Identification of constrained sequence elements across 239 primate genomes.

Noncoding DNA is central to our understanding of human gene regulation and complex diseases1,2, and measuring the evolutionary sequence constraint can establish the functional relevance of putative regulatory elements in the human genome3-9. Identifying the genomic elements that have become constrained specifically in primates has been hampered by the faster evolution of noncoding DNA compared to protein-coding DNA10, the relatively short timescales separating primate species11, and the previously limited availability of whole-genome sequences12. Here we construct a whole-genome alignment of 239 species, representing nearly half of all extant species in the primate order. Using this resource, we identified human regulatory elements that are under selective constraint across primates and other mammals at a 5% false discovery rate. We detected 111,318 DNase I hypersensitivity sites and 267,410 transcription factor binding sites that are constrained specifically in primates but not across other placental mammals and validate their cis-regulatory effects on gene expression. These regulatory elements are enriched for human genetic variants that affect gene expression and complex traits and diseases. Our results highlight the important role of recent evolution in regulatory sequence elements differentiating primates, including humans, from other placental mammals.

Black Titanium Oxide/Activated TaS2 Flakes Photoelectrode for Plasmon Assisted Hydrogen Evolution at Neutral pH at High Current Density.

A heterojunction photo-electrode(s) consisting of porous black titanium oxide (bTiO2) and electrochemically self-activated TaS2 flakes is proposed and utilized for hydrogen evolution reaction (HER). The self-activated TaS2 flakes provide abundant catalytic sites for HER and the porous bTiO2, prepared by electrochemical anodization and subsequent reduction serves as an efficient light absorber, providing electrons for HER. Additionally, Au nanostructures are introduced between bTiO2 and TaS2 to facilitate the charge transfer and plasmon-triggering ability of the structure created. After structure optimization, high HER catalytic activity at acidic pH and excellent HER activity at neutral pH are achieved at high current densities. In particular, with the utilization of bTiO2@TaS2 photoelectrode (neutral electrolyte, sunlight illumination) current densities of 250 and 500 mA cm-2 are achieved at overpotentials of 433, and 689 mV, respectively, both exceeding the "benchmark" Pt. The addition of gold nanostructures further reduces the overpotential to 360 and 543 mV at 250 and 500 mA cm-2, respectively. The stability of the prepared electrodes is investigated and found to be satisfying within 24 h of performance at high current densities. The proposed system offers an excellent potential alternative to Pt for the development of green hydrogen production on an industrial scale.

Diversification of the terrestrial frog genus Anomaloglossus (Anura, Aromobatidae) in the Guiana Shield proceeded from highlands to lowlands, with successive loss and reacquisition of endotrophy.

Two main landscapes emerge from the Guiana Shield: the highlands to the west called the Pantepui region and the Amazonian lowlands to the east, both harbouring numerous endemic species. With 32 currently recognized species, the genus Anomaloglossus stands out among Neotropical frogs as one that diversified only within the Guiana Shield both in the highlands and the lowlands. We present a time-calibrated phylogeny obtained by using combined mitogenomic and nuclear DNA, which suggests that the genus originates from Pantepui where extant lineages started diversifying around 21 Ma, and subsequently (ca. 17 Ma) dispersed during the Miocene Climatic Optimum to the lowlands of the eastern Guiana Shield where the ability to produce endotrophic tadpoles evolved. Further diversification within the lowlands in the A. stepheni group notably led to an evolutionary reversal toward exotrophy in one species group during the late Miocene, followed by reacquisition of endotrophy during the Pleistocene. These successive shifts of reproductive mode seem to have accompanied climatic oscillations. Long dry periods might have triggered evolution of exotrophy, whereas wetter climates favoured endotrophic forms, enabling colonization of terrestrial habitats distant from water. Acquisition, loss, and reacquisition of endotrophy makes Anomaloglossus unique among frogs and may largely explain the current species diversity. The micro evolutionary processes involved in these rapid shifts of reproductive mode remain to be revealed.

Multilocus approach reveals distinct evolutionary units of the South American apapa Pellona flavipinnis (Valenciennes, 1837) (Clupeiformes, Pristigasteridae).

