Ancient DNA of the pygmy marmoset type specimen Cebuella pygmaea (Spix, 1823) resolves a taxonomic conundrum.

The pygmy marmoset, the smallest of the anthropoid primates, has a broad distribution in Western Amazonia. Recent studies using molecular and morphological data have identified two distinct species separated by the Napo and Solimões-Amazonas rivers. However, reconciling this new biological evidence with current taxonomy, i.e., two subspecies, Cebuella pygmaea pygmaea (Spix, 1823) and Cebuella pygmaea niveiventris (Lönnberg, 1940), was problematic given the uncertainty as to whether Spix's pygmy marmoset ( Cebuella pygmaea pygmaea) was collected north or south of the Napo and Solimões-Amazonas rivers, making it unclear to which of the two newly revealed species the name pygmaea would apply. Here, we present the first molecular data from Spix's type specimen of Cebuella pygmaea, as well as novel mitochondrial genomes from modern pygmy marmosets sampled near the type locality (Tabatinga) on both sides of the river. With these data, we can confirm the correct names of the two species identified, i.e., C. pygmaea for animals north of the Napo and Solimões-Amazonas rivers and C. niveiventris for animals south of these two rivers. Phylogenetic analyses of the novel genetic data placed into the context of cytochrome b gene sequences from across the range of pygmy marmosets further led us to re-evaluate the geographical distribution for the two Cebuella species. We dated the split of these two species to 2.54 million years ago. We discuss additional, more recent, subdivisions within each lineage, as well as potential contact zones between the two species in the headwaters of these rivers.

Taxonomic diversity of Cebuella in the western Amazon: Molecular, morphological and pelage diversity of museum and free-ranging specimens.

OBJECTIVES: We investigated the diversity of the pygmy marmoset, Cebuella pygmaea, by comparing genetic, morphological and pelage traits of animals from Peru and Ecuador. MATERIALS AND METHODS: We extracted DNA from museum specimen osteocrusts and from fecal samples collected from free-ranging individuals. We sequenced the mtDNA cytochrome b gene and the control region from samples collected at 13 different sites and used Bayesian inference and Maximum Likelihood to identify distinct clades. We took measurements of the crania of a subset of these specimens (n = 26) and ran a logistic regression to determine if any of the cranial measurements (n = 22) could predict a specimen's clade. In addition, we examined the pelage patterns of the museum specimens and photographs taken of free-ranging individuals and divided them into pelage types based on coloration of the underbelly. RESULTS: We identified two divergent clades, and two distinct groups with clear geographic boundaries within one of those clades. Two measurements of the zygomatic bone perfectly predicted a given individual's mtDNA clade. We found four distinct pelage patterns in our samples, but these patterns are variable within clades and among individuals within the same population. CONCLUSION: These analyses indicate that the two recognized subspecies of pygmy marmoset should be elevated to the species level (C. pygmaea and C. niveiventris) based on molecular and cranial differences but not on pelage patterns. We provide evidence on the geographic limits of the two clades and identify regions where additional sampling is required to better define the geographic distribution of the two clades.

What is in a genus name? Conceptual and empirical issues preclude the proposed recognition of Callibella (Callitrichinae) as a genus.

In a recent article, Silva et al. (Zool Scr 47:133-143, 2018) proposed the relocation of the dwarf marmoset, Mico humilis, to the so far unrecognized genus Callibella. We contend that a taxonomic scheme that recognizes Callibella as if it were a valid genus is inadequately supported, and to some extent contradicted, by the ecological and morphological information provided by the authors. We discuss why the criterion of sympatry, invoked by Silva et al. to justify the recognition of Callibella at the genus level, is uninformative for taxonomic decisions above the species level. We also show that the morphological characteristics used by Silva et al. to separate Mico humilis from the other Mico are individually variable and present in every analyzed species of the genus. Moreover, we demonstrate that the Evolutionarily Distinct and Globally Endangered (EDGE) metric, employed by those authors to attempt to justify their taxonomic proposition, makes no sense in a taxonomic context. Conceptually, the use of autapomorphies and plesiomorphies to justify using Callibella goes against one of the main objectives of a meaningful classification, that is, to allow for all kinds of inferences based on previous observations (i.e., to be inductively projectible). Based on these arguments, we demonstrate that regarding Callibella as a subgenus of Mico is the most suitable way of making the Linnean taxonomy of marmosets congruent with the phylogenetic information available for the group.

Functional characterization of enhancer evolution in the primate lineage.

