﻿Karyotype description and comparative chromosomal mapping of rDNA and U2 snDNA sequences in Eigenmannialimbata and E.microstoma (Teleostei, Gymnotiformes, Sternopygidae)

﻿Abstract The genus Eigenmannia Jordan et Evermann,1896 includes electric fishes endemic to the Neotropical region with extensive karyotype variability and occurrence of different sex chromosome systems, however, cytogenetic studies within this group are restricted to few species. Here, we describe the karyotypes of Eigenmannialimbata (Schreiner et Miranda Ribeiro, 1903) and E.microstoma (Reinhardt, 1852) and the chromosomal locations of 5S and 18S rDNAs (ribosomal RNA genes) and U2 snDNA (small nuclear RNA gene). Among them, 18S rDNA sites were situated in only one chromosomal pair in both species, and co-localized with 5S rDNA in E.microstoma. On the other hand, 5S rDNA and U2 snRNA sites were observed on several chromosomes, with variation in the number of sites between species under study. These two repetitive DNAs were observed co-localized in one chromosomal pair in E.limbata and in four pairs in E.microstoma. Our study shows a new case of association of these two types of repetitive DNA in the genome of Gymnotiformes.


Introduction
The order Gymnotiformes is an endemic freshwater group inhabiting the Neotropical region and consisting of species capable of emitting low voltage continuous electric discharges (Alves-Gomes 2001; Lavoué et al. 2012). Among them, Eigenmannia is the most species-rich genus of the family Sternopygidae (Gymnotiformes), with 27 recognized species (Fricke et al. 2021). On the other hand, Eigenmannia is not a monophyletic assembly, and it is considered taxonomically ambiguous due to little morphological variation between species, which makes it difficult to define species-specific diagnostic characters (Alves-Gomes 1998;Albert 2001;Peixoto and Ohara 2019).
The physical mapping of repetitive sequences in gymnotiform species has provided important data on the structure and organization of the genome that has allowed us to understand the processes of karyotypic evolution that these species have experienced, recognizing Robertsonian rearrangements as the most frequent mechanisms of chromosomal variability in Gymnotiformes Giora and Fialho 2009;Nagamachi et al. 2010;da Silva et al. 2014;Utsunomia et al. 2014Utsunomia et al. , 2018Suárez et al. 2017;Rodrigues et al. 2021). The mapping of ribosomal DNA genes (18S rDNA and 5S rDNA) has been widely used in molecular cytogenetics of Gymnotiformes, where the evidence provided by several studies has made it possible to establish two distribution patterns of these sequences: i) 18S rDNA loci located on a single chromosome pair and ii) 5S rDNA sites located in multiple chromosomal pairs, which may be associated with transposable elements or U2 snDNA (small nuclear RNA gene) sequences (Scacchetti et al. 2011(Scacchetti et al. , 2012Utsunomia et al. 2014;da Silva et al. 2016;Araya-Jaime et al. 2017b;Sochorová et al. 2018;Rodrigues et al. 2021).
Recently, the mapping of genes belonging to the U snDNA family increased the knowledge about the dynamics of tandemly repeated multigene families in vertebrates. This multigene family harbors genes coding for nine types of non-coding RNAs; namely U1, U2, U4, U4 atac, U5, U6, U6 atac, U11 and U12; which constitute a portion of the RNA-protein complex of the spliceosome (Valadkhan 2005;Matera and Wang 2014). In fish cytogenetics, the use of these repetitive markers is relatively recent, with data being reported for several groups, including Characiformes (Silva et al. 2015a;Santos et al. 2017;Serrano et al. 2017), Batrachoidiformes (Ubeda-Manzanaro et al.  (Xu et al. 2017), Cypriniformes (Sember et al. 2018), among others. In gymnotiform fish genomes, the cytogenetic reports of these sequences are restricted to U2 snDNA, recognizing two general chromosomal patterns: i) grouped in a single pair of chromosomes or ii) scattered throughout the genome and, in some cases, associated with 5S rDNA Araya-Jaime et al. 2017b). In this way, the U snDNA sequences represent a good repetitive marker to provide information on the evolutionary relations between closely related species, infer the homology between certain chromosomes present in different lineages, and trace the origin and evolution of specific chromosomes, in the context of the great karyotype diversity found among Gymnotiformes.
With the aim of expanding our knowledge about the chromosomal structure and the dynamics of repetitive DNA sequences in the Eigenmannia genome, we present for the first time the karyotype and chromosomal location of three repetitive DNA classes (18S and 5S rDNA and U2 snDNA) in E. microstoma and E. limbata from the Sao Francisco and the Amazon River basin, respectively. Our results show a new case of physical association between the 5S rDNA and U2 snDNA in Gymnotiformes.

