An Introduction to the Callithrix Genus and Overview of Recent Advances in Marmoset Research

We provide here a current overview of marmoset (Callithrix) evolution, hybridization, species biology, basic/biomedical research, and conservation initiatives. Composed of two 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.

reach to the far north of Rio de Janeiro state [1,12]. An unconfirmed Callithrix flaviceps occurrence was reported in the municipality of Varre-Sai, northern Rio de Janeiro state [13].
The northern distribution limit of Callithrix kuhlii, or Wied's marmoset, occurs at the Contas river in Bahia state and the southern limit is the Jequitinhonha river in Minas Gerais state [1]. There are recent reports of C. kuhlii to the north beyond the Contas river, in the region of Valença, Bahia [12], and within the vicinity of Todos os Santos Bay, Bahia [14, pers. obs.,VB and IOS]. The eastern limit of C. kuhlii's distribution reaches the Atlantic Ocean, and the Cerrado likely terminates the species' natural distribution to the west [15].
Callithrix geoffroyi, or Geoffroy's tufted-ear marmoset, generally occurs in the Atlantic Forest at altitudes below 500-700 m [1]. The southern bank of the Jequitinhonha river and eastern bank of the Araçuai river represent upper limits of the C. geoffroyi range [1]. Callithrix geoffroyi occurrences have also been recorded in the Espinhaço Mountains, Minas Gerais state (see Figure 1), which are located at the western Atlantic Forest limit [16]. Additionally, the species has been observed in the region between the Jequitinhonha and Doce rivers, in the north and northeast of Minas Gerais state, but it is not clear whether C. geoffroyi was introduced there or occurs there naturally [15,17]. While C.
geoffroyi is considered a lowland species, it has been observed in the Serra do Cipó National Park, Minas Gerias state at 1274 m above sea level [1,12]. The species has also been observed in a semi-arid region of northeastern Minas Gerais state on the western bank of the Araçuai river in Berilo municipality [pers. obs., JM].
Callithrix penicillata, or the black-tufted marmoset, has the widest Callithrix geographic distribution, with an area of about 1,300,000 km 2 [15,16,18]. The northern C. penicillata range seems limited by the Grande and São Francisco rivers [15,16]. The eastern limits of the C. penicillata range seem bounded by the Araguaia river's course through Goiás, Mato Grosso and Tocantins states. This species can also be found in the north of São Paulo state, north of the Tietê and Piracicaba rivers [8], as well as eastern Minas Gerais and southern Bahia states, where C. penicillata has contact zones with C. geoffroyi, C. aurita, C. flaviceps and probably C. kuhlii [1,15,16].
For the currently recognized C. penicillata distribution, several unfilled gaps still exist in the occurrence of this species, particularly within the Caatinga biome. Although the southern portion of the Caatinga is recognized by Rylands et al. [1] as part of the natural distribution of C. penicillata, we have observed occurrences of C. penicillata in northern parts of the Caatinga (Figure 3) (pers. obs., LCMP and JM). The dataset of da Rosa et al. [19] lists several occurrences of C. penicillata as 'alien' in the northern Caatinga where no Callithrix are thought to occur naturally. We, however, would like to note that the Caatinga sensu stricto is one of the youngest Brazilian vegetation formations, originating about 15-10 MYA [20]. Further, the C. penicillata Caatinga clade and the C. jacchus clades represent the two youngest clades within the phylogeny shown in Figure 2. Thus, these occurrences of C. penicillata in the Caatinga may be part of a natural expansion of this marmoset species from the Cerrado into the Caatinga.
Callithrix jacchus, or the white-tufted marmoset, occurs in northeastern Brazil [1]. The southern portion of its geographic distribution extends to the northern bank of the São Francisco river and the west bank of the Rio Grande river [1]. Whereas the northern and eastern portion of the C. jacchus natural distribution is limited by the Brazilian Atlantic coast, the species' western limits are less clear [1]. Currently recognized limits of the natural C. jacchus distribution are at the boundary between Tocantins and Maranhão states, but the species may also occur in the northeast of Tocantins state [1].

NATURAL CALLITHRIX HYBRIDIZATION
Callithrix species are a relatively recent primate radiation, and secondary contact between Callithrix species at the borders of their natural geographical distributions can result in natural hybridization ( Figure 1). One natural hybrid zone exists in the mountains of Espírito Santo state where hybrids of C. flaviceps and C. geoffroyi occur in an area of overlap between altitude limits for each parental species [5]. A number of natural hybrid zones occur at major river boundaries between C. penicillata's natural distribution and that of other congeners (Figure 1) [21]. Callithrix aurita and C. flaviceps hybridize naturally in a region that roughly overlaps the borders of Minas Gerais, Rio de Janeiro, and Espírito Santo states. Climatic factors that shape C. flaviceps' natural distribution and past climatic fluctuations may determine locations of natural C. aurita and C. flaviceps hybrid zones [22].
We have observed natural C. jacchus and C. penicillata hybrids along the banks of the São Francisco river between Petrolina, PE and Juazeiro, BA [4,21] as well as in Xique-Xique, BA (pers. obs., LCMP). Natural hybrids between the two species have also been observed at the northern tip of the Espinhaço mountain range where there is a mixture of Cerrado, Caatinga, and semi-deciduous forests (pers. obs., LCMP).

