# Chromosomal Polymorphism in Cercopitheci: Cytogenomics Provides Evidence for Reticulate Evolution and Incomplete Reproductive Isolation

Lauriane Cacheux, Michèle Gerbault-Seureau, Peggy Motsch, Bertrand Bed'hom, Florence Anne Richard

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Lauriane Cacheux, Michèle Gerbault-Seureau, Peggy Motsch, Bertrand Bed'hom, Florence Anne Richard. Chromosomal Polymorphism in Cercopitheci: Cytogenomics Provides Evidence for Reticulate Evolution and Incomplete Reproductive Isolation. International Journal of Primatology, In press, 10.1007/s10764-025-00483-5 . hal-05043984

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# Chromosomal Polymorphism in Cercopithecini: Cytogenomics Provides Evidence for Reticulate Evolution and Incomplete Reproductive Isolation

Lauriane Cacheux¹ · Michèle Gerbault-Seureau¹ · Peggy Motsch²,⁴ · Bertrand Bed'Hom¹ · Florence Anne Richard¹,³

Received: 8 August 2024 / Accepted: 8 January 2025
© The Author(s) 2025

## Abstract

Reticulate evolution is considered a widespread phenomenon in primates, as hybridization shaped evolutionary histories in all primate radiations, including Catarrhini. While genomic data suggest extensive ancient introgression among guenon lineages (tribe Cercopithecini), field and related genetic studies report extant hybridizations, along with fertility of guenon hybrids. By combining classical and molecular cytogenomics, we characterized chromosome organization for 12 guenon individuals, belonging to three species that associate in polyspecific groups: *Cercopithecus cephus*, *C. nictitans* and *C. pogonias*. Our results reveal multiple chromosomal heterozygosities in both the morphology of metaphase chromosomes and the molecular structure of centromeres, suggesting that chromosomal heterozygosity may be common in guenon genomes. Furthermore, we found similar chromosomal polymorphisms, involving similar chromosome and centromere variants, in distinct species. Our cytogenomic analysis also confirmed the hybrid origin of three individuals: two F1 hybrids between *C. cephus* and *C. nictitans*, and one backcross hybrid *C. nictitans* x (*C. cephus* x *C. nictitans*). Together, our results provide further possible evidence for reticulate evolution and incomplete reproductive isolation, and identify guenons as a good system to untangle the evolutionary dynamics of primate centromeres. This study provides a foundation for future research regarding the influence of hybridization on chromosomal reorganization, reproductive isolation, and speciation.

**Keywords** Chromosome · Centromere · Heterozygosity · Hybridization · Hybrid fertility · Guenon · Speciation

Handling Editor: Joanna M. Setchell

Lauriane Cacheux and Michèle Gerbault-Seureau contributed equally to this work.

Extended author information available on the last page of the article
Introduction

Reticulate evolution refers to evolutionary changes that arise from horizontal genetic transfer, induced by various mechanisms, including hybridization (Gontier, 2015). Hybridization is considered to be a driving force in mammal and primate evolution, as the exchange of genetic material between species can promote phenotypic diversity and provide novel adaptive variations (Adavoudi & Pilot, 2022; Arnold & Meyer, 2006; Wu et al., 2023; Zinner et al., 2011). Reticulate evolution has been highlighted in all primate radiations—strepsirrhines, tarsiers, catarrhines, and platyrrhines—and produces discordance among molecular phylogenies, because genomic regions that introgress during hybridizations suggest different evolutionary histories compared with those that never intermix (Cortés-Ortiz, 2017). For example, difficulties in resolving phylogenetic relationships have suggested horizontal transfers within the Cercopithecini, or African guenon monkeys (Dutrillaux et al., 1980; Guschanski et al., 2013; Lo Bianco et al., 2017; Sineo, 2012; Tosi et al., 2005a, 2008, 2017). Analyses of whole-genome sequencing data have then revealed hybridizations during guenon evolutionary history, providing further evidence for ancient gene flow across taxa (Ayoola et al., 2021; Jensen et al., 2023, 2024; Svardal et al., 2017; van der Valk et al., 2020).

The Cercopithecini diverged 10 to 15 million years ago and form the largest clade of Catarrhini with more than 30 identified species (Guschanki et al., 2013; IUCN, 2024; Kuderna et al., 2023). This tribe displays a pronounced diversity of color patterns, social behaviors, ecological traits, and chromosome morphologies (Allen et al., 2014; Dutrillaux et al., 1980; Enstam & Isbell, 2007; Glenn & Cords, 2002; Grubb et al., 2003). In addition to past hybridization events, the Cercopithecini show multiple cases of contemporary hybridization (de Jong & Butynski, 2010; Detwiler et al., 2005; Tosi et al., 2005b). Field reports mention viable hybrids that retain some adaptive advantages from both parental species, with accompanying infants suggesting hybrid fertility. For example, fertile *Cercopithecus ascanius* x *C. mitis* hybrids occur in East African forests, although the parental species belong to evolutionary distinct species groups within arboreal guenons and diverged from each other more than 5 million years ago (Aldrich-Blake, 1968; Detwiller, 2002, 2019; Kuderna et al., 2023; Struhsaker et al., 1988). Interbreeding between *C. cephus* and *C. nictitans*, species with a similar divergence pattern to *C. ascanius* and *C. mitis*, also occurs in Gabon's forests (Kuderna et al., 2023; Tutin, 1999).

