Gymnotus coatesi (Gymnotiformes): A Case of Colocation of Multiple Sites of 18S rDNA with Telomeric Sequences

Background

Gymnotus (Gymnotidae, Gymnotiformes) is a monophyletic genus^1,2 of electric fishes that are distinguished from other gymnotiform by possessing a facing-up mouth, eyes in a lateral position on the head, a long body cavity, precaudal vertebrae with a modal value of 31–51, and other notable characteristics. ¹ These fishes are distributed extensively in the neotropical region; they have been recorded from the south of Mexico to the north of Argentina, with the highest diversity found in the Amazon basin. ³ Thirty-seven species of this genus have been formally described ⁴ ; of them, 28 were analyzed ⁵ and classified into six clades: Gymnotus pantherinus, Gymnotus coatesi, Gymnotus anguillaris, Gymnotus tigre, Gymnotus cylindricus, and Gymnotus carapo. Gymnotus is the Gymnotiform genus whose members have been subject to the most cytogenetic studies, mostly focusing on members of the clade G. carapo. The diploid numbers in this genus vary from 2n = 34 in Gymnotus capanema ⁶ to 2n = 54 in G. carapo, ⁷ Gymnotus mamiraua, ⁸ Gymnotus paraguensis, ⁹ and Gymnotus inaequilabiatus. ¹⁰ Huge karyotypic variations have been observed, along with differences in the locations and amounts of constitutive heterochromatin and nucleolus organizer regions (NOR). G. coatesi is a small and rare species that was originally described by LaMonte in Para state, Brazil, in 1935, ¹¹ and occurs along the main stretch of the Amazon River. ¹² G. coatesi belong to the G. coatesi clade, ⁵ together with Gymnotus stenoleucus, Gymnotus coropinae, Gymnotus javari, and Gymnotus jonasi. Of the members of this clade, only G. coatesi has not yet been the subject of a cytogenetic study. Here, we describe for the first time the karyotype of G. coatesi.

Materials and Methods

Five specimens of G. coatesi (three male and two female) were collected in Igarapé Cururutuia, Bragança, Pará, Brazil, S01°04′42.0″ W046°44′18.6″ (Fig. 1). Chromosomes were prepared from kidney tissues as described, ¹³ with some modifications. The prepared chromosomes were analyzed by Giemsa staining, C-banding, ¹⁴ and AgNO₃ staining. ¹⁵ Fluorescence in situ hybridization (FISH) was performed following the protocol of Ref., ¹⁶ using telomeric sequence, TTTAGG (All Telomere probes, Oncor) probes, 18S rDNA probe, ¹⁷ and 5S rDNA probe obtained from G. carapo. The probes were labeled with biotin or digoxigenin by nick-translation, and the signals were detected with avidin (Cy3 or FITC) or anti-digoxigenin-FITC. The karyotypes were classified as described by Ref. ¹⁸

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FIG. 1.

Gymnotus coatesi specimen (above) and place the specimens were collected in Bragança, Para (below).

Results

The analyzed specimens of G. coatesi showed 2n = 50 and a karyotypic formula (KF) = 24 m/sm +26st/a, with heteromorphism observed in the sizes of chromosome pairs 1, 3, and 5, and the short arms of pairs 8, 9, and 15 (Fig. 2a, b). There was no evidence of morphologically differentiated sex chromosomes in our meiotic analyses (Supplementary Fig. S1; Supplementary Data are available online at www-liebertpub-com.web.bisu.edu.cn/zeb).

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FIG. 2.

G. coatesi karyotype. (a) Giemsa staining; (b) C-banding; (c) 5S rDNA; (d) Ag-NOR. NOR, nucleolus organizer regions.

Constitutive heterochromatin was found in the centromeric/pericentromeric regions of all chromosomes: pair 1 has a large heterochromatic block along almost all of the short arm and a small band in the interstitial region of the long arm; pairs 6, 10, 12, 13, and 16 have heterochromatic bands in the proximal regions of their long arms; pair 9 has heterochromatic bands in the proximal regions of their short arms; and pairs 2, 14, and 15 have heterochromatic bands in the interstitial regions of their long arms (Fig. 2b).

The 5S rDNA probe hybridized to the interstitial region of the long arm of pair 17 (Fig. 2c). G. coatesi was found to have multiple NOR sites, including nine active sites on eight chromosomes, with pair 5 showing sites in the distal regions of both arms (Fig. 2d). The 18S rDNA probe hybridized to 21 sites on 19 chromosomes (Fig. 3a). The telomeric probe hybridized specifically to the distal regions of all chromosomes (Fig. 3b). All of the 18S rDNA signals colocalized with the telomeric signals (Fig. 3c).

