Cytogenetic study in natural hybrids of Callithrix ( Callitrichidae : Primates ) in the Atlantic forest of the state of Rio de Janeiro , Brazil

1. Departamento de Genética, Instituto de Biologia, Universidade Federal Rural do Rio de Janeiro/UFRRJ, Rodovia BR-465, km 7, 23890-000 Seropédica, RJ, Brasil. (denisemn@ufrrj.com) 2. Departamento de Patologia e Clínica Veterinária, Faculdade de Veterinária, Universidade Federal Fluminense/UFF, Rua Vital Brazil Filho, 64, 24230-340 Niterói, RJ, Brasil. (anaferreira@pesquisador.cnpq.br) 3. Serviço de Criação de Primatas, Centro de Criação de Animais de Laboratório, Fundação Oswaldo Cruz (CECAL/FIOCRUZ), Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brasil. (bibi@fiocruz.br) 4. Centro de Primatologia do Rio de Janeiro, CPRJ/INEA, Estrada do Paraíso, s/n, 25940-000 Guapimirim, RJ, Brasil. (pissinatticprj@globo.com) 5. Médica Veterinária Autônoma. (julianecarelli@gmail.com) 6. Universidade Estácio de Sá (UNESA), Estrada Borda do Mato, 850, 22783-320 Vargem Pequena, RJ, Brasil. (carlos.verona@ensp.fiocruz.br)

The common marmoset C. jacchus originally inhabited dry thorn scrub (Caatinga) and nearby regions of Atlantic forest in northeastern Brazil (MitterMeier et al., 1988;rylandS et al., 1993), south to the state of Bahia.For at least one century it was introduced in other regions of the Atlantic forest, particularly in southeastern Brazil, establishing itself in areas occupied by other Callithrix species (coiMbra-Filho, 1990;brandão & deVelley, 1998;ruiz-Miranda et al., 2000).
Callithrix penicillata occurs in seasonal environments of gallery forests, forest patches and savanna (Cerrado) in central Brazil (herShkoVitz, 1977).This species, as in the previous case, is invading and possibly replacing other species in many regions of the east and south, in the states of Minas Gerais, Espírito Santo, São Paulo and Rio de Janeiro (rylandS et al., 1993; rocha et al., 2004).The other four species occur in the Atlantic forest of eastern and southeastern Brazil: C. kuhli and C. geoffroyi inhabit lowlands while C. aurita and C. flaviceps occur in the mountainous rain forests above 400-500m.
Typical of the Atlantic forest of Rio de Janeiro, C. aurita also occurs in the east and northeast of São Paulo and in the southern part of Minas Gerais (coiMbra-Filho, 1986;brandão & deVelley, 1998).This species is considered vulnerable, according to the International ABSTRACT.In the Atlantic forest of Rio de Janeiro, Callithrix aurita (É.Geoffroy in Humboldt, 1812) is a native species vulnerable to extinction and C. jacchus (Linnaeus, 1758) and C. penicillata (É.Geoffroy, 1812) are invasive species.The major threats to the native species are habitat degradation and hybridization, although there are currently no genetic data about natural hybrids available.Previous studies have revealed that species of the Callithrix genus are extremely homogeneous in their karyotypes with the exceptions of the morphology and size of the Y chromosome and its nucleolar organizer region (NOR) banding pattern.Three male marmosets captured in the wild in Guapimirim municipality, Rio de Janeiro, Brazil, considered as possible hybrids between C. aurita and C. jacchus or C. penicillata on the basis of pelage pattern, were cytogenetically studied.Metaphase chromosomes were obtained by using short-term lymphocyte cultures and Ag-NOR staining was performed.The hybrids karyotypes were 2n=46, 14 uni-and 30 bi-armed autosomes, a median size submetacentric X and NOR bearing autosomes, being compatible with that observed for the genus.In the three individuals studied, Y chromosomes were similar to those found for C. aurita, without NORs.The data obtained suggest the involvement of C. aurita in natural hybridization with one of the invasive species.We discuss the possible consequences of this hybridization.
Iheringia, Série Zoologia, Porto Alegre, 101(3): 156-160, 30 de setembro de 2011 Union for Conservation of Nature (IUCN, 2008), and is also listed in the Convention on International Trade in Endangered Species (CITES, 2009) and in the Brazilian Institute for the Environment's list of nationally threatened species (IBAMA, 2003), due to its small population size and ongoing decline.The widespread degradation of the Atlantic forest and the hybridization with invasive species (C.jacchus and C. penicillata) are the major threats to C. aurita, although the extent of the latter remains obscure (rylandS et al., 2009). coiMbra-Filho et al. (1993) reported the occurrence of experimental hybridization in captivity among Callithrix species which resulted in fertile individuals with several distinct phenotypes.There is also evidence of natural hybridizations at the distributional limits of the Callithrix species or due to introduction (in the case of C. jacchus and C. penicillata) (rylandS et al., 2000).The present study examines three male Callithrix captured in the wild in Guapimirim municipality, state of Rio de Janeiro, Brazil, which were considered possible hybrids between C. aurita and C. jacchus or C. penicillata according to indistinct pelage.
Many invasive species take opportunistic advantage of other forms of ecosystem change, such as habitat disturbance, rather than being the direct cause of the decline of native species (didhaM et al., 2005).For the establishment of conservation management plans to protect the vulnerable species, different causal mechanisms that lead to interspecific breeding must be distinguished.The genetic confirmation of the hybridization is imperative to clarify if there is a directional pattern in the hybridization and also in the survival and reproduction of the hybrids.
From the cytogenetic aspect, the Callithrix species represent a homogeneous clade with the exceptions of morphology and size of the Y chromosome, and their Ag-NOR pattern (nagaMachi & Ferrari, 1984;ardito et al., 1987).
Based on chromosomal differences already reported for Callithrix species, our objectives were: to analyze the morphology of the Y chromosome of the three possible hybrids, comparing its morphology with that previously observed for C. aurita, C. jacchus and C. penicillata; to identify the paternal species involved in hybridization; and to examine if the Y chromosome of the individuals studied present secondary constriction similar to that reported for C. jacchus, which would indicate paternity by a male of this species.

