Evolution of rDNA-Linked Segmental Duplications as Lineage-Specific Mosaics in Great Apes

Abstract

Background/Objectives: Segmental duplications (SDs) are major drivers of genome evolution and structural variation in primates, particularly within acrocentric chromosomes, where rDNA arrays and duplicated sequences are densely clustered. However, the evolutionary dynamics of rDNA-linked SDs across great ape lineages have remained poorly characterized due to longstanding technical limitations in genome assembly. Here, we investigate the organization, copy number variation, and evolutionary conservation of acrocentric SDs in great apes by integrating fluorescence in situ hybridization (FISH) with comparative analyses of telomere-to-telomere (T2T) genome assemblies. Methods: Using eight human-derived fosmid probes targeting SD-enriched regions flanking rDNA arrays, we analyzed multiple individuals from chimpanzee, bonobo, gorilla, and both Bornean and Sumatran orangutans. Results: Our FISH analyses revealed extensive lineage-specific variation in SD copy number and chromosomal distribution, with pronounced heteromorphism in African great apes, particularly gorillas, and more conserved patterns in orangutans. Several SDs showed fixed duplications across species, while others exhibited high levels of polymorphism and individual-specific organization. Conclusions: Comparison with T2T assemblies confirmed consistent genomic localization for a subset of probes, whereas others displayed partial discordance, highlighting the persistent challenges in resolving highly repetitive and structurally dynamic regions even with state-of-the-art assemblies. Genome-wide analyses further revealed species-specific enrichment of SDs on rDNA-bearing chromosomes, with chimpanzees and bonobos showing higher proportions than gorillas, and contrasting patterns between the two orangutan species. Overall, our results demonstrate that rDNA-linked SDs represent highly dynamic genomic compartments that have undergone differential expansion and remodeling during great ape evolution. These regions contribute substantially to inter- and intra-species structural variation and provide a mechanistic substrate for lineage-specific genome evolution, underscoring the importance of integrating cytogenetic and T2T-based approaches to fully capture the complexity of duplicated genomic landscapes.

1. Introduction

Segmental duplications (SDs), also known as low-copy repeats (LCRs), are large genomic regions (>1 kb) that share a high degree of sequence identity (>90%) [1].

Their high sequence similarity predisposes SDs to non-allelic homologous recombination (NAHR) during meiosis, acting as hotspots for rapid genomic changes [2,3]. Dosage-sensitive genes located within SDs may give rise to clinical phenotypes such as developmental delay, intellectual disability, and cardiovascular risk when rearrangements occur and affect their copy number, underscoring the biomedical relevance of SD variation [4,5,6,7,8].

The recombination events associated with SDs do not merely have adverse effects on genomes: they also play a key role in shaping species-specific traits and driving adaptive evolution. By promoting the emergence of species-specific expansion of gene families, SDs have significantly influenced the genomes of great apes, contributing to inter- and intra-specific variation [9,10,11]. Several gene families are ubiquitously shared across humans and great apes, including those that contributed to species-specific immune adaptation (HLA) [12], while others are specifically duplicated and expanded in the human lineage, leading to neocortex development and improvement in cognitive abilities, including SRGAP2, TBCID3, and NOTCH2NL [13,14,15,16,17,18], or in great apes, such as LRPAP1 in gorillas or EIF4A3 in chimpanzees and bonobos [19,20,21,22]. More specifically, it has been observed that western chimpanzees, bonobos, and Sumatran orangutans, likely due to demographic factors like bottlenecks, exhibit very high levels of polymorphic duplications, the accumulation of which has subsequently contributed to the expansion of gene families and species-specific differentiation among these species [11].

Despite their importance in both biomedicine and evolutionary biology, SDs have long remained poorly characterized [8], considering that the standard techniques used for genome assembly, based on short reads, have often run into difficulties in resolving such complex structures due to their repetitive nature and subsequent high sequence identity, leading to misassemblies and gaps corresponding to these regions [1,23,24,25]. Recent advances and methodological improvements in sequencing technologies [26] have encompassed previous limitations and made it possible to obtain a Telomere-to-Telomere (T2T) human genome assembly (T2T-CHM13) from a complete hydatidiform mole [27], which yielded an unprecedented overall picture of SDs in the human genome, providing insights into their quantity and organization [8].