Arboreal guenon species commonly engage in polyspecific associations that maximize foraging success and reduce predation risk (Buzzard, 2010; Cords, 1990; Enstam & Isbell, 2007; Mencia et al., 2021). Associations between *Cercopithecus cephus*, *C. nictitans*, and *C. pogonias* are well documented. In the forests of West Africa (Fig. S1), polyspecific groups tend to explore larger feeding areas and detect a wider range of predators, against which *C. cephus* can be actively defended by larger *C. nictitans* (Gautier-Hion, 1988; Gautier-Hion et al., 1983). Such persistent associations may encourage hybridization between species, preventing reproductive isolation (Detwiller, 2019).
Comparative cytogenomics shows that the Cercopithecini underwent karyotype modifications through evolution: multiple chromosome fissions increased the diploid number in current species from 48 to 72 (Dutrillaux et al., 1980, 1982; Finelli et al., 1999; Moulin et al., 2008; Stanyon et al., 2005). Some homologous chromosomal segments are fissioned more or less across species, making cytogenomic characterization of hybrids theoretically achievable (Dugoujon, 1982; Muleris et al., 1984). The centromere is an essential structure that ensures chromosome segregation during cell divisions, and the formation of new centromeres has been proposed to have occurred in association with guenon chromosome fissions (Dutrillaux et al., 1980, 1982; Finelli et al., 1999; Moulin et al., 2008; Stanyon et al., 2005). Studies of alpha satellite DNA, i.e., the main tandemly repeated DNA element in primate centromeres, revealed the existence of multiple families of sequences in Cercopithecini (Cacheux et al., 2016, 2018). The distribution of alpha satellite families on guenon chromosomes supports an evolutionary model based on human data, called the “progressive proximal expansion model” (Cacheux et al., 2016, 2018; Schueler et al., 2001, 2005). This model suggests that new alpha satellite families expand at the centromere core during evolution, splitting and displacing older families onto each chromosome arm.

In this study, we examined chromosomal heterozygosity and polymorphism in guenons. We use the terms “chromosomal heterozygosity” to refer to the presence of two variants of a homologous chromosome within the same guenon genome and “chromosomal polymorphism” to refer to the presence of two or more variants of a homologous chromosome in a population, species, or group of species. We combined classical and molecular cytogenomic approaches to characterize chromosome organization for 12 guenon individuals belonging to the species *Cercopithecus cephus*, *C. nictitans*, or *C. pogonias*. Three of the individuals showed a hybrid phenotype between *C. cephus* and *C. nictitans*. Based on the hypothesis that patterns of chromosomal polymorphism should support reticulate evolution in Cercopithecini, in accordance with field and genomic studies, we predicted that we would find chromosomal heterozygosities that 1) relate to metaphase chromosome morphology and centromere molecular structure, 2) involve similar chromosome and centromere variants in distinct species, and 3) make it possible to identify F1 and backcross hybrid specimens cytogenetically.

# Methods

## Cercopithecini Living Cells

We studied fibroblast samples obtained from the “Tissues and Cryopreserved Cells from Vertebrates” collection (TCCV, RBCell), held by the National Museum of Natural History (MNHN) in Paris, France. We originally amplified these samples from skin biopsies that we collected during veterinary health checks and under anesthesia, from 12 individuals hosted by the International Center for Medical Research in Franceville (CIRMF, Gabon) in 2005, 2012, and 2013 (ethical permit n° FR1207510445-I):
- Five *Cercopithecus cebus*: MNHN-TCCV-1048 (CIRMF id: CCE02 Ndjinki, 2012); MNHN-TCCV-1053 (CIRMF id: CCE05 Clopin, 2012); MNHN-TCCV-1054 (CIRMF id: CCE04 Toby, 2012); MNHN-TCCV-1090 (CIRMF id: CCE06 Lilou, 2013); MNHN-TCCV-1091 (CIRMF id: CCE01 Simba, 2013)

- Three *C. nictitans*: MNHN-TCCV-1058 (CIRMF id: CNI01 Clown, 2012); MNHN-TCCV-1061 (CIRMF id: CNI02 Michou, 2012); MNHN-TCCV-1089 (CIRMF id: CNI03 Miro, 2013)

- One *C. pogonias*: MNHN-TCCV-0348 (CIRMF id: CPO01 Lutin, 2005)

- Two individuals that, based on external phenotype, were F1 hybrids *C. cebus* x *C. nictitans*: MNHN-TCCV-1071 (CIRMF id: CHY3 Killer, 2012); MNHN-TCCV-1072 (CIRMF id: CHY4 Yodou, 2012) (Figs. S2 and S3)

- One individual that, based on external phenotype, was a backcross hybrid *C. nictitans* x (*C. cebus* x *C. nictitans*): MNHN-TCCV-1060 (CIRMF id: CHY1 Casquette, 2012) (Fig. S2 and S3).

The *Cercopithecus cebus* and *C. nictitans* individuals were part of a CIRMF program rescuing guenons that had been orphaned by hunting. They all were collected independently from each other and were consequently considered as unrelated individuals. These orphans were subsequently reintroduced into Lékédi reserve (Gabon) (Dilger et al., 2013). The F1 and backcross hybrids lived in a semifree-ranging colony of *Allochrocebus solatus* at CIRMF.

Cell Cultures, Metaphase Preparations, and Chromosome Banding

We performed cell cultures and metaphase preparations according to Moulin et al. (2008). Briefly, we grew fibroblast cultures at 37 °C in Dulbecco's modified eagle medium (Gibco), supplemented with 10% fetal bovine serum (FBS, Cytiva), 2% AmnioMAX (Gibco) and 0.2X antibiotic/antimycotic solution (Cytiva). We used fluorodeoxyuridine (0.06 µg/ml, Sigma) overnight to synchronize cultures. We added bromodeoxyuridine (0.02 mg/ml, Sigma) for the last 8 h of culture to incorporate late-replicating DNA. We added colchicine (0.04 µg/ml, Sigma) for the last 3 h of culture to stop the cell cycle at metaphase. We used hypotonic shock with FBS 15% and KCl 181 µg/ml for 10 min at 37 °C. Then, we fixed the cells in a solution of acetic acid (1 volume) and ethanol (3 volumes), spread them on slides, and dried and stored the slides at -20 °C. We applied R-banded chromosomes stained with Giemsa (RBG), R-banded chromosomes stained with propidium iodide (RBP), and C-banded chromosomes stained with Giemsa (CBG) methods according to Popescu et al. (2000). We karyotyped metaphases using Ikaros3 software (Metasystems). We visualized at least 20 metaphases for each individual and each banding experiment, all of which confirmed the patterns we describe. We retrieved homologies between guenon and human chromosomes from Moulin et al. (2008), where needed for interpretation and comparison among guenon species.
# Fluorescence in Situ Hybridization Experiments