MATERIAL AND METHODS
Three male marmosets considered as possible hybrids between Callithrix aurita and C. jacchus or C. penicillata on the basis of pelage pattern were cytogenetically studied.The individuals were captured in the wild in Guapimirim (22 o 32'14" S, 42 o 58'55" W), in the state of Rio de Janeiro at an altitude of 40 m, in the forest near the Rio de Janeiro Primate Centre (CPRJ/ INEA), Brazil, where these species are kept in captivity.The capture location is part of the Atlantic forest, a highly threatened ecosystem, classified as a global hotspot of biodiversity due to its exceptional concentration of endemic species and the loss of more than 70% of its primary vegetation (MyerS et al., 2000).
Metaphase chromosomes were obtained by using the lymphocyte culture method (Moorhead et al., 1960).Blood (2mL) was collected at CPRJ/INEA by femoral puncture in heparinized syringes.Chromosomes were analyzed by conventional staining with 3% Giemsa in phosphate buffer pH 6.8 and examined at a 100x magnification.Nucleolar organizing region (NOR) characterization was performed following howell & black (1980).The karyotypes of the three possible hybrids were compared to those previously described by nagaMachi (1995) and nagaMachi et al. (1997) (Fig. 1).

RESULTS
The karyotypes of the three possible hybrid marmosets showed the same pattern observed in the genus Callithrix.They presented 2n=46, with 14 uni-and 30 bi-armed autosomal pairs, a median submetacentric X and a diminutive uni-armed Y chromosome, similar to that found in C. aurita (Fig. 2).
The Ag-NOR staining marked secondary constrictions located in the short arms of six acrocentric autosomes (Fig. 3).Active NOR was not found on the Y chromosome differing from that observed in C. jacchus (bedard et al., 1978;nagaMachi & Ferrari, 1984;ardito et al., 1987).

