**Abstract**

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.

**Keywords:** FISH, Biogeography, Satellite DNA, rDNA 5S, *C*0*t-1*

## **1. Introduction**

Repetitive DNA sequences are broadly distributed in eukaryotes genomes [1] 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 [2].

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 [5–7].

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 [14]. 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 [15]. 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 [16].

Neotropical electric fish species, order Gymnotiformes, have shown their heterochromatin to be preferentially distributed in the pericentromeric regions of their chromosomes [13, 17, 18].

The investigation of repetitive sequences in *Gymnotus* seems promising for understanding the chromosomal evolutionary dynamics in the genus. DNA probes and chromosomal painting were applied to investigate chromosomal rearrangements in two species of the *G. carapo* complex, and rearrangements were found between the two species, involving several pairs of chromosomes, corroborating the existence of cryptic species in this group, in addition to the recent speciation between them [19, 20]. Analysis of the satellitome of some species by Next Generation Sequencing (NGS) revealed sets of conserved satellite sequences, but the CA, GA and GAG motifs when mapped revealed a useful band for identification of homologous chromosomes [21].

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 [22]. 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 *G. carapo* with a diploid number of 54 chromosomes, which have from 14 to 19 pairs identified with this sequence [21]. This situation is totally different from the pattern observed for other species within the group and shared only by species carrying 2n = 54 chromosomes.

## **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].

**17**

**Figure 1.**

*represented schematically along with each species.*

*Composition and Nature of Heterochromatin in the Electrical Fish (Knifefishes)…*

The Gymnotiformes order comprises electric fish or knifefishes. Endemic to the Neotropical region [28], which are widely distributed, from the Pampas in Argentina to Chiapas, Mexico and reach their greatest diversity and abundance in the Amazon basin [29–31]. 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

Regarding the karyotype, *Gymnotus* is also a diverse group, with a diploid number ranging from 34 chromosomes, verified in *G*. *capanema* [33] to 54, found in several species of the *G. carapo* group [18, 21, 23–34]. Sexual chromosomal heteromorphism has already been recorded in some, and all records follow a turnover

Among the *Gymnotus*, an intriguing fact has been reported for species that have 2n = 54 chromosomes, present in the *carapo* Clade: *G*. cf. *carapo*, *G*. *inequilabiatus*, *G. paraguensis* and *G. mamiraua*. These species share the diploid number and also a large number of 5S rDNA sites [13], but they are geographically separated in different hydrographic basins. Though, *G*. cf. *carapo*, *G*. *inequilabiatus*, *G. paraguensis* occur in the Paraná-Paraguay basin, and *G. mamiraua* is distributed in the Amazon basin (**Figure 1**). The number of 5S DNAr sites in *G. paraguensis*, *G. inaequilabiatus* and *G*. cf. *carapo* are 19, 17 and 15 bearing pairs, respectively [36–38], and 14 pairs

For *G. paraguensis* and *G. mamiraua*, the 5S rDNA are present in the pericentromeric region and when sequenced, a transposition element similar to Tc1 was identified in the NTS (non-transcribed spacer), suggested as one of the mechanisms used for the transposition of this rDNA in chromosomes [22, 36]. In *G*. *inequilabiatus*, which has 17 sites of 5S rDNA, the sequencing of some clones with classes I and II revealed sequences of the TATA type in NTS, which are associated

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

*Location of species with 2n = 54 chromosomes in the Amazon and Paraná-Paraguay basins: a)* G. mamiraua *in the Amazon basin; b)* G. carapo*; c)* G. paraguensis *and d)* G. inaequilabiatus*. The pairs with 5S rDNA are* 

model, with little heterochromatin and of recent origin [24, 35].

with the regulation of the expression of the 5S rDNA gene [37].

*DOI: http://dx.doi.org/10.5772/intechopen.97673*

brackish or salt water [21].

in *G. mamiraua* [22].

