In this study, genetic differentiation between karyomorphs A (2n = 42) and D (2n = 39/40) of the wolf fish Hoplias malabaricus, which is comprised of several cryptic species that present a wide variety of diploid chromosome numbers and sex chromosome systems, resulting in the identification of seven distinct karyomorphs (A-G), was investigated using a combination of molecular and cytogenetic tools. Deep sequence divergences for both karyomorphs were observed and indicate a long period of reproductive isolation between karyomorphs A and D. Additionally, one individual with 61 chromosomes was identified, which, as far as is known, is the first case of natural triploidy resulting from the hybridization between these highly differentiated karyomorphs of H. malabaricus. Molecular and cytogenetic analyses revealed that this allotriploid specimen carries two sets of maternal chromosomes from karyomorph D (2n = 40) and one set of chromosomes from karyomorph A (n = 21). Moreover, ribosomal sites and active nucleolus organizer regions from both parental contributors were found in the triploid hybrid. Considering the significant genetic distances between karyomorphs A and D, one of the primary reasons for the lack of recurrent reports of hybridization in the H. malabaricus species complex may be due to post-zygotic barriers, such as hybrid sterility or unviability.

Cheirodontinae comprises a small-sized fish group with a wide geographical distribution throughout Central and South America, mainly in Brazilian Basins. The species Serrapinnus notomelas is widely distributed in the Upper Parana River Basin. In this work, chromosomal mapping of 5S ribosomal RNA (rRNA) gene was performed trough fluorescence in situ hybridization (FISH) in speciemens from three distinct localities belonging to two basins in the São Paulo State. All populations presented 5S clusters in two chromosome pairs. One additional pair was detected in samples from the Rio Paraitinguinha (Tietê Basin), and two additional pairs were detected in the population from Córrego Campo Novo (Tietê Basin). Analyses with partial sequences of COI were performed to verify the interelationship among the studied specimens, revealing Córrego Campo Novo population as a sister-group to the clade formed by the two other populations. The samples from Rio Paraitinguinha and Recanto dos Cambarás presented two distinct haplotypes each, while five haplotypes were observed in the Córrego Campo Novo population, sugesting that this could be older than the other populations. None of the nine haplotypes were shared among the three populations. The similarities and differences observed among the three populations using cytogenetic data and COI analysis are not related to geographic distances that separate the samples, suggesting that the origin of the Rio Paraitinguinha and Recanto dos Cambarás populations may be related to faunal exchanges. Additionally, the present data suggest that the analyzed populations of S. notomelas may be on independent evolutionary trajectories but in a very initial diversification stage. Moreover, the use of integrative information, such as molecular and chromosomal data in the analysis of population divergence and evolutionary trajectories in freshwater fishes is reinforced.