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. 2026 Feb 20;34(1):2. doi: 10.1007/s10577-026-09793-7

Emergence of satellite DNAs suggests centromeric repositioning as a driver of karyotypic variation of the freshwater darter characines (Apareiodon affinis)

Marina Soares Ribas 1, Matheus Azambuja 2, Viviane Nogaroto 2, Marcelo Ricardo Vicari 1,2,✉
PMCID: PMC12923391  PMID: 41718797

Abstract

Repetitive DNA sequences actively contribute to karyotype diversification by accumulating mutations, exhibiting susceptibility to DNA double-strand breaks that promote chromosomal rearrangements, and reshuffling within centromeric heterochromatin, among other processes. Parodontidae shows a conserved diploid number of 54, with predominantly metacentric and submetacentric chromosomes. Apareiodon affinis, from the Lower Paraná River, was described with three karyomorphs due to interpopulation structural variation, characterized by four to sixteen acrocentric chromosomes. However, the mechanisms driving chromosomal variation and the contribution of satellite DNA to these processes remain poorly understood. In this study, we characterized the A. affinis satellitome to assess the role of satellite sequences in the diversification of acrocentric chromosomes. A total of 48 satellite DNAs were identified, and 16 of them were mapped in situ. The data demonstrated that intragenomic homogenization mechanisms led to alterations in satellite sequence dominance, and intergenomic mechanisms contributed to the diversification of orthologous sequences when compared with other Characiformes families. In situ localization revealed that most satellites in the centromeric regions of acrocentric chromosomes were absent from the centromeres of meta/submetacentric chromosomes. The satellite AafSat01-200, which is highly diversified and the most abundant in the A. affinis genome, exhibits exclusive centromere localization on acrocentric chromosomes. Our results indicate the presence of distinct monomers with centromeric function in A. affinis and identify AafSat01-200 as the principal element involved in the centromeric repositioning mechanism that led the expansion of acrocentric chromosomes in this lineage.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10577-026-09793-7.

Keywords: Centromere, Centromeric repositioning, Chromosomal rearrangements, Karyotype evolution, Pericentric inversion

Introduction

The genomic content of eukaryotic species is enriched by repetitive elements (Biscotti et al. 2015). According to their origin and function, repetitive sequences are classified into gene families, transposable elements (TEs), and satellite DNAs (satDNA) (Nei and Rooney 2005; Wicker et al. 2007; Garrido-Ramos 2017). TEs and satDNA are currently considered key elements for understanding structural and functional characteristics in eukaryotic genomes (Garrido-Ramos 2017; Wells and Feschotte 2020). Structurally, repetitive DNAs organize essential regions of chromosomes, such as telomeres and centromeres (Slijepcevic 2016; Vicari et al. 2022; Enriquez and Nechemia-Arbely 2025). Additionally, repetitive sequences are considered hotspots for mutations and genomic variations, triggering polymorphisms, chromosomal rearrangements, and the origin and differentiation of sex and supernumerary chromosomes (O’Neill et al. 2004; Farré et al. 2011; Utsunomia et al. 2019; Wolf et al. 2024; de Oliveira et al. 2025).

From a functional perspective, repetitive sequences participate in various mechanisms of gene regulation, including transcriptional and post-transcriptional processes, as well as chromatin modification (Cech and Steitz 2014; Peschansky and Wahlestedt 2014; Zhang et al. 2019; Wells and Feschotte 2020). Given the importance of the repetitive fraction for genomic organization and function, satDNA characterization has gained prominence, as these sequences may act as regulatory sites, serve as hotspots for DNA double-strand breaks (DSBs), and exhibit transcriptional activity that leads to the generation of non-coding RNAs (Kuhn 2015; Garrido-Ramos 2017; Lower et al. 2018; Goes et al. 2023).

Advances in genomic analyses have led to a reevaluation of satDNA concept (Garrido-Ramos 2015, 2017; Šatović-Vukšić and Plohl 2023). The complete set of these sequences within a genome is referred to satellitome (Ruiz-Ruano et al. 2016). SatDNA is traditionally defined as long arrays of highly similar monomers arranged in tandem repeats (Richard et al. 2008; Plohl et al. 2012; Garrido-Ramos 2015, 2017). In general, satDNAs are located in the pericentromeric and subtelomeric regions of chromosomes and constitute a major component of heterochromatin (Plohl et al. 2012; Hartley and O'Neill 2019). Satellitomes analysis in different organisms have revealed that they can represent a highly diverse group, varying in numerous features, such as monomer length and complexity, copy number, epigenetic modifications, chromatin state, and chromosomal distribution, among others (Šatović-Vukšić and Plohl 2023).

According to satDNA library hypothesis proposed by Fry and Salser (1977), organisms from related species or genera share a common set of satellites due to their shared ancestry. Thus, differences in the size and abundance of satDNA sequences are expected to reflect evolutionary processes occurring both within lineages and among species (Fry and Salser 1977). Supporting this hypothesis, closely related species share a common satellite DNA library, although they exhibit different patterns of sequence accumulation and chromosomal distribution across lineages (Meštrović et al. 1998, 2006; Cesari et al. 2003; Koukalova et al. 2010; del Bosque et al. 2014; de Silva et al. 2017; Samoluk et al. 2017; Goes et al. 2022).

