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Karyotype variability in six Amazonian species of the family Curimatidae (Characiformes) revealed by repetitive sequence mapping

Abstract Fishes of the Curimatidae family represent one of the most important freshwater ichthyofauna groups of Central and South America, with 117 recognized species distributed in eight genera. In this study, six species - Curimata inornata, Curimatella dorsalis, and Psectrogaster falcata collected from the Lower Araguaia River, Pará, Brazil; Curimata vittata, Curimatella meyeri, and Psectrogaster rutiloides collected from the Catalão Lake, Amazonas, Brazil - were cytogenetically analyzed, investigate the occurrence and distribution of repetitive DNA classes in the karyotypes. All species had 2n=54 metacentric/submetacentric chromosomes. Despite the conservative diploid number, we observed variations in the karyotypic structure among species. Ribosomal DNA (rDNA) 18S and 5S were found in single or multiple sites, with the first report of synteny in Curimatella dorsalis, and the occurrence of several interstitial telomeric sequences (ITSs) in species of the genera Curimatella and Psectrogaster. Interspecific karyotypic diversity both concerning structure and location/position of the nucleolar organizer regions (NOR) and ribosomal DNA, suggesting the occurrence of several non-Robertsonian rearrangements driving the evolution of this family.

The present study aims to investigate the chromosomal composition and structure of the karyotypes of six Amazonian Curimatidae species. The results were compared with the data available in the literature to infer the hypothetical chromosomal rearrangements involved in the chromosomal evolution process.
At least 30 metaphase spreads of each individual were analyzed to confirm the diploid number and karyotype structure. The chromosomes were classified as metacentric (m) and submetacentric (sm) (Levan et al., 1964).
The six species analyzed presented 2n=54 and FN=108 (Fundamental number) (Figure 1, Table 2), it is highlighted that the karyotype of Psectrogaster falcata is presented here for the first time. CH was observed in pericentromeric blocks in all chromosomes of the six species, except in pairs 5 and 18 of P. falcata. Furthermore, additional blocks located in the terminal portions of several chromosomes of the six species were also observed. C. meyeri showed interstitial blocks in the long arms of pair 5; and pairs 2, 19, and 21 in P. rutiloides (Figure 1).
Five species presented NOR in only one chromosome pair in the terminal portion of the short arms: C. inornata, P. falcata, and P. rutiloides (pairs 20, 13, and 16, respectively), and in the end of the long arms in C. dorsalis and C. meyeri (pairs 2 and 9, respectively). C. vittata exhibited NORs in two chromosome pairs (multiple NORs) in the terminal portion of the long arms (pairs 20 and 21). The six species showed the NORs colocalized with heterochromatic blocks (Figure 1, box Ag-NOR).
The rDNA mapping corroborates the NORs in all the species studied, including an additional site observed in the end of the short arm of pair 7 in C. meyeri, which is also colocalized to the constitutive heterochromatin (Figure 2, 18S). The 5S rDNA sequences mapping revealed a species-specific pair with interstitial signals: pair 9 in C. inornata, pair 25 in C. vittata, pair 2 in C. dorsalis, pair 26 in C. meyeri, and pair 24 in P. falcata. P. rutiloides presented 5S sites in two pairs: pair 5 in the terminal portion of the short arm and interstitial in pair 22. C. dorsalis showed synteny of 5S and 18S (Figure 2). Telomeric sequences (TTAGGG) n were located in the terminal region of all chromosomes of the six species. Additionally, interstitial telomeric sequences (ITSs) were observed in several chromosomes of Curimatella and Psectrogaster species, with some conspicuous blocks (Figure 2 Chromosomal evolution of the family Curimatidae was defined as being highly conservative chromosome morphology and diploid number: 2n=54 m-sm, FN=108 for the majority of the species (Table 1). These traits, considered plesiomorphic for the family, were also evidenced in the species analyzed here in. According to Oliveira et al. (1988) and De Oliveira et al. (2009), this conservative chromosomal structure may be related to the ecological characteristics of these fishes, that is, high vagility and large shoal formation, allowing high rates of gene flow and genetic diversity (Landínez-García and Marquez, 2018). However, this apparent conservation is revealed when other cytogenetic markers, such as repetitive DNA sequences (e.g., ribosomal and telomeric) are applied.
