Whole chromosome painting in two populations of the weakly electric fish Gymnotus mamiraua (Teleostei, Gymnotiformes)

Abstract

Techniques such as whole chromosome painting (WCP) have effectively expanded our understanding of the evolution and organization of the genome in fish, allowing us to obtain more detailed information about several evolutionary questions. The genus Gymnotus is the most cytogenetically studied among the Gymnotiformes, having a significant karyotypic diversity. Molecular cytogenetic studies in the genus Gymnotus show a dynamic reorganization in the karyotype, in addition to the presence of different sex chromosome systems. In this study, with whole chromosome probes from the karyotype of Gymnotus carapo orientalis sensu stricto cytotype 2n = 42, we analyzed the karyotype of Gymnotus mamiraua 2n = 54, from two localities, and compared with previous studies, to obtain a better understanding of the homologies and chromosomal differences among the species of this genus. The results shows both populations with no difference detected by chromosome painting, and overall, a larger number of signals were observed with the fluorescent probes, since GMA has a higher diploid number. Using chromosomal painting on a greater number of species will make it possible to construct a map of homologies between them that will help define the phylogeny of the group.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-09770-w.

Introduction

In recent years, there has been growing momentum for the application of advanced molecular cytogenetic tools in studies across various fish groups¹. In taxa with complex taxonomy and systematic challenges, comparative cytogenetic approaches offer an integrative means to refine existing hypotheses on evolutionary relationships^2,3.

Molecular cytogenetic methods, including Whole Chromosome Painting (WCP) have been effective in expanding the understanding of the evolution and organization of the genome in fish, allowing us to obtain more detailed information about several of its evolutionary questions^4–11. These techniques have gained prominence in resolving several issues in the area of comparative cytogenetics of fish, allowing access to insights on a more precise scale on a series of evolutionary issues, such as: (i) divergence in the genome between closely related species^{4,6,7,9,12–14}, (ii) evolution and composition of sex chromosomes^9,13–16 and (iii) origin of B chromosomes^1,17–19.

Chromosome painting defines ancient syntenies shared between widely divergent species and reveals chromosomal rearrangements between species. Syntenic regions point to a likely ancestral chromosome structure, while chromosomal rearrangements indicate the dynamics and mechanisms of chromosomal change during the evolution of species²⁰.

Gymnotus (Gymnotidae, Gymnotiformes) is a monophyletic genus of neotropical freshwater electric fish, with 51 valid species divided into six subgenera: Gymnotus, Tijax, Tigre, Lamontianus, Tigrinus and Pantherus^21–26. Gymnotus is the most cytogenetically studied among the Gymnotiformes with a great karyotypic diversity. The diploid numbers range from 2n = 34 in Gymnotus capanema²⁷ to 2n = 54 in Gymnotus carapo orientalis, G. cuia, G. inaequilabiatus, G. mamiraua and G. paraguensis^28–39. Molecular cytogenetic studies in the genus Gymnotus show a dynamic reorganization, including inversions that can be observed through the position of repetitive DNA, the dispersion of transposable elements through association with repetitive sequences, in addition to the presence of different sex chromosome systems^{30,33,35,36,40–45}.

Whole chromosome probes from the karyotype of Gymnotus carapo orientalis (GCA₄₂) sensu stricto cytotype 2n = 42, were produced using flow cytometry⁴. Comparative genomic mapping using these probes was carried out on the karyotype of a few species: G. carapo orientalis (2n = 40 and 2n = 42)⁴, G. capanema (GCP₃₄, 2n = 34)⁵, G. arapaima (GAR_44, 2n = 44)⁸ and G. c. occidentalis “Catalão” (GCC₄₀, 2n = 40)¹¹, showing significant chromosomal rearrangement among these karyotypes. We used the same set of probes produced⁴ to analyze the karyotype of G. mamiraua (GMA₅₄, 2n = 54) and compare it with previous studies, to obtain a better understanding of the homologies and chromosomal differences between the species of this genus.