Using the same methodologies, diploid genomes of non-human primates were also fully sequenced, enabling novel perspectives for comparative analyses and a better understanding of previously uncharacterized regions from an evolutionary perspective. These high-resolution techniques have led to a more detailed characterization of duplicated genes in each primate lineage, and their association with fixed chromosomal rearrangements, such as inversions and other large-scale structural variants, that contribute to distinguishing human and non-human primate genomes [28].

Acrocentric chromosomes represent a hotspot for clustering of SDs, as a significant enrichment has been observed within the short arm of human acrocentric chromosomes 13, 14, 15, 21, and 22 [8,20]. The p-arms share a common genomic architecture composed primarily of rDNA, SDs, and satellite sequences, which are highly similar to one another. The domain encompassing the interval between the telomere and rDNA harbors a symmetric domain of inverted SDs. In contrast, the stretch within the proximal short arm, across rDNA and the centromere, is more structurally complex and rearranged [27]. Evolutionary analyses suggest that acrocentric short arms, thanks to their large block of SDs, have undergone extensive remodeling through SD-mediated recombination during nucleolar organization, leading to species-specific expansions of duplicated genes and a significant degree of heteromorphism across individuals. Additionally, in humans, chromosomes 13, 14, and 21 harbor the largest and most highly similar blocks of SDs among the acrocentrics, promoting non-allelic homologous recombination and making them particularly prone to involvement in Robertsonian translocations [8,27,29,30].

In this study, we deeply characterize segmental duplications in great apes, leveraging the new T2T assemblies, with particular emphasis on regions that have recently been extensively studied in the human genome. Comparative analysis of human and great ape genomes enables the distinction between conserved acrocentric SDs and those that underwent lineage-specific duplications, contributing to the characterization of distinct regulatory landscapes observed among primates.

2. Materials and Methods

2.1. Cell Lines

Metaphase spreads were obtained from different great ape species following standard cytogenetic protocols. Specifically, we analyzed lymphoblastoid cell lines from Pan troglodytes (PTR8 and PTR12), Pan paniscus (LB502 and Ulindi), Pongo abelii (PAB20), and Pongo pygmaues (PPY8), as well as skin-derived fibroblast cell lines from Gorilla gorilla (GGO5 and GGO9).

2.2. FISH Characterization

FISH experiments were conducted to investigate SD variability in great apes. Eight probes (

Table 1. Acrocentric segmentally duplicated probes [8] used to characterize acrocentric SDs variability in the human genome.

Clone Name	Mapping on T2T CHM13v2.0	Genes
174222_ABC10_2_1_000044420800_E22	chr13:12885129-12928710	FRG1BP-1
174222_ABC10_2_1_000044616600_A12	chr14:7053457-7120716	LINC01262-1
173650_ABC10_2_1_000043644100_E7	chr14:8616873-8659155	AC138907.8-1, AC138907.9-1, AC138907.10-1
174552_ABC10_2_1_000044788300_M21	chr15:1-55353	BNIP3P41-1, CHM13_T0067447
174222_ABC10_2_1_000044587300_G6	chr21:9899228-9964348	FRG1BP-1, AC079801.1-201
171417_ABC10_2_1_000045520200_K20	chr22:4701792-4772966	NA
174222_ABC10_2_1_000044559800_C15	chr22:6266411-6315769	SLC9B1P4-1, ACTR3BP6-1
174552_ABC10_2_1_000044707300_L19	chr22:11387009-11432887	AC138907.8-1
174222_ABC10_2_1_000044587300_G6	chr21:9899228-9964348	FRG1BP-1, AC079801.1-201

) derived from the human ABC10 fosmid library [31] were used to systematically characterize SD variation, which arises when chromosome organization differs among individuals, as in Vollger et al. (2022) [8]. These probes were selected from regions flanking the rDNA (p arms of human acrocentric chromosomes) and are characterized by SDs showing >95% sequence identity with homologous regions on other human acrocentric chromosomes [8,27]. For each clone, gene content was obtained using hs1 Human: CAT + Liftoff gene annotations, as available from Nurk et al. (2022) [27] (Table 1).

