Emergence of Mansonella sp. in free-ranging primates in southern Brazil.

Mansonellosis is a neglected and emerging tropical disease. Among all zoonotic filarial diseases, it is probably the most prevalent and least studied, with approximately 114 million people infected. The parasites of Mansonella spp. are among the most common blood parasitemias and are widely found in Africa and Latin America. Through molecular analysis of blood samples from free-ranging primates Sapajus nigritus (n 33) and Alouatta guariba clamitans (n 5) in the southern states of Brazil (Santa Catarina and Rio Grande do Sul), we identified samples positive for Mansonella perstans in two specimens of A. guariba clamitans. A fragment of 578 bp from the ITS intergenic region (5.8S-ITS2-28S) was targeted for an initial PCR screening. Subsequently, positive samples were subjected to other PCR assays targeting a fragment of the 12S and the 18S genes. This is the first record of molecular detection of the agent in this host in the Pampa Biome. With a wide distribution across Brazil and Argentina, these primates may represent a potential wild reservoir for the zoonotic agent of mansonellosis. Entomological and transmission studies are essential to avoid the urbanization of mansonellosis and to understand the cycles of agents in different environmental scenarios.

A New Circular Single-Stranded DNA Virus Related with Howler Monkey Associated Porprismacovirus 1 Detected in Children with Acute Gastroenteritis.

Putative replication-associated protein (REP) and capsid-like (CAP) proteins are encoded by circular single-stranded DNA viruses (CRESS DNA), which have been found in samples from most eukaryotic groups. However, the details of these viruses' life cycles and their significance in diseases have yet to be established. We presented and analyzed two full-length CRESS DNA genomes acquired from two children diagnosed with acute gastroenteritis (GI) in the northeast state of Tocantins, Brazil, using next-generation sequencing and a virus-like filtration approach. Both sequences (named SmaCV3BR08 and SmaCV3BR291) are closely similar to a prior CRESS DNA sequence discovered in the feces of a new world monkey (Alouatta caraya) from the United States in 2009 and termed Howler monkey-associated porprismacovirus 1 (Genbank ID: NC 026317). According to our comparative study, these porprismacovirus genomes deviate by 10% at the nucleotide level. For comparative reasons, the divergence between our sequences (SmaCV3BR08 and SmaCV3BR291) and a porprismacovirus recently identified in a human fecal sample from Peru is 37%. These data suggest that there is a great diversity of porprismacoviruses in South America, perhaps more than two species. In addition, the finding of closely related sequences of porprismacoviruses in humans and native monkeys highlights the zoonotic potential of these viruses.

Sex chromosome systems in Neotropical Primates: What have we learnt so far from cytogenetics and genomics?

Neotropical Primates (Platyrrhini) show great diversity in their life histories, ecology, behaviour and genetics. This diversity extends to their chromosome complements, both to autosomes and to sex chromosomes. In this contribution, we will review what is currently known about sex chromosomes in this group, both from cytogenetic and from genomic evidence. The X and Y chromosomes in Neotropical Primates, also known as New World Monkeys, have striking structural differences compared with Old World Monkeys when Catarrhini sex chromosomes are considered. The XY bivalent displays a different meiotic behaviour in prophase I, and their Y chromosome shows extensive genomic differences. Even though the most widespread sex chromosome system is the XX/XY and thus considered the ancestral one for Platyrrhini, modifications of this sexual system are observed within this group. Multiple sex chromosome systems originated from Y-autosome translocations were described in several genera (Aotus, Callimico and Alouatta). In the howler monkeys, genus Alouatta, an independent origin of the sexual systems in South American and Mesoamerican species was postulated. All the above-mentioned evidence suggests that the Y chromosome of Platyrrhini has a different evolutionary history compared with the Catarrhini Y. There is still much to understand regarding their sex chromosome systems.

Phylogenetic relationships, population demography, and species delimitation of the Alouatta belzebul species complex (Atelidae: Alouattinae).

Howler monkeys (genus Alouatta) exhibit the most extensive distribution among platyrrhines, comprising Mesoamerican and South American species groups, with the South American group including the Brazilian endemic A. belzebul species complex encompassing A. belzebul, A. discolor, and A. ululata. We herein analyzed their phylogenetic relationship, nucleotide and haplotype diversity, and population demography based on the mitochondrial gene cytochrome b. The phylogenetic and median-joining network analyses distinguished A. discolor, distributed in the west bank of the Xingu River, from A. belzebul on the east bank. This river is a zoogeographic barrier for these species. We did not find evidence of phylogenetic structure between the A. belzebul populations of opposite banks of the Tocantins River, likely related to the changes in the position of this river to the northeast in the late Pleistocene. The A. belzebul along this river showed great morphologic and haplotype diversity, and A. belzebul from the Amazon have kept a larger population size than A. discolor. We herein describe the karyotype of A. discolor, which was similar to those described for A. ululata and A. belzebul. Our results showed two well-defined and supported clades for A. discolor and A. belzebul. However, a new assessment of A. ululata across a large distribution of sampling is required due to the lack of a clear phylogenetic structure.

