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

Abstract

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.

Figure 1

DAPI Karyotype of Gymnotus carapo sensu stricto: cytotype with 2n = 42 (30 M/SM +12 ST/A).

Figure 2

Bivariate flow karyotype showing 4 Regions: R1, R2 with 3 subregions (S2A, S2B, S2C), R3 with 4 subregions (S3A, S3B, S3C, S3D) and R4 with 4 subregions (S4A, S4B, S4C, S4D).

Figure 3

Hybridization experiments on same species metaphases: a) Region 1 (pair 20, NOR-bearing chromosome); b) Region 2 (pairs 1, 2, 3 and 16); c) Region 3 (pairs 4, 5, 6, 7, 8, 17, 18 and 19); d) Region 4 (pairs 9, 10, 11, 12, 13, 14, 15 and 21); arrows: NORs; e) Multicolor FISH with probes from the four regions: Region 1 = white; Region 2 = purple; Region 3 = red; Region 4 = green. All the chromosomes were counterstained with DAPI; probes (in red) from "a" to "d", stained with Cy3; 3e) red: Cy3; green: FITC; purple: Cy5; white: a mix of green and purple.

Figure 4

Examples of same species hybridization with subregions hybridizing fewer chromosomes: a) S2B (pairs 2 and 16); b) S3C (pair 7); c) S4B (pairs 9, 10, 15 and 21); d) S3B (pairs 7 and 19). All the chromosomes were counterstained with DAPI; probes stained with Cy3.

Figure 5

Cross species hybridization with R3 (red) and R4 (green) for the precise definition of which chromosome or chromosome segment in the 2n = 40 genome corresponds to each chromosome from R3 (pairs 4, 5, 6, 7, 8, 17, 18, 19) and R4 (pairs 9, 10, 11, 12, 13, 14, 15, 21) from cytotype 2n = 42. All the chromosomes were counterstained with DAPI; red probes: Cy3; green probes: FITC.

Figure 6

Examples of cross species hybridizations. Chromosome numbers refer to the original pairs in Gymnotus 2n = 42 a) R1 (pair 20 red); b) S2B + R2 (pairs 1 and 3 green; pairs 2 and 16 yellow) c) S2B + S2C (pair 1 red, pair 2 green and pair 16 yellow) d) S3C (pair 7 red); e) S3A + S3B (pair 19 green, pair 7 yellow, pair {5, 6, 17} red); f) S4C + R4 (pairs 9 and 14 green; pairs 10, 11, 12, 13, 15 and 21 yellow). Arrows on b, c, e and f point to the NOR region where nonspecific hybridizations occur. All the chromosomes were counterstained with DAPI; red probes: Cy3; green probes: FITC.

Figure 7

DAPI karyotype (mounted from the metaphase of Figure 5) of Gymnotus carapo sensu stricto, cytotype with 2n = 40 (34 M/SM + 6 ST/A) showing the results of comparative mapping with the chromosomes from the 2n = 42 karyotype (numbers on the right side).