The advent of next-generation sequencing (NGS) techniques, together with the development of bioinformatics tools, has enabled the analysis of the satellitome, gene families, microsatellites, and TEs in “non-model” species (Novák et al. 2010, 2013, 2017; Ruiz-Ruano et al. 2016; Azambuja et al. 2022; Glugoski et al. 2022; de Oliveira et al. 2024, 2025). Parodontidae (Characiformes) groups three genera: Parodon, Saccodon, and Apareiodon, with 32 valid species (Fricke et al. 2025). Its representatives are characterized by a shared diploid chromosome number (2n) of 54 (Bellafronte et al. 2011). However, despite sharing the same 2n, parodontids vary in chromosome morphology, abundance, and distribution of repetitive elements, as well as in the differentiation of sex chromosomes, with female heterogamety reported in some species (Schemberger et al. 2011; Traldi et al. 2020; Nirchio et al. 2021; Azambuja et al. 2023).

The darter characines A. affinis has the La Plata River (Buenos Aires, Argentina) as its type locality but is distributed throughout the entire hydrographic system of the Lower and Upper Paraná River (Pavanelli 2003). It is one of the most intriguing species of Parodontidae in terms of karyotypic plasticity, exhibiting four karyomorphs and distinct molecular operational taxonomic units (do Nascimento et al. 2018). Specimens from the Upper Paraná River (karyomorph A) have a multiple sex chromosome system (♂ 2n = 54, ZZ - 50 m/sm + 4 st; ♀ 2n = 55, ZW1W2 - 49 m/sm + 6 st), while representatives from the Lower Paraná River exhibit demes with a variable karyotype formula and no sex chromosome heteromorphism (do Nascimento et al. 2018). In representatives from the Lower Paraná River, variation in the number of acrocentric chromosomes and structural polymorphisms in the rDNA clusters have been observed, as follows: Uruguay River 46 m/sm + 4 st + 4 a (karyomorph B); Cuiabá River 42 m/sm + 2 st + 10 a (karyomorph C); and Paraguay River 36 m/sm + 2 st + 16 a (karyomorph D) (do Nascimento et al. 2018). Regarding the distribution of satDNA, a single sequence isolated from Parodon hilarii and named pPh2004 (Vicente et al. 2003) was mapped to the chromosomes of A. affinis, showing preferential centromeric location in acrocentric chromosomes of karyomorphs C and D (do Nascimento et al. 2018).

In this context, we characterized the satellitome of A. affinis karyomorph D (Paraguay River) and performed comparative in situ localization across the three karyomorphs distributed in the Lower Paraná River to evaluate the role of satDNA in the structural organization and chromosomal evolution of the species.

Material and methods

Satellitome characterization of A. affinis

Total DNA from a male of A. affinis sampled in Paraguay River (karyomorph D) was extracted from the liver tissue using the Reliaprep gDNA Tissue Miniprep System kit (Promega, Madison, WI, USA). The DNA quality was checked on a 1% agarose gel and quantified using the BioPhotometer D30 (Eppendorf, Hamburg, Germany). The DNA was sequenced on the Illumina MiSeq platform using a paired-end strategy (2 × 100 bp, 10 million raw reads).

Data from the DNA sequencing of A. affinis were used to prospect satDNA. The preprocessing steps for the raw reads followed the default parameters recommended by Novák et al. (2020) for repetitive DNA analysis of unassembled sequence reads. The reads were pre-processed using the FASTQ paired-end reads tool (QC > 10, 100 bp) for quality control and adapter removal, avoiding selective removal of specific classes of repetitive DNA, and generating an interlaced reads library. Two random subsets of 1,000,000 reads each were then obtained using the Read sampling tool. Next, the Tandem Repeat Analyzer (TAREAN) software (Novák et al. 2017), available on the Galaxy server (Novák et al. 2013), was used to prospect the satellite sequences. The satDNA nomenclature followed the system proposed by Ruiz-Ruano et al. (2016), which consists of the species acronym followed by “Sat” (AafSat), a number representing the sequence’s relative genomic abundance, and the length of the consensus sequence.

The obtained satDNA sequences were submitted to comparative analysis in the CENSOR (Kohany et al. 2006), Basic Local Alignment Search Tool for nucleotides (BLASTn) (Altschul et al. 1990), and Dfam (Storer et al. 2021) databases, to identify satellites previously characterized in other fish species, and to remove other sequences frequently misidentified as satellites, such as gene families.

The homology among the satDNAs was determined with RepeatMasker 4.1.3 software (Smit et al. 2013−2015), using the Search engine Crossmatch and the “rm_homology_v2.py” script (https://github.com/fjruizruano/ngs-protocols/blob/master/rm_homology_v2.py, to group the sequences into variants, families, and superfamilies, as proposed by Ruiz-Ruano et al. (2016). The “rm_homology_v2.py” script was also used to identify the presence of the pPh2004 satellite among the satellites identified in A. affinis. The abundance and divergence of each satellite were determined using RepeatMasker by masking the A. affinis genomic library (2 × 5,000,000 reads) against the catalog of satellites obtained. Repeat landscape graphs were generated, based on the Kimura-2-parameter (K2P) nucleotide substitution model, using the calcDivergence-fromAlign function.pl script with RepeatMasker (Smit et al. 2013−2015) for the A. affinis genome, and individually for the AafSat01-200, AafSat02-2918, AafSat03-235, and AafSat11-227 satellites.

