Genomic skimming and nanopore sequencing uncover cryptic hybridization in one of world's most threatened primates.

The Brazilian buffy-tufted-ear marmoset (Callithrix aurita), one of the world's most endangered primates, is threatened by anthropogenic hybridization with exotic, invasive marmoset species. As there are few genetic data available for C. aurita, we developed a PCR-free protocol with minimal technical requirements to rapidly generate genomic data with genomic skimming and portable nanopore sequencing. With this direct DNA sequencing approach, we successfully determined the complete mitogenome of a marmoset that we initially identified as C. aurita. The obtained nanopore-assembled sequence was highly concordant with a Sanger sequenced version of the same mitogenome. Phylogenetic analyses unexpectedly revealed that our specimen was a cryptic hybrid, with a C. aurita phenotype and C. penicillata mitogenome lineage. We also used publicly available mitogenome data to determine diversity estimates for C. aurita and three other marmoset species. Mitogenomics holds great potential to address deficiencies in genomic data for endangered, non-model species such as C. aurita. However, we discuss why mitogenomic approaches should be used in conjunction with other data for marmoset species identification. Finally, we discuss the utility and implications of our results and genomic skimming/nanopore approach for conservation and evolutionary studies of C. aurita and other marmosets.

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).

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.

Phylogeny of the jacchus group of Callithrix marmosets based on complete mitochondrial genomes.

OBJECTIVES: Two subgroups make up the marmoset genus Callithrix. The "aurita" group is composed of two species, whereas evolutionary relationships among the four species of the "jacchus" group remain unclear. To uncover these relationships, we first sequenced mitochondrial genomes for C. kuhlii and C. penicillata to complement data available for congeners. We then constructed a phylogenetic tree based on mtDNA heavy chain protein coding genes from several primates to untangle species relationships and estimate divergence times of the jacchus group. MATERIALS AND METHODS: MtDNA genomes of C. kuhlii and C. penicillata were Sanger sequenced. These Callithrix mitogenomes were combined with other publically available primate mtDNA genomes. Phylogenies were produced using maximum likelihood and Bayesian inference. Finally, divergence times within the jacchus group of marmosets were estimated with Bayesian inference. RESULTS: In our phylogenetic tree, C. geoffroyi was the sister to all other jacchus group species, followed by C. kuhlii, while C. jacchus and C. penicillata diverged most recently. Bayesian inference showed that C. jacchus and C. penicillata diverged approximately 0.70 MYA and that the jacchus group radiated approximately 1.30 MYA. DISCUSSION: Callithrix nuclear and mtDNA phylogenies frequently result in polytomies and paraphyly. Here, we present a well-supported phylogenetic tree based on mitochondrial genome sequences, which facilitates the understanding of the divergence of the jacchus marmosets. Our results demonstrate how mitochondrial genomes can enrich Callithrix phylogenetic studies by alleviating some of the difficulties faced by previous mtDNA studies and allow formulation of hypotheses to test further under larger genomic-scale analyses.

The source-filter theory of whistle-like calls in marmosets: Acoustic analysis and simulation of helium-modulated voices.

Whistle-like high-pitched "phee" calls are often used as long-distance vocal advertisements by small-bodied marmosets and tamarins in the dense forests of South America. While the source-filter theory proposes that vibration of the vocal fold is modified independently from the resonance of the supralaryngeal vocal tract (SVT) in human speech, a source-filter coupling that constrains the vibration frequency to SVT resonance effectively produces loud tonal sounds in some musical instruments. Here, a combined approach of acoustic analyses and simulation with helium-modulated voices was used to show that phee calls are produced principally with the same mechanism as in human speech. The animal keeps the fundamental frequency (f0) close to the first formant (F1) of the SVT, to amplify f0. Although f0 and F1 are primarily independent, the degree of their tuning can be strengthened further by a flexible source-filter interaction, the variable strength of which depends upon the cross-sectional area of the laryngeal cavity. The results highlight the evolutionary antiquity and universality of the source-filter model in primates, but the study can also explore the diversification of vocal physiology, including source-filter interaction and its anatomical basis in non-human primates.

Translocation and radio-telemetry monitoring of pygmy marmoset, Cebuella pygmaea (Spix, 1823), in the Brazilian Amazon.

