Although fishes exhibit the greatest biodiversity among the vertebrates, a large percentage of this fauna is still underexplored on evolutionary cytogenetic questions, particularly the miniature species. The Lebiasinidae family is a particular example for such case. This study is the first one presenting differential cytogenetic methods, such as C-banding, repetitive DNAs mapping, comparative genomic hybridization (CGH), and whole chromosome painting in lebiasinid species. Pyrrhulina australis and Pyrrhulina aff. australis were deeply investigated concerning their chromosomal patterns and evolutionary relationships. These species have a very similar morphology, but they can be distinguished by a longitudinal midlateral faintly dark stripe exclusive for Pyrrhulina aff. australis. Both species presented 2n = 40 chromosomes (4st +36a), without heteromorphic sex chromosomes. However, despite their morphological and karyotype resemblance, it was evidenced that both species have already gone through a significant genomic divergence, thus corresponding to distinct evolutionary units. Furthermore, to give additional support to some proposals on evolutionary relationship among Lebiasinidae with other fish families, a chromosomal comparative approach with Erythrinus erythrinus, a representative species of the Erythrinidae family, was also performed. In addition to have similar karyotype structure, mainly composed by acrocentric chromosomes, both species share uncommon genomic similarities, such as (i) syntenic location of 5S and 18S rDNA sequences; (ii) huge dispersion of multiple 5S rDNA sites in the karyotypes; and (iii) complex association between 5S rDNA and Rex3 elements. CGH experiments, despite reinforcing some shared genomic homologies, also highlighted that both Pyrrhulina and Erythrinus have a range of nonoverlapping species-specific signals. The overall chromosomal data proved to be effective markers for the cytotaxonomy and evolutionary process among Lebiasinidae fishes.

The Erythrinidae family (Teleostei: Characiformes) is a small Neotropical fish group with a wide distribution throughout South America, where Hoplias malabaricus corresponds to the most widespread and cytogenetically studied taxon. This species possesses significant genetic variation, as well as huge karyotype diversity among populations, as reflected by its seven major karyotype forms (i.e., karyomorphs A-G) identified up to now. Although morphological differences in their bodies are not outstanding, H. malabaricus karyomorphs are easily identified by differences in 2n, morphology and size of chromosomes, as well as by distinct evolutionary steps of sex chromosomes development. Here, we performed comparative genomic hybridization (CGH) to analyse both the intra- and inter-genomic status in terms of repetitive DNA divergence among all but one (E) H. malabaricus karyomorphs. Our results indicated that they have close relationships, but with evolutionary divergences among their genomes, yielding a range of non-overlapping karyomorph-specific signals. Besides, male-specific regions were uncovered on the sex chromosomes, confirming their differential evolutionary trajectories. In conclusion, the hypothesis that H. malabaricus karyomorphs are result of speciation events was strengthened.

The main objectives of this study were to test: (1) whether the W-chromosome differentiation matches to species' evolutionary divergence (phylogenetic concordance) and (2) whether sex chromosomes share a common ancestor within a congeneric group. The monophyletic genus Triportheus (Characiformes, Triportheidae) was the model group for this study. All species in this genus so far analyzed have ZW sex chromosome system, where the Z is always the largest chromosome of the karyotype, whereas the W chromosome is highly variable ranging from almost homomorphic to highly heteromorphic. We applied conventional and molecular cytogenetic approaches including C-banding, ribosomal DNA mapping, comparative genomic hybridization (CGH) and cross-species whole chromosome painting (WCP) to test our questions. We developed Z-and W-chromosome paints from T. auritus for cross-species WCP and performed CGH in a representative species (T. signatus) to decipher level of homologies and rates of differentiation of W chromosomes. Our study revealed that the ZW sex chromosome system had a common origin, showing highly conserved Z chromosomes and remarkably divergent W chromosomes. Notably, the W chromosomes have evolved to different shapes and sequence contents within~15-25 Myr of divergence time. Such differentiation highlights a dynamic process of W-chromosome evolution within congeneric species of Triportheus.