CALLITHRIX AURITA
The total population size of adult C. aurita ( Figure 4) is estimated to be 10,000 individuals, and the species is currently listed as Endangered [10]. Callithrix aurita social groups usually possess a single male-female breeding pair, although groups with multiple breeding females have been noted [23,24]. Group size varies from 5-11 individuals [7,25]. Population density for C. aurita also varies from 2.8 individuals/km² in the Serra do Brigadeiro State Park, MG [26] to 14.76 individuals/km² in the Pouso Alegre Municipal Natural Park, MG [27]. The home range size for a C. aurita group is approximately 11 hectares [28]. The suitability of a given habitat for C. aurita, considering topography and forest composition, may ultimately determine population density within a given region [6]. While conducting linear transect surveys, Norris et al. [6] estimated a general population of 1892 individuals in an 250.7 km 2 area of Atlantic Forest in southern São Paulo state. Several morphological differences distinguish C. aurita from other Callithrix species.
Callithrix aurita is perhaps the largest Callithrix species considering body weight and overall body size ( Figure 5 and Table 1). Callithrix aurita shares morphological similarities with C. flaviceps [e.g., 29], likely due to the close evolutionary relationship between them [5]. For example, Souza [29] noted a larger brain case size in the aurita group, relative to the jacchus group, as well as retrusions of the upper and lower jaws. Differences in cranial morphology between C. aurita and C. flaviceps include greater lateral expansion of the parietal bone and elongation of the occipital bone in C. aurita but more lateral nasal compression and larger incisors in C. flaviceps [29].
Morphological differences between the aurita and jacchus groups are likely related to stronger morphological specialization for gumnivory in the latter group [28,29]. In a 17 ha forest fragment in Minas Gerais state, C. aurita consumed 50.5% gums, 11% fruits, and 38.5% prey [30]. Gums are generally accessed by C. aurita from sources that do not require gouging [10]. The species also consumes fungi fruiting bodies from bamboo of the Poaceae family [10].

CALLITHRIX FLAVICEPS
Callithrix flaviceps ( Figure 6) is one of the least studied Callithrix taxa. The species is listed as Critically Endangered [11], as a total of approximately 2000 adult C. flaviceps individuals likely remain in the wild [11]. In secondary forests, C. flaviceps occupies areas sized between 15 ha to 35.5 ha [31][32][33], but in denser forests, 138.5 ha home ranges have been observed [34]. These home range sizes are among the largest for any Callithrix species [32,33]. On average, C. flaviceps travels 1222.5 m per day, but food scarcity during the dry season likely causes the species to have longer daily travel than in the rainy season [7]. The size of C. flaviceps social groups is between 3-15 members [31].
Although social groups tend to have one reproductive female, there are occasionally two or more reproductive females and several adult helpers that care for offspring [35].
The C. flaviceps dentition, like that of C. aurita, is less specialized for gummivory than that of the jacchus group [36], and the species' consumption of fruits, insects and fungi varies seasonally [34].
Callithrix flaviceps uses a "scan and pounce" approach to foraging for small prey [37]. The consumption of fungi is especially high in this species, and can compose up 65% of the total items consumed [34,38]. Gum consumption by C. flaviceps is mostly opportunistic, as this species exploits gums from tree holes made by wood-digging insects [7].

CALLITHRIX GEOFFROYI
Callithrix geoffroyi is characterized by a white face and black voluminous ear tufts. The species appears to be the largest jacchus group for average body weight and size ( Figure 5 and Table   1). One C. geoffroyi social group in an Atlantic Forest patch was composed of a single female, two males, and two juveniles [39]. The same group had a total home range size of 23.3 ha, but that varied between 4.9 ha in the rainy season and 7.2 ha in the dry season [39], which is likely related to distribution of nutritional resources. The group's daily ranging distances varied from 480 to 1980 meters [39]. In a 110 ha forest fragment in Espírito Santo state, major food categories for one C.
In general, C. geoffroyi seems highly flexible in the types of habitats that it can occupy.
Forested environments occupied by C. geoffroyi include dense and semi-deciduous forests of the Atlantic Forest as well as deciduous Caatinga forests [15]. The species is tolerant of secondary habitats, for example being able to utilize forest fragments surrounded by eucalyptus monoculture [40,41]. Across all of its range, the species also occurs commonly in urban areas, where it occupies orchards, backyards and forest fragments [pers obs, JM,VB].

CALLITHRIX JACCHUS
Callithrix jacchus (Figure 7) is one of the smallest Callithrix species (Table 1, Figure 5), and several of its biological traits seem associated with a high level of specialization for exudivory [28].
Exudivory adaptations of C. jacchus include chewing muscles for production of relatively large tree gouging gapes [42] and an enlarged caecum as part of a digestive system that optimizes digestion of tree gums [43]. Further, Souza [29] attributed the compressed brain case and protruding zygomatic arches of the jacchus group to tree gouging adaptations. Callithrix jacchus and its sister species, C. penicillata, also share derived dentition patterns [36], that also likely represent exudivory adaptations.
Few Callithrix species have home ranges as small as those of Callithrix. In comparing Atlantic Forest living marmosets and Caatinga living marmosets, the former had a mean group composition of 8.3 individuals while Caatinga mean group size was 5.9 [44]. When comparing the animals' home range between these two environments, Atlantic forest living animals had a slightly smaller mean home range (4.1 ha) than Caatinga living animals (mean 5.3 ha) [44]. Castro and Araújo [45] found that C. jacchus feed on fruit, gums, and invertebrates, but fruit and gum consumption were negatively related and influenced by seasonality. In contrast, Abreu et al. [46] did not observe a seasonal difference in gum consumption in Caatinga living individuals. However, insect consumption was significantly higher in the rainy season.
Common marmosets are highly adaptive [44,46,47] and their inhabitance of the Caatinga represents an ecological challenge related to heat stress and limited resources like water [48]. The species' adaptability warrants the question as to what makes their ecological success possible. The species' funnel-shaped teeth and ability to cling vertically on tree trunks facilitate exudate extraction [49]. Having a cooperative breeding system and production of two litters per year [50] coupled with complex cognitive abilities [51] certainly contribute to C. jacchus' ecological success. However, behavioural adjustments might be the key factor for navigating the challenges of the Caatinga [46,47].
Behavioral modification for dealing with heat stress can be observed by Caatinga common marmosets who rest twice as much as Atlantic Forest marmosets [47], live in smaller groups and appear to have a higher ratio of surface area to body mass [49]. Additionally, Caatinga marmosets eat cactus, which requires cognitive skills to access the resource while avoiding injury [46].

