Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 78(2): 45-52, 2025 Firenze University Press https://riviste.fupress.net/index.php/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-3718 Citation: da Silva Frade, L. F., Vas- concelos dos Santos, C. E., Ribeiro de Almeida, B. R., Nagamachi, C. Y., Pieczarka, J. C., Santos do Nascimen- to, L. A., Martins, C., Lima Cardoso, A. & Coelho Rodrigues Noronha, R. (2025). Divergence in the chromosomal distribution of repetitive sequences in Neotropical cichlid species of the genus Lugubria. Caryologia 78(2): 45-52. doi: 10.36253/caryologia-3718 Received: August 10, 2025 Accepted: October 3, 2025 Published: December 20, 2025 © 2025 Author(s). This is an open access, peer-reviewed article pub- lished by Firenze University Press (https://www.fupress.com) and distrib- uted, except where otherwise noted, under the terms of the CC BY 4.0 License for content and CC0 1.0 Uni- versal for metadata. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID LFSF: 0000-0001-9706-4817 CEVS: 0009-0007-9839-6697 BRRA: 0000-0002-6080-5826 CYN: 0000-0003-1516-2734 JCP: 0000-0003-2951-8877 LASN: 0000-0001-9947-4078 CM: 0000-0003-3534-974X ALC: 0000-0002-7287-5039 RCRN: 0000-0001-9150-8544 Divergence in the chromosomal distribution of repetitive sequences in Neotropical cichlid species of the genus Lugubria Luan Felipe da Silva Frade1, Carlos Eduardo Vasconcelos dos Santos1, Bruno Rafael Ribeiro de Almeida2, Cleusa Yoshiko Nagamachi3, Julio Cesar Pieczarka3, Luís Adriano Santos do Nascimento5, Cesar Mar- tins4, Adauto Lima Cardoso4, Renata Coelho Rodrigues Noronha1,* 1 Laboratory of Genetics and Cell Biology, Center for Advanced Studies in Biodiversity, Federal University of Pará, Belém, Pará, Brazil 2 State University of Pará, Campus Cametá. Av. Inácio Moura - bairro São Benedito, Cametá - PA, 68400-000, Brazil 3 Cytogenetics Laboratory, Center for Advanced Studies in Biodiversity, Federal Univer- sity of Pará, Belém, Pará, Brazil 4 Integrative Genomics Laboratory, Institute of Biosciences, Paulista State University, Botucatu, São Paulo, Brazil 5 Amazon Oil Laboratory, Federal University of Pará, Belém 66075-110 Pará, Brazil *Corresponding author. E-mail: renatarcrn@gmail.com Abstract. Cytogenetic studies provide valuable insights into the evolutionary dynam- ics of fish genomes, particularly in groups with high species diversity and ecological relevance. Among Neotropical cichlids, chromosomal data have revealed both conser- vation patterns and significant structural variations, reflecting intense karyotypic diver- sification. In this context, mapping repetitive DNA sequences has proven useful in aiding understanding of genomic organization and chromosomal evolution. However, information remains scarce for several cichlid genera. The present study investigated the chromosomal distribution of repetitive sequences, such as 18S and 5S ribosomal genes, as well as telomeric sequences, in three Amazonian species of Lugubria: L. cinc- ta, L. strigata, and L. lugubris. The results revealed a diploid number of 2n = 48, along with variations in the karyotypic formula among the species. Mapping of repetitive sequences revealed distinct patterns of 18S rDNA distribution, with clusters located on different chromosome pairs. Conversely, the 5S rDNA showed a conserved posi- tion on a subtelocentric/acrocentric pair in all three species. Furthermore, the presence of interstitial telomeric sequences in L. cincta and L. strigata indicates greater genomic plasticity in these species, suggesting more pronounced chromosome dynamics in the genus Lugubria. These data contribute to the understanding of chromosomal evolution and diversification in this diverse group of Neotropical cichlids and may aid in future cytotaxonomic studies. Keywords: repetitive DNAs, neotropical cichlids, ribosomal genes. https://riviste.fupress.net/index.php/caryologia https://doi.org/10.36253/caryologia-3718 https://doi.org/10.36253/caryologia-3718 https://www.fupress.com https://creativecommons.org/licenses/by/4.0/legalcode https://creativecommons.org/publicdomain/zero/1.0/legalcode https://orcid.org/0000-0001-9706-4817 https://orcid.org/0009-0007-9839-6697 https://orcid.org/0000-0002-6080-5826 https://orcid.org/0000-0003-1516-2734 https://orcid.org/0000-0003-2951-8877 https://orcid.org/0000-0001-9947-4078 https://orcid.org/0000-0003-3534-974X https://orcid.org/0000-0002-7287-5039 https://orcid.org/0000-0001-9150-8544 mailto:renatarcrn@gmail.com 46 Luan Felipe