Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 77(4): 33-42, 2024 Firenze University Press https://riviste.fupress.net/index.php/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-3148 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Getlekha, N. & Sribenja, K. (2024). Robertsonian rearrangements in the genome of the azure damselfish, Chrysiptera hemicyanea (Perciformes, Pomacentridae). Caryologia 77(4): 33-42. doi: 10.36253/caryologia-3148 Received: November 1, 2024 Accepted: March 31, 2025 Published: July 15, 2025 © 2024 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. Robertsonian rearrangements in the genome of the azure damselfish, Chrysiptera hemicyanea (Perciformes, Pomacentridae) Nuntaporn Getlekha1, Kamika Sribenja2,* 1 Department of Biology, Faculty of Science and Technology, Muban Chombueng Rajab- hat University, Chombueng, Ratchaburi 70150, Thailand 2 Department of General Science, Faculty of Science and Technology, Muban Chom- bueng Rajabhat University, Chombueng, Ratchaburi 70150, Thailand *Corresponding author. E-mail: kamikasri@mcru.ac.th Abstract. Cytogenetic studies on the azure damselfish, Chrysiptera hemicyanea (Weber, 1913), revealed a karyotype with 2n = 31 chromosomes (17 metacentric, 2 submetacentric, and 14 telocentric; FN = 52). The study found Robertsonian poly- morphisms, which involve small heterochromatic regions at the centromeres. Nucleo- lar organizer regions (NORs) were observed near the ends of the long arms on the large metacentric chromosome pair (pair 2). FISH analysis, which detects specific DNA sequences, revealed notable variability in the distribution of ribosomal DNA (5S and 18S rDNAs) along the chromosomes. Specifically, the 18S rDNA was located at the ends of the long arms on the large metacentric chromosomes (pair 2), while the 5S rRNA genes were found near the centromere of another large metacentric chromo- some pair (pair 3). The analysis also showed that repetitive DNA sequences (CA)15, (GA)15, and (CAA)10 were spread across the subtelomeric and telomeric regions of various chromosomes. The study suggests that the structure of the karyotype and chromosome number are linked to the Robertsonian rearrangements observed, high- lighting their significant role in the evolutionary changes in the karyotype of the genus Chrysiptera. Keywords: azure damselfish, Chromosome, ribosomal DNA, Robertsonian rearrange- ments. INTRODUCTION When comparing the karyotypic (chromosomal) diversity of freshwater and marine fish, freshwater species exhibit more chromosomal changes. This increased diversity in freshwater environments is due to the numerous physical barriers present in continental waters, which limit gene flow and lead to greater karyotypic variation. In contrast, marine environments present complex and harder-to-define barriers, shaped by dynamic factors like ocean currents and winds, which can facilitate wider dispersal of species and lead to less distinct karyotypic differentiation. Genetic connectivity among marine fish popula- https://riviste.fupress.net/index.php/caryologia https://doi.org/10.36253/caryologia-3148 https://doi.org/10.36253/caryologia-3148 https://www.fupress.com https://creativecommons.org/licenses/by/4.0/legalcode https://creativecommons.org/publicdomain/zero/1.0/legalcode mailto:kamikasri@mcru.ac.th 34 Nuntaporn Getlekha, Kamika Sribenja tions, linked to changes in their karyotypes, is influenced not only by physical and ecological conditions but also by the pelagic larval stage, which is crucial for their disper- sal (Brum & Galetti, 1997; Molina & Galetti, 2004; Mar- tinez et al., 2015; Santos et al., 2024). Among Perciformes, the Pomacentridae family, which includes damselfish and clownfish, is particularly notable due to its close associa- tion with coral reefs (Allen and Werner 2002; Bellwood and Wainwright 2002; Tang et al., 2021). This family dis- plays significant diversity in forms and biological traits, making it a useful model for studying how the pelagic (open water) period of larvae affects karyotypic evolution (Molina and Galetti, 2004; McCord et al., 2021). As we advance into the era of genomic natural histo- ry, where genomic technologies offer significantly greater detail and