The research team, led by Carlos Araya-Jaime and colleagues, examined Eigenmannia limbata from the Amazon River basin and Eigenmannia microstoma from the São Francisco River basin. These two species had never been cytogenetically characterized before, making the study a first for both. Using classical staining techniques alongside fluorescence in situ hybridization, or FISH, the researchers mapped the chromosomal positions of three important repetitive DNA families: the 18S and 5S ribosomal RNA genes and the U2 small nuclear RNA gene.

Both species turned out to share the same diploid chromosome number of 38, a count that has been consistently reported across most Eigenmannia species studied to date. However, the internal structure of their karyotypes diverged significantly. E. microstoma carries eight metacentric, ten submetacentric, and twenty acrocentric chromosomes, yielding a fundamental number of 56 chromosome arms. E. limbata, by contrast, has eight metacentric, four submetacentric, and twenty-six acrocentric chromosomes, for a fundamental number of just 50. Neither species showed morphologically differentiated sex chromosomes, which is notable given that other members of the genus have evolved a bewildering array of sex chromosome systems, including XY, ZW, Z0, and even multiple sex chromosomes.

The differences in chromosome arm counts between the two species point to Robertsonian rearrangements as a likely driver of karyotypic evolution in Eigenmannia. These are fusions or fissions of chromosomes at the centromere that change the number of chromosome arms without altering the total chromosome count. The researchers suggest that such rearrangements may serve as a postzygotic reproductive isolation mechanism, effectively building genetic walls between populations. Low population sizes and limited mobility in these fish could facilitate the fixation of chromosomal polymorphisms, allowing distinct karyomorphs to emerge even within what appears to be a single species.

When it came to the ribosomal DNA genes, the two species showed both shared and divergent patterns. The 18S rDNA sites, which mark the nucleolar organizer regions responsible for producing the structural RNA of ribosomes, were located on a single chromosome pair in both species. This is consistent with what has been observed across most of the family Sternopygidae and appears to represent an ancestral condition. However, the researchers noted considerable size variation in the NOR region between the two species, with E. limbata carrying a substantially larger 18S rDNA cluster than E. microstoma. This kind of heteromorphism can arise through tandem duplication of ribosomal genes via mechanisms such as unequal crossing over during meiosis.

The 5S rDNA sites told a much more dynamic story. In E. limbata, these sites appeared on just two chromosome pairs, but in E. microstoma they were scattered across eleven pairs. Multiple 5S rDNA sites are a widely recognized feature within Gymnotiformes, having been documented in Gymnotus, Eigenmannia, Sternopygus, and Archolaemus. Ribosomal DNA loci are considered hotspots for chromosomal rearrangements because their long stretches of tandemly repeated sequences and high transcriptional activity make them prone to breakage and non-allelic homologous recombination. Additionally, transposable elements or other repetitive DNAs can insert into the non-transcribed spacers of 5S rDNA units, further driving the spread of these clusters across the genome.

Perhaps the most intriguing finding concerns the U2 small nuclear RNA gene, a component of the spliceosome that processes messenger RNA. The researchers detected U2 snDNA sites on three chromosome pairs in E. limbata and on four pairs in E. microstoma, marking the first report of multiple U2 snDNA sites within the genus Eigenmannia. Even more remarkably, these sites were co-localized with 5S rDNA in one chromosome pair in E. limbata and in all four pairs in E. microstoma. This physical association between two different classes of repetitive DNA represents a new case of such linkage in Gymnotiformes and suggests that the 5S rDNA and U2 snRNA partnership may be a characteristic feature of the Eigenmannia genome.

Previous work on E. aff. trilineata had described only a single U2 snDNA site co-localized with 5S rDNA, making the multiple co-localized sites observed here particularly noteworthy. Among other gymnotiform fishes, most Gymnotus species carry only a single U2 snDNA-bearing chromosome pair, with multiple sites having been reported only in G. pantal and Archolaemus janae. None of those species exhibits co-localization between U2 snDNA and other sequences, underscoring the uniqueness of the pattern found in Eigenmannia.

The researchers also noted that the 5S rDNA probe used in their FISH experiments was prepared from E. microstoma genomic DNA, and the possibility that the 300 base pair fragment contains inserts of other repeats in its non-transcribed spacer region cannot be ruled out. Such inserts could have promoted the spreading of 5S rDNA clusters or generated additional non-5S rDNA signals in E. microstoma, potentially explaining the much higher number of signals detected in that species compared to E. limbata. Without sequencing the probe fragment, the authors acknowledge they cannot directly evaluate the contribution of admixed repetitive sequences.

Beyond the specific findings for these two species, the study reinforces the broader principle that cytogenetic features, both conventional and molecular, serve as powerful markers for taxonomic diagnosis and biodiversity characterization in Gymnotiformes. The genus Eigenmannia is taxonomically ambiguous due to minimal morphological variation between its 27 recognized species, making chromosomal markers especially valuable for distinguishing species and understanding their evolutionary relationships. As mapping techniques continue to reveal the hidden architecture of repetitive DNA in electric fish genomes, each new species examined adds another piece to the puzzle of how these remarkable animals have diversified across the Neotropical region.

Subject of Research: Comparative chromosomal mapping of rDNA and U2 snDNA in two Eigenmannia electric fish species

Article Title: Karyotype description and comparative chromosomal mapping of rDNA and U2 snDNA sequences in Eigenmannia limbata and E. microstoma ( Teleostei , Gymnotiformes , Sternopygidae )

Article References: Araya-Jaime, C. A., Silva, D. M. Z. D. A., da Silva, L. R. R., do Nascimento, C. N., Oliveira, C., & Foresti, F. (2022). Karyotype description and comparative chromosomal mapping of rDNA and U2 snDNA sequences in Eigenmannia limbata and E. microstoma (Teleostei, Gymnotiformes, Sternopygidae). Comparative Cytogenetics, 16(2), 127-142. https://doi.org/10.3897/compcytogen.v16i2.72190

Image Credits: AI Generated

DOI: 10.3897/CompCytogen.v16i2.72190

Keywords: Eigenmannia, Gymnotiformes, cytogenetics, karyotype, rDNA, U2 snDNA, FISH, sex chromosomes, repetitive DNA, Sternopygidae, chromosomal evolution, electric fish