Gymnotus (Gymnotiformes) is a monophyletic genus of freshwater electric fishes (Albert, 2001; Lovejoy et al., 2010; Tagliacollo et al., 2016) distributed throughout South America (Albert et al., 2005). It represents the most specious genus (40 species; Ferraris et al., 2017) and the widest distribution in the order, with prevalence in the Amazon basin, where several species of Gymnotus co-occur in sympatry (Albert and Crampton, 2003; Crampton et al., 2005).

Based on the integrated data from DNA sequencing of six genes, coupled with 223 morphological characters and with Model-Based Total Evidence phylogenetic analyses, Tagliacollo et al. (2016) divided the genus into six clades: G. pantherinus, G. coatesi, G. anguillaris, G. tigre, G. cylindricus, and G. carapo. The Gymnotus carapo group is regarded as monophyletic and is located in a derived position within the genus (Albert, 2001; Lovejoy et al., 2010; Tagliacollo et al., 2016). Craig et al. (2017) described seven subspecies for G. carapo.

Cytogenetics has been an important tool in cytotaxonomy and has proved to be very useful in understanding the evolutionary processes behind the diversification of Gymnotus. The Gymnotiformes order has considerable variation, not only in diploid number (from 2n = 24 in Apteronotus albifrons, Howell, 1972; Almeida-Toledo et al., 1981; Mendes et al., 2012; to 2n = 74 in Rhabdolichops cf eastward, Suárez et al., 2017) but also in the karyotype formula and location of repetitive sequences (Fernandes et al., 2005; Almeida-Toledo et al., 2007; Silva et al., 2009; da Silva et al., 2013; Jesus et al., 2016; Araya-Jaime et al., 2017; Batista et al., 2017; Sousa et al., 2017; Takagui et al., 2017). Recently, fluorescence in situ hybridization (FISH), has played an important role in understanding the genome structure of fish species (Yi et al., 2003; Cabral-de-Mello and Martins, 2010; Martins et al., 2011; Vicari et al., 2011; Gornung, 2013; Knytl et al., 2013; Yano et al., 2017) and molecular cytogenetic studies in Gymnotiformes have shown dynamic reorganization, including pericentric inversions observed through repetitive DNA position (Fernandes et al., 2017), sequence dispersion via transposable elements and the association between different repetitive sequences (Utsunomia et al., 2014; da Silva et al., 2016; Machado et al., 2017) and the presence of different sex chromosome systems (Margarido et al., 2007; Henning et al., 2008, 2011; da Silva et al., 2011, 2014; Almeida et al., 2015). This evolutionary plasticity of the karyotype is seen in Gymnotus (Table 1), a genus that has high interspecific variability in chromosome numbers (Figure 1, Table 1), ranging from 2n = 34 in Gymnotus capanema (Milhomem et al., 2012a) to 2n = 54 in G. carapo (Foresti et al., 1984), G. mamiraua (Milhomem et al., 2007), G. paraguensis (Margarido et al., 2007) and G. inaequilabiatus (Scacchetti et al., 2011). Gymnotus arapaima is located within the G. carapo clade, with 2n = 44 (24 m/sm + 20 st/a; Milhomem et al., 2012b).

Whole chromosome painting (WCP) techniques use specific painting probes of whole chromosomes, chromosomes arms or chromosome regions to find homologous segments in other species (Yang and Graphodatsky, 2017) and Nagamachi et al. (2010) produced whole chromosome probes from G. carapo (GCA, 2n = 42) by chromosome sorting using flow cytometry and made a comparative genomic map against the chromosomal background of the cytotype with 2n = 40 chromosomes. The results uncovered a high degree of chromosomal repatterning between these cytotypes, with only eight pairs showing conserved synteny (GCA 1, 2, 6, 9, 14, 19, 20, 21). Nagamachi et al. (2013) used the same set of probes for G. capanema (GCP, 2n = 34) and the results showed that the degree of genomic reorganization was much higher, with only four pairs (GCA 6, 7, 19, 20) showing conserved synteny with GCA 2n = 42 and three pairs (GCA 6, 19, 20) with GCA 2n = 40. Of these, GCA 7 and 19 are associated with other chromosomes in the karyotype of GCP. The study of Milhomem et al. (2013), with the probe derived from the NOR bearing par of GCA, 2n = 42, shows that there is a possible synapomorphy of the NOR bearing par within the G. carapo clade.