statistical power, a reference genome will be crucial for enhancing our understanding of animal biol- ogy (Hotaling et al., 2021). Currently, there are 14 refer- ence genomes available for Pomacentrids (Roberts et al., 2023), genetic variation in reef fish populations has been well-documented through studies of allozymes (variant forms of enzymes) and mitochondrial DNA (Knowlton et al., 1993; Shulman and Bermingham, 1995; Molina and Galetti, 2002; Limon et al., 2023). Despite this vari- ation at the genetic level, many reef fish species main- tain relatively stable chromosomal structures, even over extensive geographical ranges. A key factor in karyotypic differentiation among fish is Robertsonian chromosomal rearrangements, such as centric fusion (where two acro- centric chromosomes fuse at their centromeres) and fis- sion (where a single chromosome splits into two). These rearrangements contribute significantly to chromosom- al diversity and differentiation in various fish groups (Molina and Galetti, 2002; Getlekha et al., 2017). In Pomacentridae reef fish, Robertsonian chromo- somal rearrangements, particularly centric fusion, have been observed in various contexts. For example: Das- cyllus: Centric fusion appears as a polymorphic trait, meaning different individuals or populations within the genus may exhibit this rearrangement (Ojima and Kashi- wagi, 1981; Takai A., 2012; Getlekha et al., 2016a; Getle- kha et al., 2017). Chromis: This genus also shows centric fusion, indicating its role in karyotypic diversity (Molina and Galetti, 2002). Chrysiptera: Centric fusion has been established in the derived karyotypes of specific species, highlighting its role in evolutionary changes (Takai and Ojima, 1995; Galetti et al. 2000; Hardie and Hebert, 2004; Molina and Galetti 2004). Moreover, new fish sex chromosomal systems have been associated with Robert- sonian rearrangements (Brum et al., 1992). These rear- rangements might contribute to the formation of geneti- cally distinct populations by impeding gene flow. In this study, we investigated the karyotype, het- erochromatin pattern, and nucleolar organizer regions of the fish species C. hemicyanea. We employed several methods to analyze and identify chromosomal features: Microsatellite Sequences: Specific microsatellite mark- ers, including (CA)15, (GA)15, and (CAA)10, were used to examine chromosomal patterns and identify specific chromosomes involved in the rearrangements. Repetitive DNA Probes: Fluorescence in situ hybridization (FISH) with probes for 18S rDNA and 5S rDNA was used to localize ribosomal RNA genes and further characterize chromosomal structures. These techniques helped iden- tify chromosomes involved in Robertsonian rearrange- ments, contributing to a deeper understanding of the chromosomal evolution and structure in C. hemicyanea. MATERIAL AND METHODS For the cytogenetic study, ten male and ten female azure damselfish (Chrysiptera hemicyanea) samples were collected from the Gulf of Thailand (Pacific Ocean). Chromosomes were prepared using the techniques out- lined by Getlekha et al. (2016a). The method for detect- ing nucleolar organizer regions (Ag-NORs) was based on Howell and Black (1980), while the visualization of heterochromatin bands (C-bands) followed the method described by Sumner (1972). Fluorescence in situ hybridization (FISH) was car- ried out on the chromosomes of C. hemicyanea following the procedure described by Martins and Galetti (1998). For this, two DNA sequences, 5S rDNA and 18S rDNA, were used as probes. The sequences were amplified from nuclear DNA using PCR and derived from the genome of the fish species Hoplias malabaricus, which belongs to the Erythrinidae family. The probes were cloned into plas- Figure 1. General characteristic of the azure damselfish (Chrysip- tera hemicyanea (Weber, 1913)). 