da Silva Frade et al. INTRODUCTION The Cichlidae family is one of the largest and most diverse families of freshwater fish in the world, and its geographic distribution spans most continents, includ- ing North, Central, and South America (Turner, 2007; Fricke et al. 2024). Neotropical cichlids constitute a prominent group within this family, comprising a wide variety of species adapted to different aquatic environ- ments (Kullander, 2003; Kullander et al. 2010; Chakra- barty, 2004; Genner, 2023) and exhibiting a wide range of complex reproductive strategies, including territo- rial behavior and pronounced parental care (Balshine & Abate, 2021). Due to the rapid adaptive radiation of these species, cichlids have been considered important models for evolutionary studies (Matschiner et al. 2020; Singh et al. 2022). Neotropical cichlids, in particu- lar, have been the subject of numerous scientific stud- ies not only because of their diversity and intriguing behavior, but also because many species are threatened with extinction due to the degradation of their natural habitats and the introduction of exotic species into their ranges (ICMBio, 2018). The study of the genetic charac- teristics of this group has enabled a more comprehensive understanding of their evolutionary history, supporting insights into the kinship relationships among different species and genera, as well as contributing to taxonomic classification and understanding of their evolutionary trajectories (Arbour & López-Fernández, 2014; Torres- Dowdall et al. 2021). The genus Lugubria comprises 16 large species dis- tributed throughout the Amazon Basin, the Orinoco River, and Guiana. These species exhibit changes in body coloration related to sexual maturity throughout their life cycle (Varella et al., 2023). To date, chromo- somal data are available for only four members of this genus, and these data remain largely limited to classical analyses. In general, Lugubria exhibits a diploid num- ber of 2n = 48, the absence of heteromorphic sex chro- mosomes, and 18S rDNA and 5S rDNA sites typically located on a single chromosome pair. However, inter- population variations in the karyotypic formula have been recorded for Lugubria johanna, Lugubria cincta, and Lugubria lugubris, suggesting a high rate of chromo- somal rearrangements in this genus (Frade et al., 2019; Paiz et al., 2024). Repetitive DNA refers to segments of DNA that occur in multiple copies within an organism’s genome (Lower et al., 2019; Kejnovský & Jedlička, 2022). These sequences can be organized in tandem, forming highly repetitive regions—such as telomeric and centromeric sequences—which are involved in chromosome pro- tection and proper segregation during cell division, or they may be dispersed throughout the genome, such as transposons and retrotransposons, which can influence genome evolution and are associated with chromosomal rearrangements and genetic diversification (Ayarpadikan- nan & Kim, 2014; Kejnovský & Jedlička, 2022; Šatović- Vukšić & Plohl, 2023). The function of repetitive DNA in the genome has been the subject of intense investigation because, although many of these elements do not encode proteins, they play essential roles in gene expression reg- ulation, chromosomal structure, and genomic stability (Bernstein & Allis, 2005; Liao et al., 2023). Ribosomal DNA (rDNA) is organized as tandem repeat arrays that encode crucial structural and function- al components of the ribosome and is typically found as 45S and 5S clusters within eukaryotic genomes (Mishra et al., 2021). The process of rDNA evolution involves mutations, recombination, and concerted evolution, pro- moting both homogenization and diversity, and through these processes, enabling adaptation and stability within species over time (Wang et al., 2023). Telomeric DNA, in turn, plays essential roles in maintaining genomic stabil- ity, protecting against the loss of genetic information, and preventing cellular aging (Alanazi, Parkinson, & Haider, 2024). The study of telomeric regions is fundamental for understanding chromosome structure and behavior dur- ing cell division, as well as for identifying potential chro- mosomal rearrangements and anomalies. The evolution of these sequences involves recombination and mutation events, along with variations in telomeric length, which may be associated with mechanisms of adaptation and speciation (Belyayev et al., 2023). This work presents the description of new cyto- types and the mapping of ribosomal