Extensive Karyotype Reorganization in the Fish Gymnotus arapaima (Gymnotiformes, Gymnotidae) Highlighted by Zoo-FISH Analysis

The genus Gymnotus (Gymnotiformes) contains over 40 species of freshwater electric fishes exhibiting a wide distribution throughout Central and South America, and being particularly prevalent in the Amazon basin. Cytogenetics has been an important tool in the cytotaxonomy and elucidation of evolutionary processes in this genus, including the unraveling the variety of diploid chromosome number (2n = from 34 to 54), the high karyotype diversity among species with a shared diploid number, different sex chromosome systems, and variation in the distribution of several Repetitive DNAs and colocation and association between those sequences. Recently whole chromosome painting (WCP) has been used for tracking the chromosomal evolution of the genus, showing highly reorganized karyotypes and the conserved synteny of the NOR bearing par within the clade G. carapo. In this study, painting probes derived from the chromosomes of G. carapo (GCA, 2n = 42, 30 m/sm + 12 st/a) were hybridized to the mitotic metaphases of G. arapaima (GAR, 2n = 44, 24 m/sm + 20 st/a). Our results uncovered chromosomal rearrangements and a high number of repetitive DNA regions. From the 12 chromosome pairs of G. carapo that can be individually differentiated (GCA1–3, 6, 7, 9, 14, 16, and 18–21), six pairs (GCA 1, 9, 14, 18, 20, 21) show conserved homology with GAR, five pairs (GCA 1, 9, 14, 20, 21) are also shared with cryptic species G. carapo 2n = 40 (34 m/sm + 6 st/a) and only the NOR bearing pair (GCA 20) is shared with G. capanema (GCP 2n = 34, 20 m/sm + 14 st/a). The remaining chromosomes are reorganized in the karyotype of GAR. Despite the close phylogenetic relationships of these species, our chromosome painting studies demonstrate an extensive reorganization of their karyotypes.


INTRODUCTION
Gymnotus (Gymnotiformes) is a monophyletic genus of freshwater electric fishes (Albert, 2001;Lovejoy et al., 2010;Tagliacollo et al., 2016) distributed throughout South America . 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.
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.
To find out the corresponding segments between GAR and GCA (2n = 42), we used dual-color FISH with probes from R3 and R4. The other non-hybridized chromosomes or segments correspond to R1 (GAR 19, Milhomem et al., 2013) or R2. For a more refined identification of the chromosomes from R2, R3 and R4, we employed dual-color FISH using probes from the subregions as specified in Table 2 of Nagamachi et al. (2010),  Table 1). It was included only species with know phylogenetic relationships, based on data from Albert et al. (2005) and Tagliacollo et al. (2016). G. capanema was included in the G. carapo clade based on Milhomem et al. (2012a), but has unclear place within the clade.  with some modifications related to the identification of the chromosomes of S3B made in Nagamachi et al. (2013). With those experiments (as illustrated in Figure 2) it was possible to identify individually GCA pairs 1-3, 6, 7, 9, 14, 16, and 18-21, while it was not possible to distinguish the pairs [4,8], [10,11], [5, 17], and [12, 13, 15].
at 37 • C. The probes were prepared following Nagamachi et al. (2010), denatured for 15 min at 70 • C and applied onto a slide with chromosomes that were previously denatured at 70 • C for 4 min in 70% formamide/2× SSC [pH 7.0]. The hybridization lasted 72 h at 37 • C. The slides were washed once in a solution of 50% formamide/2× SSC, once in 2× SSC and once in 4× Tween, 5 min each. The dual-color FISH experiments were made with probes that were either directly labeled or biotinylated detected with avidin, (Vector Laboratories, Burlingame, CA, USA) linked to Cy3 or FITC (Amersham, Piscataway, NJ, United States). DAPI (4 ′ ,6-diamidino-2-phenylindole) was used as a counterstain.

Microscopy and Image Processing
Image acquisition was made using the software Nis-elements in the microscope Nikon H550S. Chromosomes were morphologically classified according to Levan et al. (1964). The karyotype was organized according to Milhomem et al. (2012b).

RESULTS
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).

DISCUSSION
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(Milhomem et al., , 2012a. Whole chromosome probes from GCA 2n = 42 have been used for comparative genomic mapping (CGM) of the karyotype FIGURE 3 | Haploid karyotype of G. arapaima (GAR) arranged from mitotic chromosomes after dual-color hybridization with probes derived from Region 3 (R3, red) and Region 4 (R4, green) from the Gymnotus carapo (GCA) chromosome complement. Regions R1 and R2 were not subjected to FISH analysis and, therefore, the equivalent homeologous parts on GAR chromosomes are DAPI-stained (blue) only. For each of the 22 GAR chromosome pairs, the DAPI-only stained homolog is depicted on the left, while the dual-color FISH hybridization pattern is present on the right. The correspondence to G. carapo (GCA) homeologous chromosomes is indicated by chromosome pair numbers on the left side of the DAPI-stained GAR chromosomes, while the correspondence to the particular GCA regions (R1-4) is indicated on the right side of FISH-painted chromosomes. *Repetitive sequences.
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 , 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.

AUTHOR CONTRIBUTIONS
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.