35Robertsonian rearrangements in the genome of the azure damselfish, Chrysiptera hemicyanea mid vectors using competent Escherichia coli DH5α cells (Invitrogen, San Diego, CA, USA). After cloning, the 5S and 18S rDNA probes were labeled with Spectrum Green- dUTP and Spectrum Orange-dUTP, respectively, follow- ing Roche’s protocols (Mannheim, Germany) for nick translation. Additionally, Sigma (St. Louis, MO, USA) syn- thesized the microsatellites (CA)15, (GA)15, and (CAA)10, which were directly tagged with Cy3 at the 5’ end. High stringency conditions were used for fluores- cence in situ hybridization (FISH) to ensure precise results. Initially, metaphase chromosomal slides were treated with 40 μg/ml RNase for 1.5 hours at 37°C to eliminate background interference and remove RNA. The hybridization solution was prepared with 10% dextran sulfate to enhance probe penetration, 2.5 ng/ μl of labeled probes (microsatellites and rDNA), 2 μg/ μl salmon sperm DNA to block non-specific bind- ing, and 50% deionized formamide to minimize non- specific interactions. This mixture was then applied to the slides. To prepare the chromosomes for hybridiza- tion, the chromosomomal DNA was denatured in a solution of 70% formamide/2x SSC at 70°C for 4 min- utes to prepare them for hybridization. The slides were then incubated overnight at 37°C in a moist cham- ber with 2x SSC buffer to allow the probes to bind to their target sequences. To reduce non-specific binding, excess unbound probes were removed by washing the slides first at 65°C for 5 minutes with 2x SSC and then at room temperature for 5 minutes with 1x SSC. The slides were mounted in Vectashield antifade solution to prevent photobleaching and enhance fluorescence, and counterstained with DAPI to visualize the DNA. This careful preparation ensured that the FISH analy- sis could detect microsatellite and rDNA sequences with high specificity and clarity. After that, metaphase spreads were captured with a CoolSNAP camera and analyzed using Image Pro Plus 4.1 software. Imaging was performed with an Olympus BX50 microscope. RESULTS The azure damselfish (Chrysiptera hemicyanea) has a karyotype with 31 chromosomes and a fundamen- tal number of 52 in both male and female, including 17 metacentric, 2 submetacentric, and 14 acrocentric chro- mosomes. Notably, the karyotype features large meta- centric chromosomes that are nearly twice as large as the others. Nucleolar organizer regions (Ag-NORs) were located at the ends of the long arms on one pair of these large metacentric chromosomes. The C-banding analysis showed small blocks of heterochromatin primarily at the centromeres of most chromosomes. FISH analysis showed that 18S rDNA clusters were located at the telomeric ends of two large metacentric chromosome pairs (pair No. 2). On the other hand, 5S rDNA genes were exclusively present in the pericentro- meric regions of two different large metacentric chromo- somes (pair No. 3). These results offer valuable informa- tion about the genomic arrangement of the azure dam- Figure 2. Karyotypes of Chrysiptera hemicyanea were analyzed using several methods: Giemsa staining, Ag-NOR banding (high- lighted in the boxes), C-banding, and fluorescence in situ hybridi- zation with 5S and 18S rDNA probes. Chromosomes involved in centric fusions are indicated in the larger boxes. Scale bar = 5 μm. 36 Nuntaporn Getlekha, Kamika Sribenja selfish, particularly in relation to the placement of ribo- somal DNA sequences on its chromosomes. Microsatellite sequences in C. hemicyanea are dis- tributed unevenly across its chromosomes, as revealed by chromosomal mapping. Strong hybridization sig- nals were observed in both the telomeric and interstitial regions of many chromosomes, indicating a broad dis- tribution. Specifically, the (CA)15 microsatellite sequence was primarily found on metacentric chromosome No. 6. Conversely, the (GA)15 sequence, though weaker than (CA)15, was distributed throughout the intersti- tial regions of all chromosomes. The (CAA)10 sequence showed strong signals on several chromosomes. No sig- nificant differences were observed between males and females, suggesting that microsatellite distribution is similar in both sexes. These findings, which highlight the varied and uneven distribution of microsatellite sequences, provide valuable insights into the genomic organization and diversity of C. hemicyanea. DISCUSSION Recent genetic research on marine fish has shed light on how speciation occurs, emphasizing the impor- tance of chromosome rearrangements in this process (King, 1987; Crandall et al., 2019). These chromosomal changes