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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.

Comparative Cytogenetics and Neo-Y Formation in Small-Sized Fish Species of the Genus Pyrrhulina (Characiformes, Lebiasinidae)