Probe synthesis for in situ localization

The 19 most abundant satellite sequences recovered from the A. affinis genome were selected for in situ localization assays. For 17 of the identified sequences, primers were designed using the Primer3Plus software (Untergasser et al. 2007) (Supplementary Material, Table S1) for sequence isolation by Polymerase Chain Reaction (PCR). AafSat09-33 and AafSat12-52 probes were produced by adding a 3'-end biotin label during oligo synthesis. The PCRs were composed of 0,2 - 40 ng of genomic DNA template (Table S1), 0,4 μM of forward and reverse primers, 0,2 mM dNTPs, 1 mM MgCl2, 1X Taq reaction buffer (Tris 200 mM, pH 8,4, KCl 500 mM), and 1 U of Taq DNA Polymerase (Invitrogen). PCRs were performed with the following parameters: 95 ºC for 10 min, followed by 35 cycles (95 ºC for 1 min, 55.1–60.2 ºC for 40 s - details in Table S1, and 72 ºC for 30 s), and a final extension at 72 ºC for 10 min (Table S1). PCR products were verified on a 1% agarose gel by comparing the amplicon size and the ‘ladder’ pattern characteristic of tandem repeats. Finally, the probes were synthesized in Nick Translation reactions using the Biotin16 NT labeling mix (Jena Bioscience, Jena, Germany) and Digoxigenin NT Labeling kits (Jena Bioscience).

Biological samples and chromosome preparations

Cytogenetic preparations of A. affinis from different tributaries of the Lower Paraná River hydrographic system (Uruguay, Cuiabá, and Paraguay rivers) were used (Supplementary Material, Table S2). Mitotic preparations were obtained from the anterior kidney according to the protocol described by Bertollo et al. (2015).

Fluorescence in situ Hybridization (FISH)

FISH was performed according to the protocol described by Pinkel et al. (1986), with the following hybridization conditions: 200 ng of probe, 50% formamide, 2XSSC (Saline-Sodium Citrate), 10% dextran sulfate, at 37 °C for 16 h. Signals were detected using Alexa Fluor 488 Streptavidin (Molecular Probes, Eugene, USA) and anti-digoxigenin rhodamine Fab fragments (Roche Applied Science, Penzberg, Germany). Metaphases were counterstained with 0,2 µg/mL of 4,6-diamidino-2-phenylindole (DAPI) in VECTASHIELD mounting medium (Vector Laboratories, Burlingame, USA) and analyzed under a fluorescence microscope (Leica DM 2000) coupled with a DFC3000 G CCD camera (Leica). Apareiodon affinis chromosomes were identified according to the arm ratio rule proposed by Levan et al. (1964), classified as metacentric (m), submetacentric (sm), subtelocentric (st), and acrocentric (a), and arranged into karyomorphs B, C, and D, according to do Nascimento et al. (2018).

Non-B motifs annotation in the satDNAs

A-phased repeats (APR), direct repeats (DR), inverted repeats (IR), mirror repeats (MR), short tandem repeats (STR), and Z-DNA motifs were annotated with Non-B DNA Motif Search Tool (Cer et al. 2013).

Results

After two iterations of the TAREAN tool, 48 high-confidence and 30 low-confidence satDNA sequences for A. affinis from the Paraguay River (karyomorph D) were identified (GenBank accession: PX685767-PX685814). Sequences classified as low-confidence were removed from the analysis because they were unlikely to represent satDNAs. The Repeat Unit Lengths (RUL) ranged from 17 to 2,918 bp, with an average size of 460 bp (Table 1). The length distribution of consensus sequences showed that long satDNA (>100 bp) is prevalent in the genome, particularly in 37 of the 48 families. The A+T content of the satDNA ranged from 44.7 to 70%, with an average value of 58.12%, indicating the predominance of A/T-rich families (Table 1). Homology analysis of the sequences grouped 16 monomers into four superfamilies (SF) (Table 1). SF1 was represented by seven satellites: AafSat05-1323, AafSat16-1702, AafSat23-1019, AafSat27-938, AafSat34-457, AafSat42-418, and AafSat44-186, while SF2 grouped two sequences, AafSat03-235 and AafSat11-227. The satDNAs AafSat08-434, AafSat10-143, AafSat13-158, and AafSat15-814 were grouped into SF3, and SF4 was represented by the satellites AafSat21-678, AafSat31-401, and AafSat38-436 (Table 1). The homology search between the pPh2004 satellite and the satDNA of A. affinis revealed a 73.74% sequence identity and 98% coverage with AafSat01-200 (Table 2). A sequence similarity search identified 13 satellites in A. affinis, with small segments (14 to 45% coverage) exhibiting high identity (>80%) with satellite sequences from other Characiformes species (Table 2). The satellitome of A. affinis represents 4.84% of its genome. The Repeat landscape graph for the satDNA showed a higher abundance of satellites with lower divergence values (Fig. 1). Comparative landscape analysis revealed that the satellites AafSat01-200 and AafSat11-227 (Fig. 2a), as well as satellites AafSat02-2918 and AafSat03-235 (Fig. 2b), have diverged recently. Both AafSat01-200 and AafSat02-2918 exhibited a consistent expansion phase and became dominant elements in the genome. Concomitant with the expansion of these satellites, AafSat11-227 and AafSat03-235 stopped accumulating additional copies in the genome (Fig. 2a, b).