The fosmid clones were extracted using the PureLink Quick Plasmid Miniprep Kit (Cat. no. 7326100; Thermo Fisher Scientific Inc., Waltham, MA, USA). One hundred nanograms of each DNA probe was labeled by nick-translation with Cy3-dUTP and subsequently precipitated by ion-exchange alcohol precipitation with human Cot-1 DNA [32]. After denaturation for 2 min at 70 °C and overnight hybridization at 37 °C, post-hybridization washes were performed at 60 °C in 0.1× SSC (three times, high stringency). At the end, the slide was stained with DAPI, producing a Q-banding pattern. The fluorescence signals from Cy3 and DAPI were detected separately using specific filters on a Leica DMRXA epifluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a cooled CCD camera (Princeton Instruments, Trenton, NJ, USA) and recorded as grayscale images. Adobe Photoshop™ was used to pseudo-color the acquired images and merge them. Non-informative nuclei and signal precipitates that did not map to chromosomes were removed from the images. Signals located consistently on the same chromosome over 20 metaphases for each experiment were considered relevant to chromosomal probe localization and fully preserved. For final figure preparation, the processed images were imported into Adobe Illustrator™ 2024 to assemble supplementary panels.

2.3. Probes Mapping on T2T-GAs Assemblies

The sequence of each probe was aligned against the following great ape reference genomes: NHGRI_mPanTro3-v2.1_pri (GCF_028858775.2), NHGRI_mPanPan1-v2.1_pri (GCF_029289425.2), NHGRI_mGorGor1-v2.1_pri (GCF_029281585.2), NHGRI_mPonAbe1-v2.1_pri (GCF_028885655.2), NHGRI_mPonPyg2-v2.1_pri (GCF_028885625.2). The alignment was performed using the minimap2 [33] command, and -x asm20-t 50–eqx-s 500-N 1000-p 0.01 parameters.

The percentage identity for each probe was calculated as the number of matches divided by the query length, and the probe coverage was defined as the difference between the query start and end coordinates, normalized by the query length.

2.4. Circular Plot for SD Visualization

Circular plots were generated using the circlize [34] package in R to visualize SDs annotated on primary haplotypes using SEDEF from Yoo et al. (2025) [28], on great ape reference genomes (https://github.com/marbl/T2T-Browser; accessed on 2 June 2025). SDs were filtered based on length (>20 kb) and chromosomal locations, considering only those mapping on rDNA-bearing chromosomes [28,35]. Specifically, the analyzed chromosomes were chr14_hsa13, chr15_hsa14, chr17_hsa18, chr22_hsa21, and chr23_hsa22 for Pan troglodytes (PTR) and Pan paniscus (PPA); chr22_hsa21 and chr23_hsa22 for Gorilla gorilla (GGO); and chr11_hsa2B, chr12_hsa2A, chr13_hsa9, chr14_hsa13, chr15_hsa14, chr16_hsa15, chr17_hsa18, chr22_hsa21, and chr23_hsa22 for Pongo pygmaeus (PPY) and Pongo abelii (PAB) (see Table S1 for species-specific chromosome nomenclature). SD datasets were further filtered by length, using a threshold of >20 kb, to improve visual clarity while still allowing direct comparison between the large-scale SDs and the unfiltered data.

3. Results

To comprehensively characterize human acrocentric segmentally duplicated regions in great apes, we selected eight probes from the human ABC10 fosmid library [8,31] whose genomic locations fall within SD-enriched acrocentric regions (

Table 2. Clones used for FISH experiments, along with the naming convention adopted in figures and tables (ID), their chromosomal mapping in the human genome (HSA) according to Vollger et al. (2022) [8] based on the T2T CHM13v2.0/hs1 assembly, and the chromosomes (reported using human homolog nomenclature; see Table S1 for corresponding species-specific nomenclature) on which alignments with >96% sequence identity were detected in the T2T assemblies of non-human primates (AG18354_PTR, PR00251_PPA, Jim_GGO, AG06213_PAB, AG05252_PPY). NA indicates the absence of hits with >96% identity; additional results with <96% identity are reported in Table S2.

