American Journal of Primatology 65:73–85 (2005) RESEARCH ARTICLE Comparative Chromosome Painting in Aotus Reveals a Highly Derived Evolution AURORA RUIZ-HERRERA1, FRANCISCA GARCÍA1,2, MARISOL AGUILERA3, MONTSERRAT GARCIA1,2, and MONTSERRAT PONSÀ FONTANALS1,2n 1 Departament de Biologia Cellular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, Barcelona, Spain 2 Institut de Biotecnologia i Biomedicina (IBB), Universitat Autònoma de Barcelona, Barcelona, Spain 3 Grupo BIOEVO, Universidad Simón Bolı´var, Caracas, Venezuela The genus Aotus represents a highly diverse group with an especially intricate taxonomy. No standard cytogenetic nomenclature for the genus has yet been established. So far, cytogenetic studies have characterized 18 different karyotypes with diploid numbers ranging from 46 to 58 chromosomes. By combining G-banding comparisons and molecular cytogenetic techniques, we were able to describe the most likely pattern of chromosome evolution and phylogenetic position of two Aotus karyomorphs (KMs) from Venezuela: Aotus nancymai (KM3, 2n ¼ 54) and Aotus sp. (KM9, 2n ¼ 50). All of the proposed Platyrrhini ancestral associations (2/16, 3/21, 5/7, 8/18, 10/16, 14/15) were found in the Aotus KMs studied, except 2/16 and 10/16. In addition, some derived chromosomal associations were also detected in both KMs (1/3, 1/16, 2/ 12, 2/20, 3/14, 4/15, 5/15, 7/11, 9/15, 9/17, 10/11, and 10/22). Although some of these associations have been found in other New World monkeys, our results suggest that Aotus species have undergone a highly derived chromosomal evolution. The homologies between these two Aotus KMs and human chromosomes were established, indicating that KM3 has a more derived karyotype than KM9 with respect to the ancestral Platyrrhini karyotype. Am. J. Primatol. 65:73–85, 2005. r 2005 Wiley-Liss, Inc. Contract grant sponsor: DGI; Contract grant number: BXX2000-0151; Contract grant sponsor: Ministerio de Ciencia y Tecnologı́a; Contract grant sponsor: AIRE; Contract grant number: 140122; Contract grant sponsor: DURSI, Generalitat de Catalunya; Contract grant sponsor: DID, Universidad Simón Bolivar; Contract grant number: G-026; Contract grant sponsor: Universitat Autònoma de Barcelona. n Correspondence to: Montserrat Ponsà Fontanals, Departament de Biologia Cellular, Fisiologia i Immunologia, Unitat de Biologia Cellular, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193-Cerdanyola del Vallès, Barcelona, Spain. E-mail: Montse.Ponsa@uab.es Received 26 May 2004; revised 16 September 2004; revision accepted 3 November 2004 DOI: 10.1002/ajp.20098 Published online in Wiley InterScience (www.interscience.wiley.com). r 2005 Wiley-Liss, Inc. 74 / Ruiz-Herrera et al. INTRODUCTION The owl monkey (Aotus) is the only genus in the subfamily Aotinae (F. Cebidae, Platyrrhini, Primates) and is considered to be currently undergoing subspeciation [Ma, 1981]. This primate genus is widely distributed in Central and South America, from western Panama through the northern part of Argentina. The taxonomy of the genus Aotus is especially intricate because there is no consensus about the exact number of species and subspecies. However, different revisions of the genus are considering nine or 10 different species based on phenotypic and karyotypic characters and geographic distribution (Table I). From the cytogenetic point of view, the genus Aotus represents a highly diverse group in which many specific chromosomal variations have been described [Ma, 1981, 1983; Mudry et al., 1984; Pieczarka et al., 1993]. Given this diversity in karyotypes, cytogenetic studies based on G- and C-banding comparisons have been extremely useful for clarifying the taxonomy of this group during the past three decades [Hershkovitz, 1983; Ma, 1981; Ma et al., 1976a]. So far, 18 different karyotypes have been characterized, with chromosome diploid numbers ranging from 46 to 58 chromosomes [Torres et al., 1998]. In addition, some authors have reported a sex determination XX/‘‘XO’’ for the A. infulatus, A. azarae, and A. nigriceps species [Ma, 1981; Ma et al., 1976a, 1980; Mudry et al., 1984; Pieczarka & Nagamachi, 1988] due to a translocation of chromosome Y in one autosome. Although great efforts have been made to construct