Table 1.

Characteristics of the 48 satDNA obtained from the A. affinis (karyomorph D) genome

Satellite Ab SF RUL A + T (%) Div (%) Satellite Ab SF RUL A + T (%) Div (%)
AafSat01-200 1.7 200 58.5 7.06 AafSat25-38 0.034 38 57.9 2.17
AafSat02-2918 0.6 2.918 58.7 2.40 AafSat26-66 0.032 66 63.6 4.49
AafSat03-235 0.48 2 235 64.7 6.24 AafSat27-938 0.031 1 938 52 0.92
AafSat04-176 0.26 176 65.9 5.74 AafSat28-493 0.028 493 63.7 10.73
AafSat05-1323 0.2 1 1323 49.8 12.67 AafSat29-629 0.027 629 56.4 7.27
AafSat06-343 0.19 343 60.9 7.91 AafSat30-69 0.026 69 59.4 4.15
AafSat07-1242 0.16 1242 44.7 9.39 AafSat31-401 0.025 4 401 50.9 2.15
AafSat08-434 0.13 3 434 59.2 3.81 AafSat32-219 0.024 219 58.4 8.94
AafSat09-33 0.11 33 63.6 1.75 AafSat33-41 0.023 41 65.9 7.30
AafSat10-143 0.11 3 143 55.2 1.76 AafSat34-457 0.021 1 457 51.2 1.15
AafSat11-227 0.1 2 227 63.4 6.88 AafSat35-220 0.018 220 55.5 6.14
AafSat12-52 0.1 52 63.5 2.42 AafSat36-474 0.018 474 60.3 2.98
AafSat13-158 0.077 3 158 51.9 8.51 AafSat37-50 0.018 50 70 2.32
AafSat14-342 0.076 342 61.7 7.91 AafSat38-436 0.018 4 436 61.5 2.46
AafSat15-814 0.071 3 814 55.5 2.29 AafSat39-44 0.018 44 56.8 2.88
AafSat16-1702 0.068 1 1702 51.3 0.55 AafSat40-376 0.017 376 54.3 5.08
AafSat17-217 0.058 217 61.8 3.14 AafSat41-323 0.016 323 68.1 4.85
AafSat18-179 0.051 179 57 14.97 AafSat42-418 0.016 1 418 56.5 2.10
AafSat19-214 0.046 214 58.9 4.30 AafSat43-725 0.015 725 64.4 1.15
AafSat20-901 0.038 901 64.6 1.35 AafSat44-186 0.013 1 186 57 3.26
AafSat21-678 0.037 4 678 52.4 6.08 AafSat45-33 0.013 33 66.7 2.06
AafSat22-1176 0.036 1176 59 1.99 AafSat46-30 0.012 30 46.7 3.82
AafSat23-1019 0.035 1 1019 54.3 5.88 AafSat47-171 0.011 171 49.7 0.95
AafSat24-533 0.034 533 59.3 3.99 AafSat48-17 0.011 17 47.1 1.72
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Ab relative abundance; SF superfamily; RUL repeat unit length; A + T% AT content; Div divergence

Table 2.

Genetic similarity results among satellites of A. affinis and other Characiformes

A. affinis satellite Aligned family Species Coverage Identity
AafSat01-200* pPh2004 Parodon hilarii 98.00% 73.74%
AafSat05-1323 CmoSat049-938 Cyphocharax modestus 18.00% 95.60%
AafSat16-1702 PseSat020-1284 Pyrrhulina semifasciata 17.00% 100%
AafSat20-901 PseSat033-880 Pyrrhulina semifasciata 17.00% 83.16%
AafSat21-678 MmaSat060-1683 Megaleporinus macrocephalus 26.00% 98.91%
AafSat22-1176 HmaSat034-632 Hoplias malabaricus 19.00% 91.49%
AafSat23-1019 PseSat62-469 Pyrrhulina semifasciata 14.00% 100%
AafSat27-938 MmaSat094-450 Megaleporinus macrocephalus 16.00% 98.73%
AafSat31-401 MmaSat082-626 Megaleporinus macrocephalus 45.00% 100%
AafSat34-457 SatCE06 Cobitis elongatoides 16.00% 100%
AafSat40-376 MmaSat102-330 Megaleporinus macrocephalus 41.00% 98.09%
AafSat42-418 CmoSat089-1371 Cyphocharax modestus 18.00% 98.68%
AafSat44-186 CmoSat059-853 Cyphocharax modestus 39.00% 100%
AafSat47-171 PmaSat47-568 Pyrrhulina marylinae 44.00% 98.68%
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*Manually generated in BLASTn using AafSat01-200 as the query and the pPh2004 sequence from Vicente et al. (2003) as the subject

Fig. 1.

Fig. 1

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Repeat landscape graphs showing the abundance (Y-axis) and K2P divergence (X-axis) profiles of the A. affinis karyomorph D satellitoma

Fig. 2.