CALLITHRIX KUHLII
The home range of C. kuhlii varies from 10 to 58.3 ha [52], and daily ranging distance is estimated to be as far as 1498 m, which is the longest among Callithrix species [53]. In this species, group size averages 4.3 individuals, with 1 breeding female and 1-2 adult males [52]. The diet of Wied's marmoset is heavily fruit-and nectar-based, with invertebrates and small vertebrates complementing the diet [53]. In many regions, C. kuhlii co-occurs with golden-headed lion tamarins (Leontopithecus chrysomelas) although it is not clear whether there is a niche overlap of the two species [52].
Callithrix kuhlii utilizes various habitats that include restinga forest, riverside forests, secondary forests, mangroves, fruit groves, coconut and palm oil palm trees [52]. In the region of Itabuna and Ilhéus, Bahia state, the species also occupies old growth forests where cocoa is cultivated [52]. The species has a preference for occupying degraded areas, close to wood edges [52]. The species is common in urban areas of Southern Bahia state, where social groups can be seen in backyards, orchards and public squares [54]. Urban C. kuhlii groups travel along poles, electrical wires, and houses [54]. At dusk the animals retreat to clumps of bromeliads and vines which is believed to be an anti-predatory and thermoregulatory strategy [52].

CALLITHRIX PENICILLATA
Like its sister species, C. penicillata is among the smallest Callithrix species (Table 1, Figure   5). The home range of C. penicillata varies between 2.0 ha [55] to 18.5 ha [56], and social groups are composed of 2-19 individuals [28,57]. Callithrix penicillata exploits many sources of food such as fruit, buds, flowers, leaves, young stems, small prey, and bird eggs [28,56]. Additionally, this species also heavily exploits tree gums, which seems to be advantageous in adapting to degraded environments and secondary forests [28]. Given the close evolutionary relationship between C.
jacchus and C. penicillata [2], the latter likely possesses similar adaptations for gumnivory as the former.
Cosmopolitan C. penicillata populations have significantly expanded their ranges beyond their natural distribution [2], likely in part due to illegal pet trafficking of the species between northeastern and southeastern Brazil [58]. The species has been introduced to portions of Bahia, Espírito Santo, Minas Gerais, Rio de Janeiro, Paraná, Santa Catarina, São Paulo, and Rio Grande do Sul states [58,59, pers obs. JM,VB,IOS]. Mathematical models predict that the expansion of C. penicillata may be greater than expected, and that this species may even replace marmosets native to the Atlantic Forest [59][60][61].
While deforestation threatens other marmoset species, C. penicillata may have instead benefited through the opening of new niches favoring a population expansion of the species [28].
Additionally, C. penicillata shows great flexibility in occupying altered areas and secondary vegetation [62]. Other anthropogenic factors that may favor the range expansion of C. penicillata, especially within urban areas, include planting of exotic trees and reductions of predators such as constrictor snakes, birds of prey, and wild cats.

REPRODUCTION and GROWTH PATTERNS in CALLITHRIX
Small body size and a fast life history, characteristics that make marmosets prized primate biomedical models (e.g., pediatric obesity [63]; Zika infection during pregnancy [64]), are related to the evolution of Callithrix reproductive function. Four marmoset traits related to size and life history are likely inextricably linked: reproductive suppression, miniaturization, production of litters, and chimerism. However, it is important to note that, as biomedical models, marmosets have a delayed period of embryogenesis relative to humans [65]. Understanding this difference is extremely important to ensuring appropriate use of marmosets to model pregnancy and development [64].
Evidence for positively selected growth factor-insulin like growth factor axis genes that may have driven miniaturization in C. jacchus has been found at GHSR, IGF2, IGF1R, IGFBP2, and IGFBP7 [66,67]. Further, Callithrix growth patterns are likely tied to selection in marmosets for routine ovulation of multiple ova that leads to dizygotic twinning [68]. Altered placental and fetal growth patterns in marmosets may provide protection against discordant growth and placental pathologies of gestating litters in a simplex uterus [67]. The four positively selected genes with a likely role in regulating ova number and multiple gestations in C. jacchus are GDF9, BMP15, BMP4, and WFIKKN1 [67].
Several factors associated with small body size, twin-rearing, and the fact that marmosets do not display anovulation have led to a marmoset social structure in which generally one female reproduces regardless of the number of other adult females within the group. This reproductive suppression has been the subject of extensive study over the past 30 years, but the mechanisms behind it are still somewhat unclear [68]. Impaired ovulatory function, which can be maintained for up to several years, is associated with suppressed pituitary secretion of luteinizing hormone and chorionic gonadotropin. Suppressed pituitary function is in turn likely associated with enhanced negativefeedback sensitivity to low levels of estrogen or blunted responsiveness to increasing estrogen. Some marmoset daughters escape suppression while in the presence of their mothers and ovulate, though they often display impaired luteal function, which raises questions about the fertility of these females.
However, even if daughters ovulate, as long as a social group remains intact, with only the daughter's father and brothers and potential breeding partners, these females rarely become pregnant.

CALLITHRIX CHIMERISM
Perhaps the most poorly understood Callithrix derived trait is chimerism, which occurs when a single organism possesses individual cells with >1 distinct genotype. Marmoset litters usually consist of twins or triplets that share a bidiscoid placenta with a fused chorion and vascular anastomoses [69].
Fusion of the chorionic membranes during embryonic development may allow blastocyst stem and precocial cells to cross the anastomoses, rendering littermates chimeric [70]. Marmosets are therefore hematopoietic chimeras with blood derived tissues containing cells of the individual as well as their littermates [71]. However the selection pressures that may have led to this trait remain obscure.
Benirshke et al. [71] noted that approximately 90% of C. jacchus pregnancies result in chimerism, but the degree of marmoset chimerism within a tissue and the extent of the tissues involved are not resolved. An evaluation of microsatellites from C. kuhlii pregnancies with known parentage and samples from multiple tissue lineages estimated that all tissues could be chimeric, but bloodderived tissues were significantly more likely to be chimeric than other tissues [70]. Accordingly, Ross et al. [70] showed that blood-derived tissues from deceased and living C. kuhlii being between approximately 50%-100% chimeric. Malukiewicz et al. [4] found similar results for C. jacchus when comparing blood and skin tissues via microsatellite genotyping. In another chimerism tissue analysis based on the SRY gene concluded that germ line cells were not chimeric and only hematopoietic lineages were chimeric [72]. Therefore, the contribution of embryonic cells to the overall phenotypic impact of chimerism on individual marmosets remains unknown.
An interesting question is how chimerism in marmosets impacts different levels of gene regulation. With respect to the epigenome, it is known that both underlying genetic signatures and environmental factors influence epigenetic changes [73,74]. Thus, in marmosets, the complication of how chimerism levels vary between tissue types, may result in different genetic and epigenetic signatures across tissues. Further, the external signals that chimeric cells provide to one another within particular tissue environments may promote additional epigenetic changes. This idea has led to the hypothesis that chimerism may be a mechanism by which founder populations can rapidly increase their genetic diversity and gene expression variance, making them more adaptable to a range of novel environments [75]. However, while chimerism may affect such phenotypic plasticity through variation in genetic and epigenetic regulation, this relationship has not explicitly been tested in marmosets.
In most cases of biomedical modeling, it remains unclear whether chimerism may play a role in the variability found between individuals. However, chimerism in marmosets may be particularly well suited to questions designed to take advantage of the close relatedness of multizygotic litters using techniques such as adoptive transfer or paired design using litter mates as controls versus treatment subjects in immunology studies. Additionally, marmosets may be a natural model in which to study the impact of genomic conflict for questions of stem cell transfer, maternal-fetal microchimerism, or transplants.