Dispersal patterns in black howler monkeys (Alouatta pigra): Integrating multiyear demographic and molecular data.

Dispersal is a fundamental process in the functioning of animal societies as it regulates the degree to which closely related individuals are spatially concentrated. A species' dispersal pattern can be complex as it emerges from individuals' decisions shaped by the cost-benefit tradeoffs associated with either remaining in the natal group or dispersing. Given the potential complexity, combining long-term demographic information with molecular data can provide important insights into dispersal patterns of a species. Based on a 15-year study that integrates multiyear demographic data on six groups with longitudinal and cross-sectional genetic sampling of 20 groups (N = 169 individuals, N = 21 polymorphic microsatellite loci), we describe the various dispersal strategies of male and female black howler monkeys (Alouatta pigra) inhabiting Palenque National Park, Mexico. Genetically confirmed dispersal events (N = 21 of 59 males; N = 6 of 65 females) together with spatial autocorrelation analyses revealed that the dispersal pattern of black howlers is bisexual with strong sex-biases in both dispersal rate (males disperse more often than females) and dispersal distance (females disperse farther than males). Observational and genetic data confirm that both males and females can successfully immigrate into established groups, as well as form new groups with other dispersing individuals. Additionally, both males and females may disperse singly, as well as in pairs, and both may also disperse secondarily. Overall, our findings suggest multiple dispersal trajectories for black howler males and females, and longer multiyear studies are needed to unravel which demographic, ecological and social factors underlie individuals' decisions about whether to disperse and which dispersal options to take.

La dispersión es un proceso fundamental en el funcionamiento de las sociedades animales, ya que regula el grado en que los individuos parentados se concentran espacialmente. El patrón de dispersión de una especie puede ser complejo ya que surge de las decisiones de los individuos conformadas por las compensaciones de costo-beneficio asociadas con permanecer en el grupo natal o dispersarse. Dada esta posible complejidad, la combinación de información demográfica a largo plazo con datos moleculares puede proporcionar información importante sobre los patrones de dispersión de una especie en particular. Basado en un estudio de 15 años que integra datos demográficos de seis grupos sociales con muestreo genético longitudinal y transversal de 20 grupos (N = 169 individuos, N = 21 loci de microsatélites polimórficos), describimos las diversas estrategias de dispersión de machos y hembras del mono aullador negro (Alouatta pigra) que habitan el Parque Nacional Palenque, México. Los eventos de dispersión confirmados genéticamente (N = 21 de 59 machos; N = 6 de 65 hembras), junto con los análisis de autocorrelación espacial revelaron que el patrón de dispersión de los monos aulladores negros es bisexual con fuertes sesgos sexuales en ambas tasas de dispersión (los machos se dispersan más a menudo que las hembras) y distancia de dispersión (las hembras se dispersan más lejos que los machos). Los datos de observación y genéticos confirman que tanto machos como hembras pueden inmigrar con éxito a grupos ya establecidos, así como formar nuevos grupos con otros individuos que se están dispersando. Además, tanto los machos como las hembras pueden dispersarse individualmente, así como en parejas, y ambos también pueden dispersarse secundariamente. En general, nuestros hallazgos sugieren múltiples trayectorias de dispersión para aulladores negros de los dos sexos, y se necesitan más estudios para desentrañar qué factores demográficos, ecológicos y sociales subyacen en las decisiones de los individuos sobre si dispersarse y qué opciones de dispersión tomar.

Co-structure analysis and genetic associations reveal insights into pinworms (Trypanoxyuris) and primates (Alouatta palliata) microevolutionary dynamics.

