Introduction
============

Genomic instability in mammals has become a growing area of interest in evolutionary cytogenetics. Special attention was paid to the relationship between chromosomal instability and rearrangements, which are considered to occur in fragile regions ([@evy119-B47]; [@evy119-B42]).

Currently, it is accepted that chromosomal breakage is required before the occurrence of chromosomal rearrangements ([@evy119-B21]; [@evy119-B23]). These rearrangements are generally unviable, but they may be stable if the genetic information remains balanced as is the case for reciprocal translocations or inversions. When these new chromosomal forms occur in the germ line, they may result in new species or polymorphic variants ([@evy119-B42]). Different hypotheses have been proposed to account for the molecular mechanisms underlying this process, and although one of them was experimentally confirmed ([@evy119-B502]; [@evy119-B501]; [@evy119-B39]), the topic is still controversial (Garcia-Sagredo 2008).

Many studies in different genera and species of nonhuman primates showed that the biomarkers fragile sites (FS) and intrachromosomal telomeric sequences (ITS) may be associated with different degrees of genomic instability ([@evy119-B2]; [@evy119-B43], [@evy119-B43]). Moreover, other researchers suggested that a large proportion of chromosome bands involved in evolutionary rearrangements would correspond to some fragile and heterochromatin regions ([@evy119-B22]; [@evy119-B43]; [@evy119-B45]; [@evy119-B24]). The frequency of Sister Chromatid Exchange (SCE) is a biomarker for the detection of symmetric exchanges between chromatids of the same chromosome known to occur naturally during normal DNA replication. Currently, the most accepted hypothesis states that SCE arises through homologous recombination when Holliday junctions are resolved and DNA strands are exchanged at a given frequency ([@evy119-B58] and references therein). An association between the occurrence of SCE and chromosomal rearrangements implicated in the evolutionary process was suggested by the fact that the former is considered an indicator of genomic instability (Garcia-Sagredo 2008). Although both DNA repair and rearrangements take place in different contexts, they share, to some extent, the same underlying mechanisms.

The three genera of Platyrrhini of interest display large karyotypic diversity, including not only the chromosome formula but also types of rearrangements and amount of heterochromatin ([@evy119-B33]; [@evy119-B35]; [@evy119-B51]; [@evy119-B17]; [@evy119-B8], [@evy119-B9]; [@evy119-B11]). Although the genus *Cebus* presents rearrangements of inversion-type, it has a highly conserved karyotype in terms of euchromatin compared with the common ancestor of Platyrrhini. These primates have only two chromosome numbers, 2*n* = 54 and 2*n* = 52, with the most important cytogenetic feature being the large proportion of constitutive extracentromeric heterochromatin (∼15% of the genome in all the species examined) ([@evy119-B15]; [@evy119-B50]; [@evy119-B1]; [@evy119-B37]). The karyotypes of *Ateles* species have a large number of inversions and fusions; this genus exhibits a limited range of variation in chromosome number, with 2*n* = 32 in *A. paniscus* and 2*n* = 34 in *A. chamek*. In contrast, the chromosome number in genus *Alouatta* (with 12 described species), ranges from 2*n* = 44 in *A. seniculus* to 2*n* = 58 in *A. pigra* ([@evy119-B52]). This genus has a distinctive sex chromosome system in the male, originated from translocations involving autosomes and a sexual chromosome ([@evy119-B40]; Mudry et al. 1998, [@evy119-B30]; [@evy119-B52]).

The aims of this work were to characterize the spontaneous occurrence of SCEs in *Alouatta caraya, Ateles chamek, Ateles paniscus*, and *Cebus cay*, and to assess the possible association between the localization of SCEs and the regions with rearrangements implicated in chromosome evolution. Our hypothesis is that the SCE frequency in each species is significantly higher in rearranged regions than in conserved regions.