Clone Name	ID	HSA	PTR	PPA	GGO	PAB	PPY
174222_ABC10_2_1_000044420800_E22	E22	13	hsa13, hsa14, hsa18, hsa20, hsa21, hsa22	hsa13, hsa14, hsa18, hsa20, hsa21, hsa22	hsa20	NA	NA
174222_ABC10_2_1_000044616600_A12	A12	14	hsa4, hsa13, hsa15, hsa18, hsa21, hsa22	hsa4, hsa15	hsa2B, hsa2A, hsa4, hsa13, hsa15	hsa4	hsa4
173650_ABC10_2_1_000043644100_E7	E7	14	hsa9	hsa2B, hsa9	hsa7, hsa13, hsa15, hsa18	NA	NA
174552_ABC10_2_1_000044788300_M21	M21	15	hsa4	hsa4	hsa1, hsa4	hsa4	hsa4
174222_ABC10_2_1_000044587300_G6	G6	21	hsa13, hsa14, hsa18, hsa20, hsa21, hsa22	hsa13, hsa14, hsa18, hsa20, hsa21, hsa22	hsa20	NA	NA
171417_ABC10_2_1_000045520200_K20	K20	22	NA	NA	NA	NA	NA
174222_ABC10_2_1_000044559800_C15	C15	22	hsa10, hsa16, hsa22, hsaY	hsa10, hsa16, hsa22, hsaY	hsa16	NA	NA
174552_ABC10_2_1_000044707300_L19	L19	22	hsa3	hsa3	hsa2B, hsa3, hsa15	hsa3	hsa3

and Figure S1). We individually tested eight different great ape cell lines: two PTR (PTR8 and PTR12), two PPA (LB502 and Ulindi), two GGO (GGO5 and GGO9), and two orangutans, one Sumatran individual (PAB20) and one Bornean individual (PPY8). In parallel, we aligned all probe sequences against the T2T genome assemblies for each species (Table S2). We distinguished hybridization patterns as homomorphic or heteromorphic based on their presence across all homologous chromosomes and individuals within each species (Table S3).

Specifically, the ABC10_2_1_000044420800_E22 probe, mapping to the proximal domain between the centromere and rDNA array on the short arm of human chromosome 13, detected eight copies in chimpanzees (four of which were heteromorphic), four copies in bonobos (one heteromorphic), three copies in gorillas (one heteromorphic), and a single copy in both Bornean and Sumatran orangutans (Figure S1; Tables S3 and S4).

Although both ABC10_2_1_000044616600_A12 and ABC10_2_1_000043644100_E7 probes localize to the proximal p arm of human chromosome 14, between rDNA array and the centromere, they showed markedly different patterns. The region targeted by ABC10_2_1_000044616600_A12 (closer to the rDNA) showed extensive duplication in Pan and Gorilla lineages, with seven copies in chimpanzees (six heteromorphic), four in bonobos (one heteromorphic), and four in gorillas (three heteromorphic); conversely, it is single-copy in orangutans (Figure S2; Tables S3 and S4). ABC10_2_1_000043644100_E7 (proximal to the alpha-satellite centromeric array) predominantly mapped as a single-copy region across all great apes, except in gorillas, where two heteromorphic loci were detected (Figure S2; Tables S3 and S4).

The ABC10_2_1_000044788300_M21 probe, derived from the distal domain between the rDNA cluster and the telomere of the short arm of human chromosome 15, produced a single hybridization signal in all examined species, with heterozygosity observed in only one bonobo individual (Ulindi) (Figure S3; Tables S3 and S4).

The ABC10_2_1_000044587300_G6 probe, mapping to the region between the centromere and rDNA array on the p arm of human chromosome 21, revealed multiple duplications in all great ape lineages. Specifically, seven copies (oneheteromorphic) were detected in chimpanzees, three (two heteromorphic) in bonobos, four (three heteromorphic) in gorillas, two in Sumatran orangutan, and three in Bornean orangutan (Figure S4; Tables S3 and S4).

Finally, three probes mapping to the short arm of human chromosome 22, ABC10_2_1_000045520200_K20, ABC10_2_1_000044559800_C15, and ABC10_2_1_000044707300_L19, displayed distinct duplication profiles. The ABC10_2_1_000045520200_K20 probe, flanking the rDNA repeats in the distal domain, identified extensive copy number expansion, with six copies in chimpanzees, five in bonobos (one heteromorphic), three in gorillas (all heteromorphic), six in Sumatran orangutan (two heteromorphic), and eight in Bornean orangutan (five heteromorphic) (Figure 1, Figure S5; Tables S3 and S4). In contrast, the ABC10_2_1_000044559800_C15 probe detected fixed duplications in African great apes (four copies in chimpanzees and bonobos, three in gorillas). In contrast, it identified single-copy sequences in both Bornean and Sumatran orangutans. Finally, the ABC10_2_1_000044707300_L19 probe mapped as a single-copy region in all tested species, except in gorillas, where three additional heteromorphic loci were observed (Figure S5; Tables S3 and S4).