CALLITHRIX COGNITION
Cognition studies is one of the growing areas of research for common marmosets (reviewed in [51]). Although laboratory studies dominate C. jacchus research from the last five years, field studies should gain more attention because of their complementary value in relation to laboratory studies [51].
In this regard, field studies can have particular importance in areas that need integration between cognition and ecology. For instance, a recent study showed that common marmosets use spatial cognitive abilities to effectively obtain food in nature [76]. Studies like this, in which wild animals face the constraints and limitations imposed by the environment, can help us better understand the selective pressures that ultimately shaped their cognitive abilities.
Common marmosets are a particularly promising model for studying evolutionary and functional questions in primate personality due to their explorative and highly social character, and adaptability to different natural environments [44,51]. Marmosets display personality traits when assessed with tools such as personality tests [e.g., 77] and behavioural observations [e.g., 78]. In particular, marmosets exhibit short-term and long-term consistency of non-social and social personality traits [79]. Personality structure under captive and wild conditions is rather similar [79].
Inoue-Murayama et al. [78] found that C. jacchus sociability was positively linked with increased subjective well-being and cortisol levels, whereas neuroticism and dominance were associated with certain genetic polymorphisms. Interestingly, group level similarity in marmoset personality has been found [77], possibly as a product of shared social environment, genetics or social facilitation. Future studies are needed to further explore links between inter-individual behavioural and cognitive variation in this species.

CALLITHRIX GENOMICS and GENETICS CALLITHRIX GENOMIC ASSEMBLIES
A high-quality genome is required for animal models, and in 2014 C. jacchus became the first New World monkey whose genome was sequenced and assembled. Sanger sequences from a female marmoset were generated by an international consortium and assembled into extended continuous arrays of nucleotides (contigs). The published assembly (designated CalJac3 in the NCBI genomics database) consisted of 2.26 billion base pairs covering all Callithrix chromosomes [66]. However, the large number of continuous sequences were broken by gaps so that no chromosome sequence was complete end-to-end. Nonetheless, this genome allowed analysis of marmoset twinning genes [66,67], and detection of primate-specific constrained elements [80]. Tables 2 and 3 list several genomic assemblies now available for C. jacchus, some of which have been published [e.g., 81], as well as one unpublished assembly for the C. penicillata genome. An unpublished C. jacchus genome assembly by the Vertebrate Genomes Project is based on a novel technique called trio-binning in which the reads from parental and maternal haplotypes are assembled separately, resulting in a diploid assembly [82]. The most recent C. jacchus assembly is designated cj1700_1.1, which has now replaced calJac3 as the reference genome for that species. Cj1700_1.1 has a contig N50 (defined as the length of an individual contig for which half of all the available sequence occurs in contigs larger than that figure) of 25.2 megabases, an approximately 860-fold improvement over calJac3 in terms of contiguity. An unpublished genomic assembly for C. penicillata has recently been sequenced and annotated (pers. obs., Malukiewicz). The total length of this C. penicillata genome is 2.6 GB and the contig N50 is 21.6 megabases, which are similar assembly characteristics as that of Cj1700_1.1 (unpublished data, Malukiewicz et al.).
More accurate and (largely) complete reference genome sequences for marmosets are opening tremendous opportunities for novel research. For example, CRISPR/Cas9 gene knock-in [83] ha generated transgenic marmosets to model human disease. Further, the newer Callithrix genomic assemblies contain less gaps and better assembly of duplicated regions, which will provide better genomic annotation, leading to improved analysis of assays that require alignment of short reads to a reference genome (RNA-seq, ATAC-seq, ChIP-seq, etc). The diploid assemblies will facilitate the study of haplotype structure and structural variation in marmosets [84].

CALLITHRIX FUNCTIONAL GENOMICS and EPIGENOMICS
Marmoset epigenetics studies have examined DNA methylation patterns in a wide sampling of tissues like bone [85] and placental tissue [86]. DNA methylation variation can be associated with variation in transient traits like body weight, and when such changes in gene regulation occur in developmentally important tissues like the placenta, they can potentially impact phenotypic development in offspring [86]. Interestingly, Housman et al. [85] found that DNA methylation variation in marmosets is not strongly associated with variation in static traits like bone morphology.
This study also noted higher levels of DNA methylation heterogeneity as compared to other nonhuman primate species, which led these researchers to speculate whether such differences might be due to chimerism. Such epigenetic results suggest that it is particularly relevant to examine to what degree chimerism affects epigenetic variation within specific tissues or cell types, as levels of chimerism are known to vary across different marmoset tissues [4,70,72].
Brain development, evolution, and plasticity is one area of marmoset functional genomics that is being explored. Current work has focused on characterizing candidate gene expression patterns in brain tissues [87,88], but some research has also assayed candidate gene expression responses following severe psychological perturbations such as parental separation [89]. Interestingly, while most brain regions show conserved levels of candidate gene expression between marmosets and mice, some areas of the brain do show marmoset-specific expression patterns, such as the early visual cortical area and afferent areas of the hippocampus [87]. Now is a particularly relevant time to pursue functional genomics research, especially in primates [90], as technologies can characterize whole transcriptomes and epigenomes at both bulk and single-cell resolutions. Already, some comparative cross-species studies that include marmosets have begun utilizing these single-cell methods [91,92]. Further, these technologies can be used on primary ESCs and iPSCs are ideal for functional genomics research, especially in the context of disease, as they are immortalized and self-renewing, have the potential to differentiate into cells from any germ layer, and can be readily used in controlled experiments [93]. Another benefit is the relative ease with which these cell lines can be genetically modified [83].
Finally, consolidating functional genomics data for better access and comparison is essential in these efforts. Larger consortia that compile large -omics datasets for marmosets are being built [96,97].
For instance, functional genomics data from marmoset brain tissues have been collected and incorporated into the NIH Brain initiative [98]. This initiative is of particular relevance as the common marmoset has been identified as an important model organism for neuroscience research that can bridge the gap between mice and humans [96,99]. Such foundational databases have enabled subsequent explorations such as single-cell RNA-seq of C. jacchus and other mammals have identified primate-specific interneuron subtypes [100] and common and divergent features of preimplantation development [101].