Results

Karyotypic data and chromosomal mapping in Gymnotus mamiraua

Gymnotus mamiraua (GMA₅₄), from the Mamiraua Reserve, has 2n = 54 and a karyotypic formula (KF) consisting of 42m/sm + 12st/a as already described^31,34 G. mamiraua (GMA₅₄) from Catalão Lake has 2n = 54 and a karyotypic formula (KF) consisting of 32m + 6sm + 16st as already described³⁶. Both karyotypes were organized according to³¹ for easier comparison (Fig. 1).

Fig. 1.

Chromosome mapping in G. mamiraua species from Mamiraua Reserve. (A,B) and Catalão Lake. (C,D). GMA₅₄ karyotype, dual-color FISH using probes from the R3 (green) and R4 (red) regions (A,C). On the right, the equivalent chromosomes of each region of the GCA₄₂ flow karyotype are represented. Chromosomal segments hybridizing with 2 colors indicated repetitive DNA sequences. The chromosomes or segments in blue (DAPI) represent the NOR-bearing chromosome (GCA₄₂) and the chromosomes corresponding to R2. (B, D) The identification on the left refers to the chromosomes of GCA₄₂⁴. Bar: 10 μm.

No differences were observed between individuals from the two sampled localities. The karyotype of GMA₅₄ hybridized with probes from regions 3 (green) and 4 (red) of GCA₄₂ is shown in Figs. 1, 2 and Supplementary Fig. S1. This experiment allows us to define the chromosomes in the GMA₅₄ karyotype that correspond to the four regions of the GCA₄₂ flow karyotype⁴, with no sharing of chromosomes between these regions. Sequences hybridized by both probes (yellow) represent repetitive DNA sequences common to them. The chromosomes that remained stained with DAPI (blue) represent the NOR-carrying pair (R1, GCA₄₂ 20) and the R2 pairs (GCA₄₂ 1–3, 16).

Fig. 2.

Chromosome painting experiments in Gymnotus. Ideograms of the karyotype of G. carapo (2n = 42), represented by (A–J), and ideograms of the karyotype of G. mamiraua (2n = 54), represented by (L–U): (A) The four chromosomal regions (R1, R2, R3, R4) as defined⁴. (B–J) Two-color FISH experiments, based on^4,5, to identify chromosomal homology with GCA₄₂. (M–U). Corresponding dual-color FISH experiments for GMA₅₄. (B) R3 × R4 to determine the region to which each chromosome belongs. (L) Definition of the NOR pair. (C,D,N,O) S2A, S2B, and S2C represent sub-regions within region 2; (E,F,G,P,Q,R) S3A, S3B, S3C, and S3D represent sub-regions within region 3; (H,I,J,S,T,U) S4A, S4B, S4C, and S4D represent sub-regions within region 4.

All experiments were done according to⁴ (Fig. 2). The results show that, of the 12 GCA₄₂ pairs that are individualized (1–3, 6, 7, 9, 14, 16 and 18–21), five (GCA₄₂ 7, 14, 19, 20, and 21) remained conserved on GMA₅₄. The GCA₄₂ 20 probe hybridized only to the GMA₅₄ 24 chromosomal pair. Six pairs of the individualized chromosomes (GCA₄₂ 1, 2, 3, 6, 9, and 16) were fissioned and present two signals in the GMA₅₄ karyotype (GCA₄₂ 1: GMA₅₄ 3 + 27; GCA₄₂ 2: GMA₅₄ 1 + 8; GCA₄₂ 3: GMA₅₄ 9 + 10; GCA₄₂ 6: GMA₅₄ 5 + 19; GCA₄₂ 9: GMA₅₄ 6 + 14; GCA₄₂ 16: GMA₅₄ 17 + 23). Two pairs in GMA₅₄ fusions were detected (GMA₅₄ 1: GCA₄₂ 2 + 19: GMA₅₄ 6: GCA₄₂ 9 + 12,13,15). GCA₄₂ {5,17} probes revealed two signals in GMA₅₄ (2p and 18), GCA₄₂ {10,11} probes revealed two signals in GMA₅₄ (pairs 13 and 22) and GCA₄₂ {12,13,15} probes show hybridization in 3 pairs (6q, 16 and 20) of GMA₅₄ (Figs. 1 and 3).

Fig. 3.