Two groups of pygmy marmoset (Cebuella pygmaea) were rescued along the left bank of the Madeira River during the formation of Santo Antônio Hydroelectric Dam reservoir in the state of Rondônia, Northern Brazil. Reintroduction of both groups occurred in areas of open Tropical rainforest located within the project´s Permanent Preservation Area. A post-release monitoring was conducted for three months using radio-telemetry. Individuals of each group remained together and settled in stable home ranges near their respective release sites. The mortality rate of translocated animals was about 7%. This seems to be the first report documenting the complete group translocation of C. pygmaea and the first to successfully employ radio-telemetry techniques in monitoring this species. This study demonstrated the feasibility of translocation and the use of radio-telemetry in monitoring C. pygmaea.

Translocation and radio-telemetry monitoring of pygmy marmoset, Cebuella pygmaea (Spix, 1823), in the Brazilian Amazon / Translocação e monitoramento por radiotelemetria do mico-leãozinho, Cebuella pygmaea (Spix, 1823), na Amazônia brasileira

Two groups of pygmy marmoset (Cebuella pygmaea) were rescued along the left bank of the Madeira River during the formation of Santo Antônio Hydroelectric Dam reservoir in the state of Rondônia, Northern Brazil. Reintroduction of both groups occurred in areas of open Tropical rainforest located within the project´s Permanent Preservation Area. A post-release monitoring was conducted for three months using radio-telemetry. Individuals of each group remained together and settled in stable home ranges near their respective release sites. The mortality rate of translocated animals was about 7%. This seems to be the first report documenting the complete group translocation of C. pygmaea and the first to successfully employ radio-telemetry techniques in monitoring this species. This study demonstrated the feasibility of translocation and the use of radio-telemetry in monitoring C. pygmaea.

Dois grupos de mico leãozinho (Cebuella pygmaea) foram resgatados ao longo da margem esquerda do Rio Madeira durante o enchimento do reservatório da Usina Hidrelétrica Santo Antônio, em Rondônia, norte do Brasil. A reintrodução de ambos os grupos deu-se em áreas de floresta ombrófila densa aberta dentro da Área de Preservação Permanente do empreendimento. Após a reintrodução, empregou-se a técnica de radiotelemetria para o monitoramento dos animais por aproximadamente três meses. Cada um dos grupos manteve-se coeso e estabeleceu suas áreas de vida em regiões próximas àquelas onde foram soltos. A taxa de mortalidade foi de aproximadamente 7%. Este parece ser o primeiro trabalho a documentar a translocação de grupos completos de C. pygmaea e o primeiro a empregar com sucesso a radiotelemetria no monitoramento desta espécie. Este estudo, portanto, demonstra a factibilidade da translocação e do uso da radio-telemetria para monitorar C. pygmaea.

Reporting social behaviours of mixed-species troops formed by Callithrix jacchus and Callithrix penicillata (Primate, Callitrichidae).

In New World primates, mixed-species troops have been reported. Here, we analysed the performance of affiliative and agonistic behaviours of Callithrix jacchus and Callithrix penicillata living in mixed groups. For this purpose, we recorded the interaction of the individuals from two groups located in Bauru city, in the state of São Paulo (Brazil). Our data show that in both groups, affiliative behaviours appeared more frequently than agonistic ones. We concluded that there is cohesion inside the mixed-species troops observed. We suggest that a deeper knowledge about the social behaviour of mixed-species troop species certainly may be useful in projects linked with the management of the impact caused by them.

Reporting social behaviours of mixed-species troops formed by Callithrix jacchus and Callithrix penicillata (Primate, Callitrichidae) / Comportamento social em grupos mistos de Callithrix jacchus e Callithrix penicillata (Primate, Callitrichidae)

In New World primates, mixed-species troops have been reported. Here, we analysed the performance of affiliative and agonistic behaviours of Callithrix jacchus and Callithrix penicillata living in mixed groups. For this purpose, we recorded the interaction of the individuals from two groups located in Bauru city, in the state of São Paulo (Brazil). Our data show that in both groups, affiliative behaviours appeared more frequently than agonistic ones. We concluded that there is cohesion inside the mixed-species troops observed. We suggest that a deeper knowledge about the social behaviour of mixed-species troop species certainly may be useful in projects linked with the management of the impact caused by them.

Grupos mistos em espécies de primatas do Novo Mundo têm sido relatados. Aqui, nós avaliamos a emissão de comportamentos afiliativos e agonísticos em grupos mistos formados por Callithrix jacchus e Callithrix penicillata. Para tanto, registramos a interação de dois grupos, localizados no município de Bauru, Estado de São Paulo (Brasil). Nossos dados mostram que nos dois grupos a emissão de comportamentos afiliativos foi maior do que a de comportamentos agonísticos, indicando que há coesão dentro dos grupos mistos estudados. Sugerimos que um conhecimento mais elaborado sobre o comportamento social de grupos mistos pode ser útil em projetos de impactos causados por espécies invasoras.