## Whole Chromosome Painting

To confirm the chromosomal patterns we observed using R-banding techniques, we performed whole chromosome painting with probes corresponding to human chromosomes 4, 7, 20, and 21 (HSA4, HSA7, HSA20, and HSA21; Cambio-Adgenix) following Gerbault-Seureau et al. (2004). We chose HSA4 due to its homology to *Cercopithecus cephus* first pair of chromosomes (CCE1) and *C. nictitans* 15th and 26th pairs of chromosomes (CNI5 and CNI26). We chose HSA7 because of its homology to CCE2, CNI8, and CNI25. We chose HSA20 and HSA21 because of their homology to CCE7 and CNI6 (Dutrillaux et al., 1980, 1982; Moulin et al., 2008). Briefly, we heated metaphase preparations for 2 min at 70 °C in 70% formamide, Saline-Sodium Citrate (SSC) 2X, then hybridized them for 48 h at 37 °C, and finally treated them twice in SSC 2X at 40 °C for 10 min. We revealed hybridized probes in green by indirect detection with goat anti-biotin antibodies (10 µg/ml, Vector laboratories) followed by FITC-conjugated anti-goat antibodies (5 µg/ml, P.A.R.I.S.). We counterstained chromosomes in red with propidium iodide (2 µg/ml). For dual-color FISH detection, we revealed probes first with goat anti-biotin (10 µg/ml, Vector Laboratories) and mouse antidigoxigenin (1 µg/ml, Boehringer) antibodies, then by FITC-conjugated antigoat antibodies (5 µg/ml, P.A.R.I.S.), and finally by rhodamine-conjugated antimouse antibodies (20 µg/ml, Boehringer). We counterstained chromosomes in blue with DAPI (4 µg/ml).

## Alpha Satellite DNA Hybridization

We performed fluorescence in situ hybridization (FISH) on metaphase preparations by using three LNA-modified oligonucleotide probes (Eurogentec) described in Cacheux et al., (2016, 2018). Probe sequences and labels are as follows: C1b (3'digoxygenin) 5'AcTgCtCtGtGtTcTGtTa 3'; C2b (5'Cy5) 5'AcTgCtTtGtGtTcTGtTa3'; Cx (3'biotin) 5'tctcagaaagctt3' with LNA nucleotides written in lower case and classic nucleotides in upper case. Probes C1b and C2b were designed to target two main families of alpha satellite DNA (C1 and C2) identified in *Allochrocebus solatus* and *Cercopithecus pogonias* genomes. Probe Cx was designed to target all alpha satellite families identified in *A. solatus* and *C. pogonias* genomes. We performed FISH experiments according to Cacheux et al., (2016, 2018). Briefly, we heated metaphase preparations for 3 min at 85 °C and hybridized them for 1 h at 37 °C in a Thermobrite apparatus (Leica Biosystems), then treated them twice in SSC 2X at 63 °C for 5 min. We revealed hybridized probes by direct or indirect detection using rhodamine-conjugated antidigoxigenin (1 µg/ml, Roche) or Alexa 488-conjugated streptavidin (1 µg/ml, Life Technologies). We counterstained chromosomes in either blue with DAPI (4 µg/ml) or red with propidium iodide (2 µg/ml).
## Image Acquisition and Analysis

We performed FISH observations with epifluorescent microscopes (DMRB, Leica Microsystem and Axio Observer Z1, Zeiss) and captured images using cooled CCD cameras (ProgRes MFcool, Jenoptik and ORCA R2, Hamamatsu). We reconstructed color-combined images by using ImageJ (Abràmoff et al., 2004) and karyotyped metaphases by using Isis 5.3 software (Metasystems). We visualized at least 20 metaphases for each individual and each FISH experiment, all of which confirmed the described patterns.

## Results

### A Similar Pattern of Heterozygosity in Centromere Positioning is Observed in *Cercopithecus cephus* and *Cercopithecus nictitans* Individuals

Three of five studied *Cercopithecus cephus* individuals showed a canonical R-banded species karyotype (2n=66; Figs. 1a and c). The chromosomes of the two other specimens were similar but showed one notable difference: the seventh pair of chromosomes, homologous to fused human chromosomes 20 and 21 (CCE7, i.e., hHSA20-21), showed a heterozygous centromere position (Fig. 1d; complete karyotype in Fig. S4).

We observed a similar pattern among *Cercopithecus nictitans* individuals: two of three studied specimens possessed a canonical R-banded species karyotype (2n = 70, Figs. 1b and e), whereas one specimen showed a heterozygous centromere position on hHSA20-21 (sixth pair of chromosomes, i.e., CNI6, Fig. 1f; complete karyotype in Fig. S4) (Table 1).

Whole chromosome painting, using probes corresponding to HSA20 and HSA21, confirmed these heterozygosities in both species. While the primary constriction was close to the interface between hHSA20 and hHSA21 on the canonical chromosome, the centromere was displaced toward hHSA20 on the chromosome variant (Fig. 2).

