of these monkeys. Using the matrix representation method this "parasite-tree" is combined, using parsimony analysis, with several conflicting molecular or morphological hypothesis of the phylogeny of the host group. The results are discussed with respect to previously published classification, or composite computations of the phylogeny, of the Neotropical monkeys. Comparison of different hypothesis makes apparent several stable groups: (i) the Callithrichidae + Saimiri, (ii) the Atelidae/Alouattidae, (iii) the Pitheciidae, and (iv) the Alouattidae/Atelidae + Pitheciidae. In addition, the parasite and the molecular trees support close relationships between Callimico and Callithrix/Cebuella. The study also makes apparent that the parasite tree generally portrays the results of other studies, both when they are congruous and when they are conflicting. This is interpreted to be additional evidence for close coevolution between the Platyrrhini and their specific pinworms. Because, whatever the combination of data being considered no consensus can be found on the exact position of Aotus and Callicebus, and because it is likely that the earliest radiation of the Platyrrhini could be comparable to an evolutionary burst, which renders identification of homologous characters difficult, it is suggested that, possibly, not enough discriminating tracks of the evolutionary paths have been conserved to allow to resolve this uncertainty in the future.

The anterior dentition of adapids and anthropoid origins.

A recent discussion of the anterior teeth of adapids led to the conclusion that similarities to anthropoids are convergent. It is shown here that alternative functional and phylogenetic interpretations can be supported. These alternative interpretations are based on a different perspective on the anterior dentitions of extant and fossil anthropoids, which cannot be characterized by a single pattern of morphology and occlusion. More importantly, when this interspecific variation is placed within a phylogenetic framework it reveals persistent evolutionary trends that can be extrapolated back to infer a modest range of probable conditions in an Eocene anthropoid ancestor. The morphology and occlusion of adapid anterior teeth fall within this extrapolated range. This is compatible with an adapid ancestry for anthropoids.