*Composition and Nature of Heterochromatin in the Electrical Fish (Knifefishes)… DOI: http://dx.doi.org/10.5772/intechopen.97673*

The Gymnotiformes order comprises electric fish or knifefishes. Endemic to the Neotropical region [28], which are widely distributed, from the Pampas in Argentina to Chiapas, Mexico and reach their greatest diversity and abundance in the Amazon basin [29–31]. 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 [21].

Regarding the karyotype, *Gymnotus* is also a diverse group, with a diploid number ranging from 34 chromosomes, verified in *G*. *capanema* [33] to 54, found in several species of the *G. carapo* group [18, 21, 23–34]. Sexual chromosomal heteromorphism has already been recorded in some, and all records follow a turnover model, with little heterochromatin and of recent origin [24, 35].

Among the *Gymnotus*, an intriguing fact has been reported for species that have 2n = 54 chromosomes, present in the *carapo* Clade: *G*. cf. *carapo*, *G*. *inequilabiatus*, *G. paraguensis* and *G. mamiraua*. These species share the diploid number and also a large number of 5S rDNA sites [13], but they are geographically separated in different hydrographic basins. Though, *G*. cf. *carapo*, *G*. *inequilabiatus*, *G. paraguensis* occur in the Paraná-Paraguay basin, and *G. mamiraua* is distributed in the Amazon basin (**Figure 1**). The number of 5S DNAr sites in *G. paraguensis*, *G. inaequilabiatus* and *G*. cf. *carapo* are 19, 17 and 15 bearing pairs, respectively [36–38], and 14 pairs in *G. mamiraua* [22].

For *G. paraguensis* and *G. mamiraua*, the 5S rDNA are present in the pericentromeric region and when sequenced, a transposition element similar to Tc1 was identified in the NTS (non-transcribed spacer), suggested as one of the mechanisms used for the transposition of this rDNA in chromosomes [22, 36]. In *G*. *inequilabiatus*, which has 17 sites of 5S rDNA, the sequencing of some clones with classes I and II revealed sequences of the TATA type in NTS, which are associated with the regulation of the expression of the 5S rDNA gene [37].

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

#### **Figure 1.**

*Location of species with 2n = 54 chromosomes in the Amazon and Paraná-Paraguay basins: a)* G. mamiraua *in the Amazon basin; b)* G. carapo*; c)* G. paraguensis *and d)* G. inaequilabiatus*. The pairs with 5S rDNA are represented schematically along with each species.*

*Cytogenetics - Classical and Molecular Strategies for Analysing Heredity Material*

and has structural functions [5–7].

heterochromatin regions [16].

their chromosomes [13, 17, 18].

of homologous chromosomes [21].

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

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 [14]. 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 [15]. The mapping of Non-long terminal repeat (non-LTR) retrotransposable elements, the Rex in the fish species, for example, demonstrated strong FISH signals in

Neotropical electric fish species, order Gymnotiformes, have shown their heterochromatin to be preferentially distributed in the pericentromeric regions of

The investigation of repetitive sequences in *Gymnotus* seems promising for understanding the chromosomal evolutionary dynamics in the genus. DNA probes and chromosomal painting were applied to investigate chromosomal rearrangements in two species of the *G. carapo* complex, and rearrangements were found between the two species, involving several pairs of chromosomes, corroborating the existence of cryptic species in this group, in addition to the recent speciation between them [19, 20]. Analysis of the satellitome of some species by Next Generation Sequencing (NGS) revealed sets of conserved satellite sequences, but the CA, GA and GAG motifs when mapped revealed a useful band for identification

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 [22]. 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 *G. carapo* with a diploid number of 54 chromosomes, which have from 14 to 19 pairs identified with this sequence [21]. This situation is totally different from the pattern observed for other species within the group and shared only by species carrying 2n = 54 chromosomes.

**2. Biogeography of electric fish and repetitive DNA sequences**

to enrichment and contact of organisms [26, 27].

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

**16**

chromosomes would facilitate the process of pseudogenization and the association with transposable elements [39].