The discovery and characterization of cryptic diversity is important for conservation and management, especially for ichthyofauna, whose diversity is underestimated and understudied. Cryptic diversity is especially common in widely distributed species, and Pellona flavipinnis is one such species. Thus, the aim of the present study was to investigate and test whether P. flavipinnis harbours cryptic diversity. In this study we used the COI and control region sequences and microsatellite loci of 86-114 specimens from 11-12 locations throughout the Amazon basin, depending on the molecular marker used. We also included two COI GenBank sequences from the type locality of the species, the Paraná River. The results from COI sequences showed that P. flavipinnis from the Amazon basin presented two spatially structured lineages differentiated from P. flavipinnis from the Paraná River by 10.6%-9.8% (depending on the lineages) and 45 mutational steps. The genetic distance between the Amazon lineages was 2.4% using COI, with high population differentiation values (ФST = 0.8686 and ФST = 0.8483 for COI and control region, respectively). Among the five species delimitation methods employed, three indicated two lineages in P. flavipinnis in the Amazon basin, and all five methods indicated that the Amazonian lineages are different from that of Paraná. Results from microsatellite loci also showed that P. flavipinnis from the Amazon basin is composed of two evolutionary units. The results of 13 morphometric measurements indicated that there are no differences in shape between the P. flavipinnis lineages in the Amazon basin. The present findings suggest that there are two sympatric lineages of P. flavipinnis in the Amazon basin.

Two hundred and five newly assembled mitogenomes provide mixed evidence for rivers as drivers of speciation for Amazonian primates.

Mitochondrial DNA remains a cornerstone for molecular ecology, especially for study species from which high-quality tissue samples cannot be easily obtained. Methods using mitochondrial markers are usually reliant on reference databases, but these are often incomplete. Furthermore, available mitochondrial genomes often lack crucial metadata, such as sampling location, limiting their utility for many analyses. Here, we assembled 205 new mitochondrial genomes for platyrrhine primates, most from the Amazon and with known sampling locations. We present a dated mitogenomic phylogeny based on these samples along with additional published platyrrhine mitogenomes, and use this to assess support for the long-standing riverine barrier hypothesis (RBH), which proposes that river formation was a major driver of speciation in Amazonian primates. Along the Amazon, Negro, and Madeira rivers, we found mixed support for the RBH. While we identified divergences that coincide with a river barrier, only some occur synchronously and also overlap with the proposed dates of river formation. The most compelling evidence is for the Amazon river potentially driving speciation within bearded saki monkeys (Chiropotes spp.) and within the smallest extant platyrrhines, the marmosets and tamarins. However, we also found that even large rivers do not appear to be barriers for some primates, including howler monkeys (Alouatta spp.), uakaris (Cacajao spp.), sakis (Pithecia spp.), and robust capuchins (Sapajus spp.). Our results support a more nuanced, clade-specific effect of riverine barriers and suggest that other evolutionary mechanisms, besides the RBH and allopatric speciation, may have played an important role in the diversification of platyrrhines.

Out of the shadows: Multilocus systematics and biogeography of night monkeys suggest a Central Amazonian origin and a very recent widespread southeastward expansion in South America.

Night monkeys (Aotus, Cebidae) are a widely distributed genus of Neotropical primates with a poorly understood taxonomy and biogeography. The number of species in the genus varies from one to nine, depending on the author, and there are at least 18 known karyotypes, varying from 2n = 46 to 2n = 58. Historically, night monkeys are divided into two species groups: red- and grey-necked groups from south and north of the Amazon-Solimões River, respectively. Here, we used 10 nuclear and 10 mitochondrial molecular markers from a wide taxonomic and geographic sample to infer phylogeny, divergence times, and biogeography of the genus. For phylogenetic reconstruction we used Maximum Likelihood (ML) and Bayesian Inferences (BI). Biogeographic models were generated using the 'BioGeoBEARS' software. We found support for nine taxa of Aotus and rejected the existence of monophyletic "red necked" and "grey necked" species groups. We suggest a taxonomic reclassification of the genus, which is better represented by two clades named northern group, which contains Aotus miconax, A. nancymae, A. trivirgatus, A. vociferans, A. lemurinus, A. griseimembra, A. zonalis, and A. brumbacki, and southern group, which contains A. nigriceps, A. boliviensis, A. infulatus, and A. azarae. The results suggest that the most recent common ancestor of all species of Aotus arose in the central Amazon basin in the Early Pliocene. The evolutionary history of night monkeys was guided by dispersal, vicariance and founder events. The end of the Andean uplift and the subsequent changes in the Amazon landscape, as well as the Amazon-Solimões and Tapajós rivers may have played an important role in the origin and diversification of Aotus, respectively. However, most of the Amazonian rivers seem not to have been geographical barriers to dispersal of night monkeys. The herein named southern group is fruit of a very recent diversification guided by dispersal, crossing the Tapajós, Xingú, Tocantins, and Guapore rivers and reaching the Cerrado in the last 1.6 My.

