Background

Molecular data have led to a revaluation of the time for primate origins and current views suggest that paleontologists have underestimated the time depth of primate origins [1, 2]. Previously most paleontologists thought that the origin of primates was around 60 million years ago (mya) approximately at the Cretaceous/Tertiary (K/T) boundary, but there is a growing consensus that primates probably originated at least 85–90 mya. Fossil evidence indicates an African or possibly an Indo-Madagascan origin for primates, however data for a geographic origin are largely circumstantial [2, 3].

It now appears that the division of the two major branches of primates, Strepsirrhini (lemurs and lorisoids) and Haplorrhini (tarsiers, monkeys, apes and humans) would then easily predate 60 mya and probably 77 mya [2, 4]. Recent molecular contributions have reinforced the hypothesis of a monophyletic African origin of strepsirrhines [2, 4–6]. The division between lorisoids and lemuroids, the two major branches of strepsirrhines, is deep and probably established by 65 mya.

Current paleontological interpretations about anthropoid origins are dependent on where tarsiers are placed in the primate phylogenetic tree. The Strepsirrhini/Haplorhini taxonomic division of primates was based on the morphological conclusions that tarsiers were more closely related to anthropoid primates (monkeys, apes, and humans) than to the "lower" primates [7]. However, the molecular data are ambivalent on tarsier affinities and have been interpreted to link tarsiers with anthropoids [8–10], or to Strepsirrhini [11–14]. Apparently, the strepsirrhine/tarsier/anthropoid divergence was so rapid that for current molecular methods it appears as a trichotomy [2]. There are some isolated fossil teeth from Africa dated between 45 and 60 mya which may be close to the origin of anthropoids, but more complete and diagnostic remains from the Fayum (Egypt) only appear at about 35–37 mya [15] long after the probable origin of anthropoids. The Asian fossils appear to be a sister clade to these African remains and there may be a complicated pattern of multiple primate faunal exchanges between Asia and Africa.

Even if the origin of stem anthropoids has not yet been unequivocally elucidated [16–18] the anthropoid primates surely include both New World (Platyrrhini) and Old World (Catarrhini) monkeys, apes and humans.

Platyrrhine origins and taxonomy

Various hypotheses have been advanced for the origin of New World monkeys (NWM) and whether they were a mono or a polyphyletic assemblage [19]. The current consensus is that paleowinds, island-hop** and vegetation rafting, favoured a Paleogene African origin of New World monkeys [20, 9]. Various dispersal events probably occurred over a 20 million year period making repeated contributions to colonization possible and a plausible case for the multi-phyletic origin of extant NWM [21]. The dentition, particularly M3, of Branisella boliviana, at 27 mya the oldest fossil platyrrhine monkey, supports an African origin of NWM because it is similar to fossils from the Oligocene and Late Eocene of Fayum, Egypt [22]. Comparative phylogenomics demonstrates a close phylogenetic relationship between these primates, compatible with a monophyletic African origin for all New World monkeys [23, 24].

Dates for the origin of the platyrrhine/catarrhine divergence from the molecular data are considerably earlier than the oldest fossil remains and range from about 35 mya to over 50 mya with 40 mya as a fair compromise [1, 25]. A summary of molecular evidence indicates that extant platyrrhines diverged over the last 20 million years [21, 26] making it probable that the radiation of living species occurred wholly in the Neotropics to take advantage of favorable ecological opportunities. This radiation produced numerous species with a wide range of morphological, ecological and ethological adaptations. Today these monkeys range from southern Mexico to northern Argentina.

The taxonomic and phylogenetic relationships of these monkeys continue to challenge primatologists and are difficult to define on the basis of morphological characters because of problems in distinguishing homology and convergence [27]. In Platyrrhinae "sibling species" are not uncommon and paleontological data are as yet so limited that they are of little use to resolve phylogenetic relationships. We do not know much about what happened to the platyrrhines between 40 and 20 million years ago and it would be quite helpful to have further paleontological evidence to shed light on this gap [26].

Traditional taxonomies of Playrrhines and the position of Callimico goeldii

These difficulties are reflected in the large number of different phylogenetic trees and taxonomies presented by various authors over the years (fig 1). The various treatments of Callimico goeldii are explicatory. This species resembles tamarins and marmosets in small body size and claws, but shares with other NWM characters like single births and a third molar. Traditional taxonomies either recognized two or three families of NWMs. When two families were recognized (Cebidae and Callitrichidae) C. goeldii was placed into one or the other and was seen as basal to the other species [24, 28–30]. Others erected a third family (Callimiconidae) to accommodate C. goeldii [31–34]. All families were then placed in one superfamily, Ceboidea. A typical and largely followed morphological based taxonomy is that of Groves (1993)[35] with two families: 1. Callitrichidae, including the genera Callimico, Callithrix, Leontopithecus, Saguinus, and 2. Cebidae: including genera Alouatta, Aotus, Ateles, Brachyteles, Lagothrix, Callicebus, Cebus, Saimiri, Cacajao, Chiropotes, and Pithecia.