### *Cercopithecus cephus* and *Cercopithecus pogonias* Individuals Share an Unusual Pattern of Heterozygosity in Centromere Molecular Structure

Use of probes C1b and C2b on *Cercopithecus cephus* and *C. nictitans* individuals showed a cytogenomic particularity in one *C. cephus*, which otherwise showed a canonical karyotype. A single chromosome (with the exception of the Y-chromosome) did not display any FISH signal and seemed to be devoid of alpha satellite sequences (Fig. 3a). We observed the same pattern for the *C. pogonias* individual (Fig. 3b; Cacheux et al., 2018).

C-banding techniques on both specimens also showed an absence of staining on the centromere region of a single chromosome (Figs. 4a and b). We identified this chromosome by combining R-banding, C-banding, and FISH, which corresponds in
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Classical cytogenomic analysis of *Cercopithecus cephus* and *Cercopithecus nictitans* genomes. Samples were collected in 2012 at the International Center for Medical Research, Franceville, Gabon. R-banded karyotypes, displaying metaphase chromosomes for (a) *C. cephus* MNHN-TCCV-1048 and (b) *C. nictitans* MNHN-TCCV-1061. (c and e) Focus on chromosome pairs homologous to HSA20-21 (hHSA20-21, i.e., *C. cephus* 7th pair of chromosomes—CCE7—and *C. nictitans* 6th pair of chromosomes—CNI6), with centromere positions (primary constrictions) indicated by arrows. (d and f) R-banded hHSA20-21 for *C. cephus* MNHN-TCCV-1054 and *C. nictitans* MNHN-TCCV-1058, respectively. We observe a similar heterozygosity regarding centromere positioning in (d) and (f): while primary constriction is close to the interface between hHSA20 (long arm) and hHSA21 (short arm) on the canonical hHSA20-21 chromosome (left), centromere appears to be displaced toward hHSA20 on the chromosome variant (right). The number of individuals sharing the representative karyotypes in each species is indicated. Chromosomes are ordered according to Dutrillaux et al., (1980, 1982). Scale bar = 5 µm.</figcaption></figure>

each species to a single hHSA20-21 chromosome (CCE7 and CPO6; Figs. 4c and d; Fig. S5).

Additionally, we observed that HSA21 probes tended to hybridize to the centromere region of the *Cercopithecus cephus* and *C. pogonias* hHSA20-21 chromosome variant (Fig. 5). In contrast, HSA21 probes did not hybridize to the centromere region of canonical hHSA20-21 chromosome, nor did they hybridize in a nonspecific manner to any other centromere region (Figs. 2 and 5).
<table><caption>**Table 1** Chromosomal heterozygosities observed in Cercopithecini specimens from the National Museum of Natural History cell collections (MNHN, Paris)</caption><thead><tr><th>Species</th><th>Specimen</th><th>hHSA4</th><th>hHSA7</th><th>hHSA20-21</th></tr></thead><tbody><tr><td rowspan="5">C. cephus</td><td>TCCV-1048</td><td>-</td><td>-</td><td>-</td></tr><tr><td>TCCV-1053</td><td>-</td><td>-</td><td>CS heterozygosity</td></tr><tr><td>TCCV-1054</td><td>-</td><td>-</td><td>CP heterozygosity</td></tr><tr><td>TCCV-1090</td><td>-</td><td>-</td><td>-</td></tr><tr><td>TCCV-1091</td><td>-</td><td>-</td><td>CP heterozygosity</td></tr><tr><td rowspan="3">C. nictitans</td><td>TCCV-1058</td><td>-</td><td>-</td><td>CP heterozygosity</td></tr><tr><td>TCCV-1061</td><td>-</td><td>-</td><td>-</td></tr><tr><td>TCCV-1089</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. pogonias</td><td>TCCV-0348</td><td>-</td><td>-</td><td>CS heterozygosity</td></tr><tr><td rowspan="2">F1 hybrid<br>(C. cephus x<br>C. nictitans)</td><td>TCCV-1071</td><td>CCE/CNI<br>heterozygosity</td><td>CCE/CNI<br>heterozygosity</td><td>-</td></tr><tr><td>TCCV-1072</td><td>CCE/CNI<br>heterozygosity</td><td>CCE/CNI<br>heterozygosity</td><td>CP heterozygosity</td></tr><tr><td>Backcross hybrid<br>(C. nictitans x<br>F1 hybrid)</td><td>TCCV-1060</td><td>CCE/CNI<br>heterozygosity</td><td>CNI<br>homozygosity</td><td>-</td></tr></tbody></table>

Samples were collected between 2005 and 2013 at the International Center for Medical Research, Franceville, Gabon. The specimens are referred to by their MNHN collection numbers. hHSA4, 7 or 20-21 refers to chromosomes that are homologous to human chromosomes 4, 7, or 20-21. CS = centromere structure; CP = centromere positioning; CCE/CNI heterozygosity = presence of a single *C. cephus* chromosome along with fissioned *C. nictitans* homologs; CNI homozygosity = presence of two copies of fissioned *C. nictitans* homologs.
<figure><img src="image_4.png" /></figure>

**Fig. 2** Molecular cytogenomics applied to *Cercopithecus cephus* and *Cercopithecus nictitans* genomes. Samples were collected in 2012 at the International Center for Medical Research, Franceville, Gabon. Whole chromosome painting using probes corresponding to HSA20 (red) and HSA21 (green), hybridized on metaphase chromosomes (blue) from (a) *C. cephus* MNHN-TCCV-1054 and (b) *C. nictitans* MNHN-TCCV-1058. Labelled pairs of chromosomes correspond to hHSA20-21, i.e., CCE7 in (a) and CNI6 in (b). Centromere positions are indicated by arrows. Chromosome variants for centromere positioning are indicated by stars. Scale bar = 5 µm.