CALLITHRIX IMMUNOGENETICS
Although New World primate immunogenetic studies lag behind that of Old World primates, complex immunogene families like Major Histocompatibility Complex (MHC) Class I and the Natural Killer Complex (NKC) have been sequenced and annotated in C. jacchus. The MHC, an evolutionary hallmark of enormous genetic variability and genomic diversity, determines immune responsiveness and is associated with many diseases. MHC class I proteins bind pathogen-derived peptides for presentation to specialized cells for immune response initiation [102]. MHC class I proteins also interact with natural killer (NK) cell receptors to prevent NK cell destruction of healthy cells [103].
The C. jacchus MHC class I region is composed of three segments-Caja-B/C segment, the Caja-E segment, and the Caja-G/F segment [104,105]. In humans, six MHC class I loci are known as HLA (human leukocyte antigen)-A/B/C/D/E/F/G, and Caja-B/C corresponds to HLA-B/C [104] and Caja-G/F corresponds to the HLA-A/G/F region [104]. Shiina et al. [105] described Caja-B/C as a 1,079 kb fragment containing nonclassical (low polymorphism) MHC class I genes and Kono et al.
[105] characterized a 854 kb tract of the Caja-G/F segment, which harbors classical (polymorphic) MHC class I genes. Caja-B/C contains 54 genes among which there are nine functional MHC genes and four MHC pseudogenes [105]. Caja-G/F encompasses six functional MHC genes as well some non-MHC genes and MHC pseudogenes [105]. The MHC I region maps to chromosome 4 of C.
Interestingly, the human HLA-G locus shows limited polymorphism and grants immunotolerance between mother and fetus during pregnancy [106]. The homologous Caja-G locus instead shows high allelic polymorphism levels, and expression of Caja-G/F alleles in various tissues suggests that this locus has taken on the role of classical MHC I function in C. jacchus [106]. Evidence for genetic conversion in the role of Caja-G/F is seen further when comparing exon diversity between Caja-G/F and HLA-G gene associated with MHC protein binding of pathogen peptides. Caja-G/F MHC genes show a high level of polymorphism that sharply contrasts that of HLA-G MHC genes [106] Using the unpublished C. penicillata draft genome assembly mentioned earlier, we extracted genomic segments from chromosome 4 between BAT1 and CDSN as the putative C. penicillata MHC class I B/C (Cape-B/C) region and between RNF39 and ZFP57 as the putative C. penicillata MHC class I G/F (Cape-G/F) region. The limits chosen for Callithrix MHC class I regions followed designations by Shinna et al. [105] and Kono et al. [104]. Dot plots of Caja-B/C:Cape-B/C and Caja-G/F:Cape-G/F (Figure 8) show large tracts that are highly similar or identical between C. penicillata and C. jacchus. The two Caja segments are longer than the two Cape segments, but given that there are some gaps in the genomic assembly within Cape MHC class I segments (pers. obs., Malukiewicz), further sequencing is needed to determine the actual length of these segments for C. penicillata.
Following the annotation methodology of Kono et al. [104] and Shinna et al. [105], we identified 7 putative MHC class I loci within Cape-G/F and 9 putative MHC class I loci within Cape-B/C. Oxford Nanopore long-read targeted sequencing of these putative C. penicillata MHC class I loci is on-going to further characterize these genomic regions.
Two structurally unrelated classes of NK cell receptors exist in primates-the killer cell immunoglobulin-like receptors (KIR) encoded by the leukocyte receptor complex (LRC) and killer cell lectin-like receptors encoded by the natural killer complex (NKC) [107]. The KIR lineage is present in all simian primates [103], but in C. jacchus there are apparently only two KIR loci [66]. In C. jacchus, NKC genes cluster together and are orthologous to humans, but the marmoset NKC segment is 1.5x smaller [107]. Marmoset NKC genes also show moderate polymorphism [106].
Averdam et al. [106] found that the C. jacchus NKC encodes a single inhibitory heterodimer (CD94/ NKG2A) and two activating NK cell receptors, (CD94/NKG2CE and NKG2D). In humans, CD94/ NKG2A and CD94/NKG2C are ligands for HLA-E, a nonclassical MHC class I molecule that plays an important function in cell recognition by NK cells [107], but the role of Caja-E remains to be determined.