Materials and Methods
=====================

Samples and Chromosome Preparations
-----------------------------------

Metaphase spreads for G-banding and Fluorescence Plus Giemsa (FPG) techniques were prepared from cultures of phytohemagglutinin-stimulated lymphocytes and fibroblasts of 9 adults of *Alouatta caraya* (2*n* = 52)*, Ateles chamek* (2*n* = 34)*, Ateles paniscus* (2*n* = 32), and *Cebus cay* (2*n* = 54) ([table 1](#evy119-T1){ref-type="table"}). These individuals were kept in captivity at different institutions in Argentina and Brazil. Their cells were harvested by standard procedures and classical cytogenetic characterization was performed for each specimen according to modified standard protocols ([@evy119-B54]). Each monkey was identified taxonomically at the species level by analyzing metaphases treated by G-Wright and C-banding (20 spreads for each one) and using species-specific karyological patterns reported previously ([@evy119-B503]; [@evy119-B29]; [@evy119-B36]). Blood and biopsies were collected by trained veterinarians under sterile conditions. Table 1Information on the Specimens of *Alouatta caraya*, *Ateles chamek*, *Ateles paniscus*, and *Cebus cay*SpeciesNumber of SpecimensSexOriginBiological Samples*Alouatta caraya*3♂REHMBlood♀REHMBlood♀REHMBlood*Ateles chamek*3♂ZBASkin♂ZBASkin and blood♂ZBASkin and blood*Ateles paniscus*1♂CNPSkin*Cebus cay*2♂ZBASkin and blood♀ZBASkin and blood[^1]

Sequential G-FPG Banding
------------------------

G-banding with a modified Wright staining method ([@evy119-B32]) was applied to all metaphases as follows: first, slides were maintained for one week at room temperature and in the dark (or, alternatively, for 5 h at 37 °C); then they were pretreated in 2xSSC for 2 min 30 s at 65 °C, washed with distilled water and covered with a 3: 1 Wright stain: Sørensen buffer (KH~2~PO~4~ and Na~2~HPO~4~ solutions at a 2: 1 ratio) in the dark for 2 min 30 s. After recording all the available metaphases, slides were destained and treated by the Fluorescence Plus Giemsa (FPG) technique to visualize SCEs ([@evy119-B26]; [@evy119-B32]). Briefly, the slides were incubated with Hoechst 33258/2xSSC/distilled water for 20 min, irradiated with ultraviolet light for 35 min and rinsed in deionized water. Then, they were incubated in 2xSSC for 2 h at 60 °C, rinsed in distilled water, and counterstained with 4′, 6-diamidino-2-phenylindole (DAPI).

Image Acquisition and Processing
--------------------------------

The G-banding and FPG images were analyzed on a Leica DMLB fluorescence microscope and photographed with a Leica DFC 340 FX camera. These were compared with the karyotypes already described for the species of interest. Image processing was performed with Adobe Photoshop 7.0 (Adobe Systems Inc.). For each specimen, we analyzed a minimum of 20 metaphases and quantified the SCEs per chromosome and per cell. Then, we determined the location of the SCEs at a subchromosomal level, using banding patterns obtained with the sequential G-band (400 band resolution)-FPG technique.

Reconstruction of Evolutionary Chromosomal Rearrangements Described for Platyrrhini
-----------------------------------------------------------------------------------

We analyzed literature data of chromosome evolution in Platyrrhini based on chromosomal rearrangements occurring within and between families and genera so far described ([@evy119-B27]; [@evy119-B506], [@evy119-B8], [@evy119-B9]; [@evy119-B1]; [@evy119-B53]; [@evy119-B11]). The chromosomal rearrangements mentioned in the reviewed reconstructions were determined indirectly by analyzing human syntenies. This allowed us to identify the conserved and rearranged chromosomes in the three genera under study as determined in relation to the most probable common ancestor of Platyrrhini ([fig. 1](#evy119-F1){ref-type="fig"}). Finally, we compiled a database of all available information on the number of SCEs and their location in both conserved and rearranged chromosome regions for each studied genus.

![Partial phylogenetic tree of Atelidae and Cebidae, modified from [@evy119-B11]. This phylogeny shows chromosome syntenies characterizing each node. MRCA, Most Recent Common Ancestor of Atelidae and Cebidae. [Box]{.ul}: Idiogram of chromosome \#5 of *Ateles* spp., illustrating rearranged and conserved regions, and their respective number of G-bands.](evy119f1){#evy119-F1}

Statistical Analysis
--------------------

Following [@evy119-B25] approach to the statistical analysis of SCE, a Poisson test was used to evaluate association between the localization of SCEs and chromosomal regions with rearrangements involved in the evolution of each genus. All statistical analyses were performed with STATISTICA 10 (StatSoft Inc.). For each species, we identified the conserved and rearranged regions, where we determined the type of chromosomal rearrangement and quantified the total number of chromosomal bands per haploid set from the G-banding pattern (see [box in fig. 1](#evy119-F1){ref-type="fig"}). Then, we calculated the SCE frequencies for both regions.