BACKGROUND: In parasitism arm race processes and red queen dynamics between host and parasites reciprocally mold many aspects of their genetics and evolution. We performed a parallel assessment of population genetics and demography of two species of pinworms with different degrees of host specificity (Trypanoxyuris multilabiatus, species-specific; and T. minutus, genus-specific) and their host, the mantled howler monkey (Alouatta palliata), based on mitochondrial DNA sequences and microsatellite loci (these only for the host). Given that pinworms and primates have a close co-evolutionary history, covariation in several genetic aspects of their populations is expected. RESULTS: Mitochondrial DNA revealed two genetic clusters (West and East) in both pinworm species and howler monkeys, although population structure and genetic differentiation were stronger in the host, while genetic diversity was higher in pinworms than howler populations. Co-divergence tests showed no congruence between host and parasite phylogenies; nonetheless, a significant correlation was found between both pinworms and A. palliata genetic pairwise distances suggesting that the parasites' gene flow is mediated by the host dispersal. Moreover, the parasite most infective and the host most susceptible haplotypes were also the most frequent, whereas the less divergent haplotypes tended to be either more infective (for pinworms) or more susceptible (for howlers). Finally, a positive correlation was found between pairwise p-distance of host haplotypes and that of their associated pinworm haplotypes. CONCLUSION: The genetic configuration of pinworm populations appears to be molded by their own demography and life history traits in conjunction with the biology and evolutionary history of their hosts, including host genetic variation, social interactions, dispersal and biogeography. Similarity in patterns of genetic structure, differentiation and diversity is higher between howler monkeys and T. multilabiatus in comparison with T. minutus, highlighting the role of host-specificity in coevolving processes. Trypanoxyuris minutus exhibits genetic specificity towards the most frequent host haplotype as well as geographic specificity. Results suggest signals of potential local adaptation in pinworms and further support the notion of correlated evolution between pinworms and their primate hosts.

Use of genetic tools to assess predation on reintroduced howler monkeys (Alouatta caraya) in Northeastern Argentina.

Despite strong support from the media, the reintroduction of animals into natural environments does not always achieve its goal. Alouatta caraya is the primate species facing the greatest hunting pressure due to the illegal pet trade in Argentina. Confiscations of this species are common, as is the voluntary surrender of animals by owners no longer able or willing to care for them. These animals ultimately arrive at rehabilitation centers and, in many cases, are released into natural environments that may differ from the original sites where they were captured. Until recently, the lack of genetic analysis of the individuals involved led to biased relocation decisions. We followed the reintroduction of 12 A. caraya individuals in a protected area (Isla Palacio, Misiones, Argentina). The presence of potential predators such as pumas (Puma concolor) and jaguars (Panthera onca) in this area was confirmed by camera traps, footprints and feces. After the disappearance of four A. caraya at the reintroduction site, we investigated the applicability of genetic assignment tests based on genotypic data to accurately identify predated individuals. Genetic analyses allowed us to determine the predator species (P. onca) and to identify the predated individuals as two of the reintroduced animals. This procedure is promising for identifying the remains of predated individuals, and can contribute to the design of reintroduction policies based on scientific evidence.

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.

X-Linked Signature of Reproductive Isolation in Humans is Mirrored in a Howler Monkey Hybrid Zone.

Reproductive isolation is a fundamental step in speciation. While sex chromosomes have been linked to reproductive isolation in many model systems, including hominids, genetic studies of the contribution of sex chromosome loci to speciation for natural populations are relatively sparse. Natural hybrid zones can help identify genomic regions contributing to reproductive isolation, like hybrid incompatibility loci, since these regions exhibit reduced introgression between parental species. Here, we use a primate hybrid zone (Alouatta palliata × Alouatta pigra) to test for reduced introgression of X-linked SNPs compared to autosomal SNPs. To identify X-linked sequence in A. palliata, we used a sex-biased mapping approach with whole-genome re-sequencing data. We then used genomic cline analysis with reduced-representation sequence data for parental A. palliata and A. pigra individuals and hybrids (n = 88) to identify regions with non-neutral introgression. We identified ~26 Mb of non-repetitive, putatively X-linked genomic sequence in A. palliata, most of which mapped collinearly to the marmoset and human X chromosomes. We found that X-linked SNPs had reduced introgression and an excess of ancestry from A. palliata as compared to autosomal SNPs. One outlier region with reduced introgression overlaps a previously described "desert" of archaic hominin ancestry on the human X chromosome. These results are consistent with a large role for the X chromosome in speciation across animal taxa and further, suggest shared features in the genomic basis of the evolution of reproductive isolation in primates.

Positively selected variants in functionally important regions of TLR7 in Alouatta guariba clamitans with yellow fever virus exposure in Northern Argentina.

