Multiple rearrangements in cryptic species of electric knifefish, Gymnotus carapo (Gymnotidae, Gymnotiformes) revealed by chromosome painting

Background Gymnotus (Gymnotidae, Gymnotiformes) is the Neotropical electric fish genus with the largest geographic distribution and the largest number of species, 33 of which have been validated. The diploid number varies from 2n = 39-40 to 2n = 54. Recently we studied the karyotype of morphologically indistinguishable samples from five populations of G. carapo sensu stricto from the Eastern Amazon of Brazil. We found two cytotypes, 2n = 42 (30 M/SM + 12 ST/A) and 2n = 40 (34 M/SM + 6 ST/A) and we concluded that the differences between the two cryptic species are due to pericentric inversions and one tandem fusion. Results In this study we use for the first time, whole chromosome probes prepared by FACS of the Gymnotus carapo sensu strictu species, cytotype with 2n = 42. Using two color hybridizations we were able to distinguish pairs 1, 2, 3, 7, 9, 14, 16, 18, 19, 20 and 21. It was not possible to separate by FACS and distinguish each of the following chromosome pairs even with dual color FISH: {4,8}; {10,11}; {5,6,17}; {12,13,15}. The FISH probes were then used in chromosome painting experiments on metaphases of the 2n = 40 cytotype. While some chromosomes show conserved synteny, others are rearranged in different chromosomes. Eight syntenic associations were found. Conclusions These results show that the karyotype differences between these cryptic species are greater than assumed by classical cytogenetics. These data reinforce the previous supposition that these two cytotypes are different species, despite the absence of morphological differences. Additionally, the homology of repetitive DNA between the two provides evidence of recent speciation.

Research using chromosome painting in fishes is unusual. It has been used only with probes made by microdissection [13][14][15][16][17][18][19] or by CGH, the latter without defining chromosome pairs [20]. The probes made by flow cytometry have higher complexity than those made by microdissection, and are more appropriate for crossspecies hybridization [21]. However, there are no reports in the literature of FACS (Fluorescent Activated Chromo-some Sorting) generated probes for chromosome painting involving a whole fish genome.
The chromosome structure of a fish, a cold-blooded vertebrate, lacks both GC-rich and GC-poor compartments. The absence of compartmentalization of their genomes may be the reason for failure to obtain good Gbands (for revision, see [22]). Also, this could explain the difficulty in getting whole chromosome-specific probes by FACS. As well, fish chromosomes are small and there is not enough difference in size to allow the separation of each individual pair. An additional difficulty is to obtain fibroblast cultures and chromosome preparations of high quality and with sufficient metaphases.
Gymnotus (Gymnotidae -Gymnotiformes) is the Neotropical electric knifefish genus with the largest geographic distribution, occurring from southern Mexico to northern Argentina [23]. It is also the most diverse known Gymnotiformes genus, with 33 validated species, of which 18 are known to occur in the Amazon basin [24,25,23,26]. Previous cytogenetic studies show that the diploid number in this genus ranges from 2n = 39-40 (with sex chromosomes of the type X 1 X 2 Y) to 2n = 54, exhibiting variation in the karyotype formula, the quantity of heterochromatin, and the position of the Nucleolar Organization Region [27][28][29][30].
Gymnotus carapo (holotype from Suriname) was described by Linneaus in the first half of the XVIII century (Albert & Crampton, 2003). Currently it is defined as G. carapo sensu stricto and is understood as a complex of morphologically similar or cryptic species, with a large distribution area (Albert & Crampton, 2003). Cytogenetic studies of samples identified as G. carapo show different karyotypes: 2n = 54 and 2n = 52 from Southern Brazil, 2n = 48 in Amazonas, 2n = 42 in Pará (reviewed [30]). Recently we studied the karyotype of morphologically indistinguishable individuals from five populations of G. carapo sensu stricto from the Eastern Amazon of Brazil. We found two cytotypes, 2n = 42 (30 M/SM + 12 ST/A) and 2n = 40 (34 M/SM + 6 ST/A) and we concluded that the differences between these two cryptic species are due to pericentric inversions and one tandem fusion [31].
In this study we were able to use, for the first time, whole chromosome probes prepared by FACS from a fish, Gymnotus carapo sensu stricto species, with 2n = 42 cytotype. These probes were used in hybridizations on metaphases of the cytotype with 2n = 40, to determine the differences between the karyotypes of the two cryptic species.
A primary fibroblast cell line was established from an electric knifefish Gymnotus carapo sensu stricto, cytotype 2n = 42, from Santa Cruz do Arari (Marajo Island). Whole chromosome probes were made from this cell line at the Department of Veterinary Medicine, University of Cambridge, UK. The chromosome specific probes were made by degenerate oligonucleotide primed PCR (DOP-PCR) on flow-sorted chromosomes as described previously [32,10]. Briefly, the chromosomes were prepared as described and stained with Hoechst 33258 (2 mg/mL) and Chromomycin A3 (40 mg/mL) in the presence of magnesium sulfate (2.5 mmol/L) for 2 h. Sodium sulfate (25 mmol/L) and sodium citrate (10 mmol/L) were added 15 min prior to flow sorting. Chromosome sorting was performed using a dual-laser cell sorter (FACStar Plus; Becton Dickinson Immuno-Cytometry Systems). About 400 chromosomes were sorted from each peak in the flow karyotypes. Chromosomes were sorted directly into PCR tubes containing 30 μL distilled water. These samples were amplified by DOP-PCR using the primer 6 MW [32]. Primary PCR products were labelled either with biotin-16-dUTP (Boehringer Mannheim), Fluorescein isothiocyanate (FITC)-12-dUTP (Amersham) or Cy3-dUTP, by taking 1 μL of product to a second round of DOP-PCR using the same primer. The biotin probes were detected with avidin-Cy3 or avidin-Cy5.
In-situ hybridization of painting probes was performed as previously described [10]. Briefly, 14 μL of the hybridization mixture (50% formamide, 1 × SSC, 10% dextran sulfate, 5 mg salmon sperm DNA) and 1 μL of labeled PCR product were denatured at 65°C for 1 min. In-situ hybridization was performed for 48-72 h at 37°C. The hybridization signal was detected as described earlier [10]. After hybridization and washing of the slides, biotinylated chromosome paints were detected with avidin (Vector Laboratories) coupled with Cy3, CY5 or FITC (Amersham). Probes directly linked to fluorochromes were also used, especially for dual or multi-FISH experiments. DAPI was used as a counterstain. In England, FISH digital images were obtained using a cooled CCD camera (Photometrics NU200 series equipped with a Kodak KAF 1400 chip) coupled to a Zeiss Axiophot microscope. The software Smart Capture VP (Digital Scientific) was used for camera control, digital image acquisition and the merging of DAPI and the fluorochrome images of the paints. In Brazil, digital images were captured with a CCD camera AxioCam Mrm coupled to a Zeiss Axiophot microscope, using Axiovision 3.0 Software (Zeiss). The false color attribution was processed using Axiovision and the brightness and contrast corrected with Adobe Photoshop 7.1.

