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 , including the difference in the diploid number for males and females in C. c. rubicundus [10, 15–17].
Our results by chromosome painting in P. irrorata and C. c. rubicundus, as well as those from C. utahicki , 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) . 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 5a1 and 5a2. A similar fission was previously found in Atelinae , 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 , 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 [5–8, 12], and also the phenetic inferences .