Open Access Research Article Chromosome Phylogeny of the Subfamily Pitheciinae (platyrrhini, Primates) by Classic Cytogenetics and Chromosome Painting

Background: The New World monkey (Platyrrhini) subfamily Pitheciinae is represented by the genera Pithecia, Chiropotes and Cacajao. In this work we studied the karyotypes of Pithecia irrorata (2n = 48) and Cacajao calvus rubicundus (2n = 45 in males and 2n = 46 in females) by G-and C-banding, NOR staining and chromosome painting using human and Saguinus oedipus whole chromosome probes. The karyotypes of both species were compared with each other and with Chiropotes utahicki (2n = 54) from the literature. Results: Our results show that members of the Pitheciinae have conserved several chromosome forms found in the inferred ancestral Platyrrhini karyotype (associations of human homologous segments 3a/21, 5/7a, 2b/16b, 8a/18, 14/ 15a and 10a/16a). Further, the monophyly of this subfamily is supported by three chromosomal synapomorphies (2a/ 10b, an acrocentric 15/14 and an acrocentric human 19 homolog). In addition, each species presents several autapomorphies. From this data set we established a chromosomal phylogeny of Pitheciinae, resulting in a single most parsimonious tree. Conclusions: In our chromosomal phylogeny, the genus Pithecia occurred in a more basal position close to the inferred ancestor of Platyrrhini, while C. c. rubicundus and C. utahicki are closely related and are linked by exclusive synapomorphies. Pithecia comprises five species: Pithecia pithecia, Pithe-cia monachus, Pithecia irrorata (each two subspecies), Pithecia albicans and Pithecia aequatorialis (both mono-typic) [1]. Chiropotes is represented by the two species Chiropotes albinasus (monotypic) and Chiropotes satanas (three subspecies) [2], and Cacajao by Cacajao calvus (four subspecies) and Cacajao melanocephalus (two sub-species) [3]. The geographic distribution of this subfamily is restricted to the Amazon region. Recent morphological , karyotypic and molecular data pointed to a new species for Chiropotes, C. israelita, and indicated that the subspecies Chiropotes satanas utahicki should be

israelita were so far analyzed by chromosome painting using human whole chromosome probes [19]. Stanyon et al. [19] showed that Chiropotes retained all human homologous syntenic associations proposed for the ancestral Platyrrhini karyotype, but also include several derived chromosome forms that are exclusive to this genus.  With the aim to establish detailed chromosomal phylogenies of the three genera of Pitheciinae, we studied the karyotypes of the species Pithecia irrorata and Cacajao calvus rubicundus using both classic cytogenetics and chromosome painting with human and Saguinus oedipus whole chromosome probes.

Methods
Chromosome preparations were obtained from whole blood cultures of one female and two male Pithecia irrorata (PIR) individuals kept at the Parque Zoobotânico Gavião Real, Capitão Poço, Para, Brazil, of two females from the Centro Nacional de Primatas, Ananindeua, Para, Brazil, and of a male and a female Cacajao calvus rubicundus (CCR) individual kept at the Centro de Primatologia do Rio de Janeiro, Rio de Janeiro, Brazil.
G-banding, using Wright stain [20], C-banding [21] and NOR-staining [22], followed standard procedures. FISH experiments using human whole chromosome paint probes 1-22, X and Y, and S. oedipus paint probes (22 autosomes, X and Y) were performed as previously described [23][24][25]. While both human probes were hybridized for CCR and PIR, only PIR is queried with SOE paints. Paint probes were labeled by DOP-PCR [26] using Biotin-dUTP, Digoxigenin-dUTP (Roche) and TAMRA-dUTP (Biosystems/PE Applied). After classical staining, metaphases were photographed using a Zeiss III microscope with Kodak Imagelink HQ film. FISH/DAPI  6 (distal p-proximal q) and 4(p) 4 (distal p-proximal q) and 5(p) 16   stained metaphase images were captured with a CCD camera (AxioCam MR monochrome, or Photometrics C250/A, equipped with a KAF1400 chip, respectively) coupled to a Zeiss Axiophot microscope. The images were analyzed using Adobe Photoshop software version CS3 and Corel Photo Paint 10. Chromosomes were iden-tified by computer enhanced DAPI banding (Axiovision 3.0). For phylogenetic analysis a binary data matrix was established, based on the presence or absence of discrete chromosomal characters obtained by comparative analysis of both chromosome painting patterns derived from human whole chromosome probes and by G-banding patterns in the Pitheciinae species included in our study and from the literature [19]. Brachyteles arachnoides [24] and Cebus apella [25] were used as outgroups. We attributed "0" (zero) value to the absence of a character (an association or a syntenic group that is conserved or changed by chromosome rearrangements) and "1" (one) value to the presence of the character. These data were subjected to maximum parsimony analysis (PAUP 4.0 software; Phylogenetic Analysis Using Parsimony), using the exhaustive search option. The relative stability of nodes was assessed by bootstrap estimates.