Figure 1
figure 1

Four phylogenies for New World monkey genera. a) modified from Rosenberger et al 1984 [72] and b) modified from Ford 1986 [73] are based on morphological traits, while c) modified from Schneider et al., 2001 [40] and d) modified from Ray et al., 2005 [39] are based on molecular studies. See text for further details.

Full size image

Molecular tree of New World monkey phylogeny

Molecular data has led to a radical revision of the traditional divisions and assemblages of NWMs. The separation into two distinct branches of Cebidae and Callitrichidae is not supported. Instead, three clades are distinquished [36–41]:

1. Cebidae, marmosets (including Callimico) and tamarins are placed in a clade with Saimiri, Cebus and probably Aotus

2. Pitheciidae which groups Callicebus, Pithecia, Cacajao and Chiropotes.

3. Atelidae which consists of Alouatta, Ateles, Lagothrix and Brachyteles.

Indeed, Groves (2001), without providing any evident morphological justification, altered his taxonomy of NWMs probably to reflect developments in primate phylogenomics. His scheme is identical to that above, but erects Nyctipithecidae with the single genus Aotus as a fourth family.

On the other hand, the low number of nucleotidic differences found between Platyrrhine species, because the divergence between many taxa was probably short, often limits the resolution of molecular studies. There is some uncertainty as to the exact branching order, both between and within the three taxonomic divisions (fig. 1) [50]. However, the reciprocal painting in these species cannot determine if the rearrangements are homologous. The orientation and exact fusion points between HSA 10 and 11 in both taxa need to be tested with BAC FISH and sequencing to determine if this syntenic association is equivalent or different.

It can also be noted that the assignment of Aotus with the Cebidae is not as solid as for other species. A sister relation between Aotus and the Cebus/Saimiri clade is favored by parsimony analysis, but not by other analyses [41]. Saimiri was linked to Aotus by only 1 Alu insertion and then this branch to Callithrix and Saguinus by only 2 insertions [39]. Recently, a Bayesian analysis of 59.8 kbp genomic data for 13 primates concluded that the deepest node within the Cebidae was between squirrel monkeys and marmosets at 17.1 mya with 20.8 mya for crown platyrrhine node. Although Singer et al. also claim that Alu elements were able to resolve Cebidae branching, all branches were supported by a maximum of one marker; hardly reassuring [55].

The ancestral karyotype and monophyly of platyrrhines

Previous reconstructions of the ancestral primate karyotype (APK) hypothesized a diploid number from 2n = 48 to 50 [56–59]. Recent reciprocal chromosome painting has also refined the content of the APK. Both Stanyon et al. (2006)[60] and Nie et al. [61](2006) found in loriforms a syntenic association 7/16 identical to that found in the proposed ancestral eutherian karyotype [62]. Therefore 7b/16p should be included in the APK. Defined by reference to homology with the human karyotype, the genome of the last common ancestor of all living primates had the following chromosomes:

1, 2a, 2b, 3/21, 4, 5, 6, 7a, 7b/16b, 8, 9, 10, 11, 12a/22a, 12b/22b, 13, 14/15, 16a, 17, 18, 19a, 19b, 20, X and Y.

Then from the APK the origin of the anthropoids was marked by: 1) a fission of the syntenic association 7b/16b, 2) a reciprocal translocation that gave origin to 12 and 22, 3) fusion of 19p and 19q. The ancestral anthropoid karyotype common to both Old World and New World primates maintained a diploid number of 2n = 46 but with the following chromosomes: 1, 2a, 2b, 3/21, 4, 5, 6, 7a, 7b, 8, 9, 10, 11, 12, 13, 14/15, 16a, 16b, 17, 18, 19, 20, 22, X e Y.

The New World monkeys all share and are characterized by seven fissions in five chromosomes (1, 3/21, 8, 10 and 14/15) and by 4 fusions, which form syntenic associations (2b/16a, 5/7a, 8b/18 and 10b/16b). A comparison of our reciprocal painting with that available in the literature shows that all these syntenic associations have the same breakpoints and are homologous derived rearrangements (Figure 7). Additional, indirect, supporting evidence for the homology of these breakpoints across platyrrhine species comes from the multidirectional painting of Sanguinus oedipus and Lagothrix lagothricha paints on various species [63] including Atelidae [64, 65]. In all these cases the painting revealed numerous conserved autosomal syntenies compatible with an origin in a common ancestor. Therefore, our data and that of the literature support the hypothesis that the ancestral karyotype, (as reported in Stanyon et al 2003, 2004) of the New World monkeys has a diploid number of 2n = 54: 1a, 1b, 1c, 2a, 2b/16a, 3a, 3b, 3c/21, 4, 5/7a, 6, 7b, 8a, 8b/18, 9, 10a 10b/16b, 11, 12, 13, 14/15a, 15b, 17, 19, 20, 22, X and Y.

Divergence order of Platyrrhines

Molecular cytogenetic data do not provide convincing evidence on the order of divergence between platyrrhine families. However, it is suggestive that the karyoytpe found in the genus Cebus is almost identical to that of the APLK for diploid number (2n = 54) and for associations. It differs only for a pericentric inversion and heterochromatin additions. This condition is congruent with a basal position of the Cebidae.

