Access numbers in the NCBI of the
Fishes of the genus Gymnotus have been suggested as a good model for biogeographic studies in the South American continent. In relation to heterochromatin, species of this genus have blocks preferably distributed in the centromeric region. The content of these regions has been shown to be variable, with description of transposable elements, pseudogenes of 5S rDNA and satellite sequences. In G. carapo Clade, although geographically separated, species with 2n = 54 chromosomes share the distribution of many 5S rDNA sites, a unique case within the genus. Here, repetitive DNA sequences from G. sylvius (2n = 40) and G. paraguensis (2n = 54) were isolated and mapped to understand their constitution. The chromosome mapping by FISH showed an exclusive association in the centromeres of all chromosomes. However, the cross-FISH did not show positive signs of interspecific hybridization, indicating high levels of heterochromatic sequence specificity. In addition, COI-1 sequences were analyzed in some species of Gymnotus, which revealed a close relationship between species of clade 2n = 54, which have multiple 5S rDNA sites. Possibly, the insertion of retroelements or pseudogenization and dispersion of this sequence occurred before the geographic dispersion of the ancestor of this clade from the Amazon region to the hydrographic systems of Paraná-Paraguay, a synapomorphy for the group.
- Satellite DNA
- rDNA 5S
Repetitive DNA sequences are broadly distributed in eukaryotes genomes  and are classified into two categories: 1) repetitive sequences arranged in tandem as satellite, minisatellite, or microsatellite DNAs composed of hundreds of base pairs repeated a thousand times or more in each genome; and 2) moderately to highly repetitive sequences spread throughout the genome as retroelements or transposable mobile elements .
Copies of repetitive sequences are commonly associated with heterochromatin regions that can be visualized by C banding. These sequences are extremely important to the functional and structural organization of the eukaryote genome, composing, for example, the pericentromeric heterochromatin regions [3, 4]. The heterochromatin in fish chromosomes is largely located in pericentromeric regions and has structural functions [5, 6, 7].
The studies about the location of repetitive sequences on chromosomes has broadened the knowledge of the structural organization of chromatin in fish, revealing an association of ribosomal DNA, telomeric sequences, transposition elements and satellite sequences in chromosomal rearrangements and weak break points [8, 9], in the fixation of sex chromosomes [10, 11], in the expansion of heterochromatin [12, 13] and in gene regulation . This advances in molecular cytogenetics have demonstrated that repetitive DNA sequences are useful as chromosomal markers in studies of species evolution and can provided valuable information about sex chromosome systems and chromosomal rearrangements . The mapping of Non-long terminal repeat (non-LTR) retrotransposable elements, the Rex in the fish species, for example, demonstrated strong FISH signals in heterochromatin regions .
The investigation of repetitive sequences in
The ribosomal DNA mapping can also provide new answers about chromosomal evolution in the genus, and even serve as a tool for understanding geographic distribution patterns . In the species that had the 18S rDNA mapped, the proposal of only one pair carrying the conserved nucleolus organizing regions (NORs) is plausible [17, 18, 23, 24]. Regarding the 5S rDNA, the group behaves as an attractive model for evolutionary studies, showing a species-specific pattern. The evolution of this gene family receives special attention in the species that comprise the group
2. Biogeography of electric fish and repetitive DNA sequences
The complex history of the formation of the South American rivers is fundamental to explain the diversity and distribution of aquatic biota in this region. Successive continental geomorphological changes, such as the one that resulted in the formation of the Andes, associated with historic and biological factors allow the identification of patterns that led to the formation of the largest and most diverse freshwater ichthyofauna on the planet [25, 26]. Such changes alter the drainage scenario forming lakes, capture headwaters and basins of varying sizes, or even isolate populations for certain periods, favoring the diversification of biota by vicariance and allopatric, in addition to promoting subsequent drainage coalescence, leading to enrichment and contact of organisms [26, 27].
The Gymnotiformes order comprises electric fish or knifefishes. Endemic to the Neotropical region , which are widely distributed, from the Pampas in Argentina to Chiapas, Mexico and reach their greatest diversity and abundance in the Amazon basin [29, 30, 31]. Members of the Gymnotiformes order, are unique in their ability to produce and recognize electrical signals, never left the South America plate, since their electrosensory system is not capable of functioning in brackish or salt water .
Regarding the karyotype,
As a rule, studies investigating this ribosomal gene associate an increase in the number of 5S rDNA sites with the prese-nce of pseudogenes, which would originate through duplication of copies, resulting from the transposition or duplication of genomic DNA, which would be facilitated by the organization tandem of this rDNA family. In addition, locating these sequences in the terminal portion of the chromosomes would facilitate the process of pseudogenization and the association with transposable elements .
