Chromosome Painting in Cercopithecus petaurista (Schreber, 1774) Compared to Other Monkeys of the Cercopithecini Tribe (Catarrhini, Primates)

The Cercopithecini tribe includes terrestrial and arboreal clades whose relationships are controversial, with a high level of chromosome rearrangements. In order to provide new insights on the tribe’s phylogeny, chromosome painting, using the complete set of human syntenic probes, was performed in Cercopithecus petaurista, a representative species of the Cercopithecini tribe. The results show C. petaurista with a highly rearranged karyotype characterized by the fission of human chromosomes 1, 2, 3, 5, 6, 8, 11, and 12. These results compared with the literature data permit us to confirm the monophyly of the Cercopithecini tribe (fissions of chromosomes 5 and 6), as previously proposed by chromosomal and molecular data. Furthermore, we support the monophyly of the strictly arboreal Cercopithecus clade, previously proposed by the molecular approach, identifying chromosomal synapomorphies (fissions of chromosomes 1, 2, 3, 11, 12). We also add additional markers that can be useful for deciphering arboreal Cercopithecini phylogeny. For example, the fission of chromosome 8 is synapomorphy linking C. petaurista, C. erythrogaster, and C. nictitans among the arboreal species. Finally, a telomeric sequence probe was mapped on C. petaurista, showing only classic telomeric signals and giving no support to a previous hypothesis regarding a link between interspersed telomeric sequences in high rearranged genomes.


Introduction
The African Cercopithecini tribe (Gray, 1821) of the Cercopithecinae subfamily, Cercopithecidea family (Catarrhini), with numerous species and subspecies, inhabits various ecological niches which include semi-terrestrial, terrestrial, and strictly arboreal behaviors. Many researchers believe that the tribe's species have only recently undergone rapid adaptive radiation. In fact, it is thought that the ecological and morphological diversity that characterizes this group today (with many genera and species, as well as a plethora of sub-species and geographical varieties) appeared in the late Pliocene [1][2][3]. The fact that the tribe's radiation occurred so recently makes this group an ideal subject for evolutionary studies. Indeed, members are characterized by a high level of genetic variability and different adaptation strategies [4][5][6].
Chromosomal analysis [5][6][7] and data from different molecular approaches [8][9][10] have led researchers to propose subdividing the tribe into two primary clades of terrestrial and strictly arboreal monkeys. However, other morphological and chromosomal evidence does not support a phylogenetically significant separation of the tribe into two subgroups [11][12][13]; for this reason, their phylogeny remains the subject of debate [14].
The cytogenetic approach, using classic analysis or molecular approaches, has been used to characterize the genomes of different and distant taxa [23][24][25][26][27]. The molecular cytogenetic approach through chromosome painting permits the identification of chromosomal homologies at the level of whole or partial chromosomes, as well as interchromosomal rearrangements (translocations, fissions, and fusions) that have occurred during genome evolution. Furthermore, chromosome painting permits the determination of chromosomal syntenies (the localization of two or more genes on the same chromosome) that have been conserved or reshaped, identifying syntenic associations in the genomes of the species being compared. Comparative chromosome banding [6] and painting [28,29] have been used to demonstrate that Catarrhini (Old World monkeys) tend to have conservative genomes. Among the conserved Old World monkeys, the genus Cercopithecus is one of the major exceptions, characterized instead by a notable variation in diploid number (2N = 58-72) [5,[29][30][31] and a high frequency of chromosomal rearrangements and polymorphisms [4,7,14,[29][30][31].
Despite the diversity found in the genus, only a few studies have been conducted using painting approaches on the genomic structure of Cercopithecus species; comparisons of chromosomal banding patterns and of whole chromosome probe "paint" mappings have permitted researchers to propose phylogenetic relationships [6,[31][32][33][34][35], and to show that non-centromeric fissioning and centromere activation and deactivation [36] are noticeable chromosomal evolutionary mechanisms of the Cercopithecini tribe.
In this work, chromosome painting by fluorescence in situ hybridization (FISH) was applied to the arboreal species Cercopithecus petaurista (Schreber, 1774) in order to characterize its genome using whole human chromosome probes. Analyzing the results obtained in comparison with data from the literature regarding other Cercopithecini, symplesiomorphies, synapomorphies, and autapomorphies were shown. This analysis was performed in order to clarify the phylogenetic and systematic inferences regarding the tribe. Moreover, telomeric sequence probes were mapped by FISH in C. petaurista because recent works have suggested that interstitial telomeric sequences (ITSs) may be linked to the high rate of chromosome evolution in primates [37][38][39][40][41].