Phylogenetics and an updated taxonomic status of the Tamarins (Callitrichinae, Cebidae).

Traditionally, Saguinus has been organized into six taxonomic groups: bicolor, inustus, midas, mystax, nigricollis, and oedipus. After recent revisions, taxonomic reclassifications were proposed, including (1) the recognition of Leontocebus as a new genus, and (2) the subdivision of Saguinus into three subgenera. Nonetheless, the contradictory nature of these results reinforces the inconsistency concerning the monophyletic status of tamarins and its interspecific phylogeny. Therefore, in this study, we carried out phylogenetic inferences of Saguinus based on 44 molecular markers, of which 37 were from nuclear DNA and seven from mitochondrial DNA. A final dataset of 24,202 base pairs (bp) was obtained from 60 specimens of all recognized species of Saguinus and, also representatives of two main lineages of Leontocebus. Phylogenetic hypothesis was obtained from Maximum Likelihood (ML) and Bayesian inference (BI) methods. We also construct a Species Tree and a fossil-calibrated multi-locus phylogeny to estimate the time of divergence of Tamarins. Our phylogenetic results validated Leontocebus, or nigricollis group, as monophyletic, and recovered additionally three main clades within Saguinus. Same topology was obtained by the Species Tree. These clades correspond to (1) inustus + mystax groups, (2) oedipus group and (3) bicolor + midas group. Our results show support for a 10.5-million-year-old split between Leontocebus and the remaining Saguinus, followed by two other cladogenetic events, around 9.3 and 7.2 mya, which lead to the rise of the main clades of Saguinus. These phylogenetic data, in concert with the consistent morphological, ecological behavior and biogeographic evidence suggest a new classification for the Amazonian and trans-Andean tamarins. Therefore, we support the validation of Leontocebus as genus and recommend the split of Saguinus into three genera: (1) Tamarinus (inustus and mystax groups), (2) Oedipomidas (oedipus group), and (3) Saguinus (bicolor and midas groups).

Diversity, biogeography, and reproductive evolution in the genus Pipa (Amphibia: Anura: Pipidae).

The genus Pipa is a species-poor clade of Neotropical frogs and one of the most bizarre-looking due to many highly derived anatomical traits related to their fully aquatic lifestyle. With their African relatives, they form the Pipidae family, which has attracted much attention, especially regarding its anatomy, reproductive biology, paleontology and biogeography. However, the actual diversity and phylogenetic relationships within Pipa remain poorly understood, and thus so do their historical biogeography and the evolution of striking features, such as the absence of teeth and endotrophy in some species. Using short mtDNA sequences across the distribution of the genus, we identified 15 main lineages (Operational Taxonomic Units - OTUs). This more than doubles the number of the currently seven valid nominal species. Several closely related OTUs do not share nuDNA alleles, confirming species divergence. Time-calibrated phylogenies obtained from mitogenomes and from 10 nuclear loci provide highly similar topologies but strikingly distinct node ages for Pipa. High dN/dS ratios and the variation of substitution rates across the trees suggest a strong effect of saturation on fast evolving positions of mtDNA, producing a substantially shorter stem branch of Pipa. Focusing on the nuDNA topology, we inferred an early Neogene Amazonian origin of the diversification of Pipa, with an initial split between the Guiana-Brazilian Shields and Western Amazonia, a pattern observed in many other co-distributed groups. All the western species are edentate, suggesting a single loss in the genus. Each of these groups diversified further out of Amazonia, toward the Atlantic Forest and toward trans-Andean forests, respectively. These events are concomitant with paleogeographic changes and match patterns observed in other co-distributed taxonomic groups. The two Amazonian lineages have probably independently acquired endotrophic larval development.