BACKGROUND: In 2007-2009, a major yellow fever virus (YFV) outbreak in Northern Argentina decimated the local howler monkey (Alouatta) population. AIMS: To evaluate whether the surviving howler monkeys possess advantageous genetic variants inherited from monkeys alive prior to the YFV outbreak, we explored the relationship between Toll-like receptor (TLR) 7 and TLR8 gene variation and YFV susceptibility. METHODS: We used samples from Alouatta individuals in Misiones, Argentina alive before the YFV outbreak, individuals that died during the outbreak, and individuals that survived the outbreak and are alive today. We measured genetic divergence between Alouatta YFV exposure groups and evaluated Alouatta-specific substitutions for functional consequences. RESULTS: We did not find different allele frequencies in the post-YFV exposure Alouatta group compared to the pre-exposure group. We identified three nonsynonymous variants in TLR7 in Alouatta guariba clamitans. Two of these substitutions are under positive selection in functionally important regions of the gene. DISCUSSION AND CONCLUSIONS: Our results did not indicate that surviving howler monkey spossess advantageous genetic variants at greater frequency than those alive before the YFV outbreak. However, the positively selected unique coding differences in A. guariba clamitans are in the region important in pathogen detection which may affect YFV resistance. Morework is necessary to fully explore this hypothesis.

Low genetic diversity and limited genetic structure across the range of the critically endangered Mexican howler monkey (Alouatta palliata mexicana).

Genetic diversity provides populations with the possibility to persist in ever-changing environments, where selective regimes change over time. Therefore, the long-term survival of a population may be affected by its level of genetic diversity. The Mexican howler monkey (Alouatta palliata mexicana) is a critically endangered primate restricted to southeast Mexico. Here, we evaluate the genetic diversity and population structure of this subspecies based on 83 individuals from 31 groups sampled across the distribution range of the subspecies, using 29 microsatellite loci. Our results revealed extremely low genetic diversity (HO = 0.21, HE = 0.29) compared to studies of other A. palliata populations and to other Alouatta species. Principal component analysis, a Bayesian clustering method, and analyses of molecular variance did not detect strong signatures of genetic differentiation among geographic populations of this subspecies. Although we detect small but significant FST values between populations, they can be explained by a pattern of isolation by distance. These results and the presence of unique alleles in different populations highlight the importance of implementing conservation efforts in multiple populations across the distribution range of A. p. mexicana to preserve its already low genetic diversity. This is especially important given current levels of population isolation due to the extreme habitat fragmentation across the distribution range of this primate.

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.

Association between Genomic Instability and Evolutionary Chromosomal Rearrangements in Neotropical Primates.

During the last decades, the mammalian genome has been proposed to have regions prone to breakage and reorganization concentrated in certain chromosomal bands that seem to correspond to evolutionary breakpoints. These bands are likely to be involved in chromosome fragility or instability. In Primates, some biomarkers of genetic damage may be associated with various degrees of genomic instability. Here, we investigated the usefulness of Sister Chromatid Exchange as a biomarker of potential sites of frequent chromosome breakage and rearrangement in Alouatta caraya, Ateles chamek, Ateles paniscus, and Cebus cay. These Neotropical species have particular genomic and chromosomal features allowing the analysis of genomic instability for comparative purposes. We determined the frequency of spontaneous induction of Sister Chromatid Exchanges and assessed the relationship between these and structural rearrangements implicated in the evolution of the primates of interest. Overall, A. caraya and C. cay presented a low proportion of statistically significant unstable bands, suggesting fairly stable genomes and the existence of some kind of protection against endogenous damage. In contrast, Ateles showed a highly significant proportion of unstable bands; these were mainly found in the rearranged regions, which is consistent with the numerous genomic reorganizations that might have occurred during the evolution of this genus.

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.

Genetic structure in the southernmost populations of black-and-gold howler monkeys (Alouatta caraya) and its conservation implications.