CALLITHRIX VIRUSES and the VIROSPHERE CALLITHRIX as MODELS of VIRAL DISEASES
Callithrix jacchus has served as a model for several aspects of biomedical research related to viruses. In humans, Epstein-Barr virus infection may enhance risk of developing multiple sclerosis, and natural infection of C. jacchus with the related 1-herpesvirus CalHV3 has been harnessed to model human autoimmune disease [108]. Additionally, Callithrix jacchus has been used to model hepatitis A infection [109]. The species was also used for in vitro transformation of two permanent and virus producing lymphoblastoid cell lines with a nasopharyngeal carcinoma derived Epstein-Barr viral strain [110]. Other viral pathogens that have been explored in Callithrix include parainfluenza virus type 1 [111], Flaviviridae-like viruses [112], Oropouche virus [113], and Simian foamy virus [114].
More recently, Callithrix is emerging as an infection model of several arthropod borne viruses (arboviruses) of single stranded-RNA viruses from the genus Flavivirus. These viruses include dengue virus (DENV), now considered the most prevalent and quickly spreading human arboviral disease whose symptoms range from high fever to shock and death [115]. Both C. jacchus and C. penicillata are candidate systems to model DENV pathogenesis and for discovery of antiviral drugs and vaccines [116,117]. In C. penicillata, DENV infection produced marked changes in microglial cells of the central nervous system [118]. Both Callithrix species show elevated proinflammatory cytokines upon DENV infection, which may model aspects of human DENV pathogenesis. Another Flavivirus, Zika virus (ZIKV) was originally discovered in Uganda [119], but is now the newest Flavivirus arrival in the New World. Pregnant women infected with ZIKV are at risk for miscarriage and fetus microcephaly, and adults can develop neurological condition like Guillain-Barré syndrome [120]. A number of recent studies have found that C. jacchus replicates many of these human clinical and gestational symptoms [64,120].  [130]. Additionally, Terzian et al. [131] found evidence of widespread ZIKV exposure in over 50

EXPOSURE of WILD CALLITHRIX to PATHOGENIC ARBOVIRUSES
Callithrix sampled Minas Gerais and São Paulo states.
In order for a translocated arbovirus to enzootically establish itself within the Neotropics, wildlife exposure, zoonotic infection, and persistent enzootic transmission of the translocated zoonosis need to occur [132]. Callithrix characteristics that can facilitate arbovirus transmission include a great adaptability to human environments, frequent interactions with human beings in urban and peri-urban environments also occupied by anthropophilic arbovirus vectors such as Aedes aegypti and A.
albopictus [124]. The presence of short-lived, fast reproducing primates also facilitates the persistence of arbovirus transmission [132], which are certainly characteristics that define many Callithrix species and hybrids.

EXPOSURE of WILD CALLITHRIX to VIRUSES BEYOND ARBOVIRUSES
Human herpesvirus 1 (HV1) is transmissible from humans to Callithrix marmosets, which has resulted in a number of fatal epizootics events [133,134]. Transmission occurs through the close contact that these animals have established with humans, facilitated by the human habit of offering food, since the virus can be transmitted through contaminated saliva, aerosols and fomites.
Transmission can also occur spontaneously between marmosets within the same group [133,135].
Affected animals are usually found dead or with severe neurological symptoms, resulting from virusinduced encephalitis [134]. The virus also causes death from meningitis [134], erosions and ulcers in the oral cavity, in addition to hemorrhages and focal necrosis in several organs [135].
Rabies is a zoonotic disease caused by lyssaviruses, transmitted through saliva, and infection causes fatal acute encephalomyelitis. The disease is transmissible from animals to humans by bites.

Callithrix jacchus has played an important role in the transmission of rabies in northeastern Brazil,
where it maintains a distinct transmission cycle [136]. Infected marmosets often show no clinical signs, but rather sudden death preceded by sadness or weakness. A recent case of rabies in Callithrix sp. occurred in urban Niterói, Rio de Janeiro state [137]. The proximity of marmosets to urban environments and the practice of capturing and maintaining these animals as pets are two factors that greatly increase the risk of marmoset rabies transmission [138].

CALLITHRIX VIROME RESEARCH
Although the virosphere incorporates the most abundant and diverse group of organisms on Earth, it remains largely neglected, with most attention given to viruses of high biomedical relevance.
It is estimated that there are over 500,000 undiscovered animal viruses that are transferable to people [139]. Although Callithrix virome work is still at an infancy, one pioneering study helped to characterize two novel Papillomavirus genomes (CpenPV-1 and CpenPV-2) in captive C. penicillata [140]. Other viral families also have been sequenced in this work, but remain to be published. An important future role for Callithrix virome studies will be to help understand the capacity of Callithrix to be YFV hosts and their susceptibility to YFV disease. Also, virome studies can help to understand if this genus could actually represent a potential risk for a severe urban YFV outbreak [124,126] or even zoonotic virus spillover. Overall, virome studies can greatly enhance our ability to detect known and novel viral sequences and can also help to understand the entire dynamic and interactions of the Callithrix virosphere.

CALLITHRIX SOCIAL DYNAMICS
While long-term studies are still necessary to understand wild Callithrix social dynamics, some insight is available from C. jacchus groups at the Tapacurá Field Station, São Lourenço da Mata, Pernambuco (08⁰ 07' S, 34⁰ 55' W, Figure 9). A typical Tapacurá C. jacchus social group may contain eight individuals-three reproductive adults, two juveniles, two infant co-twins, and an adult or subadult that may or may not be related to other group members [141]. The three breeding adults may include a dominant female and two males that take on variable roles in offspring care and group defense. One breeding male will be the principal infant carrier, while the second breeding male aids the breeding female in choosing group feeding, resting, and sleeping sites, and in territorial defense [141]. A breeding female depends on her group's support to maintain dominant status, but the loss of the reproductive position may not result in her departure from the group. There are instances at Tapacurá where breeding females were displaced by one or two daughters, but remained in the group to suckle grandchildren along with the new dominant female(s) [141].
In regards to sexual behavior studied at Tapacurá, between 1994-1996 52 copulations were observed, of which 16 were intra-group, and only two involved non-reproductive females. Of 33 extragroup copulations, 17 were between neighboring focal study groups, and 16 were between members of a focal group and peripheral groups. To illustrate extra-group copulations, the reproductive female of one focal group was seen copulating seven times with two dominant males (2 to 3 times each) in neighboring groups. In general, sexually mature females take all opportunities to copulate, independent of their phase in the estrous cycle. Females also disperse more than males (Figure 10), leaving their natal groups to increase their chances of breeding [142].
Interestingly, the strongest social interactions within C. jacchus groups are between the male care-giver (one of the probable fathers in the group) and infants. The findings help to explain, at least in part, why males disperse less than females, as nutritional provisioning of infants by the male caregiver demands dedication and skill. On one occasion, a male carer was seen to dexterously secure the floral pedicel of a cashew fruit with both hands and, while balancing with extended legs, lifting it above his head after biting the hypocarpium several times to allow the infants to lick the juice ( Figure   11). During the infant weaning phase, the carer male also teaches infants skills needed to travel, forage, confront predators, and how to recognize, catch, and manipulate food items [141]. The male carer is the last to abandon a dead infant, which illustrates a deep social bond between a marmoset male and marmoset infants [143].