Molecular phylogeny and systematics of bald uakaris, genus Cacajao (Primates: Pitheciidae), with the description of a new species.

Bald uakaris, genus Cacajao, are Amazonian primates currently classified as one species and four subspecies based on the patterns of pelage coloration. In this study, we test if their current taxonomy is represented by the phylogenetic relationship of the main lineages retrieved from molecular data. We included, for the first time, all bald uakari taxa in a mitochondrial (cytochrome b) and genome-wide (ddRAD) phylogenetic analyses. We also examined the pattern of pelage colouration in specimens from zoological collections. Having determined the number of lineages using Maximum Likelihood and the species tree using coalescent analyses, we test their divergence time using a Bayesian approach. While the cytochrome b analysis only recovered two clades, the ddRAD analysis supported the reciprocal monophyly of five lineages of bald uakaris, with all clades including only individuals with distinct and exclusive diagnostic phenotypic characters. We found that species diversification in Cacajao occurred during the last 300 Kya and may have been influenced by the formation of rivers and flooded forests in western Amazonia. We propose that the four bald uakari subspecies currently recognised can be upgraded to species level and we describe the white uakaris from the basin of the Rio Tarauacá as a new species.

Paleoclimatic evolution as the main driver of current genomic diversity in the widespread and polymorphic Neotropical songbird Arremon taciturnus.

Several factors have been proposed as drivers of species diversification in the Neotropics, including environmental heterogeneity, the development of drainage systems and historical changes in forest distribution due to climatic oscillations. Here, we investigate which drivers contributed to the evolutionary history and current patterns of diversity of a polymorphic songbird (Arremon taciturnus) that is widely distributed in Amazonian and Atlantic forests as well as in Cerrado gallery and seasonally-dry forests. We use genomic, phenotypic and habitat heterogeneity data coupled with climatic niche modelling. Results suggest the evolutionary history of the species is mainly related to paleoclimatic changes, although changes in the strength of the Amazon river as a barrier to dispersal, current habitat heterogeneity and geographic distance were also relevant. We propose an ancestral distribution in the Guyana Shield, and recent colonization of areas south of the Amazon river at ~380 to 166 kya, and expansion of the distribution to southern Amazonia, Cerrado and the Atlantic Forest. Since then, populations south of the Amazon River have been subjected to cycles of isolation and possibly secondary contact due to climatic changes that affected habitat heterogeneity and population connectivity. Most Amazonian rivers are not associated with long lasting isolation of populations, but some might act as secondary barriers, susceptible to crossing under specific climatic conditions. Morphological variation, while stable in some parts of the distribution, is not a reliable indicator of genetic structure or phylogenetic relationships.

Genomic analyses reveal two species of the matamata (Testudines: Chelidae: Chelus spp.) and clarify their phylogeography.