Overall, through FISH analysis of these regions in non-human great apes, we were able to identify 33 different autosomic locations in PTR, of which 22 were fixed (67%), whereas 11 were heteromorphic (33%) in their presence on homologs and individuals; 23 in PPA, of which 16 are fixed (69%) and 7 heteromorphic (31%); 24 in GGO, of which 9 are fixed (37%) and 15 heteromorphic (63%); 14 in PAB, of which 12 are fixed (85%) and 2 heteromorphic (15%); and 16 in PPY, of which 11 are fixed (69%) and 5 heteromorphic (31%).

A comprehensive summary of the FISH mapping defined for each probe across the tested individuals is provided in Figure 2.

FISH results were then compared with data obtained from the mapping of each probe sequence against all great apes T2T assemblies [28], enabling the distinction of two primary outcomes from these analyses: “consistent”, if signals are present on both individuals from the same species (even if heteromorphic) and also detected in the assembly alignments, and “inconsistent” results, when this agreement is not observed (Table S3). Alignment analyses confirmed FISH results for three out of eight probes (ABC10_2_1_000044559800_C15, ABC10_2_1_000044707300_L19, and ABC10_2_1_000044788300_M21), defining the regions targeted by these probes as fully consistent. Additionally, genomic localization of the ABC10_2_1_000044420800_E22 probe is supported by sequence analyses (except for the chromosome Y signal detected on the PTR8 individual); however, it is heteromorphic by FISH. Similarly, the hybridization pattern for the ABC10_2_1_000045520200_K20 probe is mainly consistent with the alignment output, except for a single duplication in the Bornean orangutan (PPY8) that was not predicted by computational mapping.

Finally, the remaining three probes (ABC10_2_1_000044616600_A12, ABC10_2_1_000044587300_G6, and ABC10_2_1_000043644100_E7) showed partially discordant results when FISH was compared with sequence alignment.

Considering the duplications on rDNA-bearing chromosomes, the ABC10_2_1_000045520200_K20 probe localizations were consistent in chimpanzees, bonobos, and gorillas. Both orangutan species exhibit greater variability due to the absence of FISH signals on the chr13_hsa9 and chr17_hsa18 chromosomes, which were instead predicted from alignment analyses of their reference genomes.

Conversely, in the PPY8 individual, a heterozygous signal was detected on chr19_hsa17, contrary to expectations. FISH mapping of the ABC10_2_1_000044587300_G6 probe was consistent only for chimpanzees, while ABC10_2_1_000044420800_E22 was consistent within chimpanzees and bonobos, yet it still confirmed a degree of heteromorphism, as described.

We analyzed the distribution of SDs across rDNA-carrying chromosomes in great ape lineages (Figure 3). A shared pattern has been observed in chimpanzees and bonobos, with approximately 15% of SDs localized to these chromosomes, rising to 18% in chimpanzees when considering only duplications larger than 20 kb.

Gorillas showed a lower abundance of SDs mapping to rDNA-containing chromosomal regions, accounting for about 8% of all identified SDs (9% when restricting the analysis to SDs > 20 kb). Moreover, these duplications predominantly map to a specific subset of chromosomes across the genome (chr5_hsa6, chr6_hsa7, chr10_hsa12, chr11_hsa2b, chr17_hsa18, chr20_hsa19, chrX, and chrY).

The two orangutan species exhibited a consistent pattern: overall, the Bornean orangutan harbors fewer SDs than the Sumatran orangutan. Despite this, the proportion of SDs located on rDNA-bearing chromosomes was similarly high in both species, accounting for approximately 48% of SD content in PPY and 49% in PAB (both 48% when restricting the analysis to SDs > 20 kb).