PET TRADE
While it is illegal in Brazil to have native primates as pets [144,145] and in São Paulo state there is a moratorium on captive breeding of marmosets by authorized commercial breeding facilities [146], there still exists an underground Brazilian, illegal pet market [147]. Marmosets, most often C. jacchus and C. penicillata, are taken from the wild by several methods including trapping, killing of adults to take the infants, rescue of infants after habitat loss, and then either sold locally or transported to large urban areas [147][148][149]. Confiscated marmosets from animal traffickers are often stressed, malnourished and dehydrated -conditions that favor the development and transmission of diseases.
Transport stress and inadequate hygiene conditions also contribute to the development of infections.
Captured marmosets appear to be trafficked using the same illegal trade routes as other wildlife. In the case of C. penicillata, construction of roads and increased vehicular traffic between the Cerrado and southeastern Brazil, has greatly facilitated illegal trafficking of this species [58]. There are few studies that quantify the pet trade, but Callithrix individuals, especially C. jacchus and C. C. geoffroyi, C. jacchus, and C. penicillata, have high invasive potential [61,62], and are spreading throughout the southeastern Atlantic Forest due to the legal and illegal pet trades [21,58,154]. As a result, Callithrix jacchus and C. penicillata have established several allochthonous populations in southeastern Brazil [2,21,58,[150][151][152][153][154][155]. Mitogenomic data shows that source populations of allochthonous Callithrix species actually come from across broad geographic origins outside of the southeastern Brazilian Atlantic Forest [2]. As discussed below, anthropogenic hybridization between allochthonous marmosets and either other allochthonous or autochthonous congeners occurs widely across southeastern Brazil [2,4,21,[153][154][155].
Contemporary allochthonous Callithrix species are usually found in urban or peri-urban areas of the southeastern Atlantic Forest [21,58,151,152,[155][156][157]. Within these contexts, allochthonous Callithrix have frequent human contact and exposure, and receive anthropogenic food supplementation [54,58,158]. These allochthonous marmosets can also be found within or around natural reserves [13,58,151,152,155,157], and in some areas, such marmoset populations are larger than those of native, endangered callitrichids [e.g.,151]. Such introductions could alter the ecological relationships among taxa [13,159], as the main threats posed by allochthonous marmosets are competition for food resources, increased predation of native fauna, the introduction and maintenance of disease, and hybridization [160][161][162]. The threats can become exacerbated when introduced marmosets become abundant in a landscape of small fragments.

ANTHROPOGENIC CALLITHRIX HYBRIDIZATION
Due to the legal and illegal pet trades, C. jacchus and C. penicillata have established several allochthonous populations in southeastern Brazil that hybridize with allochthonous and autochthonous congeners ( Figure 11; [19,21,163]). It is highly likely that C. aurita faces competition, conservation, and genetic threats from allochthonous jacchus group species and anthropogenic hybrids [2,10,154].
Callithrix flaviceps also likely faces similar pressures. Some cases exist of native C. aurita and C.
flaviceps meeting up and interbreeding with hybrid and allochthonous Callithrix at urban fringes (reviewed in [21]). Such interactions likely facilitate gene flow from allochthonous jacchus group species into native marmoset populations in southeastern Brazil, with consequences that may include outbreeding depression, admixture, hybrid swamping, or introgressive replacement [164][165][166]. Indeed, Malukiewicz et al. [2] showed for the first time, evidence of introgression of mitochondrial DNA from allochthonous C. jacchus into the genetic background of native C. aurita from the São Paulo metropolitan area. These data also showed the first genetic evidence for cryptic hybridization within the aurita group marmosets. A few anthropogenic Callithrix hybrid zones have also been studied using mitochondrial, nuclear, and Y-chromosome markers [2,4,21,155, https://tede.ufrrj.br/jspui/handle/ jspui/3009].
Anthropogenic hybridization with C. jacchus and/or C. penicillata also represents a potential risk for genetic extinction of the other two jacchus group species, C. geoffroyi and C. kuhlii, and Silva et al. [58] recently showed that C. penicillata is encroaching on the native range of C. geoffroyi.
Anthropogenic hybridization of jacchus group species generally results in the formation of hybrid swarms, admixed populations that lost parental phenotypes and genotypes [4,21,153]. Should large numbers of exotic C. jacchus or C. penicillata ever invade native ranges of C. kuhlii or C. geoffroyi, the latter two species may be threatened with genetic swamping by the former two species, a process through which parental lineages are replaced by hybrids that have admixed genetic ancestry [169].

Further, biological invasions by other marmosets present potential conservation risks for C. kuhlii,
which is already considered Vulnerable by the IUCN Red List [170].