The matamata is one of the most charismatic turtles on earth, widely distributed in northern South America. Debates have occurred over whether or not there should be two subspecies or species recognized due to its geographic variation in morphology. Even though the matamata is universally known, its natural history, conservation status and biogeography are largely unexplored. In this study we examined the phylogeographic differentiation of the matamata based on three mitochondrial DNA fragments (2168 bp of the control region, cytochrome oxidase subunit I, and the cytochrome b gene), one nuclear genomic DNA fragment (1068 bp of the R35 intron) and 1661 Single Nucleotide Polymorphisms (SNPs). Our molecular and morphological analyses revealed the existence of two distinct, genetically deeply divergent evolutionary lineages of matamatas that separated in the late Miocene (approximately 12.7 million years ago), corresponding well to the time when the Orinoco Basin was established. As a result of our analyses, we describe the genetically and morphologically highly distinct matamata from the Orinoco and Río Negro Basins and the Essequibo drainage as a species new to science (Chelus orinocensis sp. nov.). Chelus fimbriata sensu stricto is distributed in the Amazon Basin and the Mahury drainage. Additionally, the analyses revealed that each species displays phylogeographic differentiation. For C. orinocensis, there is moderate mitochondrial differentiation between the Orinoco and the Río Negro. For C. fimbriata, there is more pronounced differentiation matching different river systems. One mitochondrial clade was identified from the Amazon, Ucayali, and Mahury Rivers, and another one from the Madeira and Jaci Paraná Rivers. The C. orinocensis in the Essequibo and Branco Rivers have haplotypes that constitute a third clade clustering with C. fimbriata. Phylogenetic analyses of the R35 intron and SNP data link the matamatas from the Essequibo and Branco with the new species, suggesting past gene flow and old mitochondrial introgression. Chelus orinocensis is collected for the pet trade in Colombia and Venezuela. However, neither the extent of the harvest nor its impact are known. Hence, it is crucial to gather more information and to assess its exploitation throughout its distribution range to obtain a better understanding of its conservation status and to design appropriate conservation and management procedures. RESUMEN: La matamata es una de las tortugas más carismáticas del mundo, ampliamente distribuida en el norte de Sudamérica. Debido a su variación morfológica geográfica, se debate sobre el reconocimiento de dos subespecies o especies. A pesar de que la matamata es universalmente conocida, su historia natural, estado de conservación y biogeografía han sido muy poco estudiados. En este estudio examinamos la diferenciación filogeográfica de las matamatas en base ​​a tres fragmentos de ADN mitocondrial (2168 pb de la región de control, la subunidad I del citocromo oxidasa y el gen del citocromo b), un fragmento de ADN genómico nuclear (1068 pb del intrón R35) y 1661 polimorfismos de nucleótido único (SNPs). Nuestros análisis moleculares y morfológicos revelaron la existencia de dos linajes evolutivos distintos de matamatas, genéticamente divergentes que se separaron en el Mioceno tardio (hace aproximadamente 12.7 millones de años), correspondiendo al tiempo en que se estableció la cuenca del Orinoco. Como resultado de nuestros análisis, describimos las genéticamente y morfológicamente distintas matamatas de las cuencas del Orinoco, Río Negro y Essequibo como una especie nueva para la ciencia (Chelus orinocensis sp. nov.). Chelus fimbriata sensu stricto se distribuye en la cuenca del Amazonas y en el drenaje del Mahury. Adicionalmente, los análisis revelaron que cada especie muestra diferenciación filogeográfica. Para C. orinocensis, hay una moderada diferenciación mitocondrial entre el Orinoco y el Río Negro. Para C. fimbriata, hay una diferenciación más pronunciada, concordando con los diferentes sistemas fluviales. Se identificó un clado de los ríos Amazonas, Ucayali y Mahury y otro de los ríos Madeira y Jaci Paraná. Las C. orinocensis de los ríos Essequibo y Branco tienen haplotipos que constituyen un tercer clado que se agrupa con C. fimbriata. Los análisis filogenéticos del intrón R35 y los datos de SNP asocian las matamatas de Essequibo y Branco con la nueva especie, sugiriendo flujo de genes pasado ​​e introgresión mitocondrial antigua. Chelus orinocensis se colecta para el comercio de mascotas en Colombia y Venezuela. Sin embargo, ni se conoce el alcance de las colectas ni su impacto. Por lo tanto, es crucial recopilar más información y evaluar su explotación en todo su rango de distribución, comprender mejor su estado de conservación y para diseñar acciones apropiadas de conservación y manejo.

Phylogenetic relationships in the genus Cheracebus (Callicebinae, Pitheciidae).

Cheracebus is a new genus of New World primate of the family Pitheciidae, subfamily Callicebinae. Until recently, Cheracebus was classified as the torquatus species group of the genus Callicebus. The genus Cheracebus has six species: C. lucifer, C. lugens, C. regulus, C. medemi, C. torquatus, and C. purinus, which are all endemic to the Amazon biome. Before the present study, there had been no conclusive interpretation of the phylogenetic relationships among most of the Cheracebus species. The present study tests the monophyly of the genus and investigates the relationships among the different Cheracebus species, based on DNA sequencing of 16 mitochondrial and nuclear markers. The phylogenetic analyses were based on Maximum Likelihood, Bayesian Inference, and multispecies coalescent approaches. The divergence times and genetic distances between the Cheracebus taxa were also estimated. The analyses confirmed the monophyly of the genus and a well-supported topology, with the following arrangement: ((C. torquatus, C. lugens), (C. lucifer (C. purinus, C. regulus))). A well-differentiated clade was also identified within part of the geographic range of C. lugens, which warrants further investigation to confirm its taxonomic status.

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.

An In Silico Comparison of Protocols for Dated Phylogenomics.