4. Discussion

Comparative analysis using a molecular cytogenetics approach enabled us to investigate the complex genomic architecture associated with rDNA-linked SDs in nonhuman great apes, offering new insights into their organization and evolutionary dynamics. Focusing on highly homologous regions that, in humans, map to the short arms of acrocentric chromosomes, we identified 35 distinct SD locations in PTR, 23 in PPA, 24 in GGO, 14 in PAB, and 17 in PPY. Despite differences in the absolute number of SD locations, a marked difference in the structural variability of these domains emerged across the examined lineages. In Pan, the proportion of heteromorphic sites is approximately 30%, reflecting the intrinsically variable nature of rDNA-related SDs, as previously observed in humans [27]. In contrast, most SD locations are fixed in orangutans (85% in PAB and 70% in PPY), whereas gorillas exhibit exceptional dynamism, with 63% of locations being heteromorphic.

Our findings reveal a slightly different distribution of these duplications compared to the pattern described in humans, in which acrocentric SDs exhibit a notable level of homology with pericentromeric domains of chromosomes 1, 3, 4, 7, 9, 16, and 20 [8]. Within the Pan lineage, we observe expansion on chr18_hsa16 for only one of the regions under analysis (embedded within clone C15). The homology with the pericentromeric domains of hsa3 and hsa4 chromosomes, instead, remains significant for most of the studied regions (except for K20, which instead maps on chr1_hsa1 of gorillas) across all species, as previously highlighted.

By leveraging recent fully resolved T2T assemblies [28] and integrating them with cytogenetic data, we found that these genomic domains do not follow a linear evolutionary trajectory across primates. Instead, they display strictly species-specific patterns of duplication, fixation, and variability. We identified three distinct evolutionary trajectories: i. The first trajectory are human lineage-specific expansions, in which domains such as that targeted by the ABC10_2_1_000044788300_M21 probe (widely dispersed in humans) [8] are present in a single copy on hsa4 in all non-human great apes. This suggests that massive dispersal to acrocentrics represents a relatively recent event in Homo, a pattern also confirmed by the E7 and L19 probes, which maintain a single copy on the hsa3 orthologue in nearly all species, with a few exceptions in gorillas. In particular, neither FISH analysis nor sequence alignment detected E7 signals on gorilla chr2_hsa3, suggesting lineage-specific evolutionary processes. ii. The second are shared duplications across lineages that may indicate ancestral duplicative events, as observed by the hybridization pattern of the C15 probe. This domain shows deep conservation on the hsa4 orthologue across all species, albeit with species-specific chromosomal localizations (terminal in Pan and interstitial in Gorilla and Pongo). Duplications on chromosomes hsa10 and hsa16 are shared between chimpanzees, bonobos, and gorillas, while the copy on hsa22 is an innovation specific to the genus Pan. iii. The final trajectory are lineage-specific differences, in which domains evolved independently and underwent unique expansions. Primary examples are represented by the loci covered by the E22 and A12 probes. Both domains have remained single-copy in orangutans, while they have experienced divergent expansions in the Gorilla and Pan lineages. Additionally, the locus covered by the G6 probe stands out as one of the most variable regions identified, having undergone extensive expansion that led to extreme diversification and inconsistent heteromorphic patterns across lineages. This locus exemplifies how these SDs can evolve rapidly and casually in a species-specific manner. Notably, the evolutionary dynamism of these regions increases with proximity to rDNA arrays, as shown by the ABC10_2_1_000045520200_K20 locus, which indicates that rDNA-based architecture can effectively provide a substrate for recurrent duplication, shuffling, and heteromorphism. This probe closely flanks the rDNA array on the distal domain of human chromosome hsa22, where its sequence is mapped. In non-human great apes, this domain localizes exclusively on rDNA-carrying chromosomes, with a few exceptions in gorillas (chr1_hsa1) and Bornean orangutan (chr19_hsa17). These observations support a model in which rDNA-associated genomic architecture provides the right substrate for recurrent duplication, reshuffling, and heteromorphism. Collectively, these SD-rich regions act as evolutionary crucibles, facilitating recombination events that generate both conserved and lineage-specific genomic innovations, thereby contributing to the structural and functional diversification of great ape genomes.