Classificação morfofuncional dos dentes de saguis-de-tufo-branco (Callithrix jacchus, Callitrichidae), saguis-de-tufo-preto (C. penicillata) e saguis-de-cara-branca (C. geoffroyi) / Morphofunctional classification of teeth of marmosets-tufted white (Callithrix jacchus, Callitrichidae), marmosets-tufted black (C. penicillata), and marmosets white-face (C. geoffroyi)

A study was conducted to analyze the morphology of the teeth of white-tufted-ear-marmoset (C. jacchus) black-tufted-ear-marmoset (C. penicillata) and marmosets-white-faced (C. geoffroyi), to compare them among themselves and with other species described in the literature. Teeth of the three species were submitted to macroscopic, microscopic and ultrastructural analyzes. The results were correlated with those of other species. We concluded that: the three species have a similar dental formula, reaching a total of 32 teeth, expressed in the formula 2x: incisors 2/2; canines 1/1, pre-molars 3/3 and molars 2/2, ich are classified as diplodont, anelodont, bunodont and brachyodont.

Realizou-se um estudo para analisar morfologicamente os dentes do saguis-de-tufo-branco (C. jacchus), saguis-de-tufo-preto (C. penicillata) e saguis-de-cara-branca (C. geoffroyi), para compara-los entre si e com outras espécies já descritas na literatura. Utilizou-se dentes das três espécies para analises macroscópicas, microscópicas e ultraestrutural e os resultados correlacionados com os obtidos com outras espécies citadas na literatura. Chegou-se a conclusão de que: as três espécies apresentaram uma fórmula dentária idêntica, chegando a um total de 32 dentes, expressa na fórmula 2x: incisivos 2/2; caninos 1/1; pré-molares 3/3 e molares 2/2, estes são classificados como diplodontes, anelodontes, bunodontes, e braquiodontes.

Comparison of racemic ketamine versus (S+) ketamine when combined with midazolam for anesthesia of Callithrix jacchus and Callithrix penicillata.

Ten common marmosets (Callithrix jacchus) and 10 black-tufted marmosets (Callithrix penicillata) were immobilized to compare the anesthetic effects of racemic ketamine and (S+) ketamine in combination with midazolam. The animals were distributed into four groups: five common marmosets (group CJR) and five black-tufted marmosets (group CPR) received 9.8 +/- 1.4 mg/kg of racemic ketamine, and five common marmosets (group CJS) and five black-tufted marmosets (group CPS) received 10.4 +/- 1.6 mg/kg of (S+) ketamine. All groups received similar dosages of midazolam (1.0 +/- 0.15 mg/kg). During immobilization, heart rates, respiratory rates, rectal temperatures, and muscle relaxation scores were recorded at 5, 10, and 20 min after initial injection. Quality of induction and quality of recovery were evaluated in each marmoset by recording physical reactions including withdrawal reflexes, involuntary movements, salivation, compulsive licking, catalepsy, and ataxia. There were no significant differences in the induction, immobilization, and recovery times between the four groups. Similarly, there were no significant differences between groups in heart rates, respiratory rates, or body temperatures, although there was a significant decrease in respiratory rates over time in group CPR. In addition, the CJR and CPS groups showed significant decreases over time in rectal temperature. Muscle relaxation was more profound in the CPR group than in the other groups. Compulsive licking, involuntary movements, salivation, and withdrawal reflexes were observed more frequently in animals given S(+) ketamine; but in general, racemic ketamine and S(+) ketamine had similar effects in all callitrichines. Further studies are required to confirm that S(+) ketamine has different potency in these species.

Systematics and evolution of the Jacchus group of marmosets (Platyrrhini).

Interspecific differentiation and geographic variation patterns in 39 skull traits of eastern Brazilian marmosets are analyzed. Eastern Callithrix taxa are distinct morphologically, and no evidence of intergradation among taxa is observed. Instead, there are sharp, stepped morphological boundaries among taxa, consistent with species-level distinction. The morphological similarity cluster diagram obtained from Mahalanobis distances is different from available molecular trees, and the general picture emerging is that the eastern Callithrix taxa should be considered as good species arising recently in South American history. In particular, C. kuhlii is morphologically distinct from other marmoset taxa, including C. geoffroyi and C. penicillata, which were previously hypothesized to be the parental populations that formed C. kuhlii by hybridization. Furthermore, C. kuhlii populations from southeastern Bahia do not overlap morphologically with any C. penicillata population, including the upper São Francisco River populations that display skin colors and pelage patterns to some extent similar to true Kuhli's marmosets. There is a negative, though insignificant, correlation between the morphological distance matrix and a Mahalanobis distance matrix estimated from nine climatic variables, a pattern opposite to that expected under a parapatric speciation model. This result, together with the lack of clinal variation in skull traits, suggests that an allopatric model of speciation might best explain eastern marmoset diversification.