In the age of genome-scale DNA sequencing, choice of molecular marker arguably remains an important decision in planning a phylogenetic study. Using published genomes from 23 primate species, we make a standardized comparison of four of the most frequently used protocols in phylogenomics, viz., targeted sequence-enrichment using ultraconserved element and exon-capture probes, and restriction-site-associated DNA sequencing (RADseq and ddRADseq). Here, we present a procedure to perform in silico extractions from genomes and create directly comparable data sets for each class of marker. We then compare these data sets in terms of both phylogenetic resolution and ability to consistently and precisely estimate clade ages using fossil-calibrated molecular-clock models. Furthermore, we were also able to directly compare these results to previously published data sets from Sanger-sequenced nuclear exons and mitochondrial genomes under the same analytical conditions. Our results show-although with the exception of the mitochondrial genome data set and the smallest ddRADseq data set-that for uncontroversial nodes all data classes performed equally well, that is they recovered the same well supported topology. However, for one difficult-to-resolve node comprising a rapid diversification, we report well supported but conflicting topologies among the marker classes consistent with the mismodeling of gene tree heterogeneity as demonstrated by species tree analyses of single nucleotide polymorphisms. Likewise, clade age estimates showed consistent discrepancies between data sets under strict and relaxed clock models; for recent nodes, clade ages estimated by nuclear exon data sets were younger than those of the UCE, RADseq and mitochondrial data, but vice versa for the deepest nodes in the primate phylogeny. This observation is explained by temporal differences in phylogenetic informativeness (PI), with the data sets with strong PI peaks toward the present underestimating the deepest node ages. Finally, we conclude by emphasizing that while huge numbers of loci are probably not required for uncontroversial phylogenetic questions-for which practical considerations such as ease of data generation, sharing, and aggregating, therefore become increasingly important-accurately modeling heterogeneous data remains as relevant as ever for the more recalcitrant problems.

A new species of Piaractus (Characiformes: Serrasalmidae) from the Orinoco Basin with a redescription of Piaractus brachypomus.

Piaractus orinoquensis, a new species of serrasalmid fish, is described from the Orinoco River basin. The new species differs from congeners by having a slenderer body, relatively smaller head and snout, more compressed mid-body, fewer scales above and below the lateral line and diagnostic molecular characters in the coI mitochondrial gene region. We also provide a re-description of Piaractus brachypomus, restricting its geographic distribution to the Amazon River basin. Both species are economically important in their respective basins and need to be independently managed as distinct species.

Phylogeny, molecular dating and zoogeographic history of the titi monkeys (Callicebus, Pitheciidae) of eastern Brazil.

The titi monkeys belong to a genus of New World primates endemic to South America, which were recently reclassified in three genera (Cheracebus, Plecturocebus and Callicebus). The genus Callicebus, which currently includes five species, is endemic to eastern Brazil, occurring in the Caatinga, Savanna, and Atlantic Forest biomes. In the present study, we investigated the validity of these species and inferred their phylogenetic relationships, divergence times, and biogeographic patterns based on the molecular analysis of a concatenated sequence of 11 mitochondrial and nuclear DNA markers, derived from 13 specimens. We ran Maximum Likelihood (ML) and Bayesian Inference (BI) analyses, and estimated genetic distances, divergence times. Ancestral areas were estimated on BioGeoBears. Our results suggest that at about twelve million years ago, the ancestor of all titi monkeys inhabited a wide area that extended from the Amazon forest to the South of the Atlantic forest. A first vicariant event originated Cheracebus in the West of the Amazon and the ancestor of Callicebus and Plectorocebus which, later were separated by a second one. The diversification of Callicebus occurred during the Plio-Pleistocene (beginning at 5 Ma) probably influenced by climatic fluctuations and geological events. Therefore, the results of the present work confirmed the existence of five species that currently inhabit forested areas under increasing threat from human activities. Thus, a reliable diagnosis of the taxonomic status of species living in endangered environments is extremely important for the development of conservation measures.

How many pygmy marmoset (Cebuella Gray, 1870) species are there? A taxonomic re-appraisal based on new molecular evidence.