BACKGROUND: Enhancers play an important role in morphological evolution and speciation by controlling the spatiotemporal expression of genes. Previous efforts to understand the evolution of enhancers in primates have typically studied many enhancers at low resolution, or single enhancers at high resolution. Although comparative genomic studies reveal large-scale turnover of enhancers, a specific understanding of the molecular steps by which mammalian or primate enhancers evolve remains elusive. RESULTS: We identified candidate hominoid-specific liver enhancers from H3K27ac ChIP-seq data. After locating orthologs in 11 primates spanning around 40 million years, we synthesized all orthologs as well as computational reconstructions of 9 ancestral sequences for 348 active tiles of 233 putative enhancers. We concurrently tested all sequences for regulatory activity with STARR-seq in HepG2 cells. We observe groups of enhancer tiles with coherent trajectories, most of which can be potentially explained by a single gain or loss-of-activity event per tile. We quantify the correlation between the number of mutations along a branch and the magnitude of change in functional activity. Finally, we identify 84 mutations that correlate with functional changes; these are enriched for cytosine deamination events within CpGs. CONCLUSIONS: We characterized the evolutionary-functional trajectories of hundreds of liver enhancers throughout the primate phylogeny. We observe subsets of regulatory sequences that appear to have gained or lost activity. We use these data to quantify the relationship between sequence and functional divergence, and to identify CpG deamination as a potentially important force in driving changes in enhancer activity during primate evolution.

Phenotypic evolution in marmoset and tamarin monkeys (Cebidae, Callitrichinae) and a revised genus-level classification.

Marmosets and tamarins (Cebidae, Callitrichinae) constitute the most species-rich subfamily of New World monkeys and one of the most diverse phenotypically. Despite the profusion of molecular phylogenies of the group, the evolution of phenotypic characters under the rapidly-emerging consensual phylogeny of the subfamily has been little studied, resulting in taxonomic proposals that have limited support from other datasets. We examined the evolution of 18 phenotypic traits (5 continuous and 13 discrete), including pelage, skull, dentition, postcrania, life-history and vocalization variables in a robust molecular phylogeny of marmoset and tamarin monkeys, quantifying their phylogenetic signal and correlations among some of the traits. At the family level, our resulting topology supports owl monkeys (Aotinae) as sister group of Callitrichinae. The topology of the callitrichine tree was congruent with previous studies except for the position of the midas group of Saguinus tamarins, which placement as sister of the bicolor group did not receive significant statistical support in both Maximum Parsimony and Bayesian Inference analyses. Our results showed that the highest value of phylogenetic signal among continuous traits was displayed by the long call character and the lowest was exhibited in the home range, intermediate values were found in characters related to osteology and skull size. Among discrete traits, pelage and osteology had similar phylogenetic signal. Based on genetic, osteological, pelage and vocalization data, we present an updated genus-level taxonomy of Callitrichinae, which recognizes six genera in the subfamily: Callimico, Callithrix, Cebuella, Mico, Leontopithecus and Saguinus. To reflect their phenotypic distinctiveness and to avoid the use of the informal "species group", we subdivided Saguinus in the subgenera Leontocebus, Saguinus and Tamarinus (revalidated here).

Complete mitochondrial genome of Callithrix kuhlii (Primates: Callitrichinae) with phylogenetic consideration.

The Wied's black-tufted-ear marmoset, Callithrix kuhlii, is a New World monkey that lives in tropical and subtropical forests of eastern Brazil. In this study, the mitochondrial genome sequence of this species is determined, which is found to be 16 500 bp in length and consists of 22 transfer RNA genes, 13 protein-coding genes, 2 ribosomal RNA genes and one D-loop control region. The overall nucleotide composition is 32.73% of A, 26.80% of T, 27.04% of C and 13.43% of G, with an AT content of 59.53%. The genome structure about gene order and composition of C. kuhlii mitochondrial genome is similar to those of most other vertebrates. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, all the other genes are encoded on the heavy strand. Phylogenetic analysis shows that C. kuhlii and C. jacchus are more closely related to each other than to other marmosets of the genus Callithrix that have its mitochondrial genome published.

Re-description and assessment of the taxonomic status of Saguinus fuscicollis cruzlimai Hershkovitz, 1966 (Primates, Callitrichinae).