Although the mechanism of origin and dispersion of these sequences in the 2n = 54 *Gymnotus* genome is still unclear, it is possible to assume that the increase in the number of 5S-like rDNA sites and fixation in the genome accompanies the migratory dispersion of these species. In the case of *Gymnotus* (2n = 54), infection by this transposable element may have probably occurred in a common ancestor for species with 2n = 54 chromosomes, before the final isolation between the Amazon and Paraná-Paraguay basins, since these species are currently found in different river basins. *Gymnotus mamiraua* was restricted to the Amazon basin show the smallest number of sites, 14 pairs, maybe the original satellite sequence like-5S rDNA; however, the isolated species in the Paraná-Paraguay basin experienced divergence from this satellite sequence, differing in number pairs with sequence, such as: *G. inaequilabiatus* (17 pairs), *G. paraguensis* (19 pairs), and *G. carapo* (15 pairs), (**Figure 2**) [13, 22].

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 *Prochilodus* are considered as indicators of this panorama, where the relationship of sister clades of that genus has been useful for understanding the separation of the basins [Magdalena (Orinoco (Amazonas-Paraná)]. The use of molecular clock methodologies, with species in the group, estimates that the isolation between the Amazonas-Paraná basins must have occurred between 2.3–4.1 million years, with an estimated coalescence between strains of 1–3-3 million years [40]. Morphological and molecular analyzes with rheophilic taxa increase the evidence of connection between these basins [41]. The final rupture of the connection between the Paraná-Paraguay and Amazonas-Orinoco basins is inferred as a recent event, with the final uplift of the Andes that changed the course of these basins and isolated them [25, 42], with final separation estimates still uncertain and very peculiar to each group of fish investigated [43].

#### **Figure 2.**

*Topology obtained by the method of clustering neighbors (neighbor joining, NJ) for species of* Gymnotus *of the* carapo *clade, using the distance model Kimura-2-parameters (K2P) for the mitochondrial gene COI. The values present in the nodes are the bootstrap values (> 50%) calculated from 1000 replicates and the diploid number superimposed on the tree. The distribution of species is indicated on the map. \* absence of coordinates, group not plotted on the map.*

**19**

*Composition and Nature of Heterochromatin in the Electrical Fish (Knifefishes)…*

In order to understand the relationships between *Gymnotus*, here, an analysis of a 556 bp (base pairs) COI mitochondrial gene fragment was conducted in 21 specimens from the *carapo* Clade from the central Amazon basin and four more specimens from a population in the Munim River, MA. Sequences deposited in the NCBI database were added to this analysis, with the access numbers described in **Table 1**. The analysis of 51 sequences resulted in eight clades (**Figure 2**). The data revealed a

**Species Voucher Locality NCBI accession number**

*G. sylvius* LBP7069 Alto Paraná GU.064995.1 *G. sylvius* LBP31958 Alto Paraná GU.701779.1 *G. sylvius* LBP7070 Alto Paraná GU.702209.1 *G. sylvius* LBP8831 Alto Paraná GU.7017821 *G. sylvius* LBP27380 Alto Paraná GU.701778.1 *G. sylvius* LBP29096 Alto Paraná GU.702207.1 *G. sylvius* LBP25852 Alto Paraná GU.701758.1 *G. sylvius* LBP31933 Alto Paraná GU.701767.1 *G. sylvius* LBP25853 Alto Paraná GU.701762.1 *G. sylvius* LBPV27382 Alto Paraná JN.988881.1 *G. sylvius* LBPV27381 Alto Paraná JN.988880.1 *G. sylvius* LBP9823 Alto Paraná GU.701780.1 *G. pantanal* MZUEL5644 Alto Paraná KF.359492.1 *G. pantanal* LBP31929 Alto Paraná GU.701776.1 *G. pantanal* LBP34742 Alto Paraná GU.701775.1 *G. pantanal* LBP34743 Alto Paraná GU.701774.1 *G. pantanal* LBP31927 Alto Paraná GU.701773.1 *G. pantanal* LBP31932 Alto Paraná GU.701763.1 *G. carapo* Mara GC7M Bacia Costeira- Maranhão XXXXXXXXX *G. carapo* Mara GC9M Bacia Costeira- Maranhão XXXXXXXXX *G. carapo* Mara GC11M Bacia Costeira- Maranhão XXXXXXXXX *G. carapo* Mara GC18M Bacia Costeira- Maranhão XXXXXXXXX *G. carapo* Cat GCAT11029 Amazônia Central XXXXXXXXX *G. carapo* Cat GCAT 11028 Amazônia Central XXXXXXXXX *G. carapo* Cat GCAT 11066 Amazônia Central XXXXXXXXX *G. carapo* Cat GCAT 11477 Amazonia Central XXXXXXXXX *G. carapo* GC2006 Peru Amazonas KF533344 *G. carapo* GC2007 Peru, Amazonas KF533345.1 *G. mamiraua* GM11078 Amazônia Central XXXXXXXXX *G. mamiraua* GM11733 Amazônia Central XXXXXXXXX *G. mamiraua* GM11731 Amazônia Central XXXXXXXXX *G. mamiraua* GM10947 Amazônia Central XXXXXXXXX *G. mamiraua* GM10948 Amazônia Central XXXXXXXXX *G. mamiraua* GM10949 Amazônia Central XXXXXXXXX *G. mamiraua* GM11730 Amazônia Central XXXXXXXXX