Classical cytogenetics
P. irrorata has 2n = 48 chromosomes, with nine metacentric or submetacentric and 14 acrocentric autosome pairs. The X chromosome is a medium sized submetacentric. The G-banded karyotype is shown in the Figure  1A. C-banding highlighted constitutive heterochromatin in the centromeres of all chromosomes and an interstitial band in the long arm of pair 18 ( Figure 1B).
C. c. rubicundus has 2n = 45 chromosomes in males and 2n = 46 in females as a result of a Y-autosomal translocation, causing a multiple sex chromosome system X 1 X 2 Y (Figure 2A). The autosomal set is composed by nine meta-or submetacentric and 13 acrocentric pairs. The X 1 chromosome is a medium sized submetacentric, homologous to the X of other primates. The X 2 chromo-some is a medium sized acrocentric, corresponding to the original homologue of the autosome translocated to the Y chromosome. Constitutive heterochromatin ( Figure 2B) was observed in centromeric regions of all autosomes. In addition, secondary C-bands were observed on pairs 1, 2 and 3, in the distal regions of chromosome pairs 4p, 5p, 6p and 4q and proximally on chromosome pair 15q.
NOR-staining was found in the proximal region of the long arm of six acrocentric chromosomes both on P. irrorata ( Figure 3A) and C. c. rubicundus ( Figure 3B).

Phylogenetic analysis
The comparative analysis of the P. irrorata, C. c. rubicundus (this study) and C. utahicki [19] karyotypes by G- Figure 5 Comparative karyotype analysis of P. irrorata, C. c. rubicundus and C. utahicki [19]by G-banding and FISH using human whole chromosome probes. The banded C. utahicki chromosomes were obtained from a previously unpublished metaphase [10].
banding and FISH with human whole chromosome probes is summarized in Figure 5. The results showed that the chromosomal differences found among the three taxa are consequence of centric fusions and fissions, pericentric and paracentric inversions, tandem fusions and a Y-autosome translocation ( Table 2).
These data were translated into a binary matrix (Additional file 1) and were then subjected to parsimony analysis. A single most parsimonious three was obtained ( Figure 6) comprising 45 steps with a consistency index of 0.978, a retention index 0.909 and a homoplasy index of 0.022.

Discussion
The G-, C-banding and NOR-staining results on P. irrorata and C. c. rubicundus obtained in this study are in agreement with previously published data [10], including the difference in the diploid number for males and females in C. c. rubicundus [10,[15][16][17].
Our results by chromosome painting in P. irrorata and C. c. rubicundus, as well as those from C. utahicki [19], demonstrated that these species conserved all human homologous syntenic associations found in the inferred ancestral New World primate karyotype (3a/21, 5/7a, 2b/ 16b, 8a/18, 14/15a and 10a/16a) [27]. The morphology and banding pattern of the 3a/21 homologues in C. c. rubicundus and C. utahicki is similar to the ancestral Platyrrhini type, while in P. irrorata it was modified by a paracentric inversion. The biarmed chromosome form 5/ 7a found in P. irrorata is also similar to the ancestral Platyrrhini type, whereas in C. c. rubicundus and C. utahicki this association is found on a presumably derived acrocentric chromosome. Cacajao further shows human chromosome 5 homologues fissioned into 5a 1 and 5a 2 . A similar fission was previously found in Atelinae [24], however, involving different break points. Therefore, these fissions represent no synapomorphy, but rather occurred independently in the two clades. Chromosome forms 2b/16b and 8a/18 each showed similar morphology compared to homologues from other Platyrrhini and are therefore considered ancestral traits. The association 14/ 15a was observed in an acrocentric chromosome in P. irrorata, while in C. utahicki it has fused with chromosome 20 forming the association 20/15/14 on chromosome 1. In C. c. rubicundus it was found fused with the human 3/20 homolog, leading to the association 3/20/15/ 14. Finally, a pericentric inversion was observed for chromosome form 10a/16a in all species analyzed here, resulting in chromosome form 16a/10a/16a/10a. This derived inversion has also been found previously in Callicebus [28], indicating that Pithecia, Chiropotes and Cacajao are sister groups of Callicebus. This observation is in agreement with recent classifications based on molecular data, supporting the classification of subfamilies Pitheciinae and Atelinae as sister groups included in the family Atelidae [8,12]. A comparative chromosome analysis of the three Pitheciinae species shows further synapomorphies shared between C. c. rubicundus and C. utahicki (fission of 5/7, resulting in separate 5a1 and 5a2 segments, and fusion 20/15/14), shared between P. irrorata and C. c. rubicundus (acrocentric 7b, acrocentric 12) and for Pitheciinae in general (fusion 2a/10b, acrocentric 15/14 and acrocentric 19). In contrast, no derived chromosome forms shared between P. irrorata and C. utahicki were found.
Our phylogenetic analysis suggests that P. irrorata, C. c. rubicundus and C. utahicki are a monophyletic group. Further, and as expected, the chromosomal data supported by exclusive synapomorphies demonstrates that C. c. rubicundus and C. utahicki are sister taxa. Moreover, P. irrorata was placed in a more basal position, having retained a karyotype closer to that of the inferred ancestral Platyrrhini. This phylogenetic arrangement supports previously published phylogenies [5][6][7][8]12], and also the phenetic inferences [10].

Conclusions
In conclusion, this comparative chromosomal analysis clarifies some intrageneric relationships within Pitheciinae, while the position of this group in relation to Callicebus and Aotus remained undefined. Additional comparative high-resolution molecular cytogenetic studies will be necessary to precisely define the rearrangements between Aotus and Callicebus to clarify their phylogenetic relationships with Pitheciinae.

Additional material
Authors' contributions LFMF carried out the chromosome painting in P. irrorata, organized the data and wrote most of the paper. PJSA carried out the conventional chromosome analysis of P. irrorata and performed the cladistic analysis. JCP participated of the techniques development and contributed to the discussion of data. EHCO carried out the conventional chromosome analysis of C. c. rubicundus. AP collected the samples, classified the species and discussed the phylogenetic implications of the data. MN and SM carried out the chromosome painting in C. c. rubicundus and discussed the phylogenetic implications of the data. CYN conceived of the study, and coordinated the study. All authors read and approved the final manuscript.