DNA and telomeric sequences in three Amazonian species of Lugubria. Stud- ying these repetitive DNA markers can help elucidate the processes that promote chromosomal diversifica- tion and genome organization through the construction of cytogenetic maps in Lugubria, as well as expand the available karyotypic data for the genus. MATERIAL AND METHODS The sample data used in this study are presented in Table 1, and the collection sites are shown in Figure 1. The specimens were cataloged in the collection of the Genetics and Cell Biology Laboratory at the Federal University of Pará. The taxonomic identification of L. cincta, L. strigata, and L. lugubris was performed based on the existing literature. Samples were collected under SISBIO license No. 89443, and the study was conducted 47Divergence in the chromosomal distribution of repetitive sequences in Neotropical cichlid species of the genus Lugubria with approval from the Ethics Committee on the Use of Animals of the Federal University of Pará (CEUA 8803211223). Chromosome preparations were obtained according to Bertollo et al. (2015). Chromosomes were classified according to Levan et al. (1964). The total genomic DNA of Lugubria was extracted using the GenElute Mammalian Genomic DNA Mini- prep kit (Sigma-Aldrich, St. Louis, MO, USA). 18S and 5S rDNA sequences for Fluorescent in situ Hybridization (FISH) were amplified by Polymerase Chain Reaction (PCR) following the protocol of Martins & Vicari (2012), using the genomic DNA of Lugubria, with the follow- ing set of primers: 18S rDNA: 18SF (5’-CCG CTT TGG TGA CTC TTG AT-3’) and 18SR (5’-CCG AGG ACC TCA CTA AAC CA-3’) (Gross et al. 2010); 5S rDNA: 5SF (5’-GCC ACA CCA CCC CTG AAC AC-3’) and 5SR (5’-GCC TAC GAC ACC TGG TAT TC-3’) (Suarez et al. 2017), and labeled with biotin using the BioNick kit (Invitrogen) following the manufacturer’s protocol. Telomeric sequences were amplified and labeled by PCR using digoxigenin-11-dUTP with complementary prim- ers (TTAGGG)n and (CCCTAA)n, without using tem- plate DNA, according to Ijdo et al. (1991). Table 1. General data of the samples analyzed in the present study. Species Sample size Sex Localization Geographical coordinates Lugubria cincta 4 Male Bujarú River 1°45’28.4”S 47°58’14.5”W Lugubria strigata 3 Male Bujarú River 1°45’28.4”S 47°58’14.5”W Lugubria lugubris 3 Male Tapajós River 4°15’56.5”S 55°58’30.0”W Figure 1. Map indicating the collection sites of the Lugubria samples analyzed in this study. 48 Luan Felipe da Silva Frade et al. FISH was performed according to Pinkel et al. (1986). The hybridization solution composed of 2 µL of probe, 50% formamide, 2xSSC, and dextran sulfate was denatured together with chromosomal DNA in a ther- moblock at 90°C for 10 min. Hybridization occurred overnight at 37°C. Probes were detected with avidin-CY3 or antidigoxigenin-FITC. Chromosomes were counter- stained with 4’,6-diamidino-2-phenylindole (DAPI) con- taining Vectashield antifading. The slides were analyzed using an Olympus BX41 microscope and photographed with a Canon Powershot A95 digital camera. FISH images were captured with an AxioCam camera coupled to a Zeiss D2 epifluorescence microscope using Zen2 software (Zeiss). Image edit- ing, including brightness and contrast adjustments and karyotype assembly, was performed using Adobe Photo- shop CS6. RESULTS Lugubria cincta, L. strigata and L. lugubris present- ed a diploid number 2n=48 (Figure 2). L. cincta and L. lugubris presented a fundamental number FN=56, while L. strigata showed FN=54. The karyotypes observed in the three species demonstrated the following karyotyp- ic formulas: L. cincta (8m/sm+40st/a), L. strigata (6m/ sm+42st/a) and L. lugubris (8m/sm+40st/a). Sex chro- mosomes with morphological differentiation were not observed in the analyzed males. FISH with the 18S rDNA probe revealed clusters in a single chromosome pair in each species. In L. cincta, 18S rDNA was observed in the terminal region of the short arm of metacentric pair 1 (Figure 2a). In L. striga- ta, this sequence showed a large cluster extending from the interstitial to the terminal region of the long arm of submetacentric pair 2 (Figure 2b). In L. lugubris, this sequence was observed in the terminal region of the long arm of subtelocentric pair 12 (Figure 2c). The 5S rDNA was detected in the karyotypes of L. cincta, L. strigata and L. lugubris in the interstitial region of the long arm of pair 15 (Figure 2). FISH with a telomeric probe demonstrated, in all species, the presence