*DOI: http://dx.doi.org/10.5772/intechopen.97673*

#### *Composition and Nature of Heterochromatin in the Electrical Fish (Knifefishes)… DOI: http://dx.doi.org/10.5772/intechopen.97673*

In order to understand the relationships between *Gymnotus*, here, an analysis of a 556 bp (base pairs) COI mitochondrial gene fragment was conducted in 21 specimens from the *carapo* Clade from the central Amazon basin and four more specimens from a population in the Munim River, MA. Sequences deposited in the NCBI database were added to this analysis, with the access numbers described in **Table 1**. The analysis of 51 sequences resulted in eight clades (**Figure 2**). The data revealed a


*Cytogenetics - Classical and Molecular Strategies for Analysing Heredity Material*

with transposable elements [39].

chromosomes would facilitate the process of pseudogenization and the association

between the Amazonas-Paraná basins must have occurred between 2.3–4.1 million years, with an estimated coalescence between strains of 1–3-3 million years [40]. Morphological and molecular analyzes with rheophilic taxa increase the evidence of connection between these basins [41]. The final rupture of the connection between the Paraná-Paraguay and Amazonas-Orinoco basins is inferred as a recent event, with the final uplift of the Andes that changed the course of these basins and isolated them [25, 42], with final separation estimates still uncertain and very

*Topology obtained by the method of clustering neighbors (neighbor joining, NJ) for species of* Gymnotus *of the* carapo *clade, using the distance model Kimura-2-parameters (K2P) for the mitochondrial gene COI. The values present in the nodes are the bootstrap values (> 50%) calculated from 1000 replicates and the diploid number superimposed on the tree. The distribution of species is indicated on the map. \* absence of coordinates,* 

peculiar to each group of fish investigated [43].

*Gymnotus* genome is still unclear, it is possible to assume that the increase in the number of 5S-like rDNA sites and fixation in the genome accompanies the migratory dispersion of these species. In the case of *Gymnotus* (2n = 54), infection by this transposable element may have probably occurred in a common ancestor for species with 2n = 54 chromosomes, before the final isolation between the Amazon and Paraná-Paraguay basins, since these species are currently found in different river basins. *Gymnotus mamiraua* was restricted to the Amazon basin show the smallest number of sites, 14 pairs, maybe the original satellite sequence like-5S rDNA; however, the isolated species in the Paraná-Paraguay basin experienced divergence from this satellite sequence, differing in number pairs with sequence, such as: *G. inaequilabiatus* (17 pairs), *G. paraguensis* (19 pairs), and *G. carapo* (15 pairs), (**Figure 2**) [13, 22]. 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 *Prochilodus* are considered as indicators of this panorama, where the relationship of sister clades of that genus has been useful for understanding the separation of the basins [Magdalena (Orinoco (Amazonas-Paraná)]. The use of molecular clock methodologies, with species in the group, estimates that the isolation

Although the mechanism of origin and dispersion of these sequences in the 2n = 54