Material and methods
Twelve individuals of Eigenmannia limbata and eight of E. microstoma, from the Amazon basin and the San Francisco River basin, respectively, were analyzed in this study ( Fig. 1). After dissection, the specimens were fixed and preserved in 70% ethanol. Finally, these specimens were deposited in the fish collection of the Laboratório de Biologia e Genética de Peixes, UNESP, Botucatu-SP. The animals were collected in accordance with Brazilian environmental protection legislation (Collection Permission MMA/IBAMA/SISBIO-number 3245) and the procedures for fish sampling, maintenance and analysis were performed in compliance with the Brazilian College of Animal Experimentation (COBEA) and approved (protocol 504) by the Bioscience Institute/ Unesp Ethics Committee on the use of Animals (CEUA).
Mitotic chromosomes were obtained by direct preparation from the cephalic kidney according to Foresti et al. (1993), and slides for conventional analysis were stained with 5% Giemsa solution in a phosphate buffer at pH 6.8. The constitutive het erochromatin (CH) was detected following Sumner (1972). Images were captured with a digital camera (Olympus DP90) in the Olympus BX6 epifluorescence photomicroscope and acquired using cellSens Dimension (Olympus, Sapporo-Japan). Image treatment, optimization of brightness and contrast was performed using the Adobe Photoshop CS6 program. The arm ratio (Levan et al. 1964) was used to classify the chromosomes as metacentric (m), submetacentric (sm), subtelocentric (st), and acrocentric (a). For counting the total number of chromosome arms or fundamental number (NF), chromosomes m, sm, st were considered bi-armed, while acrocentric chromosomes (or indistinguishable st/a) were classified as mono-armed chromosomes.

Results
The diploid chromosome number (2n) of the E. microstoma was 38 chromosomes, with a karyotype composed of 8m + 10sm + 20a chromosomes (NF = 56), while E. limbata had 2n = 38 and karyotype composed of 8m + 4sm + 26a chromosomes (NF = 50). Morphologically differentiated sex chromosomes were not found in either species (Table 1). C-banding technique revealed significant differences in the patterns of CH distribution between the analyzed species. Both species displayed pericentromeric regions of CH in all chromosomes and E. microstoma possessed additional interstitial blocks on several chromosomes (Fig. 2). The 18S rDNA site was located by FISH in a single chromosomal pair in both species, namely pair No. 10 in E. limbata and pair No. 14 in E. microstoma (Fig. 3). The 5S rDNA sites showed a considerable variation in the number and locations in analyzed species. These sites were detected in two chromosomal pairs in E. limbata and in 11 chromosomal pairs in E. microstoma (Fig. 3).
The distribution of the U2 snDNA sites was variable in terms of the number, chromosomal location, and number of co-localized sites with 5S rDNA between species. U2 snDNA sites were placed on three chromosomal pairs (11, 12 and 14) in E. limbata and in the chromosome pairs Nos 10, 12, 16 and 17 in E. microstoma (Fig. 4). These sites were co-localized with the 5S rDNA sites in the pair No. 14 in E. limbata and in all pairs in E. microstoma (Fig. 4). The location of all repetitive DNAs mapped by FISH is summarized in the ideogram presented in Fig. 5.

