Origem e diferenciação do sistema de cromossomos sexuais ZZ/ZW em Triportheus (Characiformes, Characidae): citogenética, mapeamento dos genes ribossomais e microdissecção cromossômica

Abstract

Chromosomes of distinct species of Triportheus (Characiformes, Characidae) from different hydrographic basins in Brazil, as well as in Argentina, were analyzed in the present work. Triportheus nematurus, from Piracicaba River (São Paulo State, Brazil), was analyzed for the first time, using conventional cytogenetic techniques, base-specific fluorochromes and physical mapping of 18S rDNA and 5S rDNA. The diploid number found in all species was 2n= 52 chromosomes, in both males and females. Females presented a pair of quite differentiated heteromorphic chromosomes, characterizing a ZZ/ZW sex chromosome system. The Z chromosome is the largest in the karyotype, bearing constitutive heterochromatin at pericentromeric and telomeric regions. The W chromosome is mostly heterochromatic, showing a heterogeneous heterochromatin, composed of GC- and AT-rich regions The Ag-NORs were equivalent to 18S rDNA sites detected through fluorescent in situ hybridization. The 5S rDNA is syntenic and adjacent to 18S rDNA site, characterizing an uncommon situation amongst fishes. The results obtained in T. nematurus agree with previous studies in this group, reinforcing the basal condition of the system ZZ/ZW in the phylogeny of this genus, prior to speciation events. Besides T. nematurus, other seven species/populations of Triportheus were analyzed by FISH with 18S and 5S rDNA probes. The results confirmed the presence of 18S sites on the W chromosome in all Triportheus species herein studied, even when Ag-NORs showed to be inactive. The 5S rDNA sites, not reported in this group so far, were always located on short arms of a single chromosomal pair, close to centromeres. The only exception was T. auritus, which presented ten 5S rDNA sites. Two distinct T. nematurus populations (Piracicaba River SP and Paraná River Argentina) presented synteny between 18S and 5S ribosomal genes, located on short arm of a NOR-bearing chromosomal pair. A probe for whole chromosome painting (WCP), specific to the Z chromosome of T. nematurus (Piracicaba River), was obtained by microdissection, followed by unspecific amplification via DOP-PCR (Degenerate Oligonucleotide Primed-PCR). Fluorescent in situ hybridization with such probe was performed in Triportheus species, as well as in other Characidae species belonging to genus closed to Triportheus. In male specimens of Triportheus, both Z chromosomes (1st large metacentric pair) presented conspicuous marks over their whole extension. In females, the Z chromosome showed the same pattern observed in males while the W chromosome bears only a subtle signal on the short arm, or on the long arm close to centromere, according to species. Therefore, the region of the W chromosome with homology to Z chromosome is relatively small. All the other Characidae species analyzed presented no positive signals after hybridization using Z chromosome of Tirportheus nematurus as a probe, even in those apparently closely related to this genus. Thus, these results stress out, positively, that the ZZ/ZW system should actually correspond to a synapomorphy shared by Triportheus species, likely to be unique to this Characidae group.