**18**

**Figure 2.**

*group not plotted on the map.*


#### **Table 1.**

*Access numbers in the NCBI of the* Gymnotus *species of the* carapo *clade used for genetic distance verification through DNA barcode.*

high percentage for the distance between the species identified as *G. carapo* (**Table 1**, **Figure 2**). The sequences of individuals identified as *G. carapo* do not form exclusive groups. Three clades with high rates of genetic distance were observed between *G. carapo* "Catalão" (2n = 40, XX/XY) and *G. carapo* Maranhão (2n = 42), but between *G. carapo* "Catalão" and *G. carapo* do the distance between Amazonas and Peru was 0.02% (**Table 2**). The species *G. mamiraua* forms an exclusive clade with an average intraspecific genetic distance of 0.12% and *G. inaequilabiatus* the average was 0.2% (**Table 3**). These two species showed an interspecific distance of only 0.6%.

The results with the mitochondrial DNA COI obtained here validate the species of *carapo* Clade, with high rates of genetic distance between groups of species identified as *G. carapo*, confirming the probable existence of more than one taxonomic unit in this group. However, this did not occur for two species: *G. mamiraua* from the Amazon basin and *G. inaequilabiatus* from Alto Paraná, for which a low interspecific distance was detected and with a distance of less than 1% was detected, possibly causing recurrent speciation.

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


**21**

*Composition and Nature of Heterochromatin in the Electrical Fish (Knifefishes)…*

**Gp- 1 Gp-2 Gp- 3 Gp- 4 Gp- 5 Gp- 6 Gp- 7**

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 *G. paraguensis and G. carapo* (2n = 54) from Alto Paraná are also related to the last two already mentioned (**Figure 2**). Thus, what has been verified is that the 5S rDNA has been a potential tool in helping to reconstruct the steps involved in the evolution and biogeographic history of the species of the genus *Gymnotus*, especially the *carapo* Clade, inferred both by the chromosomal mapping and by the molecular analysis of that gene.

*Matrix of the means of genetic distance (K2P) for the COI gene obtained among the different species of* 

Gp 7 *G*. *inaequilabiatus* 0.046 0.074 0.178 0.053 0.051 **0.006**

Gp 8 *G*. *ucamara* 0.179 0.159 0.051 0.183 0.181 0.178 0.174

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 *Gymnotus*, the latter has been associated with heterochromatin. The location of the heterochromatin is reported to be preferentially organized in the centromeric and pericentromeric regions [18, 19, 36, 44]. 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 [45]. 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 [46] and animals [47] to significantly expand our

In the present study, repetitive DNA sequences were isolated by C0t-1 (**Figure 3**) and mapped in two species of electric fish, *G. paraguensis* and *G. sylvius*. Our objective was identifying the heterochromatin compositions and to verify the presence of repetitive sequences originated from transposable elements. Twelve specimens (four females and eight males) of *G. paraguensis* and 21 specimens (seven females and 14 males) of *G. sylvius* collected at Piquiri River, Paraná – Brazil were analyzed. The isolated probes from *G. paraguensis* had lengths of 473 and 206 bp (**Figure 4a**) and when submitted to BLAST (http://www.ncbi.nlm.nih.gov/ blast), clone 2 was found to have 95% identity with microsatellites from *Salmo salar*. Dinucleotide repetitions were observed in this clone. The clones of *G. sylvius* had lengths of 124, 202 and 123 bp (**Figure 4b**). The results of hybridization with total C0t-1 in both species were coincident with heterochromatic sites,

The heterochromatin of *Gymnotus* has been reported to be preferentially organized in the centromeric regions [18, 19], as detected in the present study

**2.1 New repetitive sequences studies in the genus** *Gymnotus*

knowledge of the organization of their chromosomes.

according to the description by C banding [18].

