Phylogenetic studies of the genus Cebus (Cebidae-Primates) using chromosome painting and G-banding

Background Chromosomal painting, using whole chromosome probes from humans and Saguinus oedipus, was used to establish karyotypic divergence among species of the genus Cebus, including C. olivaceus, C. albifrons, C. apella robustus and C. apella paraguayanus. Cytogenetic studies suggested that the species of this genus have conservative karyotypes, with diploid numbers ranging from 2n = 52 to 2n = 54. Results Banding studies revealed morphological divergence among some chromosomes, owing to variations in the size of heterochromatic blocks. This analysis demonstrated that Cebus species have five conserved human associations (i.e., 5/7, 2/16, 10/16, 14/15, 8/18 and 3/21) when compared with the putative ancestral Platyrrhini karyotype. Conclusion The autapomorphies 8/15/8 in C. albifrons and 12/15 in C. olivaceus explain the changes in chromosome number from 54 to 52. The association 5/16/7, which has not previously been reported in Platyrrhini, was also found in C. olivaceus. These data corroborate previous FISH results, suggesting that the genus Cebus has a very similar karyotype to the putative ancestral Platyrrhini.

Cytogenetic studies on Cebus have shown that the diploid number ranges from 52 to 54 chromosomes. The species of this genus have large blocks of constitutive heterochromatin, mainly found in interstitial and distal regions, which displays intraspecific variation, few biarmed chromosomes and secondary constrictions in two acrocentric pairs [8][9][10][11][12]. To date, human chromosomal painting has been used to analyze three species of Cebus: C. capucinus [13]C. apella [14] and C. nigrivittatus [15].
Interspecies chromosomal comparisons of Cebus have been performed using G-and Q-banding patterns. These comparisons [16] suggest that C. capucinus, C. albifrons and C. apella share 19 chromosome pairs, C. capucinus and C. albifrons share 25 pairs and C. capucinus and C. apella share 20 pairs. Among these three species, the karyotype of C. capucinus most resembles the putative ancestor, as all chromosomes found in C. capucinus are observed in C. albifrons and C. apella. Furthermore, C. albifrons and C. apella seem to have been independently derived from an ancestor with a karyotype similar to C. capucinus. The C. capucinus karyotype is closer to C. albifrons than to C. apella.
Zoo-FISH comparative chromosome painting is a powerful method for detecting chromosome homologies between species and for resolving phylogenetic controversies. This study compared the chromosome homologies present among Cebus apella paraguayanus (2n = 54), Cebus apella robustus (2n = 54), Cebus albifrons (2n = 52) and Cebus olivaceus (2n = 52) using G-banding and chromosome painting with whole chromosome probes derived from humans and Saguinus oedipus. Our results were compared with previous reports to propose a phylogeny for these species, using chromosomal characters in a parsimony analysis.

Methods
Metaphasic chromosomes from four Cebus taxa (Table 1) were obtained by lymphocyte [17] and fibroblast culture. Karyotypes were organized following the protocol of Matayoshi et al. (1986) [18]. G-banding was performed using the methods of Seabright (1971) [19]. FISH experiments were performed in all species using S. oedipus whole chromosome probes [20] and 24 different whole chromosome probes taken from humans (1-22 autosomes, X and Y). Probes were organized into four pools (H1-H4 and S1-S4, for human and S. oedipus chromosome paints, respectively) as previously described [21,20]. The probes were then labeled by DOP-PCR [22] using biotin-dUTP, digoxigenin-dUTP (Roche) and TAMRA-dUTP (Applied Biosystems/PE). In situ hybridization and detection were performed using the protocols of Neusser et al. (2001) [21] and De Oliveira et al. (2005) [23]. Nomenclatures of chromosomes and chromosome segments were consistent with Neusser et al. (2001) [21] and De Oliveira et al. (2005) [23]. Human and S. oedipus probes were applied to all of the taxa except C. olivaceus, which was analyzed solely using human probes.
G-banded metaphases were captured using a Zeiss III photomicroscope with Imagelink HQ film manufactured by Kodak. FISH/DAPI metaphases were captured with a CCD camera under a Zeiss Axiophot microscope. Images were analyzed using Adobe Photoshop 7.01.
Phylogenetic analysis was performed by applying a cladistic method with parsimony criteria. A basic data matrix was built by comparing chromosomal differences among species, as determined by FISH or G-banding. Previously reported chromosome painting data was used for C. apella sp. [14] and C. capucinus [13]. Saimiri sciureus and Callithrix jacchus were used as outgroups. PAUP (Phylogenetic Analysis Using Parsimony, 4.0b1 for Microsoft Windows) software was used to build the cladogram, which was then tested using the bootstrap method [24].