a systematic relationship among the different Aotus species, a standard and uniform cytogenetic nomenclature for the genus has not yet been established. To date, three nomenclature systems have been published [Ma et al., 1976a; Reumer & de Boer, 1980; Torres et al., 1998]. Only Torres et al.’s  system accommodates all existing information based on size, chromosome morphology, and sequential banding pattern (Table I). Molecular studies have not been able to establish the exact phylogenetic position of the genus Aotus within New World monkey species [Ford, 1986; Rosenberger, 1984]. Rosenberger  considers that Aotus, together with Callicebus, is closely related to Pithecinae (Pithecia, Chiropotes, and Cacajao), whereas Ford  placed Aotus and Callicebus as a basal lineage for Callithrichidae, Pithecinae, and Atelinae. In the last revision, Schneider et al.  proposed an association among Aotus, Cebus, Saimiri, and Callithrichidae (Callithrix, Sanguinus, Leontopithecus, Cebuella, and Callimico), with Aotus being a basal lineage. With the development of molecular cytogenetic tools, a large database of comparative chromosome painting in New World monkeys (Platyrrhini, Primates) has accumulated in recent years (Cebus [Garcı́a et al., 2000; Richard et al., 1996], Ateles [Garcı́a et al., 2002; Morescalchi et al., 1997], Lagothrix [Stanyon et al., 2001], Saimiri [Stanyon et al., 2000], Callicebus [Barros et al., 2003; Stanyon et al., 2000, 2003], Callithrix [Neusser et al., 2001; Sherlock et al., 1996], Callimico [Neusser et al., 2001], Cebuella [Neusser et al., 2001], Saguinus [Müller et al., 2001], Alouatta [Consigliere et al., 1996; de Oliviera et al., 2002], and Leontopithecus [Gerbault-Serreau et al., 2004]), and has been used to infer the ancestral Platyrrhini karyotype [Neusser et al., 2001]. However, some species that are important for the delineation of the Platyrrhini phylogeny, especially those belonging to subfamilies Aotinae (Aotus) and Pithecinae (Pithecia, Chiropotes, and Cacajao), remain untested. In an attempt to clarify the chromosomal relationships among Aotus species in relation to the rest of the New World monkey species, human chromosome-specific paints have been used to 55 56 52 53;54 50 46 47;48 54 51#/52~ 49#/50~ 49#/50~ 49#/50~ 50–54 ? 58 50 A. lemurinus lemurinusa A. vociferansa nancymaia,d nigricepsa azarae boliviensisa azarae azaraea infulatusa trivirgatusa miconaxa hershkovitizib sp.c,d – K-V K-X; K-XI K-I K-VIII K-VI – – – – – – K-VIII K-IX K-IV K-III; K-IV Ma et al. [1976a] nomenclature KM KM KM KM KM – – – – – – KM KM KM KM KM 7 7 3 4 5 1 1 2 2 6 Reumer and de Boer  nomenclature KM KM KM – KM – – KM KM 8 9 10 3 4 5 KM 7 KM 6 KM 2 KM 1 Torres et al.  nomenclature b According to Hershkovitz . According to Ford . c According to Torres et al. . d Karyomorphs analyzed in this study. e 1, Ma et al. ; 2, Ma ; 3, Pieczarka and Nagamachi ; 4, Torres et al. ; 5, Ma et al. [1976b]; 6, Brumback et al. ; 7, Ma et al. . a A. A. A. A. A. A. A. A. A. A. brumbackia A. l. griseimembraa 2n Species TABLE I. Summary Data Reported for Aotus Karyotypes 2, 2, 2, 3, 4 4 3, 4 4 4 3, 4, 5, 6 3, 4, 5, 7 3, 4, 5 4 2, 3, 4, 5 3, 4, 6 2, 3, 4, 5 1, 2, 3, 4 Referencee ZOO-FISH in Aotus / 75 76 / Ruiz-Herrera et al. delineate the homologous chromosomal segments present in Aotus. In this report, for the first time, two Aotus KMs based on chromosome painting using human probes are described. In addition, chromosome rearrangements between both KMs are described, and the direction of chromosomal evolution in Aotus is also discussed. MATERIALS AND METHODS Cell Cultures and Chromosome Preparations Heparinized peripheral blood samples were taken from one male A. nancymai (2n ¼ 54) and one male Aotus sp. (2n ¼ 50) from an uknown location in Venezuela (Parque Zoológico Bararida, Barquisimeto). RPMI-1640, supplemented with phytohemagglutinin, pokeweed, 25% fetal bovine serum, Lglutamine, penicillin, streptomycin, heparin, and Hepes buffer were used for the blood cultures. After 72 hr, colcemid (10 mg/ml) was added to the cultures for the final 30 min. Cells were harvested and chromosomal preparations obtained using standard protocols. Both specimens were chromosomally characterized after sequential