De Novo Genome Assembly for the Coppery Titi Monkey (Plecturocebus cupreus): An Emerging Nonhuman Primate Model for Behavioral Research.

The coppery titi monkey (Plecturocebus cupreus) is an emerging nonhuman primate model system for behavioral and neurobiological research. At the same time, the almost entire absence of genomic resources for the species has hampered insights into the genetic underpinnings of the phenotypic traits of interest. To facilitate future genotype-to-phenotype studies, we here present a high-quality, fully annotated de novo genome assembly for the species with chromosome-length scaffolds spanning the autosomes and chromosome X (scaffold N50 = 130.8 Mb), constructed using data obtained from several orthologous short- and long-read sequencing and scaffolding techniques. With a base-level accuracy of ∼99.99% in chromosome-length scaffolds as well as benchmarking universal single-copy ortholog and k-mer completeness scores of >99.0% and 95.1% at the genome level, this assembly represents one of the most complete Pitheciidae genomes to date, making it an invaluable resource for comparative evolutionary genomics research to improve our understanding of lineage-specific changes underlying adaptive traits as well as deleterious mutations associated with disease.

Moderate evidence for heritability in the duet contributions of a South American primate.

Acoustic signals are ubiquitous across mammalian taxa. They serve a myriad of functions related to the formation and maintenance of social bonds and can provide conspecifics information about caller condition, motivation and identity. Disentangling the relative importance of evolutionary mechanisms that shape vocal variation is difficult, and little is known about heritability of mammalian vocalizations. Duetting--coordinated vocalizations within male and female pairs--arose independently at least four times across the Primate Order. Primate duets contain individual- or pair-level signatures, but the mechanisms that shape this variation remain unclear. Here, we test for evidence of heritability in two call types (pulses and chirps) from the duets of captive coppery titi monkeys (Plecturocebus cupreus). We extracted four features--note rate, duration, minimum and maximum fundamental frequency--from spectrograms of pulses and chirps, and estimated heritability of the features. We also tested whether features varied with sex or body weight. We found evidence for moderate heritability in one of the features examined (chirp note rate), whereas inter-individual variance was the most important source of variance for the rest of the features. We did not find evidence for sex differences in any of the features, but we did find that body weight and fundamental frequency of chirp elements covaried. Kin recognition has been invoked as a possible explanation for heritability or kin signatures in mammalian vocalizations. Although the function of primate duets remains a topic of debate, the presence of moderate heritability in titi monkey chirp elements indicates duets may serve a kin recognition function.

Response of Bolivian gray titi monkeys (Plecturocebus donacophilus) to an anthropogenic noise gradient: behavioral and hormonal correlates.

Worldwide urban expansion and deforestation have caused a rapid decline of non-human primates in recent decades. Yet, little is known to what extent these animals can tolerate anthropogenic noise arising from roadway traffic and human presence in their habitat. We studied six family groups of titis residing at increasing distances from a busy highway, in a park promoting ecotourism near Santa Cruz de la Sierra, Bolivia. We mapped group movements, sampled the titis' behavior, collected fecal samples from each study group and conducted experiments in which we used a mannequin simulating a human intrusion in their home range. We hypothesized that groups of titi monkeys exposed to higher levels of anthropogenic noise and human presence would react weakly to the mannequin and show higher concentrations of fecal cortisol compared with groups in least perturbed areas. Sound pressure measurements and systematic monitoring of soundscape inside the titis' home ranges confirmed the presence of a noise gradient, best characterized by the root-mean-square (RMS) and median amplitude (M) acoustic indices; importantly, both anthropogenic noise and human presence co-varied. Study groups resided in small, overlapping home ranges and they spent most of their time resting and preferentially used the lower forest stratum for traveling and the higher levels for foraging. Focal sampling analysis revealed that the time spent moving by adult pairs was inversely correlated with noise, the behavioral change occurring within a gradient of minimum sound pressures ranging from 44 dB(A) to 52 dB(A). Validated enzyme-immunoassays of fecal samples however detected surprisingly low cortisol concentrations, unrelated to the changes observed in the RMS and M indices. Finally, titis' response to the mannequin varied according to our expectation, with alarm calling being greater in distant groups relative to highway. Our study thus indicates reduced alarm calling through habituation to human presence and suggests a titis' resilience to anthropogenic noise with little evidence of physiological stress.

What makes a pair bond in a Neotropical primate: female and male contributions.