Fig. 2

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Comparative repeat landscape graphs showing the abundance (Y-axis) and K2P divergence (X-axis) profiles. In (a) AafSat01-200 (red) versus AafSat11-227 (blue); In (b) AafSat02-2918 (red) versus AafSat03-235 (blue)

Among all the mapped satellites (Table 3), AafSat01-200 is an exception to the standard signal distribution, being the only monomer with no signals detected in the Uruguay River – karyomorph B (Fig. 3a). Interestingly, the satDNAs that became dominant in the genome of A. affinis karyomorph D colonize the centromeric regions (Fig. 3). For AafSat01-200, signals were observed in the centromeric regions of acrocentric pairs 23–26 in Cuiabá River representatives – karyomorph C (Fig. 3b) and in acrocentric pairs 20–26 in the Paraguay River representatives – karyomorph D (Fig. 3c). For AafSat11-227, signals were detected in the centromeric region of acrocentric pair 27 in the karyomorph B (Fig. 3d), terminal locations in m/sm pairs 2, 3, and 5, besides on the centromeric region of acrocentric 27 in the karyomorph C (Fig. 3e), and on centromeric regions of acrocentric pairs 20, 21, 22, 23, 26, and 27 in the karyomorph D (Fig. 3f). AafSat02-2918 was found to be located in the centromeric region of m/sm pairs 9 and 15, besides acrocentric pair 26 in the karyomorph B (Fig. 3g), on the m/sm pairs 2 and 5 in the karyomorph C (Fig. 3h), and on the m/sm pairs 4, 7, 9, and 10 in the karyomorph D (Fig. 3i). AafSat03-235 showed centromeric localization only in m/sm chromosomes, specifically in pairs 5, 12, 15, 21, and 23 in the karyomorph B (Fig. 3j); pairs 12, 15, 16, and 17 in the karyomorph C (Fig. 3k); and pairs 5, 12, 15, 16, and 17 in the karyomorph D (Fig. 3l).

Table 3.

Number of sites and chromosomal locations of the most abundant satellites in A. affinis in metaphases of specimens from the Uruguay, Cuiabá, and Paraguay rivers

satDNA Uruguay River
Karyomorph B
46 m/sm +  4 st + 4a		 Cuiabá River
Karyomorph C
42 m/sm +  2 st + 10a		 Paraguay River
Karyomorph D
36 m/sm +  2 st + 16a		 Figure
AafSat01-200 ND 8a (cen) 14a (cen) 3a, b, c
AafSat11-227 2a (cen)* 6 m/sm (t) + 2a (cen) 12a (cen) 3 d, e, f
AafSat02-2918 4 m/sm + 2a (cen) 4 m/sm (cen) 8 m/sm (cen) 3 g, h, i
AafSat03-235 10 m/sm (cen) 8 m/sm (cen) 10 m/sm (cen) 3j, k, l
AafSat04-176 8 m/sm (cen) +  2 st (cen) 4 m/sm (cen) +  2 st (cen) 4 m/sm (cen) S1a, b, c
AafSat05-1323 2 m/sm (t) 2 m/sm (t) 2a (t) S1d, e, f
AafSat06-343 11 m/sm + 1a (cen)* 10 m/sm (cen) 8 m/sm (cen) S1g, h, i
AafSat07-1242 ND ND ND NS
AafSat08-434 2 m/sm (cen) 2 m/sm (cen) 2 m/sm (cen) S2a, b, c
AafSat09-33 ND ND ND NS
AafSat10-143 2 m/sm (cen) 2 m/sm (cen) 2a (t) S2d, e, f
AafSat12-52 12 m/sm (t) + 2 m/sm (cen) 16 m/sm (t) + 2a (t) 14 m/sm + 6a (t) + 8a (int) S2g, h, i
AafSat13-158 2 m/sm (t) 2 m/sm (t) 1a (prox) S3a, b, c
AafSat14-342 ND ND ND NS
AafSat15-814 2 m/sm (t) 2 m/sm (t) 2 m/sm (t) S3d, e, f
AafSat16-1702 2 m/sm (t) 2 m/sm (cen) 2a (int) S3g, h, i
AafSat17-217 2 m/sm (t) 2 m/sm (t) 2a (sub) S4a, b, c
AafSat18-179 6 m/sm (cen) 6 m/sm (cen) 6 m/sm (cen) S4d, e, f
AafSat19-214 2 m/sm (t) 2 m/sm (t) 2 m/sm (t) S4g, h, i
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*Heteromorphic pair described by do Nascimento et al. (2018)

cen = centromeric; t = terminal; int = interstitial; prox = proximal; sub = subterminal; ND = not detected; NS = not shown

Fig. 3.

Fig. 3

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Karyotypes of A. affinis from the Uruguay River – karyomorph B (a, d, g, j), Cuiabá River – karyomorph C (b, e, h, k), and Paraguay River – karyomorph D (c, f, i, l) subjected to FISH with the probes AafSat01-200 (a, b, c); AafSat11-227 (d, e, f); AafSat02-2918 (g, h, i); and AafSat03-235 (j, k, l). Bar = 10 μm