G-banding [Seabright, 1971] and C-banding [Sumner, 1972]. For the chromosome assignment and numeration, we employed the cytogenetic nomenclature revision published by Torres et al. . Fluorescence ‘‘In Situ’’ Hybridization (FISH) Whole human chromosome paints (WCPs; provided by R. Stanyon) were used for FISH on Aotus metaphases. Degenerate oligonucleotide primer PCR (DOPPCR) was performed as previously described [Stanyon et al., 2001] for the labeling of DNA with Biotin and Tamra. For two-color FISH, we combined the probes by mixing differently labeled WCPs and precipitating them with competitor DNA (Cot-1 human DNA), salmon sperm DNA, ethanol, and 3M sodium acetate overnight at –201C as previously described [Ruiz-Herrera et al., 2004]. The precipitated mix was resuspended in 14 ml hybridization buffer, denatured at 801C for 10 min and preannealed at 371C for 30 min. Chromosome preparations were denatured in 70% formamide/2 SSC at 651C for 1 min, and hybridization was performed at 371C for 72 hr. Post-hybridization washes were performed in 50% formamide/2 SSC at 451C for 10 min, followed by three washes in 2 SSC at 451C for 5 min. Chromosomes were counterstained with DAPI and observed with an Olympus BX60 microscope equipped with a CCD camera. Digital images were taken with the use of GENUS System software (version 2.75; Applied Imaging Corporation, Santa Clara, CA). The G-banding pattern was generated using the DAPI counterstain. RESULTS Karyotypes of Aotus Species The Aotus sp. specimen studied has a diploid number of 2n ¼ 50, and according to the cytogenetic analysis performed in the present study (sequential G- and C-banding), this specimen corresponds to KM9 as described by Torres et al. : 12 pairs of metacentric and submetacentric chromosomes, and 12 pairs of acrocentric chromosomes (Fig. 1). The X chromosome is metacentric, whereas the Y chromosome is acrocentric. Chromosome 13q shows an interstitial heterochromatic band, whereas chromosomes 13–19, 21, and 22 have heteromorphic, heterochromatic small p-arms. In addition, chromosome 8 shows a ZOO-FISH in Aotus / 77 Fig. 1. G-banded karyotype of Aotus sp. (KM9) showing the location of human chromosome paintings. The Aotus chromosomes are numbered below, and the homologous human chromosomes are numbered on the right. The bars located on the left of each chromosome represent the heterochromatic regions, and the interrupted black bars on the right of chromosome 7 indicate the location of NOR regions. terminal heterochromatic band in the p-arm, whereas chromosome 11 shows a pericentromeric, heterochromatic band. This KM, including the C-banding pattern, was previously described by Torres et al.  in a specimen of unknown origin, and does not correspond to any Aotus species classified by Hershkovitz . The A. nancymai specimen studied has a diploid number of 2n ¼ 54 and corresponds to KM3 as described by Torres et al. , which is equivalent to karyotype I (K-I) described by Ma  and Ma et al. [1976a], and to KM3 described by Reumer and de Boer . This karyotype has 11 pairs of metacentric and submetacentric chromosomes, and 15 pairs of acrocentric ones (Fig. 2). Whole heterochromatic p-arms were observed in chromosomes 12-15, 18, 19, and 21-23, as described by Torres et al. . The sexual chromosome pair morphology is equivalent to that of Aotus sp. FISH Homologies between the chromosomes of two Aotus KMs and those of humans were established (Figs. 1 and 2), and some examples of the chromosomal syntenies are represented in Fig. 3. All human chromosome-specific painting probes were hybridized on Aotus chromosomes, and the human chromosome Y probe was the only one that failed in giving a hybridization signal. 