Although the mechanism of origin and dispersion of these sequences in the 2n = 54
Evidence of the connection between South American watersheds is reflected in the evolutionary history of the fish. Other species corroborate the hypothesis of interconnection between these two systems. Migratory species of the genus
In order to understand the relationships between
|Species||Voucher||Locality||NCBI accession number|
|GC7M||Bacia Costeira- Maranhão||XXXXXXXXX|
|GC9M||Bacia Costeira- Maranhão||XXXXXXXXX|
|GC11M||Bacia Costeira- Maranhão||XXXXXXXXX|
|GC18M||Bacia Costeira- Maranhão||XXXXXXXXX|
|GCAT 11028||Amazônia Central||XXXXXXXXX|
|GCAT 11066||Amazônia Central||XXXXXXXXX|
|GCAT 11477||Amazonia Central||XXXXXXXXX|
|Intraspecific genetic distance|
|Gp 1 ||0.013526931|
|Gp 2 - ||0|
|Gp 3 - ||0.000893724|
|Gp 4 - ||0.000928937|
|Gp 5 - ||0.001858738|
|Gp 6 - ||0.001241471|
|Gp 7 - ||0.002136221|
|Gp 8 - ||0.003103677|
|Gp- 1||Gp-2||Gp- 3||Gp- 4||Gp- 5||Gp- 6||Gp- 7|
|Gp 1 |
|Gp 2 ||0.062|
|Gp 3 ||0.168||0.156|
|Gp 4 ||0.030||0.069||0.174|
|Gp 5 ||0.028||0.071||0.171||0.002|
|Gp 6 ||0.048||0.077||0.055||0.053|
|Gp 7 ||0.046||0.074||0.178||0.053||0.051|
|Gp 8 ||0.179||0.159||0.051||0.183||0.181||0.178||0.174|
The results with the mitochondrial DNA COI obtained here validate the species of
Cytogenetic data already pointed to the proximity between these two species, because in addition to sharing the same diploid number, both had many sites of 5S rDNA, a condition that is not common in fish. We suggest, by the results of diploid number and dispersion of 5S rDNAr, that the species
2.1 New repetitive sequences studies in the genus
The eukaryote genome is characterized by presenting nucleotide sequences with varied arrangements, generally forming two large groups, gene regions and repetitive DNA sequences. In fish of the genus
The prospection of repetitive sequences by the technique of DNA reassociation kinetics (C0t-1) proves to be a safe and fast technique for obtaining copies of highly and moderately repetitive DNA sequences . Thus, it is possible to build libraries and screening repetitive DNAs, and has been used to isolate the highly repeated fraction of the plant genome  and animals  to significantly expand our knowledge of the organization of their chromosomes.
In the present study, repetitive DNA sequences were isolated by C0t-1 (Figure 3) and mapped in two species of electric fish,
The isolated probes from
The heterochromatin of
According to Charlesworth et al.  and Topp and Dave , the regions located nearest to the centromere show fast evolutionary rates due to low recombination, initiating the accumulation of repetitive DNA sequences, which explains its specificity. This association between heterochromatin and repetitive sequences is fundamental to the organization of important chromosomal structures such as the centromere. In a study with
The repetitive elements isolated from
Sequencing analyses showed exclusive sequences for both species, and although repetitive elements in the heterochromatin regions are present in distant eukaryotes groups such as
The Y chromosome of
In addition to the two species analyzed in the present study, two other species of Gymnotiformes have had their repetitive DNA sequences analyzed and described. Claro  isolated the repetitive sequences of
More recently, the publication of Satellitome results has been awaited, a global study by NGS and bioinformatics of all satellite sequences of
Molecular studies with the multigene family 5S rDNA in electric fish (Gymnotidae) have advanced a lot in recent years. The chromosomal location and distribution have been particularly interesting, since all species of the genus
The current study was supported in part by INCT ADAPTA II funded by CNPq - Brazilian National Research Council (465540/2014-7). The authors are grateful to Miguel Airton Carvalho for his field assistance. The author M.D.S. received a scholarship from the
Conflict of interest
The authors declare no conflict of interest.
A.1 Methodology for obtaining cytochrome oxidase sequences
For the Cytochrome oxidase I gene, five specimens of
The calculation of intra and interspecific distance was performed using the Mega 5 software  with the Kimura-2-parameters evolutionary model. The identification was carried out according to the protocol established by DNA barcoding through the Neighbor Joining (NJ) analysis , which consists of looking for the tree with the lowest total sum of branches, using Kimura-2-parameters as an evolutionary model (K2P) . The topology confidence test was performed with bootstrap analysis, containing 1000 replicates. Such analyzes were performed using the Mega 5 software .
A.2 Methodology for obtaining cytogenetic material
Mitotic chromosomes were obtained according to the protocol described by Bertollo et al. . Genomic DNA extraction was performed using the chloroform-phenol method . Repetitive DNA probes were obtained using the
The sequences were aligned and edited using the CLUSTAL W program  using the following parameters: weights 6.66 and 10.0 for opening and extension of gaps, respectively, for pairwise alignments, and weights 10.0 and 15.0 for opening and extension of gaps in the multiple alignments respectively. Clustering analysis was performed using the parsimony method on the PAUP program v. 4.0b10 . The isolated fragments of repetitive DNA sequences (
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