Cytogenetic analysis
Diploid number analysis confirmed a range of 52 to 54 chromosomes in the genus Cebus. Cebus apella paraguayanus and C. a. robustus had 54 chromosomes (10 pairs were biarmed and 16 pairs were one-armed). Cebus albifrons had 52 chromosomes (9 pairs were biarmed and 16 pairs The SOE X and Y probes hybridized to the respective sex chromosomes of both Cebus species. The G-banded karyotype of C. a. paraguayanus, together with a summary of the chromosome painting results, is shown in Figure 2A. Figure 2B shows the karyotype of C. a. robustus. The C. a. paraguayanus male displayed a reciprocal translocation of segments homologous to HSA 2a e 10a, which was confirmed using the SOE 10 and SOE 15 probes (Figure 2A, inset). The two species differed in the distribution of constitutive heterochromatin, where the most obvious difference was the absence of the distal heterochromatic block in chromosome 11 of C. a. robustus (Figure 3).

Comparative analysis and phylogeny
Human and Saguinus oedipus painting results, in combination with the G-banding results, allowed for a detailed comparison of chromosome homologies among members of the genus Cebus. A basic data matrix (BDM) was built using alternative forms of the chromosomes as characters (Table 2). These characters were then used in a binary matrix (Table 3

Discussion
Many reports use morphological [25][26][27][28], molecular [29][30][31][32][33] or chromosomal [34,35,21] data to dissect the phylogenetic relationships among New World monkeys at the family level. However, ordering the species below the genus level is a fundamental step toward reorganizing the phylogenetic relationships among these taxa. This realization prompted us to analyze taxa from the genus Cebus to better understand their chromosomal divergences and to clarify their phylogenetic positions.
Our data in C. a. paraguayanus are quite similar with the one described by Garcia et al. (2000) [14] on C. apella sp. Interestingly, the 5/7a association was found in all but one member of the genus Cebus. In C. olivaceus, this association possessed an additional segment homologous to HSA16. This segment probably fused in tandem with chromosome HSA7, followed by a paracentric inversion that resulted in the association CGR 7/16/5, which has not been reported before in New World primates. The association 5/16 is found in Saimiri but not in the other members of the genus Cebus. However, the segment homologous to HSA16 has different sizes in these species, which strongly supports the occurrence of a homoplasy. Garcia  Chromosomal data were used to obtain a cladogram that reconstructed a possible sequence of chromosome rearrangements leading to karyotypical differentiation into the Cebus genus ( Figure 4). The cladogram supports the notion that the monophyly of Cebus. C. apella sp., C. a. paraguayanus and C. a. robustus are closely related, sharing two synapomorphic traits (i.e., the association 14/15/14 that resulted in a submetacentric chromosome and the pericentric inversion that corresponded to the HSA8b probe). C. capucinus occupies a more basal position, with a chromosomal composition very similar to the putative ancestral Platyrrhini karyotype, consistent with previous reports by Richard et al. (1996) [13].    14/15/14. We also identified an in tandem fusion, followed by a pericentric inversion involving the homologous human chromosomes HSA15b and HSA8b, in C.
albifrons. As C. olivaceus is closely related to C. apella, these species share the chromosomal inversion homologous to HSA20. Differentiation between C. olivaceus and C. apella is possible via a pericentric inversion in the association 14/15/14 and a Robertsonian rearrangement in the chromosomes homologous to HSA12 and HSA15b on their Figure 2c. Garcia et al. (2002) [15] left open two possibilities for chromosome 6 in the Cebus ancestral karyotype: the ancestral form could be a metacentric like in CAL, CCA, and CNI karyotypes or a submetacentric like in CAP.
Our cladistic analysis has shown that the ancestral form is the metacentric.
This study used chromosome painting in conjunction with G-banding to confirm the ability of these techniques to generate consistent and reliable data. These data were interpreted using a cladistic analysis capable of generating a cladogram with a high degree of consistency. Future studies should use molecular markers to further explore the phylogeny described here.

Conclusion
Chromosome painting in several species of Cebus allowed us to define all the rearrangements that ocurred during its speciation. It was also possible to use FISH and G-banding data, both from our results and from literature, to build a cladogram that reconstructed a possible sequence of chromosome rearrangements leading to karyotypical differentiation into the Cebus genus. Publish with Bio Med Central and every scientist can read your work free of charge