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FIG. 3.

FISH in G. coatesi: (a) 18S rDNA probe; (b) telomeric sequences; (c) Double FISH with 18S rDNA (red) and telomeric (green) probes. FISH, fluorescence in situ hybridization.

Discussion

The diploid number of G. coatesi (2n = 50, KF = 24 m/sm +26st/a) is shared with other species of this genus, such as Gymnotus sp. (2n = 50, KF = 26 m/sm +24 st/a ¹⁹ ;), Gymnotus sp. ‘‘Negro’’ (2n = 50, KF = 22 m/sm +28 st/a in females, and 21 m/sm +29 st/a in males ²⁰ ;), and two other species of the G. coatesi clade, G. javari (2n = 50, KF = 20 m/sm +30 st/a ²¹ ;), and G. coropinae (2n = 50/49, KF = 28 m/sm +22 st/a in females and 26 m/sm +23 st/a in males ²⁰ ). The interspecific differences in KFs likely reflect rearrangements that did not modify the diploid number, such as inversions and translocations.

In recent studies^2,4,5 G. jonasi is placed as the most basal specie within the G. coatesi clade, followed by G. stenoleucus. ⁵ Here, we plotted the 2n of the species in the phylogeny adapted from the data ⁵ (Fig. 4), and confirmed G. jonasi basal position, with diploid number 2n = 52 (KF = 12 m/sm +40 st/a ²² ;). This is because it shares this trait with G. pantherinus ²³ of G. pantherinus clade, which is a sister clade of G. coatesi, and with Electrophorus electricus, which is a sister taxa of genus Gymnotus.^24,25 G. stenoleucus has 2n = 48 (KF = 20 m/sm +28 st/a, ²⁶ ). We propose that the evolutionary chromosomal events comprised two fusions in G. stenoleucus, which changed this karyotype. G. coropinae, G. javari, and G. coatesi appear to have diverged from the basal diploid number by a fusion event.

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FIG. 4.

Representation of the diploid number of the species in the clade G. coatesi, clade Gymnotus pantherinus, and Electrophorus electricus plotted according to recent phylogeny in the genus. ⁵

Despite sharing its diploid number with G. coropinae and G. javari, G. coatesi has a distinct KF, suggesting that rearrangements such as pericentric inversions and translocations may have occurred in the karyotypes of these species. G. coatesi shares its pattern of centromeric constitutive heterochromatin with the other species of its clade, but G. coatesi also has noncentromeric heterochromatic blocks on several chromosomes (Figs. 2b and 5), including a very obvious example on the short arm of pair 1 (Figs. 2b and 5). Similar to G. jonasi, G. coatesi lacks the morphologically differentiated sex chromosomes found in G. coropinae ²⁰ .

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FIG. 5.

Constitutive heterochromatin distribution, Ag-NOR, 5S rDNA e 18S rDNA in the karyotype of G. coatesi.

G. coatesi has 5S rDNA sites on a single pair of chromosomes (Figs. 2c and 5). In this, it is similar to Gymnotus sylvius and G. coropinae,^20,27 but differs from G. paraguensis, which has these sequences on 19 chromosome pairs. ¹⁶

G. coatesi also differs from the other species of the genus in that many of its chromosomes carry 18S rDNA sequences (Figs. 2d, 3a, and 5). Within the genus, only two other species have been described as having multiple NOR: Gymnotus pantanal with two pairs ²⁸ and G. jonasi with three pairs. ²² The presence of a high number of 18S rDNA sites, several of which are silenced, and their colocalization with telomeric sequences make G. coatesi unique among the described members of its genus. Recent studies suggest that rDNA sequences may be linked to transposable elements, and that the insertion of these elements into ribosomal sequences could mediate the relocation, dispersion, and/or activation of these sites.^20,29–31 Thus, the numerous copies described herein may reflect gene duplication through retrotransposition or transposition.^32,33 The colocalization of telomeric and 18S rDNA sequences may be the result of inter-spacing of those sequences, as proposed in atlantic eels ³⁴ (Anguilla anguilla and Anguilla rostrara) and in the rainbow trout ³⁵ (Oncorhynchus mykiss). It is not known exactly what this colocalization means to the karyotype. However, further investigations may help clarify the evolutionary chromosomal processes that may have occurred in this genus.

Conclusions

G. coatesi has a diploid number in the range observed for its genus, but it has additional traits that distinguish it from the other species of order Gymnotiformes. These include numerous 18S rDNA sequences and their co-localization with telomeric sequences. This large number of 18S rDNA sequences might reflect the amplification and dispersion of these sequences mediated by the association of mobile elements (transposons and/or retrotransposons), which makes those sequences a target for future studies.