are thought to play a key role in the formation of new species. In freshwater fish, species with limited mobility and smaller reproductive populations often show greater karyotypic diversity, with a wider range of chromosome structures (Bertollo et al., 1979; Morei- ra-Filho and Bertollo, 1991; Sribenja and Getlekha, 2024a,b). In contrast, marine fish generally display more stable karyotypes due to fewer physical barriers, higher mobility, larger populations, and more consistent envi- ronmental conditions (Brum, 1995; Liggins et al., 2016). In some families of Perciformes, such as the Gobi- idae, distinct cytogenetic traits can help differentiate pop- ulations, with notable chromosome polymorphism linked to centric fusions and fissions (Giles et al., 1985; Vitturi and Catalano, 1989; Amores et al., 1990). However, in other fish groups, cytogenetic data may not provide clear markers for species identification, even in widely distrib- uted species (Rossi et al., 1996). This lack of clear differ- entiation due to significant chromosomal rearrangements might be countered by internal changes within linkage groups, which can contribute to post-zygotic barriers essential for speciation (Molina et al., 2002). The patterns of chromosomal rearrangements between Pomacentridae genome assemblies remain unclear. Numer- ous cytogenetic studies have explored how variations in chromosome number influence fish mobility, revealing an inverse relationship between chromosome diversity and mobility. Furthermore, chromosome rearrangements have been found to either facilitate or inhibit recombination events (Galetti et al., 2000; Molina and Galetti, 2004; Kirk- patrick and Barton, 2006; Martinez et al., 2015). Robert- sonian polymorphisms play a significant role in karyotype variation across different Perciformes families, including Pomacentridae, Gobiidae, and Cichlidae. In addition to verifying variations in chromosome number, the dot-plot Figure 3. Fluorescence in situ hybridization was used to map the chromosomes of Chrysiptera hemicyanea with di- and tri-nucleotide microsatellites. The distribution patterns for (CA)15, (GA)15, and (CAA)10 microsatellites as probes are illustrated. Scale bar = 5 μm. 37Robertsonian rearrangements in the genome of the azure damselfish, Chrysiptera hemicyanea comparison between this genome and that of the clos- est relative with a chromosome-scale assembly uncovered multiple rearrangements across all corresponding chromo- somes (Roberts et al., 2023). For instance, in the Pomacen- tridae family, most species have a relatively stable karyotype with 48 chromosomes. However, Chromosome formulae vary widely (FN = 48–90), often due to pericentric inver- sions that contribute to karyotype diversification (Takai and Ojima, 1987). In contrast, the Chrominae subfamily, which includes genera such as Acanthochromis, Azurina, Chromis, and Dascyllus, exhibits notable Robertsonian polymorphisms. Diploid numbers within this subfamily vary considerably: D. trimaculatus (47–48), D. reticula- tus (34–37), D. aruanus (27–33), C. insolata (46–47), and C. flavicauda (39) (Ojima and Kashiwagi, 1981; Molina and Galetti, 2002; Getlekha et al., 2016a,2017; Yuan et al., 2018). Although asynchronic hermaphroditism is prevalent among these species, the existence of multiple sexual chro- mosomes does not seem to correlate with the karyotypic variations observed (Ojima and Kashiwagi, 1981; Getlekha and Tanomtong, 2020). This assembly will provide a crucial basis for examining how genome structure varies at a meta- population level and how these variations influence recom- bination and adaptation. Gene f low among Pomacentridae populations mainly occurs through the drifting or active migration of pelagic larvae, as the adult fish are generally non- migratory (Allen, 1991; Robitzch et al., 2016). Current evidence does not indicate a direct relationship between the length of the larval stage and the geographic range of these fish. Some researchers suggest that the complex behaviors of fish larvae (Leis and Carson-Ewart, 1998) might result in hydrodynamic movements that could actually limit their dispersal rather than facilitate it (Doherty et al., 1994; Salas et al., 2020). Previous research on the