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.

Cytogenetic studies in Eigenmannia virescens (Sternopygidae, Gymnotiformes) and new inferences on the origin of sex chromosomes in the Eigenmannia genus.

BACKGROUND: Cytogenetic studies were carried out on samples of Eigenmannia virescens (Sternopygidae, Gymnotiformes) obtained from four river systems of the Eastern Amazon region (Para, Brazil). RESULTS: All four populations had 2n = 38, with ZZ/ZW sex chromosomes (Z, acrocentric; W, submetacentric). Constitutive heterochromatin (CH) was found at the centromeric regions of all chromosomes. The W chromosome had a heterochromatic block in the proximal region of the short arm; this CH was positive for DAPI staining, indicating that it is rich in A-T base pairs. The nucleolar organizer region (NOR) was localized to the short arm of chromosome pair 15; this result was confirmed by fluorescent in situ hybridization (FISH) with human 45S rDNA, and CMA3 staining indicated that the region is G-C rich. FISH with telomeric probes did not show any evidence of interstitial telomeric sequences (ITS). CONCLUSION: Previous studies have shown that the species Eigenmannia sp. 2 and E. virescens have differentiated sex chromosomes, and diverse sex chromosome systems have been described for E. virescens specimens obtained from different Brazilian rivers. A comparative analysis of the present data and prior reports suggests that the sex chromosomes of Eigenmannia may have arisen independently in the different populations.

Chromosomal evidence for a putative cryptic species in the Gymnotus carapo species-complex (Gymnotiformes, Gymnotidae).

BACKGROUND: In this study we examined the karyotypes of morphologically indistinguishable populations of the electric knifefish Gymnotus carapo sensu stricto from the Eastern Amazon of Brazil. These were identified unambiguously on the basis of external morphology, meristics, and pigmentation. RESULTS: Specimens from one of five localities exhibited a karyotype previously not documented for Gymnotus species in the Amazon basin: 2n = 40 (34M/SM+6ST/A). Samples from the other four localities exhibited a different karyotype: 2n = 42 (30M/SM+12ST/A), which we had previously described. Specimens from all five localities presented constitutive heterochromatin in the centromeric region of almost all chromosomes, including in the distal and interstitial regions. Staining with 4'6-Diamidino-2-phenylindole revealed C-positive banding. In both karyotypes the Nucleolar Organizer Region (NOR) was located on the short arm of pair 20, and Chromomycin A3 stained the NORs. Fluorescent in situ hybridization with telomeric probes showed an Interstitial Telomeric Sequence (ITS) in the proximal short arm of a metacentric pair in the 2n = 40 karyotype. CONCLUSION: The difference between the two karyotypes on the diploid number and chromosome morphology can be explained by rearrangements of the fusion-fission type and also by pericentric inversions. The presence of ITS in a metacentric pair of the 2n = 40 karyotype suggests that the difference in the diploid number of the karyotypes results from a fusion. The consistent 2n = 42 karyotype at four localities suggests an interbreeding population. However, because fusion-fission and pericentric inversions of this nature typically result in reproductive isolation, we speculate that the form with the 2n = 40 karyotype is a different species to that of the 2n = 42 form. Nonetheless, we did not observe evident differences in external morphology, meristics and pigmentation between the two forms, which suggest that they represent cryptic sympatric species in the G. carapo species complex. We speculate that the chromosomal speciation occurred recently, allowing insufficient time for the fixation of other differences following post-zygotic isolation.

Highest Diploid Number Among Gymnotiformes: First Cytogenetic Insights into Rhabdolichops (Sternopygidae).

We report the first comparative cytogenetic analysis of two species from electrogenic fish of genus Rhabdolichops (Sternopygidae, Gymnotiformes): Rhabdolichops troscheli and Rhabdolichops cf eastwardi. R. troscheli has 2n = 54 (fundamental number [FN] = 66), whereas R. cf. eastwardi has 2n = 74 (FN = 78). C-banding revealed centromeric constitutive heterochromatin in both species. Ag-NORs mapped on pair 6 in R. troscheli and pair 30 in R. cf eastwardi. Fluorescense in situ hybridization with 18S rDNA probes confirmed the Ag-NOR staining results and revealed additional (presumably silent) ribosomal genes on pairs 12, 13, 21, 23, 26, and 27 in R. cf eastwardi. 5S rDNA was found on the centromeres of pair 7 in both species. Telomeric probes showed only distal locations. Dispersed signal patterns were obtained using probes for retrotransposons Rex1 and Rex3. Histone H1 and H3 genes were found together on pair 6 in R. cf eastwardi. The high diploid number found in Rhabdolichops suggests that chromosome fission may have contributed to its chromosomal evolution, phylogenetic relationship of the Sternopygidae suggests that this increase in diploid number could be a synapomorphic characteristic of genus Rhabdolichops. Although both species are phylogenetically close related, their karyotype structure has undergone divergent evolutionary directions. All in all, our results strongly suggest that R. cf eastwardi experencied recent intense genome reorganization.

Are NORs always located on homeologous chromosomes? A FISH investigation with rDNA and whole chromosome probes in Gymnotus fishes (Gymnotiformes).

Gymnotus (Gymnotiformes, Gymnotidae) is the most diverse known Neotropical electric knife fish genus. Cytogenetic studies in Gymnotus demonstrate a huge karyotypic diversity for this genus, with diploid numbers ranging from 34 to 54. The NOR are also variable in this genus, with both single and multiple NORs described. A common interpretation is that the single NOR pair is a primitive trait while multiple NORs are derivative. However this hypothesis has never been fully tested. In this report we checked if the NOR-bearing chromosome and the rDNA site are homeologous in different species of the genus Gymnotus: G. carapo (2n = 40, 42, 54), G. mamiraua (2n = 54), G. arapaima (2n = 44), G. sylvius (2n = 40), G. inaequilabiatus (2n = 54) and G. capanema (2n = 34), from the monophyletic group G. carapo (Gymnotidae-Gymnotiformes), as well as G. jonasi (2n = 52), belonging to the G1 group. They were analyzed with Fluorescence in situ hybridization (FISH) using 18S rDNA and whole chromosome probes of the NOR-bearing chromosome 20 (GCA20) of G. carapo (cytotype 2n = 42), obtained by Fluorescence Activated Cell Sorting. All species of the monophyletic G. carapo group show the NOR in the same single pair, confirmed by hybridization with CGA20 whole chromosome probe. In G. jonasi the NORs are multiple, and located on pairs 9, 10 and 11. In G. jonasi the GCA20 chromosome probe paints the distal half of the long arm of pair 7, which is not a NOR-bearing chromosome. Thus these rDNA sequences are not always in the homeologous chromosomes in different species thus giving no support to the hypothesis that single NOR pairs are primitive traits while multiple NORs are derived. The separation of groups of species in the genus Gymnotus proposed by phylogenies with morphologic and molecular data is supported by our cytogenetic data.