Comparative ideogram between G. carapo and G. mamiraua. Ideogram showing chromosomal rearrangements observed by WCP R1, R2, R3 and R4. The colors represent the probes from the four regions of flow karyotype of GCA₄₂ used in the production of the probes. Each color represents a chromosome or group of chromosomes. GMA₅₄, present study. (Adapted from⁸).

Comparative analysis by chromosome painting between different Gymnotus karyotypes

Of the 12 pairs of chromosomes in GCA₄₂ that can be identified individually (1–3, 6, 7, 9, 14, 16, 18–21), only GCA₄₂ 20 and 21 remain with one signal in all species analyzed: GCA₄₀ (20, 15), GCC₄₀ (19, 3q), GCP₃₄ (15, 1), GAR₄₄ (19, 17), and GMA₅₄ (24, 12). Plotting the chromosomes on the phylogeny previously proposed²⁴ the individualized chromosome GCA₄₂ 14 is shared by GMA₅₄, GAR₄₄, GCC₄₀, GCA₄₀, and GCA₄₂. GCA₄₂ 1 and 9 are shared between GAR₄₄, GCC₄₀, GCA₄₀ and GCA₄₂. The chromosomes GCA₄₂ 2, 6 and 19 are shared among GCC₄₀, GCA₄₀, and GCA₄₂ (Fig. 4).

Fig. 4.

Phylogenetic tree with individualized chromosomes shared by the nodes. Ideogram with karyotypes in a phylogeny tree based on ²⁴, with individualized chromosomes shared by the nodes. Chromosome numbers refer to G. carapo 2n = 42 ⁴. The numbers on the right side of chromosomes show homology with the GCA₄₂ karyotype. Each color in the karyotypes (GCA₄₀, GCC₄₀, GAR₄₄, GMA₅₄ and GCP₃₄) represents the corresponding chromosome colored in GCA₄₂.

Analyzing the individualized chromosome pairs, the species GCA₄₀, GCC₄₀, and GMA₅₄ display four pairs with shared synteny (GCA₄₂: pairs 14, 19, 20, and 21), each showing a single signal, while other pairs exhibit two signals. When comparing the karyotypes of GCP₃₄ with GMA₅₄, four pairs maintain conserved synteny with a single signal each (GCA₄₂: pairs 7, 19, 20, and 21), while the remaining pairs show two signals. For grouped chromosome pairs, the karyotypes exhibit a conserved number of signals relative to GCA₄₂. In the comparison between GAR₄₄ and GMA₅₄, among the 12 individualized pairs, only three (GCA₄₂: pairs 14, 20, and 21) display a single signal, while the other pairs present two signals across both species. Grouped pairs also maintain a consistent signal count (Table 1).

Table 1.

Chromosomal homeology between G. carapo orientalis (2n = 42, 2n = 40), G. c. occidentalis “Catalão” (2 = 40), G. capanema (2n = 34), G. arapaima (2n = 44), and G. mamiraua (2n = 54).

	GCA42	GCA40	GCC40	GCP34	GAR44	GMA54
Region	2n = 42a	2n = 40a	2n = 40d	2n = 34b	2n = 44c	2n = 54e
R 1	20	20	19	15	19	24
R 2	1	1	1	5 + 9	1	3 + 27
	2	2	2	3 + 16	14 + 21	1 + 8
	3	5 + 6	16 + 17	2 + 12 + 13	13 + 18	9 + 10
	16	7 + 18	12 + 4	7 + 14	2 + 14	17 + 23
R 3	[4.8]	6 + 9 + 10	7 + 16	4 + 6	4 + 20	4 + 7 + 15 + 25 + 26*
	6	11	18	8	5q + 16	5 + 19
	7	8 + 9	3 + 4	1	3 + 16	21
	[5.17]	4, 8, 18	5 + 6	9 + 11 + 12	6 + 16	2 + 18
	18	3 + 7	12 + 13	1 + 2 + 4	15	4 + 7 + 15 + 25 + 26*
	19	19	14	2	7p + 22	1
R4	9	14	10	7p + 3	8	6 + 14
	[10,11]	5 + 12	9 + 20	10 + 17	5p + 12	13 + 22
	[12,13,15]	3, 12, 13, 16	11 + 15 + 17	1 + 2 + 6	10 + 11 + 13	6 + 16 + 20
	14	17	8	13 + 13	9	11
	21	15	3q	6	7	12

a. [4]; b. [5]; c. [8]; d. [11], e. Present work; * It was not possible to distinguish [4,8] from 18.