Discussion
The species E. limbata and E. microstoma were analyzed cytogenetically for the first time, showing the same 2n (38 chromosomes), but different NF and karyotypic  (Table 1). Previous cytogenetic studies in Eigenmannia have consistently reported this same 2n (38 chromosomes), but there are wide variations in terms of the reported karyotypic formula, NF and sex chromosome system (Almeida Toledo et al. 1984;Moysés et al. 2010;Henning et al. 2011;de Sene et al. 2014;Araya-Jaime et al. 2017b;Fernandes et al. 2020). These differences in karyotypic structure and NF can be explained by the occurrence of Robertsonian rearrangements, which may be participating as an important postzygotic reproductive isolation mechanism in Eigenmannia. This circumstance could be related to their low population sizes and low mobility, which would facilitate the fixation of chromosomal polymorphisms (Moysés et al. 2005(Moysés et al. , 2010Giora and Fialho 2009;Silva et al. 2009Silva et al. , 2015b. A single chromosome pair carrying the NOR has been reported for most of the species of the Sternopygidae family, although the chromosomal location of the NOR varies between species and populations; therefore, a simple NOR phenotype can be an ancestral feature in the genome of Sternopygidae (de Almeida-Toledo et al. 2001;dos Santos Silva et al. 2008;de Sene et al. 2014;Araya-Jaime et al. 2017b;Fernandes et al. 2020;Rodrigues et al. 2021). However, within Gymnotidae, the case of Gymonotus coatesi is reported as the only representative of this family with multiple 18S rDNA sites (Machado et al. 2017). Furthermore, a considerable variability has been observed in the size of the NOR region within Sternopygidae (dos Santos de Sene et al. 2014;Silva et al. 2015b;Fernandes et al. 2017;Rodrigues et al. 2021). Accordingly, we observed this NOR heteromorphism between E. limbata and E. microstoma, in which the NOR region of E. limbata is considerably larger than that of E. microstoma (Fig. 3). This could be a consequence of tandem duplication of ribosomal genes which could form through several mechanisms including unequal exchange of sister chromatids or unequal crossing over during meiosis (Charlesworth et al. 1994;Eickbush and Eickbush 2007;Bianciardi et al. 2012).
On the other hand, multiple 5S rDNA sites observed in E. limbata and E. microstoma (Fig. 3) appear to be a widely recognized feature within Gymnotiformes, with evidence in representatives of Gymnotus (da Silva et al. 2011;Scacchetti et al. 2011Scacchetti et al. , 2012Utsunomia et al. 2014;da Silva et al. 2016da Silva et al. , 2019, Eigenmannia (de Sene et al. 2014;Araya-Jaime et al. 2017b;Fernandes et al. 2020), Sternopygus (Fernandes et al. 2017) and Archolaemus (Rodrigues et al. 2021). Ribosomal DNA sites are considered as hot spots for chromosomal rearrangements due to their organization into long stretches of conserved tandemly repeated sequences and their high transcription activity, which means they are susceptible to chromosomal breakage and/ or non-allelic homologous recombination, increasing thus the probability of occurrence of chromosomal rearrangements, such as fusions, fissions and inversions (Rosa et al. 2012;Barros et al. 2017;Potapova and Gerton 2019;Warmerdam and Wolthuis 2019;Deon et al. 2020). Furthermore, the rDNA dynamics has been also correlated with the insertion of transposable elements, or other repetitive DNAs, into non-transcribed spacers (NTS) of 5S rDNA units, as has been observed in the genomes of G. inaequilabiatus (Scacchetti et al. 2012), G. paraguensis (da Silva et al. 2011) and G. mamiraua (da Silva et al. 2016. Thus, both mentioned mechanisms could explain the chromosomal dynamics of these sequences in gymnotiform genomes (de Sene et al. 2014;da Silva et al. 2016;Araya-Jaime et al. 2017b;Fernandes et al. 2017). In our case, given that 5S rDNA probe was prepared from the genomic DNA of E. microstomata in which we then revealed 22 signals, and that only four signals were evidenced in E. limbata, a possible explanation may be that the 300 bp long 5S rDNA fragment contains inserts of other repeats in its NTS region which might have promoted spreading of 5S rDNA clusters and/or generated additional non-5S rDNA signals in E. microstomata. In that case, only four signals in E. limbata might mean that the signal pattern is much less affected by the action and/or additional accumulation of the associated repeat(s). Although a single consistent PCR amplification product was obtained to be a template for the FISH probe preparation, thereby evidencing a lack of detectable amounts of 5S rDNA sequence variants or truncated copies, we cannot directly evaluate the possible presence and contribution of other repeats as we did not sequence the 5S rDNA fragment and consequently weren't looking for admixed repetitive sequences. We may, however, conclude that the chromosomal behavior of the 5S rDNA sites observed in this work is congruent with the patterns previously reported for Eigenmannia, such as the number of variable sites and their association with 18S rDNA and U2 snDNA clusters (de Sene et al. 2014;Araya-Jaime et al. 2017b).
The results presented here, for E. limbata and E. microstoma, represent the first case, within Eigenmannia, of multiple sites for U2 snDNA (Fig. 4), highlighting in E. microstoma the presence of three chromosomal pairs carrying U2 snDNA sites, where one of them (pair No 14) is co-localized with 5S rDNA, while in E. limbata, the four chromosomal pairs carrying U2 snRNA genes are co-localized with 5S rDNA. The previous report by Araya-Jaime et al. (2017b) described the karyotype of E. aff trilineata with a single U2 snDNA site being co-localized with 5S rDNA. These results reinforce the dynamic nature of these sequences and show that the 5S rDNA / U2 snRNA association would be a characteristic feature of the Eigenmannia genome. In other Gymnotiformes, six Gymnotus species are reported with a single U2 snRNA carrier pair, while only in G. pantal and Archolaemus janae, multiple sites for U2 snRNA have been reported Rodrigues et al. 2021). None of the species mentioned above exhibits co-localization between U2 snDNA with other sequences been reported.

Conclusion
In the present work, the cytogenetic analysis carried out in the species E. limbata and E. microstoma reinforced the chromosomal variability reported for the genus, evidencing the occurrence of notable differences between the karyotypes of the species / karyomorphs studied up to here, even though the 2n mostly observed is 2n = 38 chromosomes. The chromosomal location of the 5S and 18S rDNA clusters observed in the species studied here followed the same pattern observed in Gymnotiformes with a single NOR-bearing pair and multiple sites for 5S rDNA. On the other hand, the dynamic nature of the U2 snRNA sites stands out, together with the co-localization with 5S rDNA genes, as a characteristic feature of the Eigenmannia genome. Finally, the results presented here reinforce the postulate that cytogenetic features (conventional and molecular) could be considered as important markers for taxonomic diagnosis and for the description and characterization of the existing biodiversity in Gymnotiformes.