The satellites AafSat04-176 and AafSat06-343 exhibited centromeric localization in m/sm and st chromosomes across all three karyomorphs (Table 3, Supplementary Material, Fig. S1). In turn, the AafSat05-1323 showed terminal localization in a m/sm pair in karyomorphs B and C, and on an acrocentric pair in karyomorph D (Table 3, Supplementary Material, Fig. S1d, e, f). The AafSat08-434 exhibited centromeric localization in m/sm and st chromosomes across all three karyomorphs (Table 3, Supplementary Material, Fig. S2a, b, c). AafSat10-143 showed centromeric localization in a m/sm pair in the karyomorphs B and C, and terminal location of an acrocentric pair in the karyomorph D (Table 3, Supplementary Material, Fig. S2d, e, f). In turn, the satellite AafSat12-52 was preferentially located in the terminal region of the m/sm chromosomes, but also displayed in a m/sm pair in karyomorph B, an acrocentric pair with terminal location in the karyomorph C, and terminal and interstitial signals on acrocentric chromosomes in the karyomorph D (Table 3, Supplementary Material, Fig. S2g, h, i). The satellites AafSat13-158, AafSat15-814, and AafSat16-1702 showed hybridization signals on a single chromosome pair (Table 3, Supplementary Material, Fig. S3). AafSat17-217 showed a terminal location on the m/sm pair in karyomorphs B and C, and in a subterminal location in an acrocentric pair in karyomorph D (Table 3, Supplementary Material, Fig. S4a, b, c). Meanwhile, AafSat18-179 showed centromeric localization on the m/sm chromosome pairs, AafSat19-214 signals were identified in the terminal location of a m/sm pair (Table 3, Supplementary Material, Fig. S4d-i). Finally, no reliable signals were observed for AafSat07-1242, AafSat09-33, and AafSat14-342 (data not shown).

The annotation of non-B DNA motifs in A. affinis satDNAs exhibiting centromeric localization revealed the presence of DR, IR, MR, and STR (Table 4).

Table 4.

Annotation of the non-B motifs in the satDNA of A. affinis (karyomorph D) genome, which showed centromeric localization

Satellite Non-B motifs
AafSat01-200

-

2 DR, 6 IR, 1 MR, and 2 STR

1 IR

-

1 IR

3 IR and 1 MR

-

-

-
AafSat02-2918
AafSat03-235
AafSat04-176
AafSat06-343
AafSat08-434
AafSat11-227
AafSat12-52
AafSat18-179
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Discussion

Fish species with satellitome data have shown a high number of satDNA sequences, exceeding several dozen, a quantity higher than in other vertebrate groups (de Silva et al. 2017; Utsunomia et al. 2017, 2019; Crepaldi and Parise-Maltempi 2020; Crepaldi et al. 2021; Deon et al. 2024). In A. affinis, we identified 48 satDNAs in the genome, most of which have monomers longer than 100 bp and an A+T content higher than 50%, a characteristic commonly observed in fish satDNAs, as those observed in Characidium and Triportheus (Serrano-Freitas et al. 2020; Kretschmer et al. 2022).

The satelitome revealed an extensive homogenization of most satDNA families in the A. affinis genome, and the low number of superfamily-level satDNA groups aligns well with the satellite dominance alternation model and the main models of repetitive sequence evolution (Dover 1982, 1986; Camacho et al. 2022). The satDNA evolution models explain the homogenization of paralogous copies through molecular mechanisms such as unequal crossing-over, gene conversion, and transposition (Smith 1976; Dover 1986; Plohl et al. 2012). Thus, once a dominant and homogeneous satDNA becomes established in the genome, over time, some monomers may accumulate mutations, losing their identity as members of a satellite family or even a superfamily, reducing sequence similarity to levels below detectability unless new amplification events occur (Camacho et al. 2022). This entire turnover proposition for satDNA explains the sequence diversity in the A. affinis genome, which reflects the evolutionary history of the lineage that led to the fixation of monomers lacking superfamily identity. In contrast, others retain shared levels of sequence identity.

Conversely, as phylogenetic distance increases, the retention of orthologous satDNA copies becomes progressively rarer due to their rapid turnover (Camacho et al. 2022). In these cases, according to satDNA library hypothesis, only a residual retention of counterparts of the dominant satDNAs can be found in the genomes of other closely related groups (Fry and Salser 1977). The comparison of the A. affinis satellitome with those of other Characiformes revealed low-coverage segments of high identity. This process indicates that the divergence time among lineages, combined with the rapid evolution of satDNA, has driven substantial sequence diversification. Thus, mutations could lead to interspecific diversification of parts of the sequences, and the intragenomic processes of homogenization to which satDNAs are subjected could lead to the expansion of their copy number in a lineage-specific manner, consistent with the rapid evolution of satDNAs among the groups, which has led to satDNA variation (Smith 1976; Fry and Salser 1977; Dover 1986; Plohl et al. 2012).

The maintenance of sequence similarity levels for the replacement of the dominant satDNA is also observed between AafSat01-200 and the pPh2004 satellite. In situ localization studies have mapped pPh2004 in all Parodon species, as well as in karyomorphs A, C, and D of A. affinis (Vicente et al. 2003; Schemberger et al. 2011; do Nascimento et al. 2018). However, analysis of AafSat01-200 revealed that this satDNA diverged in karyomorphs C and D of A. affinis to the point of being considered a satellite distinct from pPh2004 (73.74% sequence homology). The localization of pPh2004 was possible due to the stringent conditions (approximately 70%) applied during the FISH procedure in those studies. Notably, it is worth emphasizing that the satDNAs AafSat01-200 and pPh2004 were not localized in situ in karyomorph B of A. affinis (do Nascimento et al. 2018; present study). This observation opens the possibility of investigating counterparts of the pPh2004 unit that may have diversified into a different satellite in this karyomorph. This hypothesis is further supported by the occurrence of AafSat01-200 in the centromeric regions of the acrocentric chromosomes that diversified in karyomorphs C and D of A. affinis, while the number of acrocentrics in karyomorph B remains low.