Discussion

Classical cytogenetic studies indicate that G. mamiraua has the highest diploid number within the genus, 2n = 54^31,34,36,39, consistent with observations in other species of the genus Gymnotus^{8,9,28–39,46}. Species with 2n = 54 currently inhabit the Amazon and Paraná-Paraguay Basins, though G. mamiraua is now restricted to the Amazon Basin³⁹. No karyotypic differences were observed between the two G. mamiraua populations analyzed. Despite the short-distance migratory behavior typical of Gymnotus species, they are broadly distributed throughout the Neotropical region, occupying a diverse range of environments^21,32.

The NOR (Nucleolus organizing regions) system observed in the species already mapped by chromosome painting G. carapo orientalis (2n = 40 and 2n = 42)⁴, G. capanema (2n = 34)⁵, G. carapo (2n = 40, 42, and 54)⁴⁷, G. mamiraua (2n = 54)⁴⁷, G. arapaima (2n = 44)⁴⁷, G. sylvius (2n = 40)⁴⁷, G. inaequilabiatus (2n = 54)⁴⁷, G. capanema (2n = 34)⁴⁷, G. arapaima (2n = 44)⁸, G. c. occidentalis “Catalão” (2n = 40)¹¹ and G. mamiraua (present work), indicate that this simple NOR system is a primitive condition for the genus Gymnotus^{31,32,39,43,46,48}, as well as in other groups of Gymnotiformes^{30,32,34,46,49}. Furthermore, the individualized pair GCA₄₂ 21 (Fig. 3) is already conserved by all species already mapped and is found in synteny with the NOR in the species analyzed within the subgenus Gymnotus, and we suggest that this pair is a synapomorphic characteristic for the group.

The GCA₄₂ probes {5, 17} show two signals for GCA₄₀, GAR₄₄ and GMA₅₄, the GCA₄₂ {10, 11} shows two signals in GCA₄₀, GCC₄₀, GCP₃₄, GAR₄₄ and GMA₅₄ and GCA₄₂ {12, 13, 15} with three signals in GCA₄₀, GCP₃₄, and GMA₅₄, indicating that these chromosomes may have retained their homology as already observed⁸. Therefore, comparative chromosome painting analysis has been successfully used to reconstruct karyotypic evolution among several groups of vertebrates^50–55.

Despite the chromosomal rearrangements between the analyzed Gymnotus species, up to the present work, these rearrangements are decisive in the construction of an ancestral karyotype, comparing the chromosomal complement of several related species²⁰. The comparative analysis with the species already mapped shows that GMA₅₄ has greater similarity with the karyotypes of G. arapaima⁸ and G. capanema⁵ with a greater number of shared homeologies, concerning cytotypes of G. carapo^4,11. These dates suggest that this similarity between GMA₅₄, GCP₃₄, and GAR₄₄ can be explained by the phylogenetic proximity between them and more distance from the other cytotypes of G. carapo.

GMA₅₄ exhibits a higher number of dissociations in GCA₄₂ chromosomes compared to other previously analyzed species. Although two species-specific fusions are observed (GMA₅₄ pairs 1 and 6), most probes in GMA₅₄ display multiple signals. With a higher diploid number (2n) than other species analyzed, GMA₅₄ karyotype is closer to those of the more basal groups within the genus. This suggests that many chromosomes observed in GCA₄₂ are likely the result of chromosome fusions that occurred during the speciation process. As more species are analyzed, we observe that certain chromosomes—such as GCA₄₂ pairs 2 and 16—have a repeated number of signals, supporting the hypothesis that these chromosomes were already fragmented in the common ancestor of the group.

Conclusion

The uniformity found on the two populations analyzed even though they are ~ 500 km away from each other, shows that the high variable karyotypes found in G. carapo are not reflected in G. mamiraua.