Black-and-gold howler monkeys Alouatta caraya, are arboreal primates, inhabitants of Neotropical forests, highly susceptible to the yellow fever virus, considered early 'sentinels' of outbreaks, and thus, of major epidemiological importance. Currently, anthropogenic habitat loss and modifications threatens their survival. Habitat modification can prevent, reduce or change dispersal behavior, which, in turn, may influence patterns of gene flow. We explored past and contemporary levels of genetic diversity, elucidated genetic structure and identified its possible drivers, in ten populations (n = 138) located in the southernmost distribution range of the species in South America, in Argentina and Paraguay. Overall, genetic variability was moderate (ten microsatellites: 3.16 ± 0.18 alleles per locus, allelic richness of 2.93 ± 0.81, 0.443±0.025 unbiased expected heterozygosity; 22 haplotypes of 491-bp mitochondrial Control Region, haplotypic diversity of 0.930 ± 0.11, and nucleotide diversity of0.01± 0.007). Significant evidence of inbreeding was found in a population that was, later, decimated by yellow fever. Population-based gene flow measures (FST = 0.13; θST = 018), hierarchical analysis of molecular variance and Bayesian clustering methods revealed significant genetic structure, grouping individuals into four clusters. Shared haplotypes and lack of mitochondrial differentiation (non-significant θST) among some populations seem to support the hypothesis of historical dispersal via riparian forests. Current resistance analyses revealed a significant role of landscape features in modeling contemporary gene flow: continuous forest and riparian forests could promote genetic exchange, whereas disturbed forests or crop/grassland fields may restrict it. Estimates of effective population size allow anticipating that the studied populations will lose 75% of heterozygosity in less than 50 generations. Our findings suggest that anthropogenic modifications on native forests, increasingly ongoing in Northeastern Argentina, Southern Paraguay and Southeastern Brazil, might prevent the dispersal of howlers, leading to population isolation. To ensure long-term viability and maintain genetic connectivity of A. caraya remnant populations, we recommend preserving and restoring habitat continuity. To conserve the species genetic pool, as well, the four genetic clusters identified here should be considered separate Management Units and given high conservation priority. In light of our findings and considering complementary non-genetic information, we suggest upgrading the international conservation status of A. caraya to "Vulnerable".

Cytogenetic Characterization of Brown Howler Monkeys, Alouatta guariba clamitans (Atelidae, Platyrrhini): Meiotic Confirmation of an X1X1X2X2X3X3/X1X2X3Y1Y2 Sex Chromosome System.

For brown howler monkeys (Alouatta guariba clamitans), diploid chromosome numbers varying from 2n = 45 to 2n = 52, with XX/XY, X1X1X2X2/X1X2Y, and X1X1X2X2X3X3/X1X2X3Y1Y2 sex chromosome systems have been described by mitotic studies but still await confirmation by meiotic analyses. We analyzed 3 male individuals sampled in the wild (in the municipality of Santa Maria, RS, Brazil) as well as 1 male and 1 female individual in captivity at the São Braz breeding center. Peripheral blood samples and testicular biopsies were taken. We found different diploid numbers for both sexes in somatic cells, 2n = 45,X1X2X3Y1Y2 in males and 2n = 46,X1X1X2X2X3X3 in females, with 4 metacentric (9-12), 7 submetacentric (1-6, 8), and 9 acrocentric autosomal chromosome pairs (13-20, 22). X1 and X2 were submetacentric chromosomes, while X3, Y1, and Y2 were acrocentric ones. Spermatocyte microspreads were examined for synaptonemal complexes. Pachytene spermatocyte analysis was done to verify the chromosome number and morphologies observed in mitotic karyotypes. Immunodetection was performed using anti-SMC3 and anti-CREST antibodies. The presence of a sex chromosome pentavalent X1X2X3Y1Y2 in the males was confirmed by C-banding in metaphase I and by immunodetection in prophase I by the clear identification of 5 centromeres. The G-banded karyotype corresponded to that previously described for A. g. clamitans in the south of Brazil (Curitiba, Parana State, and Blumenau, Santa Catarina State) and for the Misiones Province, Argentina.

Genetic, spatial, and social relationships among adults in a group of howler monkeys (Alouatta palliata) from Barro Colorado Island, Panama.

Kinship plays an important role in the social behavior of many primate species, including patterns of intra-group affiliation and cooperation. Within social groups, kinship is strongly affected by dispersal patterns, with the degree of relatedness among group-mates expected to decrease as the tendency to disperse increases. In primate species characterized by bisexual dispersal, relatedness among adult group-mates is predicted to be low, with social interactions shaped largely by factors other than kinship. To date, however, few studies have examined the role of kinship in social interactions in bisexually dispersing species. Accordingly, we collected genetic, spatial and behavioral data on all adult members (three males, six females) in a group of free-ranging mantled howler monkeys (Alouatta palliata)--a bisexually dispersing species of atelid primate--from Barro Colorado Island (BCI), Panama. Analyses of microsatellite variation revealed that relatedness was greater among adult males in this group (mean pairwise relatedness = 0.32 for males versus 0.09 for females). Relatedness among individuals, however, was not associated with either spatial proximity or frequency of social interactions. Instead, sex was a better predictor of both of these aspects of social behavior. While relatedness among adults had no discernible effect on the intra-group social interactions documented in this study, we postulate that kinship may facilitate affiliative and cooperative behaviors among male group-mates when interacting competitively with neighboring howler groups over access to food or potential mates.