The concept of species and the foundations of biology, a case study: the Callithrix jacchus group (Primates-Platyrrhini).

The axiomatic theory presented in Galleni and Forti [1999], being part of the foundational programme of Ennio De Giorgi, is based on the fundamental notions of quality, relation, operation and collection, and provides a very general axiomatization of the biological notions of living object, generation, species and speciation. Within this theoretical framework we consider here a difficult case of classification of species: the Callithrix jacchus group of the New World monkeys. Although the morphological analysis strongly suggests the individuation of six different species, nevertheless several experiments of crossing give evidence to fertility of hybrids. Since both the morphological and the hybridological criteria have shown to be of enormous importance in actual classification of species, this apparent contradiction seems very disappointing. Our axiomatization of speciation processes as operations acting in special time intervals permits to avoid the contradiction by allowing for individuals which, during such special periods, may belong to more than one species. Therefore we assume that one or more speciation processes are developing, starting from a unique protospecies and differentiating in six new ones. In order to obtain more evidence of these processes, new observations and suitable experiments are needed.

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.

Comparative chromosomal study of five taxa of genus Callithrix, group jacchus (Platyrrhini, Primates).

The karyotypes of four marmoset species of the Callithrix jacchus group (C. aurita, C. kuhlii, C. geoffroyi, and C. penicillata) were investigated. The patterns of G-, C-, and NOR-bands of these karyotypes were compared with those of C. jacchus, previously described, in order to clarify the taxonomic relationships of this species group. All species present 2n = 46, 14 uni- and 30 biarmed autosomes, a median size submetacentric X chromosome, and the same NOR-band patterns. No rearrangement or constitutive heterochromatic variation differentiate these species, which differ only in the morphology of the Y chromosome. The data obtained indicate that, from the chromosomal point stand, the marmoset species of C. jacchus group constitute a homogeneous clade.

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.

Analysis of constitutive heterochromatin by fluorochromes and in situ digestion with restriction enzymes in species of the group Callithrix argentata (Callitrichidae, Primates).

The karyotypes of the species belonging to the group Callithrix argentata (Callitrichidae, Platyrrhini) are characterized by large amounts of distal constitutive heterocharomatin (CH). The CH of the species C. argentata, C. humeralifera and C. emiliae was analyzed by banding with the restriction enzymes HinfI, MboI, aluI, RsaI, DdeI, HaeIII and MspI, as well as the fluorochromes CMA3 and DAPI. The results obtained permitted us to classify the CH of these species into three distinct types: 1) distal CH with a homogeneous response to enzymatic action, which was unchanged (HinfI, MboI, AluI, HaeIII), partially digested (DdeI) or fully digested (RsaI), being CMA3+, DAPI-; 2) centromeric CH, generally presenting a reduced band size. The varying extent of reduction, ranging from none to total, and also the variation of fluorochrome staining indicates that there is heterogeneity in this type of CH; 3) CH of the distal portion of the X chromosome of C. argentata and of the Y chromosome was CMA3- and unchanged by the enzymes, except for RsaI, which caused a reduction in size. MspI was the only enzyme unable to induce bands. Sequential C-banding permitted us to perceive banding variations that could not be observed simply by RE banding.

Chromosomal relationships and phylogenetic and clustering analyses on genes Callithrix group argentata (Callitrichidae, Primates).

The karyotypes of three species of marmosets of the Callithrix argentata group (C. argentata, C. humeralifera and C. chrysoleuca) were studied. Comparisons were made among species and with the previously described karyotypes of C. emiliae, C. mauesi (argentata group) and C. jacchus (jacchus group). Two chromosomes rearrangements differentiate the argentata (2n=44) and jacchus (2n=46) groups: fusion or fission and a paracentric invasion. The argentata group is also characterized by the addition of large amounts of distal constitutive heterochromatin (CH) in some chromosomes, while the jacchus group shows mainly centromeric heterochromatin. The five species of the argentata group differ in the amount or location of the distal CH. Interspecific differences were converted to a Basic Data Matrix (BDM), that was submitted to phenetic and cladistic analyses. For cladistic analyses C. jacchus was the outgroup. The results agree with morphological and geographical data.