This study aimed to identify species of Astyanax bimaculatus group from four Itaipu Reservoir tributaries (Paraná River Basin) by cytogenetics and molecular markers (COI) to investigate the possible occurrence of cryptic diversity in part of this basin. The four populations showed only one karyotype formula and simple AgNORs. FISH with 18S rDNA probe showed a high variation, and 5S rDNA probes evidenced simple sites in most of the specimens, although multiple sites are present in two specimens.

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... The variations of 5S and 18S cistrons generated 13 cytotypes. The molecular data did not reveal cryptic diversity in the populations; however, its grouping with 82 sequences from other stretches of the Paraná River Basin originated three haplogroups (distances of 3.12% and 8.82%) and 33 haplotypes were identified. DNA Barcode suggests that cytogenetic variations represent a high polymorphism degree, and it identified the analyzed specimens as Astyanax lacustris, which confirms the morphological identification. Our data suggest that the cryptic diversity of this group in the tributaries of the Paraná River Basin is different than the proposed by the synonymizations of A. altiparanae and A. asuncionensis to A. lacustris. This study reinforces the importance of integrative cytogenetics and molecular methods for taxonomy.