Our data set is assumed to follow a Poisson distribution, with the number of events per unit of area corresponding to the number of SCEs per unit of genome. We considered each chromosomal band in the G-banding pattern as a unit of area because the genome length in base pairs of the studied species has not been determined yet.

Based on the G-band karyotype, we defined the number of bands per haploid set (*N*), the number of metaphases analyzed (*S*) and the total number of SCEs observed (*E*) for each species. The probability of finding 0 or more SCEs (*h*) randomly distributed at one of the *N* bands of the karyotype is: $$a\left( h \right) = \ E/\left( {2 \times S \times N} \right)$$

The value of *a*(*h*) is usually \<1 because *N* is high but *E* is low. Since each band identifies a sample of 2*S* bands, the number of SCEs per band will follow a Poisson distribution with a mean µ = *E*/*N* = 2*S*×*a*(*h*) and variance σ^2^ = 2*S*×*a*(*h*)×\[1−*a*(*h*)\]. Under this distribution, the probability of finding *h* or more SCEs at a band is: $$p_{h} = \ 1 - \ \sum a_{i}$$ where *a~o~* = *e*^*−*µ^ and *a~i~* = (µ/*i*)×*a~i~*~−1~.

If there are *N* bands in the karyotype, the expected number of bands showing 0 or more SCEs (*h*) is: $$F_{h} = \ N \times p_{h}$$

If *F~h~*\< 0.05, then the probability of finding a band with *h* SCEs is \<5%. Thus, *h* is a value significant at the 5% level, and all bands in which *h* SCEs are found can be classified as unstable regions.

This statistical approach provides a simple and reliable tool not only to verify the nonrandom distribution of SCEs throughout the genome but also to determine the minimum number of SCEs required to consider a band as an unstable region ([@evy119-B25]).

Results
=======

The absolute SCE frequencies per metaphase and per chromosome pair were calculated for each individual of each species.

Considering all metaphases, in *A. caraya* (2*n* = 52) the number of SCEs per chromosome pair ranged from 0 to 10 in the male and 0 to 9 in the females, with an average of 7.1 ± 3.6 SCEs/metaphase. The SCE frequencies per chromosome pair were considerably higher in *Ateles* than in the other species, ranging from 4 to 42 in *A. chamek* and 1 to 25 in *A. paniscus*, with average values of 6.7 ± 2.7 and 4.7 ± 2.1 SCEs/metaphase, respectively. In *C. cay* the SCE frequency per chromosome pair varied from 0 to 8 in the male and 0 to 7 in the female, with an average of 5.7 ± 2.6 SCEs/metaphase.