We use the same set of probes produced by Nagamachi et al. (2010) to analyze the karyotype of G. arapaima and to compare the results with our previous studies of species in the genus Gymnotus. Our findings confirm and extend our understanding of the extensive karyotype reorganization within this genus.

The whole chromosome probes from G. carapo were hybridized to chromosomes of G. arapaima. The regions of homology (hereafter designated as R1-4) obtained with GCA (2n = 42) probes against the chromosomes of GAR are indicated on the karyotype of GAR arranged from DAPI-stained chromosomes (Figure 3). Dual color FISH with the probes of R3 (red) and R4 (green) defined the chromosome groups in GAR that corresponded to the four groups of regions in GCA (Figure 3), as R3 and R4 do not share chromosome pairs. Any chromosome segments hybridizing simultaneously with two colors indicate repetitive DNA sequences that are common to both regions. The chromosomes or segments in blue (DAPI) represent the NOR-bearing chromosomes (R1, GCA20) and the chromosomes corresponding to R2 (pairs 1–3 and 16). Table 2 shows the correspondence of the GCA (2n = 42) chromosomes with the previously published karyotypes of GCA (2n = 40) and GCP (2n = 34), and GAR (2n = 44, present study).

From the 12 chromosome pairs of G. carapo that can be individually differentiated (GCA 1–3, 6, 7, 9, 14, 16, and 18–21), six pairs (GCA 1, 9, 14, 18, 20, 21) have conserved homology within GAR. GCA 20 hybridizes to one whole chromosome, pair 19, as described by Milhomem et al. (2013). Six chromosome pairs (GCA 2, 3, 6, 7, 16, and 19) show two signals on GAR chromosomes.

The GCA probes that represent two chromosome pairs [4, 8] revealed two signals, and pairs [10, 11] and [5, 17] revealed three signals and the probe representing three pairs [12, 13, 15] also revealed three signals on GAR chromosomes.

The following associations were found: GAR 4: [10, 11]/C/6, GAR 7: 19/C/[10, 11], GAR 13: [12, 13, 15]/C/ [12, 13, 15]/^*/3, GAR 14: ^*/C/16/^*/2, GAR 16: 7/C/ [5, 17]/6/ [5, 17] (where C = centromere and ^* = repetitive sequences).

Our results demonstrate that the genomic reorganization in the analyzed species of Gymnotus is greater than that assumed by classical cytogenetics (Milhomem et al., 2008, 2012a,b).

Whole chromosome probes from GCA 2n = 42 have been used for comparative genomic mapping (CGM) of the karyotype of (i) cryptic species GCA 2n = 40 (Nagamachi et al., 2010), (ii) GCP 2n = 34 (Nagamachi et al., 2013) and, in the present work, iii) onto the karyotype of GAR 2n = 44 (Figure 4). Similar to the observations in the two previously mapped species (Nagamachi et al., 2010, 2013), GAR also presents a highly reorganized karyotype (Figures 3, 4, Table 2) in relation to GCA 2n = 42 and also in relation to GCA 2n = 40 and GCP 2n = 34. From the 12 chromosome pairs of GCA 2n = 42 that can be individually differentiated (GCA 1–3, 6, 7, 9, 14, 16, 18–21), GAR shows conserved synteny of six pairs (GCA 1, 9, 14, 18, 20, 21); five pairs (GCA 1, 9, 14, 20, 21) with the cryptic species GCA 2n = 40 and only one pair with GCP (GCA 20). On the other hand, GCA 2n = 40 shares with GCA 2n = 42, eight pairs (GCA 1, 2, 6, 9, 14, 19, 20, 21) and with GCP, three pairs (GCA 6, 19, 20) (Figure 4). It is also worth noting that the probes representing GCA [4, 8] and [12, 13, 15] show two and three signals, respectively, in three species (GCA 42, GCP and GAR) indicating that these chromosomes may have retained their homology.