78 / Ruiz-Herrera et al. Fig. 2. G-banded karyotype of A. nancymai (KM3) showing the location of human chromosome paintings. The Aotus chromosomes are numbered below, and the homologous human chromosomes are numbered on the right. The bars located on the left of each chromosome represent the heterochromatic regions. Aotus sp. (KM9). Figure 1 summarizes the hybridization results of WCPs on Aotus sp. chromosomes. The hybridization results have allowed us to identify different kinds of relationships between human and Aotus sp. chromosomes: 1) human chromosomes (6, 12, 13, 14, 17, 18, 19, 20, 21, 22, and X) represented in one Aotus sp. chromosome; 2) human chromosomes homologous to two Aotus sp. chromosomes (4, 8, 9, 10, 11 and 16); and 3) human chromosomes homologous to more than two Aotus sp. chromosomes (1, 2, 3, 5, 7, and 15). The following chromosomal associations were found in Aotus sp.: 1/3, 1/16, 2/12, 2/20, 3/21, 4/15, 5/7, 5/15, 7/ 11, 8/18, 9/15, 10/11, 10/22, 14/15, and 16/22. A. nancymai (KM3). The hybridization results of WCPs on A. nancymai chromosomes are summarized in Fig. 2. The human chromosome syntenies conserved in A. nancymai are also detected in Aotus sp., except for human chromosome 17, which is split into two different chromosomes. When the chromosomal associations are analyzed, all of those found in Aotus sp. are conserved in A. nancymai, plus associations 3/14 and 9/17 (Fig. 2). Chromosome Homologies Between the Two Aotus Species Based on G-banding comparisons and FISH results, the chromosomal homologies between Aotus sp. (KM9, 2n ¼ 50) and A. nancymai (KM3, 2n ¼ 54) have been established. The A. nancymai karyotype differs from that of Aotus sp. by diverse intra- and interchromosomal rearrangements (Fig. 4). The ZOO-FISH in Aotus / 79 Fig. 3. Examples of FISH using WCPs to metaphases of A. nancymai (KM3) (a–c) and Aotus sp. (KM9) (d–f). a: Human chromosome 9 in green, and human chromosome 15 in red. b: Human chromosome 3 in green, and human chromosome 21 in red. c: Human chromosome 7 in green, and human chromosome 5 in red. d: Human chromosome 9 in red, and human chromosome 15 in green. e: Human chromosome 3 in red, and human chromosome 21 in green. f: Human chromosome 7 in green, and human chromosome 5 in red. interchromosomal reorganizations detected were five fusion/fissions, implicating KM9 chromosomes 4, 8, 13, 21, and 24. In addition, the G-banding comparisons revealed the presence of some intrachromosomal reorganizations, with the most likely interpretation of the reorganizations being one paracentric inversion to homologue KM3 chromosome 4 with KM9 chromosome 5, and a centromeric shift to homologue KM3 chromosome 9 with KM9 chromosome 20. 80 / Ruiz-Herrera et al. Fig. 4. Presumed chromosome reorganizations between Aotus sp. (KM9) and A. nancymai (KM3) based on chromosome comparative painting and G-banding comparisons. The dark bars located on the side of each chromosome represent the heterochromatic regions. Inv: paracentric inversion. The arrowheads indicate the centromere position, whereas the arrows follow the direction of the reorganization. DISCUSSION The aims of the present work were to establish, for the first time, the chromosome homologies between Aotus and human chromosomes based on chromosome comparative painting, to contribute to the study of Aotus chromosome phylogeny, and to bring new data to the picture of Platyrrhini chromosome evolution. Implications for the Ancestral Platyrrhini Karyotype Recent publications have inferred that the ancestral Platyrrhini karyotype is that found conserved in Cebus apella and C. capucinus, which has a diploid number of 2n ¼ 54 [Neusser et al., 2001]. One of the most informative methods for interpreting comparative chromosome painting data is to analyze chromosomal syntenies. Of the proposed ancestral chromosomal associations (2/16, 3/21, 5/7, 8/ 18, 10/16, and 14/15), all were found in the Aotus KMs studied, except for 2/16 and 10/16 (Table II). In addition, some derived chromosomal associations were detected in both KMs: 1/3, 1/16, 2/12, 2/20, 4/15, 5/15, 7/11, 9/15, 10/11, 10/22, and 16/22. Associations 3/14 and 9/17 were found only in A. nancymai. Twelve human chromosomes (4, 6, 9, 11, 12, 13, 17, 18, 19, 20, 21, and 22) are conserved without disruption in the putative ancestral Platyrrhini karyotype. Eight of these (6, 12, 13, 18, 19, 20, 21, and 22) are also present in Aotus karymorphs as an undisrupted chromosome segment. Regarding ancestral chromosomes 4, 9, and 11, all are split into two different chromosomes in both Aotus karymorphs (Figs. 1 and 2) and associated with other chromosomes, ZOO-FISH in Aotus / 81 TABLE II. Chromosomal Homologies Detected by Comparative Chromosome Painting Between Aotus Species and Those of Human With