Background Callithrix marmosets are a relatively young primate radiation, whose phylogeny is not yet fully resolved. These primates are naturally para- and allopatric, but three species with highly invasive potential have been introduced into the southeastern Brazilian Atlantic Forest by the pet trade. There, these species hybridize with each other and endangered, native congeners. We aimed here to reconstruct a robust Callithrix phylogeny and divergence time estimates, and identify the biogeographic origins of autochthonous and allochthonous Callithrix mitogenome lineages. We sequenced 49 mitogenomes from four species (C. aurita, C. geoffroyi, C. jacchus, C. penicillata) and anthropogenic hybrids (C. aurita x Callithrix sp., C. penicillata x C. jacchus, Callithrix sp. x Callithrix sp., C. penicillata x C. geoffroyi) via Sanger and whole genome sequencing. We combined these data with previously published Callithrix mitogenomes to analyze five Callithrix species in total. Results We report the complete sequence and organization of the C. aurita mitogenome. Phylogenetic analyses showed that C. aurita was the first to diverge within Callithrix 3.54 million years ago (Ma), while C. jacchus and C. penicillata lineages diverged most recently 0.5 Ma as sister clades. MtDNA clades of C. aurita, C. geoffroyi, and C. penicillata show intraspecific geographic structure, but C. penicillata clades appear polyphyletic. Hybrids, which were identified by phenotype, possessed mainly C. penicillata or C. jacchus mtDNA haplotypes. The biogeographic origins of mtDNA haplotypes from hybrid and allochthonous Callithrix were broadly distributed across natural Callithrix ranges. Our phylogenetic results also evidence introgression of C. jacchus mtDNA into C. aurita. Conclusion Our robust Callithrix mitogenome phylogeny shows C. aurita lineages as basal and C. jacchus lineages among the most recent within Callithrix. We provide the first evidence that parental mtDNA lineages of anthropogenic hybrid and allochthonous marmosets are broadly distributed inside and outside of the Atlantic Forest. We also show evidence of cryptic hybridization between allochthonous Callithrix and autochthonous C. aurita. Our results encouragingly show that further development of genomic resources will allow to more clearly elucidate Callithrix evolutionary relationships and understand the dynamics of Callithrix anthropogenic introductions into the Brazilian Atlantic Forest.

Zoonotic malaria poses a unique problem for malaria control. Autochthonous cases of human malaria in the Atlantic Forest have recently been attributed to Plasmodium simium, a parasite that commonly infects non-human primates in this Brazilian biome. However, due to its close similarity at both the morphological and molecular level to Plasmodium vivax, the diagnosis of P. simium in this region remains problematic. Therefore, a diagnostic assay able to accurately identify P. simium is important for malaria surveillance. Based on mitochondrial genome sequences, primers were designed to amplify a region containing a SNP specific to P. simium. This region can then be digested with the restriction enzyme HpyCH4III, which results in digestion of P. simium sequences, but not of any other malaria parasite. Fifty-two human and monkey blood samples from different regions and infected with different Plasmodium species were used to validate this protocol. This easy and inexpensive tool can be used for the diagnosis of P. simium in non-human primates and human infections from the Atlantic Forest region to monitor zoonotic malaria transmission in Brazil.

We provide here a current overview of marmoset (Callithrix) evolution, hybridization, species biology, basic/biomedical research, and conservation initiatives. Composed of 2 subgroups, the aurita group (C aurita and C flaviceps) and the jacchus group (C geoffroyi, C jacchus, C kuhlii, and C penicillata), this relatively young primate radiation is endemic to the Brazilian Cerrado, Caatinga, and Atlantic Forest biomes. Significant impacts on Callithrix within these biomes resulting from anthropogenic activity include (1) population declines, particularly for the aurita group; (2) widespread geographic displacement, biological invasions, and range expansions of C jacchus and C penicillata; (3) anthropogenic hybridization; and (4) epizootic Yellow Fever and Zika viral outbreaks. A number of Brazilian legal and conservation initiatives are now in place to protect the threatened aurita group and increase research about them. Due to their small size and rapid life history, marmosets are prized biomedical models. As a result, there are increasingly sophisticated genomic Callithrix resources available and burgeoning marmoset functional, immuno-, and epigenomic research. In both the laboratory and the wild, marmosets have given us insight into cognition, social group dynamics, human disease, and pregnancy. Callithrix jacchus and C penicillata are emerging neotropical primate models for arbovirus disease, including Dengue and Zika. Wild marmoset populations are helping us understand sylvatic transmission and human spillover of Zika and Yellow Fever viruses. All of these factors are positioning marmosets as preeminent models to facilitate understanding of facets of evolution, hybridization, conservation, human disease, and emerging infectious diseases.