[Table 2](#evy119-T2){ref-type="table"} shows published information on chromosomal rearrangements for *A. caraya*, [table 3](#evy119-T3){ref-type="table"} for *A. chamek* and *A. paniscus*, and [table 4](#evy119-T4){ref-type="table"} for *C. cay*. Subsequently, the SCEs located in each chromosomal region (rearranged and conserved) were quantified for each species. Table 2Summary of Chromosomal Rearrangements Described for Each Chromosome Pair of *Alouatta caraya*Chromosome PairRearranged/Conserved ChromosomeType of RearrangementSCEs1Rearranged region 1Fusion 1a/5b2.51Rearranged region 2Fusion 5b/7a/5a/7a71Conserved--12Conserved--4.53Conserved--44Rearranged region 1Fission--fusion 16b/4c0.54Conserved--4.55Rearranged region 1Inversion 16a/10a4.55Conserved--06Conserved--4.57Conserved--1.58Conserved--39Conserved--2.510Conserved--011Rearranged region 1Fusion 2a/207.511Conserved--112Conserved--513Conserved--5.514Conserved--4.515Conserved--516Conserved--4.517Conserved--8.518Conserved--4.519Conserved--2.520Conserved--521Conserved--2.522Conserved--2.523Conserved--1.524Conserved--625Conserved--0.5X~1~Conserved--6X~2~[^a^](#tblfn3){ref-type="table-fn"}Rearranged region 1Translocation 7-Y0X~2~[^a^](#tblfn3){ref-type="table-fn"}Conserved--0Y~1~[^a^](#tblfn3){ref-type="table-fn"}Rearranged region 1Translocation Y-15b/3c1Y~1~[^a^](#tblfn3){ref-type="table-fn"}Conserved--0Y~2~[^a^](#tblfn3){ref-type="table-fn"}Rearranged region 1Fission 15b/3c2Y~2~[^a^](#tblfn3){ref-type="table-fn"}Conserved--0[^2][^3]Table 3Summary of Chromosomal Rearrangements Described for Each Chromosome Pair of *Ateles* sppChromosome PairRearranged/Conserved ChromosomeType of RearrangementSCEs *A. chamek*SCEs *A. paniscus*1Rearranged region 1Fission--fusion 9-18/8a5.3111Rearranged region 2Fusion 18/8a-16a/10a13.3171Rearranged region 3Inversion 16a/10a5.091Conserved--11.0152Rearranged region 1Fusion 12/15a8.062Rearranged region 2Fusions 15a/14-1a-4b/15a14.0232Conserved--10.3253Rearranged region 1Fusion 22/15b4.773Rearranged region 2Fusion 15b-3b/2a3.393Rearranged region 3Fusion 3b/2a12.7153Conserved--5.364Rearranged region 1Fusion 3c/7b8.364Rearranged region 2Translocation 1a and fusion 1a/7b11.0134/12[^a^](#tblfn5){ref-type="table-fn"}Rearranged region 3Fusion 13/3c10.3174Conserved--9.085Rearranged region 1Fusion 5b/8b13.7115Conserved--11.0176Rearranged region 1Fusion 16/2b-1b11.0126Conserved--5.797Rearranged region 1Translocation 1a y 67.797Rearranged region 2Fusion 6/1c5.3237Conserved--10.018Conserved--5.769Rearranged region 1Fusion 4c-7a/5a/7a9.0119Conserved--2.3810Rearranged region 1Translocation 6 and fusion 6--21/3a10.3510Conserved--13.72311Conserved--8.01512[^b^](#tblfn6){ref-type="table-fn"}Rearranged region 1Inversion 132.3--12[^b^](#tblfn6){ref-type="table-fn"}Conserved--8.3--13Conserved--4.3914Conserved--3.01415Conserved--3.3416Conserved--7.04XConserved--1.36YRearranged5.39[^4][^5][^6]Table 4Summary of Chromosomal Rearrangements Described for Each Chromosome Pair of *Cebus cay*Chromosome PairRearranged/Conserved ChromosomeType of RearrangementSCEs1Conserved--5.52Conserved--4.53Conserved--24Conserved--2.55Conserved--46Rearranged region 1Pericentric inversion 14/1506Rearranged region 2Pericentric inversion 14/15/140.56Conserved--07Conserved--28Rearranged region 1Pericentric inversion 8b0.58Conserved--19Conserved--0.510Rearranged region 1Pericentric inversion 20110Conserved--011Conserved--712Rearranged region 1[^a^](#tblfn8){ref-type="table-fn"}Pericentric inversion 121.512Conserved--313Conserved--2.514Conserved--215Conserved--2.516Conserved--317Conserved--618Conserved--4.519Conserved--1.520Conserved--021Rearranged region 1[^a^](#tblfn8){ref-type="table-fn"}Pericentric inversion 170.521Conserved--0.522Conserved--123Conserved--124Conserved--0.525Conserved--0.526Conserved--0.5XConserved--1YConserved--0[^7][^8]

Using the equations given in Materials and Methods, we determined the minimal number of SCEs necessary to classify a given band as significantly unstable for each species (see [Supplementary Material](#sup1){ref-type="supplementary-material"}). [Table 5](#evy119-T5){ref-type="table"} shows the percentage of significantly unstable bands in rearranged and conserved regions obtained for each species. To emphasize interspecific differences, [figure 2](#evy119-F2){ref-type="fig"} illustrates the percentage of unstable bands in conserved and rearranged regions per species, relativized to the species having the largest percentage (i.e., *A. chamek*). Table 5Minimum Number of Sister Chromatid Exchanges (SCEs) for Which a Given Band is Considered to be Statistically Unstable, and Percentage (%) of Significantly Unstable Bands in Rearranged and Conserved RegionsSpeciesMinimum Number SCEs% Unstable Bands in Rearranged Regions% Unstable Bands in Conserved Regions*P* Value*Alouatta caraya* ♂518.1851.440.044*Alouatta caraya* ♀550.0019.930.008*Ateles chamek* ♂1483.3386.250.033*Ateles paniscus* ♂986.2150.630.047*Cebus cay* ♂40.0033.450.022*Cebus cay* ♀40.0019.800.020[^9]