CALLITHRIX POPULATION DECLINES and CONSERVATION CALLITHRIX POPULATION DECLINES
All Callithrix species face population declines across their native ranges as a result of habitat loss and land conversion, principally for urbanization, agriculture, and livestock production [11,10,12,168,169]. The estimates of remaining original forest area are 63% for the Cerrado, 47% for the Caatinga, and 11-28% for the Atlantic forest [171,172]. Furthermore, between 2006 and 2015, the accumulated area of lost natural vegetation of Brazilian biomes was 300,000 km² [173]. For arboreal primates, marmosets included, continued deforestation in Brazil is the most serious threat to the persistence of populations and species [12,174].
The three most threatened Callithrix species are C. flaviceps, C. aurita and C. kuhlii [11,10,170]. Callithrix aurita has also been listed among the 25 most threatened primates on the planet [175].
Projections based on forest loss and other anthropic factors predict reductions of over 50% of C. aurita and C. flaviceps populations in the next 18 years [10,11]. For instance, forests within the geographical distribution of C. aurita were reduced by 43% between 1990 to 2008 [176]. For C. flaviceps, agricultural activities have profoundly modified the environment where the species naturally occurs, with cattle breeding and coffee plantation occupy formerly forested areas [58]. The remaining C.
flaviceps sub-populations, estimated at ~4,440 individuals, are restricted to the margins of riparian forests, and steep hillsides unsuitable for raising cattle, or planting coffee or eucalyptus [11]. Between 2015-2017, the wide-spread Brazilian YFV epidemic decimated > 90% the C. flaviceps population at RPPN Feliciano Miguel Abdala, a private reserve in Caratinga, Minas Gerais [177]. The virus may have caused other such reductions across the C. flaviceps range [11]. It is interesting to note that the global climate changes that are happening could lead to reductions in the distribution area as high as 95% for C. flaviceps and 27% for C. aurita [60]. For C. kuhlii, whose total population is estimated to be >10,000 individuals, a population decline of 30% is expected by 2031, because much of its native habitat is being converted to cattle ranches and agriculture [174].
The other three Callithrix species are of "least concern", nonetheless their populations are declining throughout their native geographic ranges [168,169,178]. These changes are likely driven by the illegal wildlife trade and agricultural/ranching activities. The urban expansion that accompanies agriculture expansion is characterized by activities that limit primate dispersal or increase mortality: building of roads, topographical changes caused by erosion, and the presence of domestic animals such as dogs that enter the forests for hunting [180]. For the three "least concern" jacchus group species, the human activities that devastated marmoset habitats also drive social groups into urbanized areas, where they are more vulnerable to disease, accidents and poaching-perhaps making urban Callithrix Forest NAP [186]. In 2018, Callithrix species became part of the NAP for the Conservation of The Primates of the Atlantic Forest and the Collared Sloth [187]. The Saguis-da-serra Conservation Program (PCSS) is an initiative to put into practice the actions foreseen by such NAPs, along with the help of several researchers and institutions.
Recently, a studbook was created to track genealogical relationships between individuals of the Brazilian captive C. aurita population [154]. The C. aurita Studbook Keeper, C. Igayara, estimates that there are approximately 33 individuals that make up the current captive population. An important objective in breeding and maintaining a captive C. aurita population is to maximize genetic diversity individuals [154,175]. Currently, no C. flaviceps are maintained in captivity (pers obs., FR de Melo).
Another important Callithrix conservation initiative has been the creation of the Center for the In the last decade, regulations for the management of fauna have emerged in Brazil [e.g., 188,189] including, more recently, the management for the control of invasive alien and allochthonous species [146,190,191]. Currently, many confiscated and apprehended animals end up in Brazilian animal triage centers [192], and these captive facilities do not have further capacity to receive a higher influx of marmosets, nor to keep them for long. In addition, most management institutions have no interest in maintaining "common" or non-threatened species. From an ecological perspective, allochthonous marmosets can be considered introduced and potential or de facto invasive species. If allochthonous marmoset species are considered exotic, then the proper course of action would be to eradicate them during the early invasion stages or control established populations. Introduced and hybrid marmosets could be also removed from areas in which they pose a direct conservation problem, but the major obstacle for this course of action is resolving the destination of these animals.
Ultimately, although these kinds of actions are supported by national Brazilian legislation and NAPs, effective action implementation runs into problems concerning funding and reaching a common consensus for the best plan of action.

CONCLUSION
Marmosets have been the quintessential laboratory primate model for a wide variety of health studies. Nowadays, with the increased adoption of the concept of One Health, and the opportunities afforded by established field sites for several species under a variety of environmental conditions, marmosets can become a model group to study the evolution and ecology of infectious diseases, the relationship between environment, health, ageing and genetics, adaptive responses to emerging diseases, immune system genetics, physiology, epidemics and other public health topics. These studies can contribute both to the understanding of the dynamics of diseases of concern to humans and to the conservation of several endangered species of marmosets and other primates with which they are sympatric. This scenario offers an unprecedented opportunity for synergy between evolutionary biology, human health, and species conservation concerns. Thus, the stage is set to establish long term scientific exchange and collaboration among scientists from various disciplines and different countries to address human and non-human primate issues of world-wide importance. Figure 1. Maps of natural Brazilian Callithrix ranges following Rylands et al. [1], and natural hybrid zones following Malukiewicz [21]. Each range is color-coded by species and respective hybrid zones            penicillata. Hybrid zone locations adapted from da Rosa et al. [19], Malukiewicz [21], and Bezerra et al. [168].    Art. 3 sets a standard for the performance of the following activities, "with the purpose of scientific or didactic, in the national territory, on the continental shelf, in the territorial sea and in the exclusive economic zone: I -collection of biological material; II -capture or marking of wild animals in situ; III -temporary maintenance of specimens of wild fauna in captivity; IV -transport of biological material; and V -conducting research in federal conservation unit or in underground natural cavity." Activities with didactic purposes are restricted to those performed in the scope of higher education. This normative instruction does not apply to the collection and transportation of material from biological species, domesticated or cultivated, except when related to research carried out in federal conservation units in the public domain, and exotic wild in exsitu conditions. When the research activities are carried out within state or municipal Conservation Units, in addition to SISBio authorization, it is also necessary to request authorization from the respective competent environmental agencies: - The objective of the Strategy is to guide the implementation of measures to prevent the introduction and dispersion and significantly reduce the impact of invasive alien species on Brazilian biodiversity and ecosystem services, control or eradicate invasive alien species. Exotic species or subspecies are considered as those occurring outside its past or present natural distribution area; including any part, such as gametes, seeds, eggs or propagules that can survive and subsequently reproduce.

NORMATIV INSTRUCTION ICMBio Nº 6/2019
It provides for the prevention of introductions and the control or eradication of exotic or invasive species in federal Conservation Units and their damping zones.
Fuidance for the management of invasive alien species in Federal Conservation Units is available in http://www.icmbio.gov.br/cbc/ publicacoes, which includes methods already approved by ICMBio and is considered the guiding document for project analysis.

RESOLUTION SMA No. 164/2018 (São Paulo)
It establishes procedures for reproduction of Callithrix specimens kept in wildlife enterprises in captivity in the State of São Paulo.
It prohibits the production of hybrid individuals of the genus Callithrix and the reproduction of individuals belonging to the species C. jacchus and C. penicillata (exception given only to scientific and commercial breeding sites using these species as matrices, but marketed animals should be sterilized). Callithrix penicillata reproduction may be authorised only for individuals of known origin and for conservation, by Zoos and scientific breeding sites for conservation purposes.