<figure><img src="image_5.png" /></figure>

**Fig. 3** FISH analysis of centromere regions from *Cercopithecus cephus* and *Cercopithecus pogonias* genomes. Samples were collected in 2005 for *C. pogonias* and 2012 for *C. cephus* at the International Center for Medical Research, Franceville, Gabon. Hybridization of probes C1b (red) and C2b (green) on metaphase chromosomes (blue) from (a) *C. cephus* MNHN-TCCV-1053 and (b) *C. pogonias* MNHN-TCCV-0348. Probes C1b and C2b target two alpha satellite families (C1 and C2) that are widespread in Cercopithecini centromere regions (Cacheux et al., 2016, 2018). Arrows point to a single unlabeled chromosome in each genome. Scale bar = 5 µm.

## Cytogenomic Characterization of F1 Hybrids *Cercopithecus cephus* x *Cercopithecus nictitans

Comparing *Cercopithecus cephus* and *C. nictitans* karyotypes, each of the two largest *C. cephus* chromosomes is homologous to two smaller chromosomes in *C. nictitans*: i.e., homologs to CCE1 and CCE2 are fissioned in *C. nictitans* (Fig. 6a).
<figure><img src="image_6.png" /><figcaption>**Fig. 4** Cytogenomic identification of a chromosome variant in *Cercopithecus cephus* and *Cercopithecus pogonias*. Samples were collected in 2005 for *C. pogonias* and 2012 for *C. cephus* at the International Center for Medical Research, Franceville, Gabon. C-banding techniques applied on metaphase chromosomes from (a) *C. cephus* MNHN-TCCV-1053 and (b) *C. pogonias* MNHN-TCCV-0348. Arrows point to a single unstained chromosome in each genome. (c) R-banded profile corresponding to C-banded *C. cephus* metaphase shown in (a). (d) Hybridization pattern of probe Cx (green) on chromosome pairs 1 to 8 (red) from *C. pogonias* MNHN-TCCV-0348 karyotype (complete karyotype in Fig. S5). Probe Cx targets all alpha satellite families previously identified in guenon centromere regions (Cacheux et al., 2016, 2018). Arrow points to hHSA20-21 (i.e., *C. pogonias* 6th pair of chromosomes—CPO6) unlabeled chromosome variant. Chromosomes are ordered according to Muleris et al. (1984). Scale bar = 5 µm.</figcaption></figure>

The homologous forms—fissioned and nonfissioned—are expected to coexist in F1 hybrid karyotypes, as half of their genome should be inherited from *C. cephus*, and half from *C. nictitans*. Indeed, cytogenomic data revealed that both apparently F1 specimens possessed a typical hybrid karyotype, with single versions of CCE1 and CCE2 associated to fissioned CNI homologs (Figs. 6b, c, and Fig. S6). We further confirmed heterozygosity in chromosome morphology in F1 hybrids using whole chromosome painting (Fig. 7).

One F1 hybrid showed a similar centromere positioning heterozygosity as described above for *Cercopithecus cephus* and *C. nictitans*, on hHSA20-21 (seventh pair on Figs. 6b, c, and Fig. S7).
<figure><img src="image_7.png" /><figcaption>**Fig. 5** Whole chromosome painting applied to *Cercopithecus cebus* and *Cercopithecus pogonias*. Samples were collected in 2005 for *C. pogonias* and 2012 for *C. cebus* at the International Center for Medical Research, Franceville, Gabon. HSA21 probes (green) hybridized on metaphase chromosomes (red) from (a) *C. cebus* MNHN-TCCV-1053 and (b) *C. pogonias* MNHN-TCCV-0348. White arrows point to canonical hHSA20-21 chromosomes (CCE7 and CPO6). Red arrows point to hHSA20-21 chromosome variants, whose centromeres were suggested as devoid of alpha satellite sequences: these centromere regions are singularly hybridized by HSA21 probes compared with any other centromere region. Scale bar = 5 µm.</figcaption></figure>

## Cytogenomics Provides Evidence for a Backcross Hybrid *Cercopithecus nictitans* x (*Cercopithecus cebus* x *Cercopithecus nictitans*)

The third hybrid specimen showed an unusual karyotype with 1) a single version of CCE1 along with fissioned CNI homologs, and 2) no CCE2 but two copies of fissioned CNI homologs (Fig. 8). These features correspond to the karyotype of a backcross hybrid with *Cercopithecus nictitans* as a parental species, as such a specimen should inherit half of its genome from the *C. nictitans* parent, and half from the F1 hybrid parent, i.e., three-quarters of its genome from *C. nictitans*, and a quarter from *C. cebus*. Chromosome segregation during meiosis in the F1 parent appears to have yielded a gamete containing one copy of CCE1 and one copy of CCE2-homologous CNI chromosomes.

We further confirmed R-banding observations using whole chromosome painting (Fig. 9). As expected, HSA4 probes hybridized on one large chromosome corresponding to CCE1, along with two smaller chromosomes corresponding to CNI homologs (Figs. 9a and b). HSA7 probes hybridized on four small chromosomes, corresponding to CNI chromosomes that are homologous to CCE2 (Figs. 9c and d).

## Discussion

We identified two sets of chromosomal heterozygosities in the centromeres of hHSA20-21 chromosomes in *Cercopithecus* species. Centromeres are chromosomal loci that are essential for chromosome segregation and cell division. These
<figure><img src="image_8.png" /><figcaption>**Fig. 6** Characterization of a F1 hybrid *Cercopithecus cephus* x *Cercopithecus nictitans* using classical cytogenomic techniques. Samples were collected in 2012 at the International Center for Medical Research, Franceville, Gabon. (a) Comparative R-banded karyotype for *C. cephus* vs. *C. nictitans*, whose chromosomes are displayed respectively on left and right of each homologous assembly. We selected metaphase chromosomes from *C. cephus* MNHN-TCCV-1048 and *C. nictitans* MNHN-TCCV-1061. (b) R-banded karyotype for F1 hybrid *C. cephus* x *C. nictitans* MNHN-TCCV-1072. (c) Focus on chromosome homologies 1 to 8 in the hybrid karyotype. Centromere positions are indicated by arrows for hHSA20-21. Chromosomes are ordered according to *C. cephus* karyotype in (a), (b), and (c). Scale bar = 5 µm.</figcaption></figure>