![Percentages (%) of significantly unstable bands in conserved (black dots on white background) and rearranged (white dots on black background) regions in *Alouatta caraya, Ateles chamek, Ateles paniscus*, and *Cebus cay*. Results for males and females of *A. caraya* and *C. cay* are shown separately. For comparative purposes, values were relativized to those obtained for *A. chamek*, which were assigned to be 100% because this species had the highest percentage of SCEs.](evy119f2){#evy119-F2}

The male and females of *Alouatta caraya* showed differences in the proportion of significantly unstable bands between conserved and rearranged regions. The male had a larger proportion of significantly unstable bands in conserved regions (chromosome pairs \# 3, 4, 6, 9, 12--18, 20, and X~1~), while in the females these were mainly detected in rearranged regions (chromosome pairs \# 1, 5, and 11) (see [Supplementary Material](#sup1){ref-type="supplementary-material"}).

In the two studied species of *Ateles*, SCE frequencies were higher in rearranged than in conserved regions. *Ateles chamek* had a large proportion of unstable bands in both rearranged and conserved regions and the SCEs were concentrated in chromosome pairs \# 1, 2, 3, 4, 5, 6, 7, 9, and 12. *Ateles paniscus* showed a higher proportion of significantly unstable bands in rearranged regions, particularly in chromosome pairs \# 1, 2, 3, 4/12, 5, 6, 7, 9, and Y (see [Supplementary Material](#sup1){ref-type="supplementary-material"}).

Both sexes of *C. cay* showed a low proportion of unstable bands, all of which were concentrated in conserved regions. These bands were found in chromosome pairs \# 1, 2, 5, 11, 15, and 16 of the male, and in \# 1, 11, 17, and 18 of the female (see [Supplementary Material](#sup1){ref-type="supplementary-material"}).

Discussion
==========

Comparative cytogenetic analysis proved to be useful to determine chromosome homeologies and to identify changes leading to the evolution of karyotypes. In this regard, FISH and derived techniques allow a more detailed study of genomic reorganizations in mammals from an evolutionary perspective. In the context of primate evolution, the analysis of homologous synteny blocks have contributed to the reconstruction of chromosome rearrangements based on an inferred ancestral karyotype. This same approach has been used to identify unstable chromosome bands involved in evolutionary reorganizations ([@evy119-B45]). Moreover, in the human genome [@evy119-B47] have reported the presence of bands indicative of chromosome instability that may accumulate evolutionary breakpoints, which are colocalized with intrachromosomal telomeric sequences (ITS) corresponding to remnants of ancestral chromosomal rearrangements.

If, as mentioned earlier, genomic instability is a requirement for the occurrence of stable rearrangements, then SCE frequency combined with other cytogenetic techniques emerges as a valuable tool to establish the relationship between genomic regions with differential stability and stable rearrangements. In our study focused on Neotropical primates, SCE showed high sensitivity and specificity for the detection of regions prone to break and rearrange. Thus, it proved to be a valuable genomic instability biomarker which provides information to be used in comparative evolutionary studies.

In *Alouatta caraya*, the chromosome pairs \#11 and \#17 showed the highest frequencies of SCEs. In pair \#11, SCEs were more frequent than expected in the band showing a rearrangement involving fusion, which is consistent with the considerable amount of evolutionary rearrangements that are likely to have occurred in this pair in different species of *Alouatta* ([@evy119-B52]). The pair \#17, which corresponds to the chromosome \#20 in the study of de Oliveira et al. (2002) because they adopted a different criterion for ordering the karyotype, shares homeology with the human chromosomes \#14 and \#15, indicating that, at least, it would have been derived from a rearrangement of the ancestral Platyrrhini karyotype ([@evy119-B11]). The high SCE frequency in pair \#17 suggests that the rearranged region is unstable ([fig. 2](#evy119-F2){ref-type="fig"}). Compared with the male, the females of *A. caraya* showed a significantly higher frequency of SCEs in the pair \#1, especially in regions with fusions as primary rearrangements. This may be related to the homeology shared by the pair \#1 with eight chromosomal segments of other species of the genus *Alouatta* ([@evy119-B52]). However, the difference between sexes may be explained by the small sample size. Overall, the sex chromosomes of both sexes of *A. caraya* exhibited a low SCE frequency ([table 2](#evy119-T2){ref-type="table"}). Notwithstanding this, the male showed a high instability in conserved regions, which is in agreement with the presence of multiple sex chromosome systems in the males of *Alouatta* species ([@evy119-B52]). A hypothetical explanation is that sex chromosomes are protected at the structural or epigenetic level, given their evolutionary importance.