genus Chrysiptera have revealed differences in the diploid chromosome num- ber and fundamental number among species, which are caused by chromosomal rearrangements (Table 1). Centric fusions likely account for the species with fewer chromosomes and distinctive metacentric chromosomes. This chromosomal variation could be a transient phe- nomenon, reflecting a specific stage in the evolutionary process within the Chrysiptera species, potentially indic- ative of karyotypic orthoselection (White, 1973; Artoni et al., 2015; Santos et al., 2024). In C. hemicyanea, the nucleolar organizer regions (NORs) reflect their evolutionary connections. Although the NORs are positioned at the ends of metacentric chro- mosomes in C. hemicyanea, their distribution pattern is quite similar to that observed in other Pomacentrid spe- cies, despite being located at a subterminal position on the metacentric chromosome (2nd pair) (Takai and Oji- ma, 1987; Takai and Ojima, 1995; Artoni et al., 2015). Chromosomal regions that are differentially stained using C-banding techniques, known as C-bands, high- light localized areas of constitutive heterochromatin. In fish, C-bands are primarily found in centromeric regions, and occasionally in telomeric and interstitial regions (Gold et al., 1986; Takai and Ojima, 1988; Kashi- wagi et al., 2005). Although there is considerable varia- tion in the distribution of C-banded heterochromatin among chromosomes and species, many fish species exhibit C-bands as small dot-like formations predomi- nantly in centromeric regions, with often weak staining. This relatively simple distribution of C-bands appears to be a fundamental characteristic in fish. In C. hemicya- nea, the distribution patterns of centromeric C-bands, appearing as small dot-like spots in centromeric and tel- omeric regions, are consistent with previous cytogenetic studies. However, C. hemicyanea also displays distinc- tive C-bands in the terminal regions of the long arms of NOR-bearing chromosomes (pair no. 2). These observa- tions suggest that C. hemicyanea has a notably differenti- ated karyotype with respect to the distribution of consti- tutive heterochromatin. Table 1. Karyotype data of some genus Chrysiptera. Species 2n NF Formula NORs Reference Chrysiptera cyanea 42 66 6m+16sm+2st+18a 2 Takai and Ojima, 1995 C. leucopoma 48 80 4m+22sm+6st+16a 2 Takai and Ojima, 1995 C. rex 36 58 12m+10sm+14st-a 2 Takai and Ojima, 1995 C. rollandi 46 50 2sm+2st+42a 2 Kasiroek et al., 2014 C. starckii 48 60 2m+10sm+36a 2 Takai and Ojima, 1987 C. hemicyanea 48 78 30sm+10st+8a 2 Takai and Ojima, 1999 31 56 8m+10sm+32a 2 present study 2n = diploid number, NF = fundamental number, NORs = nucleolar organizer regions, m = metacentric, sm = submetacentric, st = subtelo- centric, a = acrocentric chromosome. 38 Nuntaporn Getlekha, Kamika Sribenja The distribution of C-bands in pomacentrids varies widely, ranging from simple to complex patterns (Takai and Ojima, 1999). These variations highlight the signifi- cance of constitutive heterochromatin in chromosome evolution. The C-band distribution in C. hemicyanea, which exhibits G-band-like patterns (Takai and Ojima, 1999), appears to be a rare phenomenon among fish chromosomes. This pattern represents one of the most differentiated conditions not only within Pomacentridae but also among teleostean fish. The alignment of 18S rDNA probes with the Ag- NOR clusters in C. hemicyanea suggests that having ribosomal DNA clusters on a single chromosomal pair is a fundamental trait for the Pomacentridae family (Moli- na, 2000; Getlekha et al., 2016a, b). In contrast, 5S rDNA clusters were located on a different pair of metacentric chromosomes, specifically subterminally on the long arm, and did not overlap with the chromosome pair con- taining the NORs. Previous research on 5S rRNA gene mapping has been conducted in genera like Chromis, Dascyllus, Abudefduf, and Pomacentrus (Molina and Gal- etti, 2002; Getlekha et al., 2016a, b; 2018; Zhang et al., 2021). The 5S rDNA loci appear to be more conserved in Pomacentridae, usually positioned in the pericentromer- ic region of a chromosome. In summary, various FISH applications using 18S and 5S ribosomal genes have proven effective in