Materials and Methods
Skin biopsies from one male C. petaurista (CPE) individual were collected from the Bioparco di Sicilia (Carini, Italy). The experiments were carried out according to international ethical protocols.
Chromosome-specific probes from human chromosomes (1-22 probes-XY) were kindly provided by Roscoe Stanyon and were created at the National Cancer Center (Frederick, MD, USA) after DOP-PCR. A 6MW primer was used in a secondary reaction to label the chromosomal DNA with biotin-dUTP or digoxigenin-dUTP (both from Roche) for indirect detection [42,43]. About 300-400 ng of each PCR product per probe, together with 10 µg of human Cot-1 (Invitrogen, Thermofisher scientific, Van Allen Way, Carlsbad, CA, USA) were precipitated and then dissolved in 14 µL of hybridization buffer. Probe denaturation occurred at 70 • C with 70% formamide. After FISH incubation for 72 h at 37 • C, slides were washed at low stringency conditions in the presence of 70% formamide and 2XSSC. Biotinylated DNA probes were detected with avidin coupled with fluorescein isothiocyanate (FITC, Vector Laboratories Inc., USA Ingold Road, Burlingame, CA, USA). Digoxigenin-labeled probes were detected with anti-digoxigenin antibodies conjugated with Rodamine (Roche, Eugene, OR, USA). Metaphases were counterstained with DAPI (Vectashield antifade mounting medium) [44].
Telomeric motif distribution was analyzed by FISH using the FITC-labeled PNA oligonucleotide probe (Panagene, Cambridge Research Biochemicals, Belasis Court, Belasis Hall Technology Park Billingham, Cleveland, UK) Hybridization was performed following the protocols furnished by Panagene, adjusting stringency conditions. The detection of the probe signals was performed at high stringency with high temperatures at 70 • C and a low saline concentrate buffer [38].
The metaphases were analyzed under a Zeiss Axio2 epifluorescence microscope and captured using a coupled Zeiss digital camera. The DAPI inverted karyotype was obtained using the Adobe Photoshop (CC 2022 V23.3.2) program to permit the identification of chromosomes.
The C bands were obtained sequentially after FISH, according to a previously described protocol [45] that included denaturation with formamide.
Human probe 14 covers C. petaurista chromosome 11, and a very small segment falls on chromosome 17 in association with synteny 15 (Figures 1b and 2). Human probes 20/21 both fall on the same chromosome pair 9, showing a heterozygous pattern (Figures 1f,g and 2). C banding obtained after FISH showed large C bands at the centromeres of all chromosome pairs. An additional sizeable interstitial band below the centromere was present on a large submetacentric chromosome pair in C. petaurista  Biotin-labeled probes were detected with avidin-FITC (green) and digoxigenin-labeled probes with antidigoxigenin-rhodamine (red). The slides were counterstained with DAPI. Human paints are indicated by numbers in green (FITC) or red (rhodamine). Note the small human syntenic association 14/15 (asterisk) (b) and the different pattern of synteny 20 on the two homologous chromosomes (f) with signals on p and q arms as it is possible to appreciate in the DAPI chromosomes (g). Scale bar, 5 µm.

Discussion
The Cercopithecini tribe, with its chromosomal variability distributed across its different species and genera, is a prime example of genome evolution related to niche separation and geographical barriers which has been scarcely explored. Cytogenetic and molecular approaches have supported clustering the tribe into two groups: primarily terrestrial and more chromosomally conservative monkeys, and arboreal and chromosomally non-conservative guenons. However, the composition of these species groups,

Discussion
The Cercopithecini tribe, with its chromosomal variability distributed across its different species and genera, is a prime example of genome evolution related to niche separation and geographical barriers which has been scarcely explored. Cytogenetic and molecular approaches have supported clustering the tribe into two groups: primarily terrestrial and more chromosomally conservative monkeys, and arboreal and chromosomally non-conservative guenons. However, the composition of these species groups,