Cruz Lima's saddle-back tamarin Saguinus fuscicollis cruzlimai Hershkovitz, 1966, was described from a painting by Eládio da Cruz Lima in his book Mammals of Amazonia, Vol. 1, Primates (1945). The painting was of four saddle-back tamarins from the upper Rio Purus, one of them distinct and the inspiration for Hershkovitz to describe it as a new subspecies. Its exact provenance was unknown, however, and the specimen was lost. Surveys in the Purus National Forest in 2011 resulted in sightings of this tamarin along the north bank of the Rio Inauini, a left-bank tributary of the middle Purus, and also on the left bank of the Purus, north and south of the Rio Inauini. It is possible that it extends north as far as the Rio Pauini, and that S. f. primitivus Hershkovitz, 1977, occurs north of the Pauini as far the Rio Tapauá, both also left-bank tributaries of the Purus. Morphometric and molecular genetic analyses and the coloration of the pelage indicate that this tamarin differs from its neighbors sufficiently to be considered a full species. In his doctoral dissertation [2010, Taxonomy, Phylogeny and Distribution of Tamarins (Genus Saguinus Hoffmannsegg, 1807) Georg-August Universität, Göttingen], C. Matauschek found that saddle-back and black-mantle tamarins diverged from the tamarin lineage around 9.2 million years ago; time enough to warrant their classification in a distinct genus. Leontocebus Wagner, 1840, is the first name available. In this article we re-describe Cruz Lima's saddle-back tamarin. We propose a neotype with a precise locality, and make it a full species in the genus Leontocebus.

Biogeography of the marmosets and tamarins (Callitrichidae).

The marmosets and tamarins, Family Callitrichidae, are Neotropical primates with over 60 species and subspecies that inhabit much of South America. Although callitrichids exhibit a remarkable widespread distribution, attempts to unravel their biogeographic history have been limited by taxonomic confusion and the lack of an appropriate statistical biogeographic framework. Here, we construct a time-calibrated multi-locus phylogeny from GenBank data and the callitrichid literature for 38 taxa. We use this framework to conduct statistical biogeographic analyses of callitrichids using BioGeoBEARS. The DIVAj model is the best supported reconstruction of biogeographic history among our analyses and suggests that the most recent common ancestor to the callitrichids was widespread across forested regions c. 14 Ma. There is also support for multiple colonizations of the Atlantic forest region from the Amazon basin, first by Leontopithecus c. 11 Ma and later by Callithrix c. 5 Ma. Our results show support for a 9 million year old split between a small-bodied group and large-bodied group of tamarins. These phylogenetic data, in concert with the consistent difference in body size between the two groups and geographical patterns (small-bodied tamarins and large-bodied tamarins have an unusually high degree of geographic overlap for congeners) lend support to our suggestion to split Saguinus into two genera, and we propose the use of distinct generic names; Leontocebus and Saguinus, respectively.

Parallel episodes of phyletic dwarfism in callitrichid and cheirogaleid primates.

The Callitrichidae are the smallest anthropoids, whereas the Cheirogaleidae include the smallest of all primates. Using species-level analyses, we show that these are derived conditions; both neonatal and adult body mass decreased in a gradual, phyletic manner in parallel across callitrichids, and across cheirogaleids. We identify lineages with particularly rapid decreases and highlight the pygmy marmoset, Callithrix pygmaea, as a phenotypic outlier. The life-history traits associated with body-mass reduction in each clade suggest that the convergent evolution of small body size was achieved by changes in different ontogenetic stages. Body-size reduction in callitrichids appears to be almost exclusively due to alterations in prenatal growth rate, whereas body-size reduction in cheirogaleids may have been largely due to reduced duration of growth phases. Finally, we use these results to discuss some of the debates surrounding the evolution of Homo floresiensis and suggest potential parallels between the evolution of H. floresiensis and callitrichids.

Q-factor analysis as a tool for phylogenetic studies of morphometric data.

The statistical procedure of Q-factor analysis was applied with the aim to detect complex morphological characters, which clearly separate taxonomic groups. Since Q-factor analysis, unlike cluster analysis, does not prescribe a hierarchical grouping, the results can be interpreted phylogenetically using an outgroup comparison. The essential steps of the approach are demonstrated by an empirical study on the phylogeny of Callitrichinae. Except for some complications concerning the determination of the number of relevant factors and factor rotation, the procedure proved to be a suitable instrument for phylogenetic research.

Mitochondrial COII gene sequences provide new insights into the phylogeny of marmoset species groups (Callitrichidae, Primates).

