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.

On Being a Monkey's Uncle: Germline Chimerism in the Callitrichinae and the Evolution of Sibling Rivalry.

AbstractA typical monkey of the subfamily Callitrichinae has two or more cell lineages occupying its tissues: one from "itself," and one from its co-twin(s). Chimerism originates in utero when the twin placentae fuse, vascular anastomoses form between them, and cells are exchanged between conceptuses through their shared circulation. Previously it was thought that chimerism was limited to tissues of the hematopoietic cell lineage and that the germline was clonal, but subsequent empirical work has shown that chimerism may extend to many tissues, including the germline. To explore how natural selection on chimeric organisms should shape their social behavior, I construct an inclusive fitness model of sibling interactions that permits differing degrees of chimerism in the soma and germline. The model predicts that somatic chimerism should diminish sibling rivalry but that germline chimerism should typically intensify it. A further implication of the model is the possibility for intraorganismal conflict over developing phenotypes; as tissues may differ in their extent of chimerism-for example, placenta versus brain-their respective inclusive fitness may be maximized by different phenotypes. Communication between tissues in chimeric organisms might therefore be noisy, rapidly evolving, and fraught, as is common in systems with internal evolutionary conflicts of interest.

Chromosome painting of the red-handed tamarin (Saguinus midas) compared to other Callitrichinae monkeys.

Here we present, for the first time, the complete chromosome painting map of Saguinus midas, the red-handed tamarin. Chromosome banding and painting with human chromosome-specific probes were used to compare the karyotype of this species with those of four other Neotropical primates of the subfamily Callitrichinae: Leontopithecus rosalia, Callithrix geoffroyi, C. penicillata, and Mico argentatus. The chromosome painting map of S. midas was identical to that of L. rosalia and other previously studied tamarin species (genera Saguinus and Leontopithecus). The three marmoset species studied (genera Callithrix and Mico) differed in the painting pattern of four human probes (chromosomes 1, 2, 10, and 16). These paints identified the presence or absence of chromosome associations HSA 1/10 and 2/16 in these taxa. By integrating our data with those from the literature, we were able to propose an ancestral Callitrichinae karyotype. The genera Saguinus and Leontopithecus (tamarins) conserve the ancestral Callitrichinae karyotype, while Mico and Callithrix (marmosets) show more derived karyotypes due to chromosome translocations and fissions that occurred during the evolution of these taxa.

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.

Identification and characterization of a subtelomeric satellite DNA in Callitrichini monkeys.

Repetitive DNAs are abundant fast-evolving components of eukaryotic genomes, which often possess important structural and functional roles. Despite their ubiquity, repetitive DNAs are poorly studied when compared with the genic fraction of genomes. Here, we took advantage of the availability of the sequenced genome of the common marmoset Callithrix jacchus to assess its satellite DNAs (satDNAs) and their distribution in Callitrichini. After clustering analysis of all reads and comparisons by similarity, we identified a satDNA composed by 171 bp motifs, named MarmoSAT, which composes 1.09% of the C. jacchus genome. Fluorescent in situ hybridization on chromosomes of species from the genera Callithrix, Mico and Callimico showed that MarmoSAT had a subtelomeric location. In addition to the common monomeric, we found that MarmoSAT was also organized in higher-order repeats of 338 bp in Callimico goeldii. Our phylogenetic analyses showed that MarmoSAT repeats from C. jacchus lack chromosome-specific features, suggesting exchange events among subterminal regions of non-homologous chromosomes. MarmoSAT is transcribed in several tissues of C. jacchus, with the highest transcription levels in spleen, thymus and heart. The transcription profile and subtelomeric location suggest that MarmoSAT may be involved in the regulation of telomerase and modulation of telomeric chromatin.

Quantification of Myosin Heavy Chain Isoform mRNA Transcripts in the Supraspinatus Muscle of Vertical Clinger Primates.

Vertical clinging is a specialized form of locomotion characteristic of the primate family Callitrichidae. Vertical clinging requires these pronograde primates to maintain a vertical posture, so the protraction of their forelimbs must resist gravity. Since pronograde primates usually move as horizontal quadrupeds, we hypothesized that the supraspinatus muscle of vertical clingers would present specific characteristics related to the functional requirements imposed on the shoulder area by vertical clinging. To test this hypothesis, we quantified by real-time quantitative polymerase chain reaction the mRNA transcripts of myosin heavy chain (MHC) isoforms in the supraspinatus muscle of 15 species of pronograde primates, including vertical clingers. Our results indicate that the supraspinatus of vertical clingers has a specific expression pattern of the MHC isoforms, with a low expression of the transcripts of the slow MHC-I isoform and a high expression of the transcripts of the fast MHC-II isoforms. We conclude that these differences can be related to the particular functional characteristics of the shoulder in vertical clingers, but also to other anatomical adaptations of these primates, such as their small body size.

