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

Abstract

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.

Figure 1

Map showing the localities of species Gymnotus with known cytogenetic formulas. Data are taken from a review of the cytogenetic literature by Milhomem et al. (2007), from Margarido et al. (2007) and Lacerda & Maistro (2007), and from the species discussed in this paper. See Table 4 for chromosome numbers of these species, and further locality information. At some localities the coordinates are approximate because detailed information was not available from the respective papers.

Figure 2

Specimens of: A. Gymnotus carapo from Capanema, Pará, Brazil (2n = 42) (MPEG 13331); B. Gymnotus carapo from Almeirim, Pará, Brazil (2n = 40) (MPEG 13329). Scale bar = 10 mm. Specimen A was photographed fresh, while specimen B was frozen and photographed after defrosting. This partially explains the color differences.

Figure 3

Karyotype of Gymnotus carapo from Benfica, Pará, Brazil, with a diploid number of 2n = 42: A. Conventional Giemsa stained karyotype with the NOR pair (20); B. Sequenced C-banding; C. DAPI stained karyotype, the arrows indicate distal and interstitial markings; D. CMA₃ stained metaphase (the arrows indicate the NOR pair). M-SM = Metacentric – Submetacentric; ST-A = Subtelocentric – Acrocentric.

Figure 4

Karyotype of Gymnotus carapo from Almeirim, Pará, Brazil, with a diploid number of 2n = 40: A. Conventional Giemsa stained karyotype with the NOR pair (20); B. Sequenced C-banding; C. DAPI stained karyotype, the arrows indicate interstitial markings; D. CMA₃ stained metaphase (the arrows indicate the NOR pair). M-SM = Metacentric – Submetacentric; ST-A = Subtelocentric – Acrocentric.

Figure 5

Telomeric FISH from: A. Gymnotus carapo from Benfica (2n = 42); B. Gymnotus carapo from Almeirim (2n = 40). The arrows indicate interstitial marking.

Figure 6

Principal component scores from a correlation matrix of morphological and meristic data for two karyotypic forms of the Gymnotus carapo species complex from the Eastern Amazon. The first three principal components represent 39.9%, 67.7%, and 90.1% of cumulative variance respectively. In all combinations of these four axes the two karyotypes exhibited substantial overlap. The following meristic counts were eliminated from this analysis due to a lack of variance: P1R, BAN, SAL, APS, CEP (see Table 2 for abbreviations).