establishing phylogenetic relation- ships, distinguishing species, and understanding histori- cal population dynamics in both freshwater and marine environments (Artoni et al., 2015). Microsatellite sequences like (CA)15, (GA)15, and (CAA)10 show significant variation in their distribu- tion across the chromosomes of C. hemicyanea. Most of these sequences are concentrated in telomeric regions, which are rich in DNA repeats. Their clustering on spe- cific chromosomes suggests that repetitive DNA dynam- ics could influence divergence among pomacentrid fish species. Research has shown that repetitive DNAs are crucial in the evolution of genomes across different fish species (Cioffi and Bertollo, 2012; Moraes et al., 2017; Saenjundaeng et al., 2018, 2020; Sassi et al., 2019; Yano et al., 2014; Yüksel and Gaffaroglu, 2008). In this study, microsatellites (CA)15, (GA)15, and (CAA)10 are present on all chromosomes of C. hemicyanea, with notable clus- ters showing strong signals in specific regions (Figure 3). Recent and previous studies indicate that microsatellites are commonly located in heterochromatic regions of fish genomes (Getlekha et al., 2016a, b; 2018). Investigating evolutionary relationships within the genus Chrysiptera necessitates analyzing these data. Despite differences in chromosomal architecture, simi- lar microsatellite distribution patterns might indicate shared evolutionary histories. Conversely, variations in these patterns could highlight rapid changes in micros- atellite sequences (Cioffi et al., 2011; Molina et al., 2014a, b). Analyzing microsatellite sequence structures pro- vides valuable insights into the functional diversifica- tion and organization of repeated DNAs across species. However, studies examining how chromosome structure influences ecology, population dynamics, and adaptive evolution are facilitated by chromosome-scale genomes, which provide more detailed gene sequences and their locations (Roberts et al., 2023). Understanding the dis- tribution and evolution of microsatellite sequences in C. hemicyanea enhances our knowledge of genomic dynam- ics and evolutionary processes in pomacentrid fishes. This insight also aids in more thorough investigations of genetic diversity and speciation. Our focus is on Robertsonian rearrangements in the karyotypes of C. hemicyanea. According to Ojima (1983), in higher teleostean groups, including Pomacen- tridae, the average number of subtelo- and acrocentric (A-type) chromosomes is 38.3, while meta- and submeta- centric (M-type) chromosomes average 7.5. In contrast, the pomacentrids studied show an average of 30.2 A-type chromosomes and 17.1 M-type chromosomes. This indi- cates that Pomacentridae has undergone significant structural changes, with a notable transition from A-type to M-type chromosomes. CONCLUSION Teleostean fishes frequently display a notable kar- yotypic characteristic: many species possess 48 diploid chromosomes, with a majority being acrocentric. This observation suggests a conservative pattern in the evolu- tion of fish karyotypes (Ohno, 1974; Ojima, 1983). Gos- line (1971) and Ojima (1983) noted that this karyotypic feature is especially prominent in intermediate and high- er teleostean groups. Ohno (1974) proposed that a karyo- type consisting of 48 acrocentric chromosomes (48A kar- yotype) might represent the ancestral form in fish evolu- tion, as it appears across various fish families and orders. In the Pomacentridae family, diploid chromosome numbers vary from 26 to 48, and fundamental numbers range from 48 to 84. Some species within this family also exhibit the 48A karyotype. It is hypothesized that the 48A karyotype could have been ancestral for Poma- centridae, with subsequent diversification occurring pri- marily through pericentric inversions and Robertsonian rearrangements (Takai and Ojima, 1987, 1991, 1995). Furthermore, this study proposes that the karyotypic diversification in pomacentrids has been influenced not 39Robertsonian rearrangements in the genome of the azure damselfish, Chrysiptera hemicyanea only by structural chromosomal changes but also by variations in the amount and placement of constitutive heterochromatin. 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