*DOI: http://dx.doi.org/10.5772/intechopen.97673*

Gp 2 *G*. *Pantanal* 0.062

Gp 3 *G*. *carapo* "Maranhão" 0.168 0.156

Gp 4 *G*. *carapo* "Catalão" 0.030 0.069 0.174

Gp 5 *G*. *carapo* 0.028 0.071 0.171 0.002

Gp 6 *G*. *mamiraua* 0.048 0.077 **0.181** 0.055 0.053

**Interspecific diversity**

Gymnotus *from* carapo *clade.*

Gp 1 *G*. *sylvius*

**Table 3.**

#### **Table 2.**

*Intraspecific genetic distance based on mutations of the COI gene, using the K-2-P model.*

*Composition and Nature of Heterochromatin in the Electrical Fish (Knifefishes)… DOI: http://dx.doi.org/10.5772/intechopen.97673*


**Table 3.**

*Cytogenetics - Classical and Molecular Strategies for Analysing Heredity Material*

**Species Voucher Locality NCBI accession number**

*G. inaequilabiatus* MZUEL5649 Alto Paraná KF.359490.1 *G. inaequilabiatus* LBP26331 Alto Paraná GU.701766.1 *G. inaequilabiatus* LBP31931 Alto Paraná GU.701764.1 *G. inaequilabiatus* LBP25850 Alto Paraná GU.701760.1 *G. inaequilabiatus* LBP7071 Alto Paraná GU.702210.1 *G. inaequilabiatus* LBP29097 Alto Paraná GU.702208.1 *G. inaequilabiatus* LBP34744 Alto Paraná GU.701781.1 *G. ucamara* GU11575 Amazônia Central XXXXXXXXX *G*. *ucamara* GU11802 Amazônia Central XXXXXXXXX *G*. *ucamara* GU11701 Amazônia Central XXXXXXXXX *G*. *ucamara* GU11698 Amazônia Central XXXXXXXXX *G*. *ucamara* GU11574 Amazônia Central XXXXXXXXX

high percentage for the distance between the species identified as *G. carapo* (**Table 1**, **Figure 2**). The sequences of individuals identified as *G. carapo* do not form exclusive groups. Three clades with high rates of genetic distance were observed between *G. carapo* "Catalão" (2n = 40, XX/XY) and *G. carapo* Maranhão (2n = 42), but between *G. carapo* "Catalão" and *G. carapo* do the distance between Amazonas and Peru was 0.02% (**Table 2**). The species *G. mamiraua* forms an exclusive clade with an average intraspecific genetic distance of 0.12% and *G. inaequilabiatus* the average was 0.2%

*Access numbers in the NCBI of the* Gymnotus *species of the* carapo *clade used for genetic distance verification* 

(**Table 3**). These two species showed an interspecific distance of only 0.6%.

Gp 1 *- G. sytlvius* 0.013526931 Gp 2 - *G. Pantanal* 0 Gp 3 - *G*. *carapo* "Maranhão" 0.000893724 Gp 4 - *G*. *carapo* "Catalão" 0.000928937 Gp 5 - *G carapo* 0.001858738 Gp 6 - *G*.*mamiraua* 0.001241471 Gp 7 - *G*. *inaequilabiatus* 0.002136221 Gp 8 - *G*. *ucamara* 0.003103677

*Intraspecific genetic distance based on mutations of the COI gene, using the K-2-P model.*

possibly causing recurrent speciation.

**Intraspecific genetic distance**

The results with the mitochondrial DNA COI obtained here validate the species of *carapo* Clade, with high rates of genetic distance between groups of species identified as *G. carapo*, confirming the probable existence of more than one taxonomic unit in this group. However, this did not occur for two species: *G. mamiraua* from the Amazon basin and *G. inaequilabiatus* from Alto Paraná, for which a low interspecific distance was detected and with a distance of less than 1% was detected,

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

**20**

**Table 2.**

**Table 1.**

*through DNA barcode.*

*Matrix of the means of genetic distance (K2P) for the COI gene obtained among the different species of*  Gymnotus *from* carapo *clade.*

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 *G. paraguensis and G. carapo* (2n = 54) from Alto Paraná are also related to the last two already mentioned (**Figure 2**). Thus, what has been verified is that the 5S rDNA has been a potential tool in helping to reconstruct the steps involved in the evolution and biogeographic history of the species of the genus *Gymnotus*, especially the *carapo* Clade, inferred both by the chromosomal mapping and by the molecular analysis of that gene.

