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.

Although fishes have traditionally been the subject of comparative evolutionary studies, few reports have concentrated on the application of multipronged modern molecular cytogenetic techniques (such as comparative genomic hybridization = CGH and whole chromosome painting = WCP) to analyze deeper the karyotype evolution of specific groups, especially the historically neglected small-sized ones. Representatives of the family Lebiasinidae (Characiformes) are a notable example, where only a few cytogenetic investigations have been conducted thus far. Here, we aim to elucidate the evolutionary processes behind the karyotype differentiation of Pyrrhulina species on a finer-scale cytogenetic level. To achieve this, we applied C-banding, repetitive DNA mapping, CGH and WCP in Pyrrhulina semifasciata and P. brevis . Our results showed 2n = 42 in both sexes of P. brevis , while the difference in 2n between male and female in P. semifasciata (♂41/♀42) stands out due to the presence of a multiple X 1 X 2 Y sex chromosome system, until now undetected in this family. As a remarkable common feature, multiple 18S and 5S rDNA sites are present, with an occasional synteny or tandem-repeat amplification. Male- vs .-female CGH experiments in P. semifasciata highlighted the accumulation of male-enriched repetitive sequences in the pericentromeric region of the Y chromosome. Inter-specific CGH experiments evidenced a divergence between both species’ genomes based on the presence of several species-specific signals, highlighting their inner genomic diversity. WCP with the P . semifasciata -derived Y (PSEMI-Y) probe painted not only the entire metacentric Y chromosome in males but also the X 1 and X 2 chromosomes in both male and female chromosomes of P. semifasciata. In the cross-species experiments, the PSEMI-Y probe painted four acrocentric chromosomes in both males and females of the other tested Pyrrhulina species. In summary, our results show that both intra- and interchromosomal rearrangements together with the dynamics of repetitive DNA significantly contributed to the karyotype divergence among Pyrrhulina species, possibly promoted by specific populational and ecological traits and accompanied in one species by the origin of neo-sex chromosomes. The present results suggest how particular evolutionary scenarios found in fish species can help to clarify several issues related to genome organization and the karyotype evolution of vertebrates in general.

We present the first cytogenetic data for Lebiasina bimaculata and L. melanoguttata with the aim of (1) investigating evolutionary events within Lebiasina and their relationships with other Lebiasinidae genera and (2) checking the evolutionary relationships between Lebiasinidae and Ctenoluciidae. Both species have a diploid number 2n = 36 with similar karyotypes and microsatellite distribution patterns but present contrasting C-positive heterochromatin and CMA3+ banding patterns. The remarkable interstitial series of C-positive heterochromatin occurring in L. melanoguttata is absent in L. bimaculata. Accordingly, L. bimaculata shows the ribosomal DNA sites as the only GC-rich (CMA3+) regions, while L. melanoguttata shows evidence of a clear intercalated CMA3+ banding pattern. In addition, the multiple 5S and 18S rDNA sites in L. melanogutatta contrast with single sites present in L. bimaculata. Comparative genomic hybridization (CGH) experiments also revealed a high level of genomic differentiation between both species. A polymorphic state of a conspicuous C-positive, CMA3+, and (CGG)n band was found only to occur in L. bimaculata females, and its possible relationship with a nascent sex chromosome system is discussed. Whole chromosome painting (WCP) and CGH experiments indicate that the Lebiasina species examined and Boulengerella maculata share similar chromosomal sequences, thus supporting the relatedness between them and the evolutionary relationships between the Lebiasinidae and Ctenoluciidae families.