Pair living and pair bonding are rare in mammals, and the mechanisms of their maintenance remain a puzzle. Titi monkeys, a 'textbook example' for 'monogamous' primates, have strong pair bonds and extensive male care. To investigate mechanisms of pair-bond maintenance, we studied seven wild groups of red titis (Plecturocebus cupreus) in Peruvian Amazonia over a period of 14 months. We analysed pair bonds by measuring proximity, grooming and approaches/leaves within pairs, and collected data on intergroup encounters. Females contributed to grooming more than males, especially during infant dependency, when most of the grooming within pairs was done by females. Females were also more active in controlling proximity between pair mates, making most of the approaches and leaves. Males, on the other hand, invested more in territorial defences. They participated in more intergroup encounters than females and were more active during these encounters. Our data is most consistent with the 'male-services' hypothesis for pair-bond maintenance, where a female contributes more to the proximity and affiliation maintenance while a male provides beneficial services.

Adherence to Menzerath's Law is the exception (not the rule) in three duetting primate species.

Across diverse systems including language, music and genomes, there is a tendency for longer sequences to contain shorter constituents; this phenomenon is known as Menzerath's Law. Whether Menzerath's Law is a universal in biological systems, is the result of compression (wherein shortest possible strings represent the maximum amount of information) or emerges from an inevitable relationship between sequence and constituent length remains a topic of debate. In non-human primates, the vocalizations of geladas, male gibbons and chimpanzees exhibit patterns consistent with Menzerath's Law. Here, we use existing datasets of three duetting primate species (tarsiers, titi monkeys and gibbons) to examine the wide-scale applicability of Menzerath's Law. Primate duets provide a useful comparative model to test for the broad-scale applicability of Menzerath's Law, as they evolved independently under presumably similar selection pressures and are emitted under the same context(s) across taxa. Only four out of the eight call types we examined were consistent with Menzerath's Law. Two of these call types exhibited a negative relationship between the position of the note in the call and note duration, indicating that adherence to Menzerath's Law in these call types may be related to breathing constraints. Exceptions to Menzerath's Law occur when notes are relatively homogeneous, or when species-specific call structure leads to a deterministic decrease in note duration. We show that adherence to Menzerath's Law is the exception rather than the rule in duetting primates. It is possible that selection pressures for long-range signals that can travel effectively over large distances was stronger than that of compression in primate duets. Future studies investigating adherence to Menzerath's Law across the vocal repertoires of these species will help us better elucidate the pressures that shape both short- and long-distance acoustic signals.

On a new species of titi monkey (Primates: Plecturocebus Byrne et al., 2016), from Alta Floresta, southern Amazon, Brazil.

The taxonomy of the titi monkeys (Callicebinae) has recently received considerable attention. It is now recognised that this subfamily is composed of three genera with 33 species, seven of them described since 2002. Here, we describe a new species of titi, Plecturocebus, from the municipality of Alta Floresta, Mato Grosso, Brazil. We adopt an integrative taxonomic approach that includes phylogenomic analyses, pelage characters, and locality records. A reduced representation genome-wide approach was employed to assess phylogenetic relationships among species of the eastern Amazonian clade of the Plecturocebus moloch group. Using existing records, we calculated the Extent of Occurrence (EOO) of the new species and estimated future habitat loss for the region based on predictive models. We then evaluated the species' conservation status using the IUCN Red list categories and criteria. The new species presents a unique combination of morphological characters: (1) grey agouti colouration on the crown and dorsal parts; (2) entirely bright red-brown venter; (3) an almost entirely black tail with a pale tip; and (4) light yellow colouration of the hair on the cheeks contrasting with bright red-brown hair on the sides of the face. Our phylogenetic reconstructions based on maximum-likelihood and Bayesian methods revealed well-supported species relationships, with the Alta Floresta taxon as sister to P. moloch + P. vieirai. The species EOO is 10,166,653 ha and we predict a total habitat loss of 86% of its original forest habitat under a "business as usual" scenario in the next 24 years, making the newly discovered titi monkey a Critically Endangered species under the IUCN A3c criterion. We give the new titi monkey a specific epithet based on: (1) clear monophyly of this lineage revealed by robust genomic and mitochondrial data; (2) distinct and diagnosable pelage morphology; and (3) a well-defined geographical distribution with clear separation from other closely related taxa. Urgent conservation measures are needed to safeguard the future of this newly discovered and already critically endangered primate.

Duetting Patterns of Titi Monkeys (Primates, Pitheciidae: Callicebinae) and Relationships with Phylogeny.