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.

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.

Evolution of ASPM is associated with both increases and decreases in brain size in primates.

A fundamental trend during primate evolution has been the expansion of brain size. However, this trend was reversed in the Callitrichidae (marmosets and tamarins), which have secondarily evolved smaller brains associated with a reduction in body size. The recent pursuit of the genetic basis of brain size evolution has largely focused on episodes of brain expansion, but new insights may be gained by investigating episodes of brain size reduction. Previous results suggest two genes (ASPM and CDK5RAP2) associated with microcephaly, a human neurodevelopmental disorder, may have an evolutionary function in primate brain expansion. Here we use new sequences encoding key functional domains from 12 species of callitrichids to show that positive selection has acted on ASPM across callitrichid evolution and the rate of ASPM evolution is significantly negatively correlated with callitrichid brain size, whereas the evolution of CDK5RAP2 shows no correlation with brain size. Our findings strongly suggest that ASPM has a previously unsuspected role in the evolution of small brains in primates. ASPM is therefore intimately linked to both evolutionary increases and decreases in brain size in anthropoids and is a key target for natural selection acting on brain size.

Fluorescence in situ hybridization to chromosomes as a tool to understand human and primate genome evolution.

For the last 15 years molecular cytogenetic techniques have been extensively used to study primate evolution. Molecular probes were helpful to distinguish mammalian chromosomes and chromosome segments on the basis of their DNA content rather than solely on morphological features such as banding patterns. Various landmark rearrangements have been identified for most of the nodes in primate phylogeny while chromosome banding still provides helpful reference maps. Fluorescence in situ hybridization (FISH) techniques were used with probes of different complexity including chromosome painting probes, probes derived from chromosome sub-regions and in the size of a single gene. Since more recently, in silico techniques have been applied to trace down evolutionarily derived chromosome rearrangements by searching the human and mouse genome sequence databases. More detailed breakpoint analyses of chromosome rearrangements that occurred during higher primate evolution also gave some insights into the molecular changes in chromosome rearrangements that occurred in evolution. Hardly any "fusion genes" as known from chromosome rearrangements in cancer cells or dramatic "position effects" of genes transferred to new sites in primate genomes have been reported yet. Most breakpoint regions have been identified within gene poor areas rich in repetitive elements and/or low copy repeats (segmental duplications). The progress in various molecular and molecular-cytogenetic approaches including the recently launched chimpanzee genome project suggests that these new tools will have a significant impact on the further understanding of human genome evolution.

Investigation of marmoset hybrids (Cebuella pygmaea x Callithrix jacchus) and related Callitrichinae (Platyrrhini) by cross-species chromosome painting and comparative genomic hybridization.

We report on the cytogenetics of twin offspring from an interspecies cross in marmosets (Callitrichinae, Platyrrhini), resulting from a pairing between a female Common marmoset (Callithrix jacchus, 2n = 46) and a male Pygmy marmoset (Cebuella pygmaea, 2n = 44). We analyzed their karyotypes by multi-directional chromosome painting employing human, Saguinus oedipus and Lagothrix lagothricha chromosome-specific probes. Both hybrid individuals had a karyotype with a diploid chromosome number of 2n = 45. As a complementary tool, interspecies comparative genomic hybridization (iCGH) was performed in order to screen for genomic imbalances between the hybrids and their parental species, and between Callithrix argentata and S. oedipus, respectively. These genomic imbalances were confined to centromeric and telomeric heterochromatin, while euchromatic chromosome regions appeared balanced in all species investigated. When comparing marmosets and tamarins, sequence divergence of centromeric heterochromatin was already clearly noticeable. In the C. argentata and C. pygmaea genomes numerous subtelomeric regions were affected by amplification of different repetitive sequences. Cross-species FISH with a microdissection-derived C. pygmaea repetitive probe revealed species specificity of this repetitive sequence at the molecular cytogenetic level of resolution.

Cone pigment polymorphism in New World monkeys: are all pigments created equal?

