| Summary: | <i>Pseudacanthicus</i> is a genus of Neotropical fish with eight valid species, in addition to numerous lineages not formally identified. It occurs along the Amazon and Tocantins River basins, in Suriname and in the Guiana shield. There are no karyotypic data in the literature for species of this genus. Here, the karyotypes of three <i>Pseudacanthicus</i> species (<i>P. spinosus</i>, <i>P. leopardus</i> and <i>Pseudacanthicus</i> sp.) were comparatively analyzed by classical cytogenetics and fluorescence in situ hybridization using 18S and 5S rDNA probes, U2 snDNA and telomeric sequences. The analyzed species presented 52 chromosomes and KF = 18 m + 34 sm. Constitutive heterochromatin occurred in blocks on a few chromosomes. The 18S rDNA occurred in a single pair; interestingly, <i>P. leopardus</i> presented only one locus of this sequence in its diploid genome. The 5S rDNA sequence occurred in only one pair in <i>P. leopardus</i>, and in multiple sites in <i>Pseudacanthicus</i> sp. and <i>P. spinosus</i>. The snDNA U2 occurred in only one pair in all analyzed species. Telomeric sequences did not show interstitial sites. Although <i>Pseudacanthicus</i> species share the same 2n and KF, repetitive sequence analysis revealed karyotypic diversity among these species. The occurrence of DNA double-strand breaks related to fragile sites, unequal crossing over and transpositions is proposed as the mechanism of karyotypic diversification, suggesting that the conservation of the karyotypic macrostructure is only apparent in this group of fish.
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