Long-range vocal communication in socially monogamous titi monkeys is mediated by the production of loud, advertising calls in the form of solos, duets, and choruses. We conducted a power spectral analysis of duets and choruses (simply "duets" hereafter) followed by linear discriminant analysis using three acoustic parameters-dominant frequency of the combined signal, duet sequence duration, and pant call rate-comparing the coordinated vocalizations recorded from 36 family groups at 18 sites in Bolivia, Peru and Ecuador. Our analysis identified four distinct duetting patterns: (1) a donacophilus pattern, sensu largo, characteristic of P. donacophilus, P. pallescens, P. olallae, and P. modestus; (2) a moloch pattern comprising P. discolor, P. toppini, P. aureipalatii, and P. urubambensis; (3) a torquatus pattern exemplified by the duet of Cheracebus lucifer; and (4) the distinctive duet of P. oenanthe, a putative member of the donacophilus group, which is characterized by a mix of broadband and narrowband syllables, many of which are unique to this species. We also document a sex-related difference in the bellow-pant phrase combination among the three taxa sampled from the moloch lineage. Our data reveal a presumptive taxonomic incoherence illustrated by the distinctive loud calls of both P. urubambensis and P. oenanthe within the donacophilus lineage, sensu largo. The results are discussed in light of recent reassessments of the callicebine phylogeny, based on a suite of genetic studies, and the potential contribution of environmental influences, including habitat acoustics and social learning. A better knowledge of callicebine loud calls may also impact the conservation of critically endangered populations, such as the vocally distinctive Peruvian endemic, the San Martin titi, P. oenanthe.

Ambient temperature and humidity modulate the behavioural thermoregulation of a small arboreal mammal (Callicebus bernhardi).

Maintaining a constant body temperature is critical to the proper functioning of metabolic reactions. Behavioural thermoregulation strategies may minimize the cost of energetic balance when an animal is outside its thermoneutral zone. We investigated whether ambient temperature and relative air humidity influence the use of behavioural strategies by a group of Prince Bernhard's titi monkeys (Callicebus bernhardi) living in a forest fragment. We monitored a social group composed of four individuals (an adult couple and two juveniles) for 1010h from March to September 2015. We used the instantaneous scan sampling method to record the body posture, the microhabitat, and the occurrence of huddling with group mate(s) when animals were resting. We recorded ambient temperature and relative humidity in the shade every 10min with a data logger hanging at a height of approximately 5m. Daytime temperature ranged from 18.5°C to 38.5°C and relative humidity ranged from 21% to 97%. Titi monkeys avoided sunny places at higher temperatures, especially above 31°C. Minimum night temperature did not influence the choice of resting microhabitats during the first hour after sunrise. Sitting was the major resting posture during the day (62%). Titi monkeys increased the use of heat-dissipating postures at ambient temperatures >27°C. In addition, an increase in relative humidity increased the use of these postures at 26°C, 27°C, 29°C and 33°C, but caused a decrease at 24°C. On the other hand, the ambient temperature did not influence the occurrence of huddling. We conclude that microhabitat choice and postural behaviour are important for titi monkeys to prevent overheating and suggest that these behavioural adjustments might also be critical for other tropical arboreal mammals.

Phylogenetic relationships of the New World titi monkeys (Callicebus): first appraisal of taxonomy based on molecular evidence.

BACKGROUND: Titi monkeys, Callicebus, comprise the most species-rich primate genus-34 species are currently recognised, five of them described since 2005. The lack of molecular data for titi monkeys has meant that little is known of their phylogenetic relationships and divergence times. To clarify their evolutionary history, we assembled a large molecular dataset by sequencing 20 nuclear and two mitochondrial loci for 15 species, including representatives from all recognised species groups. Phylogenetic relationships were inferred using concatenated maximum likelihood and Bayesian analyses, allowing us to evaluate the current taxonomic hypothesis for the genus. RESULTS: Our results show four distinct Callicebus clades, for the most part concordant with the currently recognised morphological species-groups-the torquatus group, the personatus group, the donacophilus group, and the moloch group. The cupreus and moloch groups are not monophyletic, and all species of the formerly recognized cupreus group are reassigned to the moloch group. Two of the major divergence events are dated to the Miocene. The torquatus group, the oldest radiation, diverged c. 11 Ma; and the Atlantic forest personatus group split from the ancestor of all donacophilus and moloch species at 9-8 Ma. There is little molecular evidence for the separation of Callicebus caligatus and C. dubius, and we suggest that C. dubius should be considered a junior synonym of a polymorphic C. caligatus. CONCLUSIONS: Considering molecular, morphological and biogeographic evidence, we propose a new genus level taxonomy for titi monkeys: Cheracebus n. gen. in the Orinoco, Negro and upper Amazon basins (torquatus group), Callicebus Thomas, 1903, in the Atlantic Forest (personatus group), and Plecturocebus n. gen. in the Amazon basin and Chaco region (donacophilus and moloch groups).