Curimatids, in general, have a large amount of HC, and in Psectrogaster species for example, pericentromeric and terminal blocks were observed in several chromosome pairs (Figure 1). Beyond that, large heterochromatic blocks are often coincident or adjacent to the NORs, with interspecific and interpopulation differences, both in the number of loci (single or multiple NORs) and in the chromosomal location/ position in the karyotype (Table 1), as seen in the present study as well as in previous studies (Feldberg et al., 1992;Navarrete and Júlio-Júnior, 1997;Brassesco et al., 2004;Venere et al., 2008). These differences may be related to the repetitive and highly transcribed structure of rDNA, where the number of copies might vary owing to rearrangements of the chromosomal microstructure, such as duplications, translocations and/or inversions (Symonová et al., 2013;Goffová and Fajkus, 2021).
The mapping of the 18S rDNA sequence confirmed Ag-NOR in all species with an additional site in C. meyeri, similar situation also reported by Sampaio et al. (2016) in Steindachnerina biornata. This additional site might be related to the lack of transcriptional activity, which depends on cell activity , or simply associated with the presence of pseudogenic rDNA variants (Gong et al., 2021).
The 5S rDNA localization in interstitial region, ranging from two to four chromosomes, is a pattern found in most curimatids corroborated in the present study. However, markings in terminal chromosomal regions have also been reported in this family (Pinheiro et al., 2016;present study), again evidencing the occurrence of non-Robertsonian rearrangements in chromosome microstructure of these species.
The location of 18S and 5S rDNA in different chromosome pairs is a trait found in all curimatid species (Table 1). Interestingly, Curimatella dorsalis seems to be the first case to show synteny between these rDNAs in curimatids, which may have arisen independently during non-Robertsonian rearrangements (Symonová et al., 2013), demonstrating the dynamic nature of the 18S and 5S rDNA sites, prone to recombination events. Synteny between 18S and 5S rDNA is an atypical situation, including for the superfamily Anostomoidea (Anostomidae, Chilodontidae, Prochilodontidae and Curimatidae), which has been reported only in lineages derived of the Anostomidae (De Barros et al., 2017;Dulz et al., 2019), Prochilodontidae (Vicari et al., 2006;Terencio et al., 2012;Voltolin et al., 2013) and Curimatidae families (present study).
Chromosome mapping of telomeric sequences revealed a high degree of chromosome structure variation in Curimatella and Psectrogaster species, presenting ITSs in several chromosome pairs. ITS has been observed in several vertebrate species and is classified into short ITS (s-ITS) and heterochromatic ITS (Het-ITS) (Bolzán, 2017). In the case of the curimatids here analyzed, we classify the ITSs as Het-ITSs, since the signals are colocalized with heterochromatic blocks.
Many authors relate the presence of Het-ITSs to ancestral chromosomal fusion events and are generally associated with a reduction in diploid number (Meyne et al., 1990;Rosa et al., 2012;Schneider et al., 2013;Sember et al., 2015). Similarly, there are reports of Het-ITSs in species that present the conserved karyotype (Metcalfe et al., 2004;Di-Nizo et al., 2020), as observed in the present study, considering that 2n=54 is the ancestral diploid number for the whole superfamily Anostomoidea.
Thus, the appearance of these Het-ITSs may be related to other mechanisms, such as (1) occurrence of pericentric inversions or translocations with the insertion of s-ITSs, followed by amplification of these regions and subsequent heterochromatinization; (2) transpositions, mediated by transposable elements, which are internally reinserted into the chromosomes and undergo an amplification process; and, (3) telomeric sequences (TTAGGG) n would constitute the main repetitive motif of centromeric DNA, as observed in amphibians and marsupials (Meyne et al., 1990;Paço et al., 2012;Bolzán, 2017;Clemente et al., 2020).
Regardless of the mechanism that gave rise to Het-ITSs in the curimatids here analyzed, these sequences are an important component of the karyotype diversification. As observed in another genus of Curimatidae, in Potamorhina ITSs are involved in multiple chromosomal fissions in the ancestor of the species P. latior (2n=56, 18 pairs with ITS), P. altamazonica (2n=102), and P. squamoralevis (2n=102) (Pinheiro et al., 2016), as suggested in molecular phylogeny of Dorini et al. (2020). Thus, the Het-ITSs present in Curimatella and Psectrogaster can signal the presence of "hot spots" for the occurrence of recombination, which according to Bolzán (2017), can lead to new karyotypes and even new species.
Thus, despite the conservative diploid number for most species of the Curimatidae (2n=54), our data highlights a high level of variation in repetitive DNA sequences among species, suggesting that additional integrative analyzes, involving the mapping of other repetitive sequences classes as well as investigation in other species/populations of curimatids, will produce a more complete picture of the chromosomal evolution of this family.