Discussion
The Cercopithecini tribe, with its chromosomal variability distributed across its different species and genera, is a prime example of genome evolution related to niche separation and geographical barriers which has been scarcely explored. Cytogenetic and molecular approaches have supported clustering the tribe into two groups: primarily terrestrial and more chromosomally conservative monkeys, and arboreal and chromosomally non-conservative guenons. However, the composition of these species groups, and the relationships between the arboreal species are still unclear [7][8][9][10][11][12][13]. In order to acquire further information regarding the complex phylogenomics of this tribe, we utilized the painting approach, using the complete set of human probes on C. petaurista. C. petaurista is characterized by a forest ecology and a rather rearranged karyotype, with a diploid number of 2n = 66. The sample analyzed here confirms the species' diploid number and the GTG-banding pattern (Figure 2), in agreement with what has previously been described in the literature [6,30,46].
The ancestral mammalian syntenic association 3/21 was not found in C. petaurista, as in all Cercopithecini species and in the ancestral Catarrhine karyotype [47]. Indeed, human probes 3 and 21 were mapped onto different chromosomes ( Figure 2).
The ancestral mammal syntenic association 14/15 was fissioned in the ancestral Catarrhine karyotype [47], but is still present in the Cercopithecine species. In C. petaurista, human probes 14 and 15 covered two different bi-armed chromosomes, 11 and 17, respectively (Figures 1b and 2), and the 14/15 association does not seem to be present. However, on chromosome 17, a very small and hard to detect fragment of synteny 14 was found in association with synteny 15 (Figure 1b). This evidence is supported by what is shown in the other species here analyzed, where the 14/15 syntenic association is present and human probe 14 is split into two fragments. The different size of human synteny 14 (in association with synteny 15) in the species analyzed indicates that the fissions presumably occurred with different breakpoints and are consistent with the high level of rearrangements previously shown in other primate taxa [49,50]. Human probe 15 can also be found in a single block or split into two blocks in the analyzed taxa (Table 1). Thus, human chromosomes 14 and 15 have been subject to different chromosomal rearrangements involving different breakpoints, giving different fragments [49][50][51]. It would be useful in the future to verify the breakpoints of these syntenies in the Cercopithecini species using the BAC (bacterial artificial chromosomes) mapping approach, which can provide new insight into their chromosomal evolution [52].
The 20/21 syntenic association is an apomorphic trait of all Cercopithecini species (Table 1), also present as a polymorphism known as a "tribal-specific" polymorphism [14]. In C. petaurista, the 20/21 syntenic association is found on chromosome 9 in heterozygosity, as shown here through the paint patterns (Figures 1f,g and 2) and in agreement with previous banding, painting [4,5], and BAC [33] data mapping.
Human probes 1, 2, 3, 5, 6, 8, 11, 12 show from two to three fragments in C. petaurista; the rearrangements affecting these syntenies represent symplesiomorphies or synapomorphies linking either all members of the Cercopithecini tribe or the arboreal clade, or just a few species. These data support the previously hypothesized molecular phylogenetic groups [8] (Figure 4). In particular, human probes 1, 2, and 3 are fragmented into at least three fragments in the species of the arboreal clade (three segments in C. petaurista, C. erythrogaster, and C. nictitans stamptii, four fragments in C. wolfi and C. neglectus), but only one or two fragments are present in the terrestrial Cercopithecini C. aethiops and Erythrocebus patas (Table 1). Analyzing this evidence, it could be hypothesized that having at least three fragments of the two syntenies is a feature common to the arboreal clade ( Figure 4). Despite its highly rearranged karyotype characterized by many fissions, telomeric probe mapping on C. petaurista showed only classic telomeric signals; indeed, no ITSs far from the chromosomal ends were found (Figure 3). The hypothesis that non-centromeric fissions could have been accompanied by ITS formation in Cercopithecidae [37] is not supported by this evidence, though it is not possible to exclude the possibility that lowly amplified sequences could be present but undetectable at this level of resolution.