Notably, many of the investigated duplications overlap with annotated genes in the human genome, revealing distinct evolutionary trajectories. Probes E22 and G6, for example, intersect with the FRG1BP-1 gene. In humans, the FRG1 gene (located on the q arm of chr4) is highly duplicated, with 23 copies concentrated primarily in the acrocentric regions [27]; it is associated with facioscapulohumeral muscular dystrophy (FSHD) when a deletion of a 3.3 kb macrosatellite repeat block occurs in this region [36]. Our results indicate that FRG1 expansion has not been uniform: in orangutans, probe E22 identifies a single domain on chr3_hsa4, which may represent the ancestral copy. In contrast, the same domain is multicopied in chimpanzees, bonobos, and gorillas. In PTR, we observed an absence of signal from the chr3_hsa4 telomere, with signals instead on rDNA-bearing chromosomes and the Y chromosome, highlighting profound remodeling.

Furthermore, it has recently been shown that multiple copies of the FRG1 gene underwent pseudogenization in most lineages, including humans, while they remained functional in orangutans and gorillas, where marks of positive selection have been described [37]. Probe G6, which also harbors a long noncoding RNA (AC079801.1-201), proved to be among the most dynamic and variable loci in the entire study. Additional examples include the unprocessed pseudogene SLC9B1P4-1 and the processed pseudogene ACTR3BP6-1, both mapping to the locus covered by probe C15. The FISH pattern of this probe highlights shared signals between hsa4 orthologs in chimpanzees and bonobos (terminal) and gorillas and orangutans (interstitial). Other duplications on chromosomes hsa10 and hsa16 are shared between chimpanzees, bonobos, and gorillas, while the copy on hsa22 appears to be a Pan-specific innovation. Although the functional impact of these variations remains to be determined, their recurrence suggests species-specific effects on gene dosage and genomic stability.

A deeper analysis of the quantitative distribution of SDs on rDNA-carrying chromosomes reveals a stark contrast among genera. The Pan lineage shares a significant enrichment, with approximately 15% of SDs located on these chromosomes (18% in chimpanzees for SDs > 20 kb). In contrast, gorillas exhibit a much more limited expansion, with only 8–9% of SDs mapping to rDNA regions. These duplications in gorillas are confined to a specific subset of chromosomes, suggesting evolutionary constraints that limit their dispersal or the different nucleolar organization that limits SD exchange between the acrocentrics and the rest of the genome relative to humans or chimpanzees [35]. The Pongo lineage presents a peculiar situation: although the Bornean orangutan has fewer total SDs than the Sumatran, their content on rDNA-carrying chromosomes underwent a massive expansion in both species (up to 48% of SDs greater than 20 kb), highlighting their role as hotspots for duplications, likely driven by species-specific evolutionary dynamics.

A critical issue that has emerged is the discrepancy between FISH results and the T2T-based alignments [28]. Among the 135 predicted loci, 41 lacked high-confidence sequence alignments (>96% identity), corresponding to an overall discordance rate of ~30%. While several probes exhibited high levels of concordance (K20: 26/2, ~93% of concordance; L19: 8/1, ~89%; M21: 5/1, ~83%), others showed substantial inconsistency (E7: 2/10, ~83% of inconsistency; G6: 12/13, ~52%; A12: 13/6, ~32%). For example, the E7 probe did not produce FISH signals on the gorilla chr2_hsa3, despite alignment predictions. Similarly, the G6 probe showed inconsistencies in chimpanzees, bonobos, and gorillas. These discrepancies are not necessarily errors; they may reflect the true complexity of these genomic regions, such as recent duplications, homogenization of paralogous sequences, and polymorphic rearrangements, as well as the limitations of current methodologies, including the resolution of FISH and the challenges of accurately assigning nearly identical duplications even in high-quality assemblies. Similarly, the detection of additional signals in cytogenetic analyses compared with sequence-based alignments underscores the continued value of FISH as a complementary approach, as it can reveal regions that are still difficult to resolve by sequencing alone.

Overall, this study reveals that, while the rDNA arrays constitute a conserved structural backbone of nucleolar organizer regions [37], the surrounding proximal and distal SD-rich domains have undergone divergent, largely independent evolutionary trajectories across great apes. Our findings underscore that these regions do not evolve as a single unit but rather form a dynamic mosaic of lineage-specific architectures. Full resolution and comprehensive characterization of these domains will benefit from future fully resolved T2T sequencing of NOR-bearing chromosomes in all great ape genomes.