Repetitive DNA sequences play an important role in the structural and functional organization of chromosomes, especially in sex chromosome differentiation. The genus Triportheus represents an interesting model for such studies because all of its species analyzed so far contain a ZZ/ZW sex chromosome system. A close relationship has been found between the differentiation of the W chromosome and heterochromatinization, with the involvement of different types of repetitive DNA in this process. This study investigated several aspects of this association in the W chromosome of Triportheus trifurcatus (2n = 52 chromosomes), including the cytogenetic mapping of repetitive DNAs such as telomeric sequences (TTAGGG)n, microsatellites and retrotransposons. A remarkable heterochromatic segment on the W chromosome was observed with a preferential accumulation of (CAC)10, (CAG)10, (CGG)10, (GAA)10 and (TA)15. The retrotransposons Rex1 and Rex3 showed a general distribution pattern in the chromosomes, and Rex6 showed a different distribution on the W chromosome. The telomeric repeat (TTAGGG)n was highly evident in both telomeres of all chromosomes without the occurrence of ITS. Thus, the differentiation of the W chromosome of T. trifurcatus is clearly associated with the formation of heterochromatin and different types of repetitive DNA, suggesting that these elements had a prominent role in this evolutionary process.

The Neotropical fish, Hoplias malabaricus, is one of the most cytogenetically studied fish taxon with seven distinct karyomorphs (A-G) comprising varying degrees of sex chromosome differentiation, ranging from homomorphic to highly differentiated simple and multiple sex chromosomes. Therefore, this fish offers a unique opportunity to track evolutionary mechanisms standing behind the sex chromosome evolution and differentiation. Here, we focused on a high-resolution cytogenetic characterization of the unique XX/XYY multiple sex chromosome system found in one of its karyomorphs (G). For this, we applied a suite of conventional (Giemsa-staining, C-banding) and molecular cytogenetic approaches, including fluorescence in situ hybridization FISH (with 5S and 18S rDNAs, 10 microsatellite motifs and telomeric (TTAGGG) sequences as probes), comparative genomic hybridization (CGH), and whole chromosome painting (WCP). In addition, we performed comparative analyses with other Erythrinidae species to discover the evolutionary origin of this unique karyomorph G-specific XYY multiple sex chromosome system. WCP experiments confirmed the homology between these multiple sex chromosomes and the nascent XX/XY sex system found in the karyomorph F, but disproved a homology with those of karyomorphs A-D and other closely related species. Besides, the putative origin of such XYY system by rearrangements of several chromosome pairs from an ancestral karyotype was also highlighted. In addition, clear identification of a male-specific region on the Y chromosome suggested a differential pattern of repetitive sequences accumulation. The present data suggested the origin of this unique XYY sex system, revealing evidences for the high level of plasticity of sex chromosome differentiation within the Erythrinidae.

Channid fishes, commonly referred to as “snakeheads”, are currently very important in Asian fishery and aquaculture due to the substantial decline in natural populations because of overexploitation. A large degree of chromosomal variation has been found in this family, mainly through the use of conventional cytogenetic investigations. In this study, we analyzed the karyotype structure and the distribution of 7 repetitive DNA sequences in several Channa species from different Thailand river basins. The aim of this study was to investigate the chromosomal differentiation among species and populations to improve upon the knowledge of its biodiversity and evolutionary history. Rearrangements, such as pericentric inversions, fusions and polyploidization, appear to be important events during the karyotypic evolution of this genus, resulting in the chromosomal diversity observed among the distinct species and even among populations of the same species. In addition, such variability is also increased by the genomic dynamism of repetitive elements, particularly by the differential distribution and accumulation of rDNA sequences on chromosomes. This marked diversity is likely linked to the lifestyle of the snakehead fishes and their population fragmentation, as already identified for other fish species. The karyotypic features highlight the biodiversity of the channid fishes and justify a taxonomic revision of the genus Channa, as well as of the Channidae family as a whole, as some nominal species may actually constitute species complexes.

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Fishes exhibit the greatest diversity of species among vertebrates, offering a number of relevant models for genetic and evolutionary studies. The investigation of sex chromosome differentiation is a very active and striking research area of fish cytogenetics, as fishes represent one of the most vital model groups. Neotropical fish species show an amazing variety of sex chromosome systems, where different stages of differentiation can be found, ranging from homomorphic to highly differentiated sex chromosomes. Here, we draw attention on the impact of recent developments in molecular cytogenetic analyses that helped to elucidate many unknown questions about fish sex chromosome evolution, using excellent characiform models occurring in the Neotropical region, namely the Erythrinidae family and the Triportheus genus. While in Erythrinidae distinct XY and/or multiple XY-derived sex chromosome systems have independently evolved at least four different times, representatives of Triportheus show an opposite scenario, i.e., highly conserved ZZ/ZW system with a monophyletic origin. In both cases, recent molecular approaches, such as mapping of repetitive DNA classes, comparative genomic hybridization (CGH), and whole chromosome painting (WCP), allowed us to unmask several new features linked to the molecular composition and differentiation processes of sex chromosomes in fishes.