Trachelyopterus Valenciennes 1840 species exhibit striking morphological and cytogenetic similarities, leading to persistent taxonomic challenges. This research focuses on Trachelyopterus galeatus Linnaeus 1766 and Trachelyopterus porosus Eigenmann & Eigenmann 1888, both widely distributed throughout South America and often sympatric, facilitating cytogenetic comparisons. These taxonomic entities are noteworthy for their extensive geographical ranges within the genus. We examined two populations of T. galeatus and T. porosus collected from sympatric sites in the Amazon and Pantanal regions. Both species had the same diploid number and simple Ag-NORs. The 18S rDNA sites were found in only one subtelocentric chromosome pair. Meanwhile, the 5S rDNA sites were found on two distinct chromosomal pairs, with differences in the chromosomal morphology and site position among the species, constituting the most efficient chromosomal marker to distinguish them. The 5S rDNA pattern differed between species but remained consistent between populations of the same species. Minor differences were observed between the T. galeatus populations, probably related to chromosomal rearrangements. In contrast, despite the considerable geographical distance, no cytogenetic differences were detected among the T. porosus populations. Overall, the congruence between cytogenetic and morphological characteristics, combined with our findings from sympatric samples and existing data from geographically separated populations of Trachelyopterus, indicates that the cytogenetic is a promising tool for species differentiation and for delving into the cytotaxonomic and evolutionary aspects of Auchenipteridae.
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