The differentiation of acrocentric chromosomes in A. affinis, evidenced by shifts in the m/sm and st/a ratios across populations in the Lower Paraná River basin, has been proposed to result from the influence of repetitive DNA sequences. do Nascimento et al. (2018) suggested that the expansion of acrocentric chromosomes in this species was driven by structural rearrangements, particularly pericentric inversions or centromeric repositioning. Our data showed that satellites AafSat01-200 and AafSat11-227, as well as AafSat02-2918 and AafSat03-235, have undergone shifts in genome dominance in the A. affinis karyomorph D. While AafSat02-2918 and AafSat03-235 are preferentially centromeric in m/sm chromosomes, the AafSat01-200 is exclusively centromeric in acrocentrics in the karyomorphs C and D, opening a perspective on its role in centromeric repositioning (Fig. 4). Centromeric repositioning refers to the emergence of a new centromere, with significant implications for chromosome stability and function (Montefalcone et al. 1999; Amor et al. 2004; Schubert 2018). Schubert (2018) proposed that the gradual loss of an ancestral centromere, accompanied by the emergence of a new one on the same chromosome, can be inferred from the absence at the new locus of the repetitive sequences characteristic of the original centromere. Thus, concomitant with the stabilization in the copy number of AafSat11-227, the satellite AafSat01-200 underwent a marked expansion in the genome of A. affinis karyomorph D, characterized by a higher number of copies with fewer accumulated divergences. According to this evidence, the satellite AafSat01-200 could have acquired a centromeric function in the acrocentric chromosomes, rapidly becoming the dominant one due to satDNA homogenization mechanisms in A. affinis karyomorph D, a process also recognized in other organisms (Dover 1982; Camacho et al. 2022).

Fig. 4.

Fig. 4

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Proposals of centromere repositioning in acrocentric chromosomes of A. affinis karyomorph D. Within A. affinis karyomorph D, the satellite DNA families AafSat01-200 and AafSat11-227, as well as AafSat02-2918 and AafSat03-235, have experienced a reorganization of their genomic dominance. AafSat02-2918 and AafSat03-235 remain preferentially centromeric in the m/sm chromosomes, whereas AafSat01-200 is exclusively centromeric in acrocentrics of both karyomorphs C and D. AafSat11-227, however, is found in terminal regions of the m/sm chromosomes in karyomorph C and at the centromeric region of acrocentric pair 27 in all three karyomorphs. The absence of AafSat11-227 on pairs 23–26 in karyomorph C indicates that inversions cannot account for the origin of these acrocentric chromosomes. Conversely, these patterns are consistent with centromere repositioning, in which an ancestral centromere is progressively lost as a new one emerges on the same chromosome, typically revealed by the absence of the original centromeric repeats at the new locus. The shift in dominance toward the AT-rich satellite AafSat01-200 in karyomorph D, reflected by its increased copy number and low sequence divergence, supports the hypothesis that this satellite may have acquired a centromeric role in the acrocentric chromosomes

In addition, whereas AafSat01-200 has preferential loci in the centromeric regions of acrocentrics, the AafSat02-2918 and AafSat03-235 satellites are widely distributed across the centromeric regions of m/sm chromosomes. The nucleotide divergence graph shows that AafSat02-2918 is in the process of increasing the number of copies with low or no divergence, while AafSat03-235 shows a greater number of copies with higher divergence values. By overlapping the divergence graphs of satellites AafSat01-200, AafSat11-227, AafSat02-2918, and AafSat03-235, it is possible to suppose that the reorganization of the centromeric regions in acrocentric chromosomes was accompanied by a restructuring of the centromeres in some m/sm chromosomes. In addition, these two satellites do not account for the centromeric organization of all m/sm and st chromosomes in karyomorph D. Although centromeres share the standard function of ensuring accurate chromosome segregation, they differ remarkably among species in both sequence composition and structural organization (Enriquez and Nechemia‑Arbely 2025).

In some equid lineages, Robertsonian rearrangements have generated chromosomes with satellite-free centromeres (Cappelletti et al. 2022, 2025). In several of these cases, satellite-free neocentromeres have been proposed to undergo a subsequent “satellitization” process, in which newly recruited satellite repeats become incorporated into the developing centromeric domain (Nergadze et al. 2018). There is currently no evidence that A. affinis experienced satellite-free neocentromeres during lineage differentiation. Nonetheless, a process analogous to “satellitization” can be inferred from the origin and expansion of AafSat01-200, an AT-rich satellite DNA localized to acrocentric centromeres, which likely contributed to the structural “maturation” of these centromeres over evolutionary time. This diversity underscores the dynamic nature of centromere evolution and suggests that different species have adopted distinct strategies to maintain centromere stability and function.