heterozygosities show 1) similar chromosome variants for centromere positioning in *C. cephus* and *C. nictitans*, and 2) similar chromosome variants for centromere molecular structure in *C. cephus* and *C. pogonias*. Additionally, we detected chromosomal heterozygosities on hHSA4 and hHSA7 that confirm the hybrid origin of three individuals: two F1 hybrids between *C. cephus* and *C. nictitans*, and one backcross hybrid *C. nictitans* x (*C. cephus* x *C. nictitans*).
<figure><img src="image_9.png" /><figcaption>**Fig. 7** Molecular cytogenomics applied to a F1 hybrid *Cercopithecus cebus* x *Cercopithecus nictitans*. The sample was collected in 2012 at the International Center for Medical Research, Franceville, Gabon. (a) Whole chromosome painting using HSA7 probes (green) hybridized on metaphase chromosomes (red) from F1 hybrid *C. cebus* x *C. nictitans* MNHN-TCCV-1072. (b) Corresponding R-banded profile. Arrows point to chromosomes that are labelled by HSA7 probes. We chose HSA7 based on its homology to CCE2 and corresponding CNI chromosomes (Moulin et al., 2008). Scale bar = 5 µm.</figcaption></figure>

Interspecific Chromosome Variant Analogies as Potential Markers for Ancient
Hybridization Events in Cercopithecini

Because horizontal transfers may be responsible for the presence of similar variants
in distinct species, the observation of similar centromere positioning and centromere
structure variants in *Cercopithecus cebus*, *C. nictitans*, and *C. pogonias* supports
existing evidence for reticulate evolution in *Cercopithecini*, whose hybridization his-
tory has been revealed in recent comparative genomic studies (Jensen et al., 2023,
2024). Using excess allele sharing as genomic signature for introgression, these
studies suggested hybridization events between *C. cebus* and *C. nictitans* lineages
(3.8 million years ago, Mya) and between *C. cebus* and *C. pogonias* lineages (4.4
Mya). Both exchange of immune genes and species diversification were detected in
association with hybridization events, emphasizing the importance of adaptative and
radiative role of introgression during guenon evolution (Jensen et al., 2023). The
latter study also highlighted the complementary role of incomplete lineage sorting
in shaping guenon genomes, suggesting that hybridization and incomplete lineage
sorting may have acted together and be the source of extant chromosomal polymor-
phism in guenons. However, we must interpret these patterns with caution, as the
presence of similar variants in distinct species also may be explained via conver-
gence, and hHSA20-21 may be a highly dynamic chromosome.

Our findings regarding centromere positioning heterozygosity on hHSA20-21 are
analogous to cases reported in two further *Cercopithecus* species: *C. albogularis*
and *C. petaurista* (Sineo, 1990; Tolomeo et al., 2020). As these species diverged
about 5 Mya (Perelman et al., 2011; Springer et al., 2012) and are not known to
hybridize with each other, long-term maintenance of polymorphism was proposed
a

<figure><img src="image_10.png" /></figure>

b

<figure><img src="image_11.png" /><figcaption>**Fig. 8** Characterization of a backcross hybrid *Cercopithecus nictitans* x (*Cercopithecus cephus* x *Cercopithecus nictitans*) using classical cytogenomic techniques. The sample was collected in 2012 at the International Center for Medical Research, Franceville, Gabon. (a) R-banded karyotype for backcross hybrid *C. nictitans* x (*C. cephus* x *C. nictitans*) MNHN-TCCV-1060. Chromosomes are ordered according to *C. cephus* karyotype. (b) Focus on chromosome homologies 1 to 5 within the hybrid karyotype. Scale bar = 5 µm.</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>**Fig. 9** Molecular cytogenomics applied to a backcross hybrid *Cercopithecus nictitans* x (*Cercopithecus cephus* x *Cercopithecus nictitans*). The sample was collected in 2012 at the International Center for Medical Research, Franceville, Gabon. (a) Whole chromosome painting using HSA4 probes (green) hybridized on metaphase chromosomes (red) from backcross hybrid MNHN-TCCV-1060. (b) Corresponding R-banded profile. Arrows point to chromosomes that are labelled by HSA4 probes. We chose HSA4 based on its homology to CCE1 and corresponding CNI chromosomes (Moulin et al., 2008). (c) Whole chromosome painting using HSA7 probes (green) hybridized on metaphase chromosomes (red) from backcross hybrid MNHN-TCCV-1060. (d) Corresponding R-banded profile. Arrows point to chromosomes that are labelled by HSA7 probes. We chose HSA7 based on its homology to CCE2 and corresponding CNI chromosomes (Moulin et al., 2008). Scale bar = 5 µm.</figcaption></figure>

to explain the existence of similar chromosome variants in both species. Following
these reports, whole genome studies revealed ancient gene flow between *C. albogu-
laris* and *C. petaurista* lineages, suggesting that centromere positioning variants
might have been exchanged 3.8 Mya (Jensen et al., 2023). As for *C. cephus* and *C.
nictitans*, the persistence of polymorphism over four million years is still needed to
explain current chromosomal patterns. Intrachromosomal inversions are responsible
for the emergence of centromere positioning variants, and a hypothesis places these
rearrangements as vectors for reduced recombination and subsequent maintenance
of distinct favorable allelic combinations (Tolomeo et al., 2020; Wellenreuther &
Bernatchez, 2018). The persistence of chromosomal polymorphism in populations
may also be explained by selective advantages of balancing selection (negative
frequency-dependent selection, selection varying in space, varying in time, between the sexes, between different life stages or between fitness components) or heterozygote advantages (Hedrick, 2012). Heterozygote advantage might explain the intriguing prevalence of heterozygosity in extant guenon genomes: in our study, six of 12 unrelated individuals display a chromosomal variant coupled to heterozygosity. A larger number of guenon specimens and karyotypes should be studied to confirm the prevalence, while additional genomic studies are expected to shed light on the selective advantages, if any, associated with centromere positioning and centromere structure polymorphisms.

