The W chromosome of Megaleporinus trifasciatus was isolated in order to analyze its behavior in the karyotype of this and other species of the family, including forms with differentiated and undifferentiated sex chromosomes. The chromosome was microdissected, and the WMt probe was prepared for the chromosome painting procedure. M. trifasciatus was also cross-hybridized (cross-FISH) using existing probes available for M. macrocephalus (WMm) and M. elongatus (WMe). Two Leporinus species and Semaprochilodus taeniurus, representing a clade close to the Anostomidae, were also cross-hybridized with the objective to better understand the evolution of the sex chromosomes. In the metaphase of female M. trifasciatus, the WMt probe highlighted the whole long arm of the W chromosome and a small, distal portion of the long arm of the Z chromosome. In males, the probe highlighted the distal portion of the long arm of the Z chromosomes. The hybridization of female M. trifasciatus with the WMe and WMm probes revealed a pattern similar to that encountered using the WMt probe. The WMt, WMm, and WMe probes revealed broad similarities among the species of the genus Megaleporinus, which has a ZZ/ZW system of sex chromosomes, with only minor alterations becoming apparent when analyzed separately.

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