Results
Flow sorting of Gymnotus carapo sensu stricto, cytotype 2n = 42: Characterization and chromosome identification.   Table 1. Apart from the homologous sequences, the probes also hybridized to non-specific regions, mainly in centro-meric, interstitial, pericentromeric areas and/or the arms of some chromosomes, including the NOR. These areas have highly repetitive DNA.

Discussion
Gymnotus carapo sensu stricto with 2n = 42 from Marajó Island (Pará-Brazil) is the first fish whose genome was used to generate FACS whole chromosome-specific probes covering the whole karyotype. This is a landmark in the cytogenetic studies of the family Gymnotidae (Gymnotiformes) and the fish in general.
The genome organization of fishes, with its small-sized chromosomes and non-compartmentalization of the chromosomes in GC-or AT-rich sequences (for review, see [22]), makes it difficult to isolate single chromosomes  Table 2.
The mapping of these probes in the 2n = 40 G. carapo cytotype shows surprising and very interesting results. Using classical cytogenetics we supposed that the differences between these karyotypes resulted from a tandem fusion, to explain the diploid number difference, and several pericentric inversions to explain the differences in the karyotypic formula [31]. However, the cross-species FISH made clear that genomic rearrangements were much more frequent than supposed by classical cytogenetics. From the original 21 chromosome pairs of the 2n = 42 G. carapo cytotype, only seven chromosomes (pairs 1,2,9,14,19,20,21) have their synteny conserved in the 2n = 40 cytotype. The remaining chromosomes which were individually identified (pairs 3, 7, 16, 18) are all involved in rearrangements with other chromosomes (Figure 7, Table 3). From the chromosomes that remained in groups, the {4,8} group of chromosomes paint a single chromosome (pair 10) and two other chromosomal segments (6q distal and 9q); the {5,6,17} group of chromosomes paint two single chromosomes (pairs 4 and 11) and  two segments (8q and 18q distal); the {10,11} group of chromosomes paint two segments (5p+q proximal and 12q); the {12,13,15} group of chromosomes paint chromosomes 13 and 16 and two other chromosomal segments (3q and 12p).
These results show the great importance of comparative genomic mapping using FACS generated whole chromosome-specific probes. In cold-blooded vertebrates like fishes, whose chromosomes do not have a G-banding pattern, the number of rearrangements that differentiate karyotypes is likely to be underestimated. Probably many species with similar 2n and fundamental numbers have many rearrangements, like translocations, that do not change the diploid number and chromosome morphology.
The large genomic reorganization found between the two population cytotypes of Gymnotus carapo sensu stricto here studied confirms the hypothesis that they are really different species. The fact that the external morphology, the meristic data and their pigmentation do not allow their distinction [31], suggests that their speciation has been a recent event, in which chromosomal reorganization had a major role.
The paint probes from the 2n = 42 genome also hybridize to highly repetitive DNA regions in the 2n = 40 genome, indicating that this DNA is homologous in both species. Normally, satellite DNA has great sequence divergence between closely related species, despite having great similarity within the repeats of one species [34][35][36]. The homology of this DNA in both species is additional evidence for recent speciation.
The production of FACS whole chromosome probes from other fishes will be important for understanding chromosomal evolution in vertebrates. Certainly this approach is revealing a wealth of new data in these organisms.

Conclusion
The whole chromosome probes in Gymnotus carapo sensu stricto with 2n = 42 here described and obtained by FACS were used in a cross-species experiment. The chromosome painting demonstrated the large genomic reorganization found between the two cytotypes.