Although the available literature does not provide any relationship between rearranged regions in *A. caraya* and the Platyrrhini ancestor, it may seem that rearranged regions in Y~1~ and Y~2~ sex chromosomes are prone to breakage and rearrangement. Furthermore, in *A. caraya*, we observed a considerable difference in the instability of conserved regions between male and females. Further analysis is required to shed light on the possible reasons for this result.

Both species of the genus *Ateles* showed higher chromosome instability than did the other genera under study. Particularly in *A. chamek*, we detected a large proportion of bands showing a high degree of instability, both in conserved (86.3%) and rearranged (83.3%) regions. Average SCE frequency was lower in *A. paniscus* than in *A. chamek*, but SCE distribution was highly associated with chromosomal rearrangements implicated in the evolution of this genus. Most of the rearranged regions (86.2%) showed a significantly high instability ([table 5](#evy119-T5){ref-type="table"}). This result is in line with the several chromosomal rearrangements accumulated during the evolution of *Ateles* genome ([fig. 2](#evy119-F2){ref-type="fig"}). Moreover, the different percentage of unstable regions recorded for this genus may be due to the drastic reduction in the chromosome number occurring in the ancestors of Atelidae (2*n* = 62) and *Ateles* (2*n* = 34), which implies an important genomic reorganization. In both *A. chamek* and *A. paniscus*, SCE frequency in the chromosome pair \#8 was noticeably lower than expected from random (see [Supplementary Material](#sup1){ref-type="supplementary-material"}), possibly because the nucleolar organizer regions (NOR) are located in this pair. These regions would confer protection against breakage and chromatid exchanges, as we previously proposed for the sex chromosomes of *A. caraya*. It is worth mentioning that in both species the Y chromosome showed the highest degree of instability among the smaller chromosomes of the complement. The SCE frequency in the chromosome Y was higher in *A. paniscus* than in *A. chamek*, in agreement with the study of [@evy119-B12], who used interspecies comparative genomic hybridization (iCGH) analysis in *Ateles* sp.; these authors reported that the different species exhibited rearrangements involving the Y chromosome, with higher frequencies in *A. paniscus*. These results, together with the frequent spontaneous occurrence of SCEs observed in our study, suggest that genome instability is much higher in *Ateles* than in the other studied genera.

In both sexes of *C. cay*, the average SCE frequency was substantially lower compared with the other two genera; the SCEs were mainly located in conserved regions, of which \<20% were significantly unstable ([fig. 2](#evy119-F2){ref-type="fig"}). Our results on *C. cay* and those using genome instability biomarkers other than SCE frequency ([@evy119-B5]; [@evy119-B32]) indicated that SCEs and FS shared the same chromosomal location. This supports the proposal that chromosome organization involves not only obvious bands (e.g., G-banding patterns) but also nonvisible bands which can be partially evidenced by biomarkers such as FS, ITS, and SCE. The latter bands are especially prone to breakage and are located in chromosome regions conserved during primate evolution ([@evy119-B45]).

The fact that *Cebus* presents a highly conservative karyotype compared with the most recent common ancestor of Platyrrhini explains the high instability (i.e., the high tendency to break and reorganize) in conserved regions. Among significantly unstable chromosome pairs, \#11 showed the highest SCE frequency, most of which were located at the interface between the euchromatin and heterochromatin, suggesting that it is an unstable region. Pairs \# 4, 6, 12, 13, 17, and 19 also showed high SCE frequencies in eu-heterochromatic junctions. These results are in disagreement with those of [@evy119-B32], who found a significantly higher SCE frequency within the blocks and a nonsignificant SCE frequency between the two types of chromatin.