of these sequences at the ends of all chromosome arms, in addition to interstitial telomeric sequences (ITSs) in L. cincta, in pairs 1, 2, 3, 6, 8, 10, 14, 16 and 17 and in L. strigata, in pairs 1 and 6 (Figure 2). Figure 3 presents an idiogram showed the locations of the repetitive sequences analyzed in this study. DISCUSSION The karyotypes observed in L. cincta, L. strigata, and L. lugubris in this study corroborate those previ- ously described by other authors. Interestingly, the previ- ous populations of these species analyzed cytogenetically are located in regions distant from the sampling sites of the present study. Benzaquem et al. (2008), for example, described the karyotypes of L. cincta and L. lugubris from specimens from Lake Catalão, in Amazonas State (1,300 km away); Poletto et al. (2010) and Valente et al. (2012) used specimens of L. strigata from the Araguaia- Tocantins River (Mato Grosso State- more than 1,600 km away) to characterize the karyotype of this species. Thus, our results promote an increase in the geographic distribution of these cytotypes and reveal a great conser- vation of the karyotypic macrostructure in the members of Lugubria. Similar findings were described between two distinct populations of Lugubria johanna (Frade et al. 2019). Despite the karyotypic stability observed in Cichlidae (Majtánová et al., 2019), Paiz et al. (2024) recorded in specimens from Lake Catalão (Amazonas State) karyotypic formulas of L. cincta (2m + 4sm + 32st + 10a) and L. lugubris (6sm + 22st + 20a) divergent from the findings of the present study and Benzaquem et al. (2008); considering that fish from lake environments present a high rate of chromosomal alterations (MacGui- gan et al., 2023), this may be a case of intraspecific pol- ymorphism in the Catalão Lake region, originated by inversion-type rearrangements (considering the conser- vation of 2n = 48), maintained by intrinsic factors of this population. The distribution of rDNA sequences, specifically the 18S and 5S ribosomal RNA genes, in cichlid chro- mosomes has been the subject of extensive research. Regarding 5S rDNA, Nakajima et al. (2012) observed that, in 48 cichlid genomes analyzed, more than 52% presented clusters of these sequences located in the interstitial region of the long arm of subtelocentric/ acrocentric chromosomes, as observed in the findings of the present study, confirming the conserved behav- ior of these sequences, especially among Neotropical cichlids, which presented this pattern in 82% of cases. On the other hand, in Amazonian peacock bass spe- cies, Quadros et al. (2020) showed distinct distribution patterns of 5S rDNA located in interstitial and distal positions in different chromosome pairs. These findings collectively demonstrate conserved and variable distribu- tion patterns of 5S rDNA sequences in cichlids, reflect- ing the complex evolutionary dynamics of these genomic elements (Schneider et al., 2013). The position of the 5S rDNA sites in Lugubria shows that this sequence is 49Divergence in the chromosomal distribution of repetitive sequences in Neotropical cichlid species of the genus Lugubria highly conserved in this group. This is because, in addi- tion to having an invariable diploid number (2n=48), the location of this sequence in the interstitial region of the long arm of an st/a pair remains constant in all studies carried out to date, including the present work (Frade et al. 2019, Paiz et al. 2024) (Table 2). The presence of multiple 18S rDNA sites in cich- lid genomes, with varying numbers in different species, indicates a dynamic evolutionary process shaping the chromosomal distribution of these sequences (Gross et al. 2009; Nakajima et al., 2012; Frade et al. 2019; Nirch- io et al., 2020). The present study is the first to generate data on the physical mapping of 18S rDNA in L. strigata, locating it in pair 2 of this species. The pattern found for L. cincta is similar to that described for the specimens studied by Paiz et al. (2024). In both cases, our FISH Figure 2. Double FISH with telomeric sequence probes (green signal) and 18S rDNA sequence probes (red signal) in the karyotype of (A) L. cincta, (B) L. strigata, and (C) L. lugubris. Yellow signals represent syntenic regions. The highlighted boxes show FISH with 5S rDNA sequence probes (red signal). All chromosomes were counterstained with DAPI. 50 Luan Felipe da Silva Frade et al. data corroborate the number of Ag-NOR bands observed by Benzaquem et al. (2008) and Valente et