A comparative analysis of the WCP data described above shows that the karyotypes of both GCP and GAR are related to the karyotypes of GCA. GCP, although part of the carapo group (Milhomem et al., 2012a), has an uncertain position inside the phylogeny of the clade, while GCA and GAR are closely related. GCP and GAR do not share the same chromosome rearrangements (Table 2), meaning that these rearrangements must have occurred after their speciation. The results of the CGM suggest either a divergence prior to that of GAR or a recent divergence characterized by fast karyotype evolution and fixation of a high number of chromosomal rearrangements.

It is also clear that the karyotype of GAR is evolutionary closer to the GCA karyotype than to the GCP karyotype. However, GAR is located 2000 km away from the other species, while GCP and GCA (2n = 42) are 200 km apart (Figure 5). This might suggest that the karyotypes of GCA and GAR are more conserved while GCP changed over a shorter period of time. Another explanation for this huge differentiation of the GCP karyotype might lie in the fact that this species inhabits Rio Açaiteuazinho drainage from Northeast Para, which is not connected with the Amazon basin, while GCA and GAR are part of the same hydrographic basin, despite the long distance between them (Figure 5).

Freshwater fishes in general have a higher rate of chromosomal rearrangements than marine fishes due to the reduced flow with the natural barriers present in the freshwater environment compared to the open marine biome, with bigger populations and high potential for dispersion and higher gene flow, reducing the chance for karyotype changes to fixate in the population (Molina, 2007; Nirchio et al., 2014; Artoni et al., 2015). Lande (1977) theorizes that the rates of chromosomal rearrangement are proportional to selection and inversely proportional to the effective size of the population and Araya-Jaime et al. (2017) suggests that this could be considered a general model of chromosomal evolution within Gymnotiformes, since populations with little or no geneflow may facilitate the fixation of chromosomal rearrangements within a particular species in a shorter evolutionary time. This may be a contributory factor to speciation within the group and may also contribute to the higher number of rearrangements found. It is a valid reminder that the high number of rearrangements observed in the present study was possible through WCP, and groups with a more stable diploid number and karyotypic formula potentially could have fixed a higher number of rearrangements that did not cause major structural changes.

As Region 3 was labeled with a red fluorochrome and Region 4 with a green one, all yellow regions in Figure 3 are the result of hybridization of both probes to the same region. Although R3 and R4 do not share the same chromosome pair, they share the same or highly similar repetitive DNA. The hybridization of both probes to the same regions of GAR chromosomes confirms that this sequence is also present in this species. Since repetitive sequences evolve quickly by concerted evolution with significant differences between species (Pons and Gillespie, 2004), the presence of the highly similar repetitive DNA sequence in different species clearly shows that these species diverged recently, without sufficient time to accumulate sequence differences. Despite the huge amount of rearrangement, the repetitive DNA sequence strongly suggests that these species diverged recently and also that the rearrangements responsible for the karyotypic differences are also recent.

Taken together, the sum of the results might explain the difficulty in finding synapomorphies among the species compared so far, since most of the rearrangements might have become fixed after the species became isolated. On the other hand, because the G. carapo clade is a derived one (Tagliacollo et al., 2016, Figure 1) and because up until today there are few species of Gymnotus studied by chromosome painting, we currently cannot conclusively resolve whether the homologous chromosomes present a symplesiomorphic or synapomorphic character. An example is the NOR bearing pair that maps to GCA 20 using rDNA probes in species of the carapo group, but this location is different in species outside this group (Milhomem et al., 2013), which suggests that it is a synapomorphy. This matter will be better understood once species outside the carapo group are mapped with all the GCA whole chromosome probes.

MM, JP, FS, PO, MF-S, and CN: gave substantial contributions to the conception of the work; the acquisition, analysis, and interpretation of data for the work; participated in the draft of the work or revised it critically for important intellectual content; gave final approval of the version to be published; and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.