The pygmy marmoset, Cebuella pygmaea, the smallest of the New World monkeys, has one of the largest geographical distributions of the Amazonian primates. Two forms have been recognized: Cebuella pygmaea pygmaea (Spix, 1823), and C. p. niveiventris Lönnberg, 1940. In this study, we investigated if the separation of pygmy marmosets into these two clades can be corroborated by molecular data. We also examine and compare coloration of the pelage in light of the new molecular results. We analyzed the mtDNA cytochrome b gene and, for the first time for any Neotropical primate, we used a reduced representation genome sequencing approach (ddRADseq) to obtain data for recently collected, geographically representative samples from the Rio Japurá, a northern tributary of the Rio Solimões and from the Javarí, Jutaí, Juruá, Madeira and Purus river basins, all tributaries south of the Solimões. We estimated phylogenies and diversification times under both maximum likelihood and Bayesian inference criteria. Our analysis showed two highly supported clades, with intraclade divergences much smaller than interclade divergences, indicating two species of Cebuella: one from the Rio Japurá and one to the south of Solimões. The interpretation of our results in light of the current taxonomy is not trivial however. Lönnberg stated that the type of Spix's pygmy marmoset (type locality 'near Tabatinga') was obtained from the south of the Solimões, and his description of the distinct niveiventris from Lago Ipixuna, south of the Solimões and several hundred kilometres east of Tabatinga, was based on a comparison with specimens that he determined as typical pygmaea that were from the upper Rio Juruá (south of the Solimões). As such it remains uncertain whether the name pygmaea should be applicable to the pygmy marmosets north of the Rio Solimões (Tabatinga type locality) or south (near Tabatinga but across the Solimões). Finally, our analysis of pelage coloration revealed three phenotypic forms: (1) south of the Rio Solimoes, (2) Eirunepé-Acre, upper Juruá basin; and (3) Japurá. More samples from both sides of Solimões in the region of Tabatinga will be necessary to ascertain the exact type locality for Spix's pygmaea and to resolve the current uncertainties surrounding pygmy marmoset taxonomy.

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).

Phylogeny of the titi monkeys of the Callicebus moloch group (Pitheciidae, Primates).

Callicebus is a Neotropical primate genus of the family Pitheciidae, which currently comprises 34 recognized species. Based on their morphological traits and geographic distribution, these species are currently assigned to five groups: the C. moloch, C. cupreus, C. donacophilus, C. torquatus, and C. personatus groups, although in the past, alternative arrangements have been proposed based on the analysis of morphological data. The principal disagreements among these arrangements are related to the composition of the C. moloch group. In the present study, we tested the different taxonomic proposals for the C. moloch group, based on the molecular analysis of nuclear markers (Alu insertions and flanking regions) and three mitochondrial genes (16S, COI, and Cyt b), with a total of approximately 7 kb of DNA sequence data. Phylogenetic reconstructions based on maximum likelihood and Bayesian inference methods indicated that the species of the current C. cupreus group should be reintegrated into the C. moloch group. In addition, our results corroborated previous studies suggesting that the species of the current C. personatus group form a distinct species group. We also observed a relatively subtle level of divergence between C. dubius and C. caligatus. While the known diversity of Callicebus is considerable, these findings indicate that the relationships among groups and species may still not be completely understood, highlighting the need for further research into the biological, geographic, and genetic variability of these primates, which will be fundamental to the effective conservation of the genus. Am. J. Primatol. 78:904-913, 2016. © 2016 Wiley Periodicals, Inc.

Effects of Forest Fragmentation on Genetic Diversity of the Critically Endangered Primate, the Pied Tamarin (Saguinus bicolor): Implications for Conservation.

We analyzed DNA at 9 microsatellite loci from hair samples of 73 pied tamarins (Saguinus bicolor) located in 3 urban forest fragments and a biological reserve in the city of Manaus, Amazonas, Brazil. The forest fragments had become isolated from the continuous forest 6-15 years prior to the time of sampling. Tests for reduction in population size showed that all groups from the urban forest fragments had undergone genetic bottlenecks. Pied tamarins in this region historically formed one biological population, and the fragments were connected by high levels of gene flow. These results indicate the need to implement a conservation plan that allows for connectivity between the urban fragments, as well as protection from further constriction. Such connectivity could be achieved via the creation and protection of corridors. In addition to the current population trends explained by anthropogenic actions, the species also shows a trend of long-term demographic decline that has resulted in approximately an order of magnitude decrease and began 13 thousand years ago.