Respect to the Ancestral karyotype of New World Monkeys (NWM)n Ancestral NWM karyotype 1a 1b 1c 16b/2b 2a 3b 3a/21 3c 4 7a/5 6 7a/5 7b 8a/18 8b 9 10b 16a/10a 11 12 13 14/15 Aotus sp. (KM9) Aotus nancymai (KM3) 1a 1b/3a/21 1c/16b 12/2b 20/2a2 2a1 3b 1a 1b/3a/21 1c/16b 12/2b 20/2a2 2a1 3b 3b/14/15/14 1/3a/21 3c 4c1 (4a+4b+4c2)/15/5b 10b/11b/7a/5a (4a+4b+4c2)/15/5b 5b/15 6 10b/11b/7a/5a 7b 7b 8a/18 8b 15/9a/17b 9b/15 10b/11b/7a/5a 16a/22/10a 10b/11b/7a/5a 11a 12/2b 13 3b/14/15/14 5b/15 9b/15 15/9a/17b (4a+4b+4c2)/15/5b 14/15 16a/22/10a 16b/1c 15/9a/17b 17a 8a/18 19 20/2a2 3a/21 16a/22/10a X 1/3a/21 3c 4c1 (4a+4b+4c2)/15/5b 10b/11b/7a/5a (4a+4b+4c2)/15/5b 5b/15 6 10b/11b/7a/5a 7b/11a 7b 8a/18 8b 9a/15 9b/15 10b/11b/7a/5a 16a/22/10a 10b/11b/7a/5a 7b/11a 12/2b 13 15/14/15/14 5b/15 9b/15 9a/15 (4a+4b+4c2)/15/5b 16a/10a 16b/2b 17 16a/22/10a 16b/1c 17 8a/18 19 20 3a/21 22 X 8a/18 19 20/2a2 3a/21 16a/22/10a X n The alphabetic denomination a, b and c corresponds to the primate ancestral segments homologous to human chromosomes described in Ruiz-Herrera et al. . In the case of the homologues to human chromosomes 11 and 17, a represents the human p-arm whereas b corresponds to the human q-arm. Aotus chromosomes homologous to 5b, 7b, 14 and 15 are split into different chromosomes. 82 / Ruiz-Herrera et al. whereas ancestral chromosome 17 is split into two different chromosomes only in A. nancymai (Table II). When comparing our results with previous reports, an important feature to consider is that two of the Platyrrhini ancestral associations common to New World monkeys (2/16 and 10/16) are absent in both Aotus species studied. In the case of ancestral association 2/16, the lack of this synteny has also been observed in Callithrix jacchus [Neusser et al., 2001] and in the presumed ancestral karyotype of Alouatta [Consigliere et al., 1996; de Oliviera et al., 2002]. The separation of association 2/16 in these three different phylogenetic lineages indicate a convergence of independent fission events. In contrast, and in light of the hybridization results, Aotus is the only New World monkey in which association 10/16 has been interrupted by, in this case, the insertion of human homologous chromosome 22. Thus, the lack of association 10/16 would be considered a derived characteristic of the Aotus genus. It seems that ancestral chromosome 16 has undergone a different evolutionary history, being associated with different chromosomes (16/22/10 and 2/16), with respect to the rest of the Platyrrhini species studied so far. By analyzing previous cytogenetic studies in Aotus, we found that the morphology of the chromosomes that contain association 16/22/10 (KM9 chromosome 3 and KM3 chromosome 3), and the chromosomes that contain the association 2/16 (KM9 chromosome 9 and KM3 chromosome 8) have been conserved in other Aotus KMs [Ma, 1981; Torres et al., 1998], suggesting an ancestral characteristic of Aotus. The derived associations of New World monkeys homologous to human chromosomes 1/3, 2/12, 4/15, and 10/11 are also present in Atelinae, Cebidae, and Callithrichidae. However, some aspects must be taken into account. The 1/3 association has also been found in Callicebus lugens [Stanyon et al., 2003] and Callimico goeldii [Neusser et al., 2001], but it seems that these 1/3 associations do not represent the same segments of chromosome 3. Considering the ancestral New World monkey karyotype, and based on G-banding comparisons, Callimico goeldii would present the association 1b/3c, whereas Aotus species have the association of different segments: 1b/3a/21 (Table II). Thus, although Callimico goeldii and Aotus share the 1/3 association, it seems that the translocations are not the result of the same event. In the case of Callicebus lugens, although the authors [Stanyon et al., 2003] do not provide any karyotype with which to compare it, it seems that neither corresponds to the case of Aotus. Association 2/12 is present only in the most cytogenetically derived titi monkeys, Callicebus lugens [Stanyon et al., 2003]. In this case, there is insufficient information to define the segments involved in the association. Both of the Aotus KMs analyzed in this study share association 4/15 with Atelinae species (Lagothrix