al. (2012) for L. cincta and L. strigata, respectively. Our results revealed only one ribosomal site located in pair 12 for L. lugubris, which disagrees with the findings of Paiz et al. (2024), who showed ribosomal cistrons in several pairs of the karyotype of this species. This result highlights the occurrence of pericentric inversions during the chro- mosomal evolution of the genus Lugubria, ratifying the evolutionary trend influenced by pericentric inversions, generating chromosomes with two arms, proposed by Feldberg et al. (2003) for Neotropical cichlids. In con- trast, a large number of 18S rDNA sites were evidenced in L. johanna (Frade et al., 2019). Unlike other members of Lugubria, the colocalization of 18S rDNA and intersti- tial telomeric sequences (ITSs) may have contributed to greater plasticity of this ribosomal DNA in L. johanna, since this association can generate unstable sites in the chromosomes (Frade et al., 2019). The different distribu- tion patterns of 18S rDNA in the chromosomes of the four Lugubria species karyotypes, as demonstrated in this study and by Frade et al. (2019), allow us to differ- entiate them, contributing to their cytotaxonomy, which highlights the relevance of these markers in understand- ing the evolution and phylogeny of these species. Additionally, the presence of several ITSs in the kar- yotypes of Lugubria cincta and Lugubria strigata raises several questions and possible interpretations. First, the presence of these ITSs suggests additional genomic com- plexity in these organisms, as these sequences may play important roles in chromosome stability and gene regu- lation (Lee et al. 2021; Lu & Liu, 2024). The observed pattern of telomeric sequence distribution corrobo- rates the high variability in the distribution pattern of these repetitive sequences observed in Cichlinae species (Frade et al. 2019; Nirchio et al., 2020; Quadros et al. 2020). ITSs can arise during DNA break repair; however, when considering the evolutionary lines proposed for the karyotype of Neotropical cichlids (Feldberg et al. 2003), it can be concluded that many of the observed ITSs are artifacts of recent chromosomal rearrangements. The identification and characterization of ITSs in Lugubria can provide valuable insights into genome evolu- tion in the Crenicichlina, as well the possible mechanisms of adaptation and genetic diversification. Furthermore, such information contributes to a better understanding of the dynamics of chromosomal rearrangements, genomic plasticity, and the evolutionary processes that have shaped the genome of these fishes over time (Ocalewicz, 2013; Lafuente & Beldade, 2019; Vicari et al. 2022). In summary, the data presented in this work reveals a duality in the distribution of rDNA sequences in Neotropical cichlids, highlighting both conserved pat- terns, such as the position of 5S rDNA, and variable patterns such as the distribution of 18S rDNA and the great diversity of ITSs. Taking into account that rDNA sequences cluster during interphase to form one or more nucleoli (Cazaux et al. 2011), the location of 5S and 18S rDNA clusters in different chromosomal regions enables complex evolutionary dynamics within the Crenicichli- na, since these sequences can directly or indirectly influ- ence chromosome structure and composition, as suggest- ed by Molina & Galetti-Jr. (2002) for species of the genus Cromis, by Cazaux et al. (2011) for rodents of the genus Mus, and by Marajó et al. (2022) for species of the genus Rineloricaria. The observation of multiple 18S rDNA sites and telomeric sequences in different Lugubria spe- cies not only reflects genomic plasticity within cichlids but also suggests a fundamental role for these sequences in the genome evolution of these organisms (Ocalewicz, Figure 3. Idiogram of the metaphases of L. cincta, L. strigata, and L. lugubris indicating the proposed positioning for the telomeric sequenc- es (green / diagonal lines), 18S rDNA sequences (red / grid of squares) and 5S rDNA sequences (blue / vertical lines). Centromeres are shown in solid gray. 51Divergence in the chromosomal distribution of repetitive sequences in Neotropical cichlid species of the genus Lugubria 2013; Bolzán, 2017). Understanding the distribution of repetitive DNAs in cichlids not only contributes to our understanding of their evolutionary biology but also pro- vides insights into the mechanisms underlying cichlid diversification and adaptation. 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