Variant forms of hHSA20-21 regarding centromere positioning are very similar in *Cercopithecus cephus*, *C. nictitans*, *C. albogularis*, and *C. petaurista* (Sineo, 1990; Tolomeo et al., 2020; this study). As genomic studies have identified ancient hybridization events between *C. cephus*, *C. nictitans*, and *C. petaurista* lineages (3.8 Mya; Jensen et al., 2023), our results, combined with previous cytogenomic reports, provide further evidence suggesting that large-scale introgressions shaped guenon evolution, altogether with incomplete lineage sorting and polymorphism maintenance. It will be interesting to pursue chromosome sequence analysis and BAC-FISH techniques on heterozygous specimens, to collect complementary evidence of synteny and homology between centromere positioning variants (Tolomeo et al., 2020).

The centromere structure heterozygosity we describe in *Cercopithecus cephus* and *C. pogonias* hHSA20-21 is also analogous to one heterozygosity reported in *C. albogularis* (Hirai et al., 2000), which further supports the idea that cytogenomic data provide evidence for reticulate evolution. The description of such a heterozygosity in *C. pogonias* extends our knowledge about guenon chromosomal polymorphism to the *mona* species group (*Cercopithecus mona*, *C. pogonias*, and *C. campbelli*), which is quite divergent from the *cephus* species group (*Cercopithecus cephus*, *C. petaurista*, *C. ascanius*, *C. erythrogaster*, *C. erythrotis*) and the *mitis* species group (*Cercopithecus mitis*, *C. nictitans*, *C. albogularis*, *C. doggetti*, *C. kandti*) (Grubb et al., 2003; Lo Bianco et al., 2017). Adding the *C. pogonias* lineage to the guenon chromosome-exchange network supports the past existence of large-scale introgressions during guenon evolution. Comparative cytogenomic studies including additional species will provide a greater understanding of guenon diversification as well as chromosomal polymorphism, genome architecture and species evolution in primates.

## Cytogenomics Provides Evidence for Current Hybridization and Hybrid Fertility, Supporting Ongoing Reproductive Isolation in *Cercopithecini*

*Cercopithecus cephus*, *C. nictitans*, and *C. pogonias* individuals associate in poly-specific groups that may promote sexual intercourse and genomic exchange (Gautier-Hion, 1988; Gautier-Hion et al., 1983). In addition to past introgressions, the presence of similar centromere positioning and centromere structure variants in these species may reflect more recent hybridization phenomena. The observation of sexual interactions and the presence of a hybrid specimen in Lopé Reserve (Gabon), two decades ago, suggested the possibility of current gene flow between *C. cephus*

<figure><img src="image_13.png" /></figure>
and *C. nictitans* (Tutin, 1999). Our study provides cytogenomic evidence for such F1 hybrids *C. cephus x C. nictitans* and, through the characterization of a backcross specimen, for hybrid fertility. The achievement of meiosis in F1 hybrids *C. cephus x C. nictitans* (2n=66 and 2n=70, respectively) is remarkable, as karyotypic differences can act as prezygotic reproductive barriers (Garagna et al., 2014; Giménez et al., 2013; Mackintosh et al., 2023). Our results echo the documented fertility of wild *C. ascanius x C. mitis* F1 hybrids (2n=66 and 2n=70, respectively) (Aldrich-Blake, 1968; Detwiller, 2002, 2019; Struhsaker et al., 1988). Signatures of introgression regarding genes responsible for chromosome segregation have been reported in guenons: the homogenization of such genes may facilitate hybrid fertility and gene flow by promoting pairing and recombination across homologous chromosomes, despite karyotypic differences (Jensen et al., 2023).

While the ethics and invasiveness of the following experiments must be consid-
ered carefully, additional cytogenomics on reproductive cells might provide insights
into chromosome segregation and fertility in guenon F1 hybrids. Disrupted synapsis
has been associated with F1 hybrid sterility in mice (Bhattacharyya et al., 2014). It
would therefore be interesting to characterize synapsis and physical association of
homologous chromosomes during guenon F1 hybrid meiosis by immunostaining of
proteins of synaptonemal complexes. Crossing-overs in parental species could be
quantified using immunostaining of mismatch repair proteins, as interspecific vari-
ations regarding global recombination rate have been associated to hybrid sterility
(Balcova et al., 2016).

Additional cytogenomics could be applied to somatic cell cultures from back-
cross hybrids and parental species: comparative genome hybridization techniques,
using probes derived from total parental genomes and hybridized on a backcross
hybrid genome, might highlight interesting recombination patterns within chromo-
somes inherited from the F1 parent (Fantini et al., 2011). Finally, whole genome
sequencing could be applied to available cell cultures from backcross hybrids. The
analysis of whole genome data from backcross individuals may clarify the molec-
ular pathways that overcome sterility, while highlighting recombination patterns,
genomic loci and homozygosities that are fundamental for primate fertility, includ-
ing achievement of meiosis, fertilization, and embryonic development.