Conclusions
The data shown here confirm the monophyly of the Cercopithecini tribe as well as of the arboreal clade, and add additional markers that can be useful for deciphering Cercopithecini phylogeny, especially the relationships between arboreal species. The comparison of C. petaurista with other previously analyzed species permits us to confirm that fissions of homologues to human chromosomes 5 and 6 are symplesiomorphies linking all Cercopithecini, in agreement with the previous molecular proposed phylogeny [7][8][9][10]. Furthermore, the comparative analysis shows that human chromosome 2, 3, 11, and 12 homologue fissions are chromosomal synapomorphies, supporting the previously proposed molecular phylogenetic arboreal clade [8]; in addition, the fission of homologues to human chromosome 8 is a synapomorphy linking C. petaurista, C. erythrogaster, and C. nictitans among the arboreal species. Further analyses with a multidisciplinary approach and a wider range of samples are needed to clarify the relationships between the species of the arboreal clade.  Human paints 5 and 6 in C. petaurista are fissioned into two fragments, in agreement with what has been shown in other Cercopithecini [7,31]. These features represent chromosomal symplesiomorphies linking all the Cercopithecine clade and supporting the monophyly of the tribe (Figure 4).
Human synteny 8 is in one segment in the ancestral Catarrhine karyotype, as well as in the other Cercopithecini, except for C. petaurista, C. erythrogaster, and C. nictitans, where it is in two segments. The presence of two segments of synteny 8 is a synapomorphy linking these latter three arboreal species. Only in C. neglectus is there an association of human syntenies 1/8, a derived feature considered to be an autapomorphy (Table 1).
Human probes 11 and 12 are split into two fragments in C. petaurista (Figures 1d and 2), as well in all the arboreal species analyzed here, except for C. neglectus (Table 1). These two human chromosomes are present as a single fragment in the ancestral Catarrhine karyotype [47] as well as in other Cercopithecini species (Table 1). From this analysis, it can be hypothesized that the splitting of these syntenies into two segments can be considered a feature linking all species of the arboreal clade ( Figure 4).
On the other hand, human probes 4 and 7 are present as single fragments in C. petaurista but in diverse conditions in other taxa. Human synteny 4 is conserved as one segment in some arboreal species, such as C. erythrogaster and C. neglectus, and in the terrestrial Erytrocebus patas, as well as in the ancestral Catarrhine karyotype. It is fragmented into two or three segments in the other arboreal species, for example, C. nictititans and C. wolfi, and the terrestrial Chlorocebus aethiops. Human synteny 7 is present as one segment in the arboreal C. petaurista, C. erytrogaster, and C. neglectus, as also occurs in the ancestral Catarrhine karyotype [47]; however, it is split into two fragments in other Cercopithecini, both arboreal and terrestrial, such as C. wolfi, C. nictititans, and Erythrocebus patas, and C. aethiops, respectively. The complex conditions of human syntenies 4 and 7 can be explained as the consequence of a presumed presence of polymorphic forms in the ancestral Cercopithecini group before the splitting of the terrestrial and arboreal clades. Fissions of human syntenies 4 and 7 are not phylogenetically informative but may support the hypothesis regarding the occurrence of polymorphisms among Cercopithecus species [4,7]. In any case, it cannot be excluded that this complex condition is the result of convergent evolution [53].
Despite its highly rearranged karyotype characterized by many fissions, telomeric probe mapping on C. petaurista showed only classic telomeric signals; indeed, no ITSs far from the chromosomal ends were found (Figure 3). The hypothesis that non-centromeric fissions could have been accompanied by ITS formation in Cercopithecidae [37] is not supported by this evidence, though it is not possible to exclude the possibility that lowly amplified sequences could be present but undetectable at this level of resolution.

Conclusions
The data shown here confirm the monophyly of the Cercopithecini tribe as well as of the arboreal clade, and add additional markers that can be useful for deciphering Cercopithecini phylogeny, especially the relationships between arboreal species. The comparison of C. petaurista with other previously analyzed species permits us to confirm that fissions of homologues to human chromosomes 5 and 6 are symplesiomorphies linking all Cercopithecini, in agreement with the previous molecular proposed phylogeny [7][8][9][10]. Furthermore, the comparative analysis shows that human chromosome 2, 3, 11, and 12 homologue fissions are chromosomal synapomorphies, supporting the previously proposed molecular phylogenetic arboreal clade [8]; in addition, the fission of homologues to human chromosome 8 is a synapomorphy linking C. petaurista, C. erythrogaster, and C. nictitans among the arboreal species. Further analyses with a multidisciplinary approach and a wider range of samples are needed to clarify the relationships between the species of the arboreal clade.  Data Availability Statement: All data generated or analyzed during this study are included in this article.