In previous studies, GCA₄₂ probes have been used to compare karyotypes of different Gymnotus species. The results demonstrate that karyotypes are more rearranged than those found by classical cytogenetics. Using whole chromosomal painting on a greater number of species will make it possible to construct a map of homologies between them that will help define the group’s phylogeny and help differentiate populations from cryptic species.

Material and methods

Samples

The sample (Fig. 5) consisted of G. mamiraua (GMA₅₄) specimens previously analyzed by classical cytogenetics^31,34 and the diagnostic characters listed in⁵⁶ were used to unequivocally identify the species, with four males and two females. The specimens are deposited at the Museum of Science and Technology of the Pontifical Catholic University of Rio Grande do Sul (MCT-PUCRS): (G. mamiraua = MCP 40927).

Fig. 5.

Distribution map of samples of Gymnotus species analyzed by chromosome painting. The numbers refer to localities detailed on Table 2.

The other sample of G. mamiraua (GMA₅₄) was previously analyzed by classical cytogenetics³⁶, with three males and two females. The specimens are deposited in the zoological collection of the National Institute for Amazonian Research (Instituto Nacional de Pesquisas da Amazônia—INPA), under identification numbers INPA-46869 and INPA-46868.

JCP has a permanent field permit issued by the Chico Mendes Institute for Biodiversity Conservation (nº 13,248). The Cytogenetics Laboratory of the Universidade Federal do Pará (UFPA) has a license to transport and handle samples issued by the Ministry of the Environment (nº19/2003) and authorization for their manipulation for research nº 52/2003. Furthermore, this study is approved by the UFPA Ethics Committee (license nº68/2015). All specimens were processed in the field and euthanized with eugenol⁵⁷. All methods were performed in accordance with the relevant guidelines and regulations. We also confirm that the study is reported in accordance with ARRIVE guidelines.

Map

Map showing the sample points for Gymnotus taxa analyzed (Fig. 5). Each symbol corresponds to a distinct taxon. The numbers refer to localities detailed on Table 2. The map was made using QGIS v.3.10.7. The shapefiles containing country limits and hydrography were obtained from DIVA-GIS⁵⁸.

Table 2.

Geographic distribution of samples of Gymnotus species analyzed by chromosome painting.

Species	2n	N	Location	Coordinates
G. mamiraua	2n = 54	4M/2F	Mamiraua Reserve, AM	S03° 07′ 50.3″/W064° 48′ 26.4″
G. arapaima	2n = 40	10M/2F	(1’) Mamiraua Reserve, AM	S03° 02′ 11.8″/W064° 51′ 16.6″
G. mamiraua	2n = 54	3 M/2F	(2) Catalão Lake, AM	S03° 14′ 29.1″/W59° 53′ 50.3″
G. carapo catalão	2n = 40	1 M/6F	(2’) Catalão Lake, AM	S03° 15′ 24.2″/W59° 53′ 50.3″
G. carapo	2n = 42	2 M/2F	(3) Santa Cruz do Ariri, PA	S00° 42′ 03.2″/W049° 10′ 42.1″
G. carapo	2n = 40	1 M/6F	(4) Almerim, PA	S01° 31′ 34.2″/W052° 33′ 37.9″
G. capanema	2n = 34	4 M/2F	(5) Capanema, PA	S1° 11″ 45″ /W047° 10′ 51″

N number of specimens submitted to cytogenetic analysis, M male, F female.

Whole chromosome painting

Whole-chromosome probes of the karyotype of G. carapo orientalis sensu stricto, cytotype 2n = 42 were generate by flow cytometry⁴. The resulting flow karyogram categorizes the chromosomes into four distinct regions (no overlaping): R1 (comprising pair 21, which contains the NOR), R2 (including pairs 1–3 and 16), R3 (comprising pairs 4–8 and 17–19), and R4 (containing pairs 9–15 and 21) (Fig. 6). Within regions R2, R3, and R4, probes were further refined into sub-regions. By applying two-color Fluorescent in situ Hybridization (FISH) with probes from these sub-regions, the majority of chromosomes (pairs 1–3, 6, 7, 9, 14, 16, and 18–20) were uniquely identified. The remaining pairs were identified in groups of two or three pairs ({4,8}; {10,11}; {5,17}; {12,13,15}) (Fig. 2 and supplementary Table S1).

Fig. 6.