Cercopithecini Genomes as a System to Understand the Evolution of Primate Centromeres

The emergence of centromeres is part of the multiple chromosome rearrangements that shaped genomes during evolution (Montefalcone et al., 1999; Rocchi et al., 2012; Ventura et al., 2001). Evolutionary new centromeres (ENCs) may functionally replace older centromeres or provide a centromeric function to acentric chromosomes derived from chromosomal fission events. Accurate identification of mammalian ENCs has been achieved in *Equus caballus*, *Macaca mulatta*, and *Pongo* species, combining whole genome sequence analyses to molecular cytogenomic techniques (Locke et al., 2011; Tolomeo et al., 2017; Ventura et al., 2007; Wade et al., 2009). These centromere variants are devoid of repeated DNA elements, and
ENC seedings are considered to be epigenetic phenomena. ENCs would then slowly acquire large arrays of satellites during evolution. While numerous ENCs have been characterized in *Equus* species (Piras et al., 2010, 2022), the scarcity of available and appropriate systems restricts the understanding of centromere establishment and evolution in primate genomes. Our results, combined to previous cytogenomic findings, may suggest that Cercopithecini species are suitable candidates for the investigation of primate centromere evolution, along with their atypical chromosome evolution thought to be mainly influenced by non-centromeric fission events associated with ENC formation (Dutrillaux et al., 1980, 1982; Finelli et al., 1999; Moulin et al., 2008; Stanyon et al., 2005).

Our study shows an unusual centromere structure variant in *Cercopithecus cephus* and *C. pogonias* hHSA20-21 that does not display the classical features of repeat-rich centromeres. This variant shows no staining following C-banding or FISH of alpha satellite sequences. In contrast, this centromere is characterized by uninterrupted staining following whole chromosome painting, which further suggests the absence of repetitive DNA sequences and centromeric chromatin. This centromere variant displays characteristics that make it very similar to the one observed in *C. albogularis* (Hirai et al., 2000) and likely corresponds to an early stage ENC that has not yet acquired alpha satellite sequences (Tolomeo et al., 2020). Furthermore, FISH analysis of alpha satellite sequences in additional *C. albogularis* specimens showed very scarce signals on centromere positioning variants, suggesting an ongoing colonization of ENCs by repeated DNA sequences (Tolomeo et al., 2020). Finally, sequencing and cytogenomic studies of *Allochrocebus solatus* and *C. pogonias* genomes revealed that alpha satellite DNA diversified into multiple families of sequences during guenon evolution (Cacheux et al., 2016, 2018). The chromosomal distribution of these families provides further support to the progressive proximal expansion model, which suggests that new families of alpha satellite sequences expand at the centromere core during evolution, splitting, and displacing older families toward pericentromeres (Cacheux et al., 2016, 2018; Schueler et al., 2001, 2005). Together, these observations place the Cercopithecini as a good system to untangle the evolution of centromere regions, from initial epigenetic stages to well-established nucleotide structures. Larger-scale comparative cytogenomic studies of guenon centromeres, including sequencing, immunostaining, and FISH of identified satellite-free and satellite-less centromere variants, are expected to shed light on primate centromere evolutionary dynamics.

## Conclusions

We identified chromosomal heterozygosities in guenon species by combining classical and molecular cytogenomics. These heterozygosities involve similar chromosome and centromere variants in divergent lineages, suggesting ancient and perhaps current gene flow across taxa. Our study provides possible evidence for hybridization events and reticulate evolution of guenon species, complementing existing field and genomic data (Detwiler, 2002; Jensen et al., 2023; Struhsaker et al., 1988). The cytogenomic characterization of both F1 and backcross hybrids provides further evidence of incomplete
reproductive isolation and potential current introgression phenomena in guenons, supporting existing evidence for recent genetic exchanges (Detwiler, 2019). In addition, we identified centromeric variants that make guenons an interesting system to investigate the mechanisms and dynamics of centromere seeding and evolution on primate chromosomes. This study provides a foundation for future research regarding the influence of hybridization on chromosomal reorganization, reproductive isolation, and speciation.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s10764-025-00483-5.

**Acknowledgements** We are indebted to Florence Anne Richard to whom we dedicate this manuscript. We are grateful to the CIRMF for providing the guenon samples to the MNHN-TCCV collection. We thank Christophe Escudé, Bernard Dutrillaux, and Jérôme Fuchs for helpful discussions and suggestions about the manuscript. We sincerely thank the editor and reviewers for their careful reading of our manuscript and their many insightful comments.

**Authors' Contributions** F.A.R. conceived the study. F.A.R. and P.M. worked for the establishment of research convention between the CIRMF and the MNHN. P.M. provided the biological samples. L.C. and M.G. performed the experiments. F.A.R., L.C., M.G., and B.B. analyzed the data. L.C. drafted the manuscript. All authors except F.A.R., who passed away during the study, read, edited, and approved the final manuscript.

**Funding** Open access funding provided by Museum National d'Histoire Naturelle. This research was funded by the Action Thématique du Muséum Emergence.

**Data Availability** Data generated or analyzed during this study are included in this published article. Supplementary Material is available online.

**Conflict of Interest** The authors declare that they have no conflict of interest.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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## Authors and Affiliations

Lauriane Cacheux<fnref n="1" /> · Michèle Gerbault-Seureau<fnref n="1" /> · Peggy Motsch<fnref n="2" />,<fnref n="4" /> ·

Bertrand Bed'Hom<fnref n="1" /> · Florence Anne Richard<fnref n="1" />,<fnref n="3" />

Lauriane Cacheux
lauriane.cacheux@mnhn.fr

<footnote n="1">¹ Institut de Systématique, Evolution, Biodiversité, Département Origines et Evolution, Muséum national d'Histoire naturelle CNRS SU EPHE UA, CP39, 75005 Paris, France</footnote>

<footnote n="2">² Centre International de Recherches Médicales de Franceville, Franceville, Gabon</footnote>

<footnote n="3">³ Université de Versailles Saint-Quentin-en-Yvelines, Université Paris-Saclay, Versailles, France</footnote>

<footnote n="4">⁴ Chimfunshi Wildlife Orphanage Trust, Off T5 (Solwezi Road), Chingola, Zambia</footnote>