lagothricha [Stanyon et al., 2001], Ateles [Garcı́a et al., 2002; Morescalchi et al., 1997], and Alouatta [Consigliere et al., 1996; de Oliviera et al., 2002]). However, in contrast to what occurs in all Platyrrhini species studied, ancestral chromosome 15 has undergone a huge number of translocations in the Aotus karyotypes (Table II). The fragments involved in association 10/11 appear to be the same in the Aotus and Callicebus species studied so far (10b/11). This 10/11 association is presumably ancestral for all Callicebus [Stanyon et al., 2003]. Therefore, the presence of the derived association 10/11 in Callicebus and Aotus can be considered a landmark that relates Aotus and Callicebus phylogenetically. This fact would support the hypotheses of Ford  and Rosenberger , which closely relate Callicebus and Aotus. Nevertheless, in the light of our comparative chromosomal results, we are not able to conclude that Aotus has a basal position ZOO-FISH in Aotus / 83 in the Platyrrhini phylogenetic tree. On the contrary, Aotus species would have a derived chromosomal history, with respect to the putative New World monkey ancestral karyotype. Further comparative studies including more Aotus species and the subfamily Pithecinae would be extremely useful for discarding either Rosenberger’s or Ford’s hypothesis, and clarifying the final position of these Platyrrhini species. Mechanisms and Direction of Chromosomal Evolution in Aotus In the present study, the chromosomal reorganizations detected between the two Aotus KMs are predominantly fusion/fission events, as also happens in Cercopithecus and Alouatta [Consigliere et al., 1996; Ponsà et al., 1986]. This fact contrasts with what occurs in other Primate phylogenetic branches. For instance, in Cebus and Ateles genera, the most frequent rearrangements are inversions [Garcı́a et al., 2002]. After they compared nine different karyotypes, Ma et al.  postulated that the putative ancestral Aotus would show a karyotype of 2n ¼ 54, which would correspond to the A. nancymai KM (KM3) studied in the present work. However, the data obtained with the use of human chromosome probes revealed some support for the hypothesis that KM9 shares more ancestral chromosome characteristics with the putative New World monkey ancestral karyotype than does KM3. Based on our chromosome painting results, both KMs differ due to a different signal distribution of WCPs from human chromosomes 3, 14, 15, and 17, which are the result of interchromosomal reorganizations. Human chromosome 3 is represented as three different chromosomes in the ancestral Platyrrhini karyotype (3a/21, 3b, and 3c; Table II). The Aotus chromosomes homologous to ancestral chromosome 3b are different in both KMs; that is, KM9 has maintained the ancestral form as one whole chromosome (chromosome 8; Fig. 1), whereas within KM3 this chromosome has a derived state, as two different chromosomes (chromosomes 14 and 18; Fig. 2). Regarding chromosomes homologous to HSA 14 and 15, the situation is more complex. As has been reported in other Platyrrhini species, although association 14/15 is always present, in some cases (e.g., the Saimiri and Cebus species) it has undergone intrachromosomal reorganizations, such as inversions [Garcı́a et al., 2002]. Nevertheless, the case of Aotus is quite different because human chromosome 15 is split into five and six different chromosomes in KM9 and KM3, respectively (Table II). This feature supports the highly derived situation of Aotus regarding the ancestral New World monkey karyotype. Concerning human chromosome 17, in KM9 one whole chromosome is homologous to HSA 17 without any disruption (chromosome 21), whereas in KM3 the ancestral chromosome 17 is split into two different chromosomes (chromosomes 6 and 26). The situation present in KM9 is also conserved in all Platyrrhini species analyzed thus far. In addition, revising the classic cytogenetic data, Aotus karyotypes K-III, KV, K-VI, and K-VII [Ma, 1981] share the same chromosome morphology for chromosomes homologous to KM9 chromosome 5. In KM3, chromosome 4 shows a derived morphology, which differs from those mentioned above, by a paracentric inversion (Fig. 4). 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