A higher genomic instability at eu-heterochromatic junctions has been previously reported for other mammalian models, such as the Indian muntjac ([@evy119-B6]; [@evy119-B7]). In addition, an increased frequency of FS and ITS in heterochromatin regions or at eu-heterochromatic junctions has been observed for different species of primates ([@evy119-B28]; [@evy119-B14]; [@evy119-B2]; [@evy119-B57]; [@evy119-B31]; [@evy119-B48]; [@evy119-B10]). These results suggest an evolutionary relationship between heterochromatin distribution and regions or loci associated with chromosome breakage, such as FS and ITS. Heterochromatin has been assumed to have a genome stabilizing role through the induction of chromatin compaction and the protection against endogenous damage ([@evy119-B56]; [@evy119-B38]). Therefore, the reduced SCE frequency in *C. cay* would be due, at least partially, to the large proportion of heterochromatin in its genome (the heterochromatin of *Cebus* species so far analyzed represents between 5% and 13% of the genome) ([@evy119-B38]).

Our results provide evidence of higher genomic instability in karyotypes with numerous chromosomal rearrangements and are in agreement with studies that used instability biomarkers other than SCE ([@evy119-B55]; [@evy119-B505]; [@evy119-B43]; [@evy119-B45]). Thus, both species of *Ateles*, with numerous chromosomal rearrangements implicated in the evolution of this genus, showed a large degree of genomic instability; this is particularly the case for *A. paniscus*, which is one of the most derived species ([@evy119-B27]; [@evy119-B8]) In contrast, *C. cay* showed a considerable low genomic instability, which is consistent with its highly conserved karyotype compared with the ancestor of Platyrrhini. In regard to *A. caraya*, we found that its genomic instability and average number of chromosomal rearrangements were intermediate between those in the species of *Cebus* and *Ateles* under study.

In brief, through the use of SCE, we assume that genomes with rearrangements of evolutionary significance might provide evidence of association between genomic instability and a higher occurrence of structural chromosomal rearrangements. However, the analysis of a larger number of species belonging to a genus with a large occurrence of rearrangements (e.g., *Ateles*) may be needed to provide a more robust interpretation.

Finally, in all four species studied here (mainly *Cebus*), the use of SCE led us to characterize a large proportion of conserved regions as significantly unstable. This suggests the need to include other biomarkers such as FS and ITS to detect instability. Our approach represents a first step toward the identification of regions repeatedly involved in the chromosome evolution of the different Platyrrhini lineages.

Supplementary Material
======================

[Supplementary data](#sup1){ref-type="supplementary-material"} are available at *Genome Biology and Evolution* online.

Supplementary Material
======================

###### 

Click here for additional data file.

This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, project PIP 0572) and grants from the Secretaría de Ciencia y Tecnología, (UBACyT) of the Universidad de Buenos Aires (Project 20020130100317BA) to Marta Dolores Mudry. We thank the zoological institutions, researchers, and collaborators from different localities for issuing permits to access sampling sites, and to collect and transport biological material. We specially thank veterinarians for their assistance in capturing and handling monkeys. Biological sampling was conducted in compliance with the guidelines for the care and manipulation of animals established by the Ethical Committee of the Argentine Society of Mammalian Studies (SAREM).

[^1]: ZBA, Buenos Aires Zoological Garden; REHM, Horco Molle Experimental Park; CNP, National Center of Primates, Belém, Brazil.

[^2]: [Note]{.smallcaps}.---The last column gives the average frequency of Sister Chromatid Exchanges (SCEs) for all the individuals analyzed. References: de Oliveira et al. (2002), [@evy119-B53], and [@evy119-B11].

[^3]: The chromosomes of males (with a multiple sex chromosome system X~1~X~2~Y~1~Y~2~).

[^4]: [Note]{.smallcaps}.---The last column gives the average frequency of Sister Chromatid Exchanges (SCEs) for all the individuals analyzed. References: [@evy119-B8] and [@evy119-B11].

[^5]: The chromosome pair belongs to *Ateles paniscus* only, which was originated from the fusion of the ancestral chromosomes \#4 and \#12.

[^6]: The chromosome pair belongs to *Ateles chamek* only.

[^7]: [Note]{.smallcaps}.---The last column gives the average frequency of Sister Chromatid Exchanges (SCEs) for all the individuals analyzed. References: [@evy119-B1] and [@evy119-B11].

[^8]: Chromosomal rearrangements identified in this study.

[^9]: [Note]{.smallcaps}.---Values were considered statistically significant at the *P* \< 0.05 level.