Both Canavez et al. (1999)[36], Schneider et al. (2001)[40] and Seuanez et al (2005)[66] also placed the Cebidae as basal with the Pitheciidae and Atelidae sharing a more recent common ancestor. Analysis of primate retroviral restrictive domains also placed the Cebidae as basal [67]. Ray et al. (2005)[39] on the basis of Alu insertions places the Pitheciidae as basal and with the Cebidae and Atelidae sharing a more recent common ancestor. Steiper and Ruvolo (2003)[41] using G6PD data depending on the type of analysis put either Atelidae or Pitheciidae as basal. In a recent study of orthologous sequences of six nuclear genes compared for most platyrrhine genera, the branching order between the three clades shifted when different algorithms were used. In one Pitheciidae were basal with Cebidae and Atelidae sharing a more recent common ancestor; in the other the Cebidae were basal with Atelidae and Pitheciidae as sister clades [42]. For now, it seems that molecular studies apparently cannot yet unequivocally determine the relationship between Cebidae, Atelidae and Pitheciidae.

Conclusion

Our results support the molecular genomic assemblage of Cebidae. Five chromosome associations, phylogenetically linking tamarins and marmosets are homologous and derive from shared chromosome rearrangements in a common ancestor. Four derived homologous associations nest Callimico goeldii within the radiation of the marmosets. One derived association 2/15 may link Saimiri with these species. An apparently common breakpoint on chromosome 5q33 may link Saimiri and Aotus. A comparison of our reciprocal painting with that available in the literature shows that the great majority of syntenic associations and breakpoints found in New World monkeys are homologous. Our data support the hypothesis that the ancestral karyotype of all Neotropical primates had a diploid number of 2n = 54 with chromosomes: 1a, 1b, 1c, 2a, 2b/16b, 3a, 3b, 3c/21, 4, 5/7a, 6, 7b, 8a, 8b/18, 9, 10a 10b/16b, 11, 12, 13, 14/15a, 15b, 17, 19, 20, 22, X and Y. This suite of derived chromosome rearrangements found in all these monkeys, overwhelming supports the monophyly of NWM and shows that these primates form a tight phylogenetic and taxonomic unit. Although molecular cytogenetic data do not yet provide convincing evidence on the order of divergence between platyrrhine families, it does suggest that the conserved karyotypes found in species of the genus Cebus is congruent with a basal position of the Cebidae.

However, it should be noted that chromosome painting even with reciprocal hybridizations does not usually detail intrachromosomal rearrangements and breakpoint resolution is limited. Cloned DNA probes such as BACs, cosmids and locus specific probes, provided increased resolution and can reveal intrachromosomal rearrangments and precisely map breakpoints. Further high resolution molecular cytogenetic research, using such cloned DNA probes, will be necessary to confirm and resolve unanswered questions of New World primate evolution [68–70]. These questions are urgent today because many of these primates are highly endangered.

Methods

Cell samples, tissue culture and chromosome preparation

Metaphase preparations were obtained from established fibroblast cell lines of one male individual of Callimico goeldii and of Cebuella pygmaea, of one female individual of Callithrix argentata and of one male individual of Saimiri sciureus. The cell lines were kindly provided by S. O'Brien, Laboratory of Genomic Diversity, National Cancer Institute, Frederick MD, USA. Normal culture procedures were followed. Cultures were maintained in DMEM supplemented with 10% fetal bovine serum.

Flow sorting and in-situ hybridization

Chromosome-specific probes from the NWM were made by DOP-PCR from flow sorted chromosomes using PCR primers amplification and labeling conditions as previously described [44, 71]. Chromosome sorting was performed using a dual laser cell sorter (FACSDiVa). This system allowed a bivariate analysis of the chromosomes by size and base-pair composition. About five hundred chromosomes were sorted from each peak in the flow karyotype. Chromosomes were sorted directly into PCR tubes containing 30 μl of distilled water. The same 6MW primer was used in the primary reaction and to label the chromosomal DNA with biotin dUTP or digoxigenin-dUTP (both from Roche) in a secondary PCR for indirect detection. Direct labeling was with rodamine 110-dUTP (Perkin-Elmer) for green, Spectrum Orange (Vysis) for red and Cy5-dUTP (Amersham) for infrared as previously described [57]. In situ hybridization and probe detection were carried out following common FISH procedures. About 300–400 ng of each PCR product per probe, together with 10 μg of human Cot-1 (Invitrogen) were precipitated and then dissolved in 14 μl hybridization buffer. After hybridization and washing of the slides, biotinylated DNA probes were detected with avidin coupled with fluorescein isothiocyanate (FITC, Vector, Burlingame, CA). Digoxigenin-labeled probes were detected with antidigoxigenin antibodies conjugated with Rodamine (Roche, Eugene, Oregon).

Digital images were taken using a cooled Photometrics CCD camera coupled to the microscope. Imaging software was SmartCapture (Digital Scientific, Cambridge, UK).