DISCUSSION
The five Callitrix species, except C. flaviceps (not studied), present 2n=46, 14 acrocentric and 30 bi-armed autosomes, a median size submetracentric X chromosome, and differ only in the morphology of the Y chromosome (Fig. 1;nagaMachi et al., 1997), which in the case of C. jacchus was variable even between populations.The Y chromosome is subtelocentric in individuals from Bahia and metacentric in those from Pernambuco, probably due to a pericentric inversion (nagaMachi & Ferrari, 1984).
Analysis of the G-and C-banding patterns demonstrated a lack of any chromosomal rearrangements to differentiate their karyotypes (nagaMachi et al., 1997).
Ag-NOR staining marked secondary constrictions of the small arms of the acrocentric chromosomes of all Callithrix species studied.Callithrix jacchus differs from its congeners by being the only species that shows Ag-NOR staining on the Y chromosome (bedard et al., 1978;nagaMachi & Ferrari, 1986;ardito et al., 1987).
The karyotype of the three individuals studied  nogueira et al. was compatible with that described for the genus.The Y chromosome similar to that of C. aurita and the absence of NOR in this chromosome suggest that the paternal species of the three possible hybrids is C. aurita, and that one of the invaders, C. jacchus or C. penicillata, is the maternal species.Cytogenetic and molecular analyses conducted so far indicate that the Callithrix genus constitute a homogeneous clade with little genetic divergence among the species, which suggests that the speciation in this group is recent (Schneider, 2000) and that they can still be in a process of speciation (Veracini et al., 2002).
One piece of evidence of the close phylogenetic relation of the taxa is the fact that all species hybridize at the distributional limits of the various forms (C.(coiMbra-Filho et al., 1993).In the last case, the occurrence of those species together with C. aurita allied with its population decline can result in interspecific breeding and generation of natural hybrids.
Since the only karyotypic difference among the species is represented by the Y chromosome morphology, it does not interfere with synapse during the meiosis, allowing for the production of normal gametes.The great genetic similarity contributes to the viability and fertility of the hybrid descendents observed in captivity (coiMbra-Filho, 1978); however, its fitness in the wild in terms of survival and reproductive success has yet to be determined.
Although there are few studies on the breeding and social behavior of C. aurita, polygyny was observed as a mating system for one group studied in São Paulo, southeastern Brazil (coutinho & corrêa, 1995).Due to the population decline, hybridization can be viewed in the case of the threatened species as an alternative strategy to carry on reproduction.Otherwise, by "hybrid vigor", the hybrids can represent an additional competitor to the vulnerable species, possibly superior to the invasive species, C. jacchus and C. penicillata, which have already established themselves in the new territory.
Biological invasion is now considered to be one of the "big five" environmental issues of public concern (Sala et al., 2000), since a significant emphasis has been placed on invasive species as one of the leading causes of species decline and loss of biodiversity (wilcoVe et al., 1998;VitouSek et al., 1996).The data presented here represents the first attempt to confirm the involvement of C. aurita in interspecific breeding, although further genetic analyses are necessary to identify the other species involved and the magnitude of the involvement.
When management strategies to control biological invasion are being debated, including invasive species eradication (zaValeta et al., 2001), genetic analysis can help understand the type and the extension of interactions between native and invasive species to direct conservation action in order to accomplish effective restoration goals.

Fig. 2 .
Fig. 2. Karyotypic pattern found in all Callithrix hybrids with 2n=46, XY and the diminutive Y chromosome similar to C. aurita as described by nagaMachi et al. (1997).

Fig. 3 .
Fig. 3. Ag-NOR stained metaphase of Callithrix sp.All the hybrids have similar NOR banding pattern.The arrows indicate six NORbands in the short arms of acrocentric chromosomes.The head of the arrow indicates the diminutive Y chromosome without NOR.