The karyotypes of B. stramineus and B. turiuba were similar in terms of their acrocentric chromosome number and they shared the same FN (84). One karyotype very similar was described for B. stramineus from Mogi-Guaçu River (State of São Paulo, Brazil) with 26 m/sm and 26st/a chromosomes . By contrast, B. cf. iheringii had half the acrocentric chromosome number and the FN was higher (94) compared with the other two species. Other populations of B. aff. iheringii from rivers belonging to state of Paraná (Brazil) showed fundamental numbers 88, 90, 92 and 94 [8, 9, 11].
Further relevant features were also detected when the positive C-band heterochromatin regions were compared, including blocks in the pericentromeric region of some submetacentric pairs, which seem to be conserved among the three studied species; for example, heterochromatin blocks in pairs 5 and 6 in B. stramineus, 5 and 6 in B. turiuba, and 6 and 7 in B. cf. iheringii, as well as in pairs 11 and 13 in B. turiuba and 14 and 18 in B. cf. iheringii. Thus, the similar position of these blocks indicates that this heterochromatin could be conserved and include similar repetitive DNA in the genome these three species.
Different Ag-NOR pairs were also detected, which differed in size in B. stramineus and B. turiuba, and in morphology in B. cf. iheringii. NORs were detected using both silver impregnation and 18S rDNA probes. Only one pair carrier of NOR was detected in B. stramineus and B. cf. iheringii with both techniques, whereas in B. turiuba two pairs were detected by FISH (18S rDNA probe) indicating that in all cells the Ag-NORs in the pair 10 were inactive. Ag-NORs identified by silver impregnation only correspond to sites that were active in previous interphase, which explains the additional pair (pair 10) observed in B. turiuba when the FISH technique was employed.
The Ag-NOR size heteromorphism found in pair 24 of the B. cf. iheringii karyotype coincided with the secondary constriction (Figure 4). This may indicate a difference in 45S rDNA gene transcription, because the 18S rDNA FISH probe of these regions detected signals with the similar size. As discussed by Arruda and Morielle-Versute , the size heteromorphism of the secondary constriction and Ag-NOR located in the chromosome of pair 8 of the amphibian Leptodactylus podicipinus, can be mainly attributed to the difference in the degree of condensation between homologues chromosomes and differential gene activity of the rDNA segments (45S), taking into account that and such no heteromorphism was observed in the signal strength of fluorescence in situ hybridization with rDNA probes. This could explain the difference between the size of the Ag-NOR and the FISH (18S rDNA probe) found in the pair 24 of B. cf. iheringii in this study. Differently, other population of B. aff. iheringii (Cytotype III) from Tatupeba Stream (State of Paraná, Brazil) showed one chromosome pair with NOR-size heteromorphism after the FISH technique using 18S rDNA probe .
The chromosomal location of the 5S rDNA sequences is described for the first time in Bryconamericus and also provided a useful cytological marker to comparisons among the three species. From an evolutionary viewpoint, these data are intriguing because the insertion of transposable elements in 5S rDNA sequences could lead to the dispersion of these sequences in different chromosomes via transposition in B. stramineus, B. turiuba, and B. cf. iheringii. According Raskina et al. , transposable elements have been proposed as one of the mechanisms responsible for the process of mobility of rDNA sequences to new sites.
The 45S gene family is transcribed in the nucleolus, whereas the 5S gene family is transcribed outside the nucleolus, suggesting that functional differences may require different physical locations of these two multigenic families . Gene conversion and unequal crossing-over could be important processes in the maintenance of a conserved sequence in multiple tandem arrays . These mechanisms might be more efficient regardless of whether 45S and 5S clusters remain separated from each other instead of in a linked configuration, avoiding disruptive interferences, such as undesired translocation of 5S sequences inside the 45S arrays , and explaining why this sequences are in different chromosomes in B. stramineus and B. turiuba, such as most vertebrates, and not in synteny.
Unlike the other two species analyzed in this study, the chromosomes of B. cf. iheringii showed a physical position adjacent of the 18S and 5S rDNA genes in the pair 24. This characteristic synteny was also reported in a population of Astyanax scabripinnis, where one of the chromosome pairs that carried the 5S rDNA also carried NORs . According to Schweizer and Loidl , telomeric regions are propitious to genetic material transference due to their proximity within interphasic nucleus, promoted by the ordering of chromosomes based on the Rabl’s model. Hence, in the case of B. cf. iheringii, the 45S rDNA transfer events near 5S rDNA or vice versa could be facilitated.
Diniz et al.  showed that Triportheus nematurus (Characidae) had three pairs of NORs, two of which were co-located adjacent to the 5S rDNA. Species of the family Salmonidae, such as Salmo salar and Oncorhynchus mykiss, had syntenic 45S and 5S sites, suggesting that were likely to have co-evolved [22, 23]. The authors argued that this synteny could be attributable to a translocation of 18S rDNA sequences within the 5S rDNA or vice versa in T. nematurus, while chromosomal rearrangements in S. salar and O. mykiss may have contributed to this feature after the divergence of the two species.