Most platyrrhine monkeys have a triallelic M/L opsin gene polymorphism that underlies significant individual variations in color vision. A survey of the frequencies of these polymorphic genes suggests that the three alleles occur with equal frequency among squirrel monkeys (subfamily Cebinae), but are not equally frequent in a number of species from the subfamily Callitrichinae. This departure from equal frequency in the Callitrichids should slightly increase the ratio of dichromats to trichromats in the population and significantly alter the relative representation of the three possible dichromatic and trichromatic phenotypes. A particular feature of the inequality is that it leads to a relative increase in the number of trichromats whose M/L pigments have the largest possible spectral separation. To assess whether these trichromatic phenotypes are equally well equipped to make relevant visual discriminations, psychophysical experiments were run on human observers. A technique involving the functional substitution of photopigments was used to simulate the discrimination between fruits among a background of leaves. The goal of the simulation was to reproduce in the cones of human observers excitations equivalent to those produced in monkey cones as the animals view fruit. Three different viewing conditions were examined involving variations in the relative luminances of fruit and leaves and the spectrum of the illuminant. In all cases, performance was best for simulated trichromacies including M/L pigments with the largest spectral separation. Thus, the inequality of opsin gene frequency in Callitrichid monkeys may reflect adaptive pressures.

Cone pigment variations in four genera of new world monkeys.

Previous research revealed significant individual variations in opsin genes and cone photopigments in several species of platyrrhine (New World) monkeys and showed that these in turn can yield significant variations in color vision. To extend the understanding of the nature of color vision in New World monkeys, electroretinogram flicker photometry was used to obtain spectral sensitivity measurements from representatives of four platyrrhine genera (Cebus, Leontopithecus, Saguinus, Pithecia). Animals from each genus were found to be polymorphic for middle to long-wavelength (M/L) sensitive cones. The presence of a short-wavelength sensitive photopigment was established as well so these animals conform to the earlier pattern in predicting that all male monkeys are dichromats while, depending on their opsin gene array, individual females can be either dichromatic or trichromatic. Across subjects a total of five different M/L cone pigments were inferred with a subset of three of these present in each species.

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.

Phylogeny of lion tamarins (Leontopithecus spp) based on interphotoreceptor retinol binding protein intron sequences.

The evolutionary relationships of the lion tamarins (Leontopithecus) were investigated using nuclear interphotoreceptor retinol binding protein (IRBP) intron sequences. Phylogenetic reconstructions strongly support the monophyly of the genus, and a sister relationship between the golden lion tamarin, Leontopithecus rosalia, and the black lion tamarin, L. chrysopygus, to the exclusion of the golden-headed lion tamarin, L. chrysomelas. The most parsimonious evolutionary reconstruction suggests that the ancestral lion tamarin and the common ancestor of L. rosalia and L. chrysopygus had predominantly black coats. This reconstruction is not consistent with a theory of orthogenetic evolution of coat color that was based on coat color evolution in marmosets and tamarins. An alternative reconstruction that is consistent with metachromism requires that ancestral lion tamarins had agouti hairs.

Molecular cloning of three nonhuman primate follicle stimulating hormone beta-subunit cDNAs.

The follicle stimulating hormone (FSH) beta-subunit cDNAs were cloned and sequenced for an old world primate, the rhesus monkey (Macaca mulatta), and two New World primates, the common marmoset (Callithrix jacchus) and pygmy marmoset (Cebuella pygmaea). The cDNA and predicted amino acid sequences of the rhesus monkey FSH beta-subunit were related most closely to the human FSH beta-subunit (> 96% identity). The common and pygmy marmosets have identical FSH beta-subunit cDNAs, whereas the marmoset FSH beta-subunit diverges from the rhesus and human molecules with less than 93% identity. These results have significance for the implementation of assisted reproductive technologies in the nonhuman primate as well as the evolution of genes encoding reproductive hormones.

Multiple nuclear insertions of mitochondrial cytochrome b sequences in callitrichine primates.

We report the presence of four nuclear paralogs of a 380-bp segment of cytochrome b in callitrichine primates (marmosets and tamarins). The mitochondrial cytochrome b sequence and each nuclear paralog were obtained from several species, allowing multiple comparisons of rates and patterns of substitution both between mitochondrial and nuclear sequences and among nuclear sequences. The mitochondrial DNA had high overall rates of molecular evolution and a strong bias toward substitutions at third codon positions. Rates of molecular evolution among the nuclear sequences were low and constant, and there were small differences in substitution patterns among the nuclear clades which were probably attributable to the small number of sites involved. A novel method of phylogenetic reconstruction based on the large difference in rates of evolution at different codon positions among mitochondrial and nuclear clades was used to determine whether different nuclear paralogs represent independent transposition events or duplications following a single insertion. This method is generally applicable in cases where differences in pattern of molecular evolution are known, and it showed that at least three of the four nuclear clades represent independent transposition events. The insertion events giving rise to two of the nuclear clades predate the divergence of the callitrichines, whereas those leading to the other two nuclear clades may have occurred in the common ancestor of marmosets.

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.