Karyogram showing the regions and subregions obtained in flow cytometry. (A) Karyogram showing the four regions obtained in flow cytometry. Below is an ideogram through colors representing the probes of the four regions (R1, R2, R3, and R4) of GCA₄₂ (B) New sorting identifying the subregions within each region. Below is an idiogram where each different color represents a chromosome or group of chromosomes of GCA₄₂ (Adapted from^4,8).

Image capture

The metaphases, which were used in classical cytogenetic techniques, were captured using a Zeiss Imager D2 photomicroscope (Zeiss) and the images acquired with an Axiocam 503 camera and processed with the ZEN software (Zeiss, version 2.0.0.0). The images were edited using the Adobe Photoshop CC program (Adobe Systems, San Jose, CA, USA, version 25.10.0).

Chromosome assembly and disposition

The karyotypes were assembled and organized according to the morphological classification of chromosomes⁵⁹, with modifications, divided into two groups: metacentric + submetacentric and subtelocentric + acrocentric, arranged in pairs in decreasing order of size. The images were edited using Adobe Photoshop CC (Adobe Systems, San Jose, CA, USA, version 25.10.0).

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

2n

Diploid number

CGH

Comparative genomic hybridization

GAR₄₄

Gymnotus arapaima

GCA₄₂

Gymnotus carapo orientalis

GCC₄₀

Gymnotus carapo occidentalis “Catalão”

GCP₃₄

Gymnotus capanema

GMA₅₄

Gymnotus mamiraua

FISH

Fluorescent in situ hybridization

KF

Karyotypic formula

NOR

Nucleolus organizing regions

WCP

Whole chromosome painting

Author contributions

AMP: Conceptualization; Data Curation; Formal analysis; Investigation; Methodology; Visualization; Writing original draft; Writing review and editing. MAM: Data Curation; Formal analysis; Investigation; Methodology; Visualization; Writing original draft; Writing review and editing. FHRS: Investigation; Methodology; Visualization; Writing review and editing. PPR: Investigation; Methodology; Visualization; Writing review and editing. EF: Investigation; Methodology; Funding acquisition; Visualization; Writing review and editing. PCMO’B: Investigation; Methodology; Visualization; Writing review and editing. MAF-S: Investigation; Methodology; Resources; Visualization; Writing review and editing. JCP: Data Curation; Formal analysis; Funding acquisition; Resources; Visualization; Writing review and editing. CYN: Project administration; Data Curation; Formal analysis; Funding acquisition; Resources; Supervision; Visualization; Writing review and editing.

Funding

This research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Fundação Amazônia Paraense de Amparo à Pesquisa (FAPESPA) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) on projects coordinated by CY Nagamachi (Edital BIONORTE-CNPq, Proc 552032/2010–-7; Edital BIONORTE-FAPESPA, ICAAF 007/2011; Edital Pró-Amazônia Proc 047/2012); the FAPESPA (Edital Vale-Proc 2010/110447) and Banco Nacional de Desenvolvimento Econômico e Social-BNDES (Operação 2.318.697.0001) on a project coordinated by JCP; The Prociência UERJ and CNPq (Edital Universal 47290920096) to LG; CNPq for Productivity Grants to CYN (307170/2021-7) and to JCP (307154/2021-1); PDJ/ CNPQ Scholarship (168394/2022-7) to WOS. The article processing charge was paid by PROPESP-UFPA (Edital 06/2021-PAPQ/PROPESP). This work was funded by a joint grant between National Research Council of Brazil (CNPQ—Conselho Nacional de Desenvolvimento Científico e Tecnológico) and Amazon State Research Foundation (FAPEAM—Fundação de Amparo à Pesquisa do Estado do Amazonas) through the Program National Institutes of Science and Technology (INCT—Institutos Nacionais de Ciências e Tecnologia) to the ADAPTA Project (Environmental Adaptations, of Aquatic Organisms) coordinated by Adalberto Luis Val.

Data availability

All data generated or analysed during this study are included in this published article (and its Supplementary Information files).

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

The animal study was reviewed and approved by The Ethics Committee from Para Federal University (Comitê de Ética Animal da Universidade Federal do Pará) (Permit 68/2015).