Mitochondrial cytochrome oxidase II (COII) gene sequences (549 base pairs) were used to investigate the taxonomic relationships among 12 marmoset (Callithrix, Cebuella and Mico) taxa. A large number of substitutions were found in the third base codon positions, providing a strong phylogenetic signal in a gene coding a conserved protein. Despite the significant affinity between the 2 Amazonian genera Cebuella and Mico, found in recent molecular studies, the analysis presented here did not resolve convincingly the phylogenetic relationships between the 3 genera. Mico nevertheless formed 3 distinct clades, reflecting a basic division of species groups based on geographic distribution (east or west of the Rio Tapajós) rather than morphology (presence or absence of auricular hair). This supports the taxonomic distinction of the allopatric emiliae forms. In Callithrix, Callithrix aurita forms a distinct clade, but the remaining morphotypes form a somewhat contradictory cluster, possibly resulting from an extremely rapid radiation.

The behavioural ecology of mixed-species troops of callitrichine primates.

This review summarizes information on the behavioural ecology of mixed-species troops (interspecific associations) formed by different species of callitrichines, small New World monkeys, in western and central Amazonia. The formation of mixed-species troops is an integral part of the biology of several species of this subfamily. Niche separation between associated species is obtained through vertical segregation which results in differences in the prey spectrum. The degree of niche separation is a predictor for the stability of mixed-species troops. Individuals may benefit from the formation of mixed-species troops through increased safety from predators, increased foraging efficiency, and/or increased resource defence. Costs of mixed-species troop formation are probably very low and mainly relate to patterns of interspecific behavioural interactions. We point to gaps in our knowledge and suggest pathways for future research into mixed-species troops.

Phylogenetic analysis of twinning in callitrichinae.

The callitrichines are known for twinning and for a communal rearing system in which all or most group members help care for the offspring. The origin of twinning has been the subject of much speculation. In this study predictions from earlier hypotheses are tested on the basis of two alternative phylogenetic trees. From this analysis we infer that helping behavior and male care preceded the origin of twinning, and that these traits did not coevolve with, but might have been important prerequisites for twinning in callitrichines. Small body size does not necessarily result in twinning, although it might still have been a prerequisite for its evolution. Gum feeding was an ecological change which evolved along with twinning. If nutrition was a limiting factor in the number of off-spring produced, then the use of a new feeding resource could have been crucial for the origin of twinning in callitrichines. According to one of the two alternative solutions inferred by the total evidence tree, and in accordance with the morphological tree, semi-annual breeding appears in the marmosets together with specialization in gum feeding. The fact that gums are available for these monkeys all year may have facilitated semiannual breeding. We suggest that the exploitation of gums as a feeding resource could have been the decisive factor in the increase of the reproductive rate by twinning and by semi-annual breeding.

The place of Callimico goeldii in the Callitrichine phylogenetic tree: evidence from von Willebrand factor gene intron II sequences.

Sequences of a 0.9-kb DNA segment spanning intron 11 of the von Willebrand Factor gene (vWF) were determined for 21 individuals of 19 primate species. The results of maximum parsimony and maximum likelihood analyses of these vWF sequences are congruent with previous molecular findings from other nonlinked nuclear genomic loci which divide the platyrrhine superfamily Ceboidea into three monophyletic families: Cebidae, Atelidae, and Pitheciidae. The vWF results strongly support the taxon Callitrichinae as a monophyletic subfamily within Cebidae. The four extant callitrichine genera constitute tribe Callitrichini, and the basal branchings within this tribe first separate out Saguinus (tamarins), next Leontopithecus (lion tamarins), and last the sister genera Callimico (Goeldi's monkeys) and Callithrix (marmosets). Callithrix divides into three subclades, with pygmy marmosets (C. pygmaea) as sister of the C. argentata species group and with the C. jacchus species group as their sister. Fossil and DNA evidence place the emergence of the callitrichine clade in the basal cebid radiation at about 20 Ma (million years ago) and the three basal branchings in the callitrichin radiation at about 13 to 11 Ma. In turn, the branchings separating the three subclades of Callithrix are placed at about 5 to 4 Ma.

Phylogenetic relationships of the Callitrichinae (Platyrrhini, primates) based on beta2-microglobulin DNA sequences.