#### **2.1 New repetitive sequences studies in the genus** *Gymnotus*

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 *Gymnotus*, the latter has been associated with heterochromatin. The location of the heterochromatin is reported to be preferentially organized in the centromeric and pericentromeric regions [18, 19, 36, 44].

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 [45]. 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 [46] and animals [47] 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, *G. paraguensis* and *G. sylvius*. Our objective was identifying the heterochromatin compositions and to verify the presence of repetitive sequences originated from transposable elements. Twelve specimens (four females and eight males) of *G. paraguensis* and 21 specimens (seven females and 14 males) of *G. sylvius* collected at Piquiri River, Paraná – Brazil were analyzed.

The isolated probes from *G. paraguensis* had lengths of 473 and 206 bp (**Figure 4a**) and when submitted to BLAST (http://www.ncbi.nlm.nih.gov/ blast), clone 2 was found to have 95% identity with microsatellites from *Salmo salar*. Dinucleotide repetitions were observed in this clone. The clones of *G. sylvius* had lengths of 124, 202 and 123 bp (**Figure 4b**). The results of hybridization with total C0t-1 in both species were coincident with heterochromatic sites, according to the description by C banding [18].

The heterochromatin of *Gymnotus* has been reported to be preferentially organized in the centromeric regions [18, 19], as detected in the present study

#### **Figure 3.**

*1% agarose gel electrophoresis of DNA treated for kinetic re-association method (C0t – 1). a) after one minute in autoclave the DNA appears as a trail; b) after treatment with S1 nuclease enzyme the DNA appears with defined length between 100 and 400 bp. (methodology described in complementary material).*


#### **Figure 4.**

*a) Alignment of two isolated clones of* G. paraguensis*, b) alignment of three clones of* G. sylvius*.*

(**Figure 5a** and **b**), and inmost species of the Gymnotidae family [36, 44]. Furthermore, heteromorphisms in length between homologous chromosomes of the NOR regions were observed in *G. paraguensis*, indicating differential accumulations of heterochromatin regions (**Figure 5b**). However, when the probes of one species were hybridized with the other (cross-FISH) no positive hybridization signals were observed (data not shown).

According to Charlesworth et al. [48] and Topp and Dave [49], 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 *Oreochromis niloticus* using the GISH (Genomic *in situ* Hybridization) methodology, the heterochromatin present in pericentromeric regions was found to be species-specific [50] and composed of repetitive and transposable elements [51–53].

**23**

are not yet known.

*organizer regions in pair 25. Bar = 10* μ*m.*

**Figure 5.**

telomeric regions.

*Composition and Nature of Heterochromatin in the Electrical Fish (Knifefishes)…*

The repetitive elements isolated from *G. sylvius* and *G. paraguensis* in the present study were located in the pericentromeric region, coincident with the heterochromatin observed by C banding. High levels of specificity of the isolated probes and of the species' genomes were assumed because no signals were observed in crossed FISH analyses - confirming the heterogeneous composition of the heterochromatin of these species. Sequencing analyses showed exclusive sequences for both species, and although repetitive elements in the heterochromatin regions are present in distant eukaryotes groups such as *Drosophila* and plants [54, 55] as important structural regions of the genome, the structural functions of these sequences in *G. sylvius* and *G. paraguensis*

*Fluorescent* in situ *hybridization (FISH) performed with probes isolated for kinetic re-association of DNA (C0t–1). a)* G. sylvius *karyotype; b)* G. paraguensis *karyotype. Observe in b the heteromorphism of nucleolus* 

The Y chromosome of *Eigenmannia virescens* isolated by Henning et al. [56] had large amounts of heterochromatin and physical mapping with Y chromosome probe was performed with closely related species without differentiated sex chromosome systems, and the probes hybridized only in the centromeric and

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 [57] isolated the repetitive sequences of *G. sylvius* and *G. carapo* by enzymatic digestion with *Alu*I and *Hae*III. The isolated fragments with 300 bp showed dispersed distributions in both the species and similar locations using both enzymes. Furthermore, a transposable SINE element that labeled different regions in *G. carapo* was identified; all the sequences showed disperse labeling that was not coincident with heterochromatin regions, suggesting an important function in the

More recently, the publication of Satellitome results has been awaited, a global study by NGS and bioinformatics of all satellite sequences of *Gymnotus* species from the main clades. According to previous data released by the authors, the massive characterization of satellite DNA families processes of genetic differentiation and the dynamics of these sequences among the representatives stand out [59], which

evolution and organization of non-codifying DNA regions [58].

corroborates our findings in the present work.