Another line of evidence comes from studies in grasshoppers, where no conserved functional motifs have been identified in centromeric monomers (Camacho et al. 2022). As proposed for grasshoppers, A. affinis may also rely on a diverse set of satellite sequences to support centromeric function. This possibility is particularly evident in the m/sm chromosomes of karyomorph D, whose different centromeres appear to be colonized by distinct satellites, including AafSat02-2918, AafSat03-235, AafSat04-176, AafSat06-343, AafSat08-434, AafSat12-52, and AafSat18-179. Except for AafSat02-2918 and AafSat03-235, which show higher copy numbers and greater variability, these satellites are relatively more stable across the three karyomorphs, suggesting a longer-term presence in the lineage. In species lacking a characteristic centromeric sequence motif, it has been proposed that inverted repeats within satDNA can promote the formation of thermodynamically stable non-B DNA structures that serve as targets for centromeric protein recruitment (Koch 2000; Hall et al. 2003; Luchetti et al. 2003; Camacho et al. 2022). Given that cruciform structures within satellite monomers can generate such non-B conformations and facilitate centromere assembly (Kasinathan and Henikoff 2018; Talbert and Henikoff 2025), the non-B DNA motifs identified in AafSat02-2918, AafSat03-235, AafSat06-343, and AafSat08-434 of A. affinis support the hypothesis that these sequences may contribute to centromere function.

On the other hand, it is known that new centromeres can arise from novel DNA sequences. In such cases, centromere emergence may be accompanied, or even facilitated, by the rapid expansion of novel satDNA, as newly expanded AT-rich repeat arrays can reshape the local chromatin landscape and promote CENP-A deposition (Murillo-Pineda and Jansen 2020; Murillo-Pineda et al. 2021). In our data, we observed that the satDNAs located in the centromeres of acrocentric chromosomes in A. affinis lack detectable non-B DNA conformations. However, AafSat01-200 represents a recently amplified satellite family forming long AT-rich arrays within these centromeres. This pattern suggests that AafSat01-200 likely emerged in the A. affinis lineage and became associated with centromere function in the acrocentric chromosomes. This interpretation is further supported by the absence of evidence for pericentric inversions and by the observation that satDNAs with centromeric localization in m/sm chromosomes are not present at the centromeres of the acrocentrics. This view aligns with recent proposals that centromeres comprise multiple, functionally distinct chromatin domains, implying that the expansion of novel AT-rich satDNA during neocentromere formation may contribute not only to CENP-A recruitment but also to shaping the broader multilayered centromeric architecture (Corless et al. 2025). Functional assays such as CENP-A ChIP enrichment, combined with immuno-FISH colocalization and targeted deletion or ectopic array recruitment experiments, could directly test whether AafSat01-200 plays a centromeric role.

Conclusion

The satellitome data generated for A. affinis from the Paraguay River (karyomorph D) in this study, along with the chromosomal localization of the most abundant sequences, demonstrated that AafSat01-200 rapidly became the dominant sequence present in the acrocentric chromosomes of karyomorphs C and D. Reinforced by the lack of a consistent chromosomal signature to support pericentric inversions, the data demonstrated the involvement of the AafSat01-200 in centromeric repositioning, which could explain the increasing number of acrocentric chromosomes in these populations. Additionally, the study highlights that numerous A. affinis satellite monomers adopting non-B DNA conformations may participate in centromere organization, and it further reveals a clear distinction between the monomers located on m/sm chromosomes and those found on acrocentric chromosomes.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 580 KB) (579.6KB, pdf)

Acknowledgements

We thank the staff of the Life Sciences Core Facility (LaCTAD) at the State University of Campinas (UNICAMP) for the MiSeq DNA sequencing service. The authors are also grateful to ICMBio (Instituto Chico Mendes de Conservação da Biodiversidade, license number 15117) and the Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional (SISGEN AE12D3D) for authorizing the collection of specimens.

Abbreviations

2n

Diploid chromosome number

a

Acrocentric

APR

A-phased repeats

bp

Base pair

BLASTn

Basic Local Alignment Search Tool for nucleotides

DAPI

4,6-Diamidino-2-phenylindole

DR

Direct repeats

DSBs

Double-strand Breaks

FISH

Fluorescence in situ Hybridization

IR

Inverted repeats

m

Metacentric

MR

Mirror repeats

K2P

Kimura-2-parameter

NAHR

Non-allelic Homologous Recombination

NGS

Next-Generation Sequencing

PCR

Polymerase Chain Reaction

pPh2004

Satellite DNA from Parodon hilarii

rDNA

Ribosomal DNA

RUL

Repeat Unit Lengths

satDNA

Satellite DNA

SF

Superfamily (of satDNA)

sm

Submetacentric

SSC

Saline-Sodium Citrate

st

Subtelocentric

STR

Short tandem repeats

TAREAN

Tandem Repeat Analyzer

TEs

Transposable elements

Author contributions

MSR, MA, VN, and MRV conceived, designed, and performed the research. MSR and MA performed bioinformatics data analysis. MSR, MA, VN, and MRV designed the experiments and contributed to the writing of the manuscript. All authors read and approved the manuscript.

Funding

The Article Processing Charge (APC) for the publication of this research was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) (ROR identifier: 00x0ma614). This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant number: 313566/2023–2). The APC for this publication was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; ROR identifier: 00x0ma614).

Data availability

All data supporting the findings of this study are included in this published article and its supplementary information files.

Declarations

Ethics approval

The procedures of this study are in agreement with the Ethics Committee of Animal Usage of the Universidade Estadual de Ponta Grossa, Brazil (Protocol: 06/2019).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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