The phylogenetic relationships of callitrichine primates have been determined by DNA sequence analyses of exons 1, 2, and 3 of the beta2-microglobulin gene. Parsimony, distance, and maximum likelihood analyses of ca. 900 base pairs of 21 taxa, representing all callitrichine genera, indicated that Saguinus was the most basal offshoot. Within Saguinus, S. fuscicollis appeared as the first divergent lineage followed by an unresolved trichotomy formed by S. mystax/S. imperator, S. midas/S. bicolor, and S. oedipus. A second callitrichine lineage was formed by Leontopithecus; each of the three species studied showed identical nucleotide sequences. Callimico appeared as the sister taxon of Callithrix/Cebuella. Genetic distances within this latter group were very small, although a stronger association between Cebuella and species of the Callithrix argentata group was observed. The inclusion of Cebuella in the genus Callithrix is suggested. These studies indicated that tamarins are more plesiomorphic than marmosets in agreement with the phyletic dwarfism hypothesis.

Electrophoretic polymorphisms and their taxonomic implications in Callitrichini (Primates, Platyrrhini).

Five hundred forty-three blood samples from 15 populations of the four genera of callitrichin primates were studied electrophoretically. Polymorphism and genetic distances were estimated for 20 loci, 13 of which were polymorphic. The lion tamarin (Leontopithecus) studied here exhibited the least variability for these loci, while the monospecific Cebuella showed the most. The genetic distances observed between Callithrix and Cebuella genera support previous evidence indicating a close taxonomic relationship between them. Genetic distance values obtained in this study also support the synonimyzation of the kuhli form with Callithrix jacchus penicillata.

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

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

Sequences of the primate epsilon-globin gene: implications for systematics of the marmosets and other New World primates.

Sequences of the epsilon-globin gene were determined for five species of marmosets, along with approximately 2 kb of 5' flanking sequence. An analysis of these data, compared with those of other primates strongly supports the classification of Callithrix jacchus and C. geoffroyi into the jacchus group, and C. argentata and C. mauesi into the argentata group. The pygmy marmoset, formerly identified as Cebuella pygmaea joined strongly to the argentata group, indicating that without the pygmy marmoset the genus Callithrix would be paraphyletic. Our data support recent studies which indicate that C. pygmaea should be included in the genus Callithrix. Relationships among other primates were as indicated by previous studies of epsilon-globin sequences. Divergence times were estimated according to a local molecular clock. These calculations indicated the divergence of C. mauesi and C. argentata to be approximately 1.6-1.9 Myr (million years ago), and the most recent common ancestor of the marmosets to be between 4.5 and 4.7 Myr. The latter estimate corresponds well to the date of 4.6 Myr calculated from an independent data set.

Comparative karyology and evolution of the Amazonian Callithrix (Platyrrhini, Primates).

Chromosomal studies in three species of Amazonian Callithrix (2n=44) and data in the literature show that this group is karyomonotypic. Moreover, it is characterized by the presence of abundant heterochromatic regions, unlike the situation in congeneric forms of Callithrix of the Atlantic coast with 2n=46, and by the presence of a highly repetitive, exclusive DNA component, with a basic repeat motif of 1528bp. Karyotypic comparisons with other Callitrichids and an outgroup species showed that Callitrichids are karyologically conserved and explained several rearrangements that had presumably occurred during their phyletic radiation. Analyses of karyologic data enabled the construction of two alternative phylogenetic topologies. The lack of derived homoeologies, common to all members of the genus Callithrix grouped at present, and the fact that Amazonian species were more similar to Cebuella pygmaea (2n=44) than to their congeneric forms with 2n=46 suggested that species at present included in the Amazonian Callithrix should be grouped with C. pygmaea.

Karyotypic comparison among Cebuella pygmaea, Callithrix jacchus and C. emiliae (Callitrichidae, Primates) and its taxonomic implications.

The karyotype of Cebuella pygmaea (2n = 44) obtained by G-, C-banding, and NOR-staining is described. This species presents a heteromorphic C band in the intersticial region of the short arm of chromosome 2. The data obtained were compared with those previously described for the karyotypes of Callithrix jacchus and Callithrix emiliae. The three species differ in the amount and distribution of non-centromeric constitutive heterochromatin. The importance of the variation in constitutive heterochromatin for the phylogeny of the group is discussed. Comparison of the karyotypes in terms of G-banding patterns showed that C. pygmaea and C. emiliae differ from C. jacchus by a Robertsonian translocation and a paracentric inversion, whereas C. pygmaea and C. emiliae differ from each other by a reciprocal translocation between an acrocentric autosome and the short arm of the submetacentric chromosomes that distinguishes their karyotypes from that of C. jacchus. The possible evolutionary paths followed by the karyotypes of the three species are discussed.