*DOI: http://dx.doi.org/10.5772/intechopen.97673*

*Composition and Nature of Heterochromatin in the Electrical Fish (Knifefishes)… DOI: http://dx.doi.org/10.5772/intechopen.97673*

#### **Figure 5.**

*Cytogenetics - Classical and Molecular Strategies for Analysing Heredity Material*

*1% agarose gel electrophoresis of DNA treated for kinetic re-association method (C0t – 1). a) after one minute in autoclave the DNA appears as a trail; b) after treatment with S1 nuclease enzyme the DNA appears with* 

*defined length between 100 and 400 bp. (methodology described in complementary material).*

(**Figure 5a** and **b**), and inmost species of the Gymnotidae family [36, 44]. Furthermore, heteromorphisms in length between homologous chromosomes of the NOR regions were observed in *G. paraguensis*, indicating differential accumulations of heterochromatin regions (**Figure 5b**). However, when the probes of one species were hybridized with the other (cross-FISH) no positive hybridiza-

*a) Alignment of two isolated clones of* G. paraguensis*, b) alignment of three clones of* G. sylvius*.*

According to Charlesworth et al. [48] and Topp and Dave [49], 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 *Oreochromis niloticus* using the GISH (Genomic *in situ* Hybridization) methodology, the heterochromatin present in pericentromeric regions was found to be species-specific [50] and composed of repetitive and

tion signals were observed (data not shown).

transposable elements [51–53].

**22**

**Figure 3.**

**Figure 4.**

*Fluorescent* in situ *hybridization (FISH) performed with probes isolated for kinetic re-association of DNA (C0t–1). a)* G. sylvius *karyotype; b)* G. paraguensis *karyotype. Observe in b the heteromorphism of nucleolus organizer regions in pair 25. Bar = 10* μ*m.*

The repetitive elements isolated from *G. sylvius* and *G. paraguensis* in the present study were located in the pericentromeric region, coincident with the heterochromatin observed by C banding. High levels of specificity of the isolated probes and of the species' genomes were assumed because no signals were observed in crossed FISH analyses - confirming the heterogeneous composition of the heterochromatin of these species.

Sequencing analyses showed exclusive sequences for both species, and although repetitive elements in the heterochromatin regions are present in distant eukaryotes groups such as *Drosophila* and plants [54, 55] as important structural regions of the genome, the structural functions of these sequences in *G. sylvius* and *G. paraguensis* are not yet known.

The Y chromosome of *Eigenmannia virescens* isolated by Henning et al. [56] had large amounts of heterochromatin and physical mapping with Y chromosome probe was performed with closely related species without differentiated sex chromosome systems, and the probes hybridized only in the centromeric and telomeric regions.

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 [57] isolated the repetitive sequences of *G. sylvius* and *G. carapo* by enzymatic digestion with *Alu*I and *Hae*III. The isolated fragments with 300 bp showed dispersed distributions in both the species and similar locations using both enzymes. Furthermore, a transposable SINE element that labeled different regions in *G. carapo* was identified; all the sequences showed disperse labeling that was not coincident with heterochromatin regions, suggesting an important function in the evolution and organization of non-codifying DNA regions [58].

More recently, the publication of Satellitome results has been awaited, a global study by NGS and bioinformatics of all satellite sequences of *Gymnotus* species from the main clades. According to previous data released by the authors, the massive characterization of satellite DNA families processes of genetic differentiation and the dynamics of these sequences among the representatives stand out [59], which corroborates our findings in the present work.