BackgroundNon-human primates (NHPs) as a source for Plasmodium infections in humans are a challenge for malaria elimination. In Brazil, two species of Plasmodium have been described infecting NHPs, Plasmodium brasilianum and Plasmodium simium. Both species are infective to man. Plasmodium brasilianum resembles morphologically, genetically and immunologically the human quartan Plasmodium malariae. Plasmodium brasilianum naturally infects species of non-human primates from all New World monkey families from a large geographic area. In the family Callitrichidae only the genus Saguinus has been described infected so far. The present study describes the natural infection of P. brasilianum in tamarins and marmosets of the genera Callithrix, Mico and Leontopithecus in the Atlantic forest.MethodsOne hundred and twenty-two NHPs of the family Callitrichidae housed in the Primate Centre of Rio de Janeiro (CPRJ) were sampled in June 2015, and January and July 2016. The CPRJ is located in the Atlantic forest in the Guapimirim municipality, in the Rio de Janeiro state, where human autochthonous cases of malaria have been reported. The samples were screened for the presence of Plasmodium using optical microscopy and nested PCR for detection of 18S small subunit rRNA gene. The amplicon was sequenced to confirm the molecular diagnosis.ResultsThe frequency of Plasmodium infections detected by nested PCR in New World monkeys of the family Callitrichidae was 6.6%. For the first time, Callitrichidae primates of genera Callithrix, Mico and Leontopithecus were found naturally infected with P. brasilianum. Infection was confirmed by sequencing a small fragment of 18S rRNA gene, although no parasites were detected in blood smears.ConclusionsThe reported P. brasilianum infection in NHP species maintained in captivity suggests that infection can be favoured by the presence of vectors and the proximity between known (and unknown) hosts of malaria. Thus, the list of potential malaria reservoirs needs to be further explored.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-017-1724-0) contains supplementary material, which is available to authorized users.

Oxford, Headington, Oxford, OX3 9DU, UK 41 42 §) Contributed equally, arranged alphabetically 43 †) Corresponding authors 44 45 46 Summary 47 48 Plasmodium simium, a malaria parasite of non-human primates in the Atlantic forest region of 49Brazil was recently shown to cause zoonotic infection in humans in the region. Phylogenetic 50 analyses based on the whole genome sequences of six P. simium isolates infecting humans 51 and two isolates from brown howler monkeys revealed that P. simium is monophyletic within 52 the broader diversity of South American Plasmodium vivax, consistent with the hypothesis 53 that P. simium first infected non-human primates as a result of a host-switch from humans 54 carrying P. vivax. We provide molecular evidence that the current zoonotic infections of 55 people have likely resulted from multiple independent host switches, each seeded from a 56 different monkey infection. Very low levels of genetic diversity within P. simium genomes 57 and the absence of P. simium-P. vivax hybrids suggest that the P. simium population emerged 58 recently and has subsequently experienced a period of independent evolution in Platyrrhini 59 monkeys. We further find that Plasmodium Interspersed Repeat (PIR) genes, Plasmodium 60Helical Interspersed Subtelomeric (PHIST) genes and Tryptophan-Rich Antigens (TRAg) 61 genes in P. siumium are genetically divergent from P. vivax and are enriched for non-62 synonymous single nucleotide polymorphisms, consistent with the rapid evolution of these 63 genes. Analysis of genes involved in erythrocyte invasion revealed several notable differences 64 between P. vivax and P. simium, including large deletions within the coding region of the 65 Duffy Binding Protein 1 (DBP1) and Reticulocyte Binding Protein 2a (RBP2a) genes in P. 66 simium. Genotyping of P. simium isolates from non-human primates (NHPs) and zoonotic 67 human infections showed that a precise deletion of 38 amino acids in DBP1 is exclusively 67 present in all human infecting isolates, whereas non-human primate infecting isolates were 68 polymorphic for the deletion. We speculate that these deletions in the parasite-encoded key 69 erythrocyte invasion ligands and the additional rapid genetic changes have facilitated zoonotic 70 transfer to humans. Non-human primate malaria parasites can be considered a reservoir of 71 potential infectious human parasites that must be considered in any attempt of malaria 72 elimination. The genome of P. simium will thus form an important basis for future functional 73 characterizations on the mechanisms underlying malaria zoonosis. 74 75 76 Introduction 77 78 There are currently eight species of malaria parasites known to cause disease in humans; 79 Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale 80 curtisi, Plasmodium ovale wallikeri, Plasmodium knowlesi, Plasmodium cynomolgi and 81 Plasmodium simium. The latter three species are more commonly parasitic on non-human 82 primates and have only relatively recently been shown to infect humans 1-3 . ...