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Non-native species cause several impacts on freshwater biodiversity, but studies focusing on the Neotropical stream’s biota are still incipient. We used a data set of 586 headwater stream’s fish assemblages from the Brazilian Upper Paraná ecoregion to test whether the presence/absence of non-native species affect: species richness (S), functional diversity (MPD) and taxonomic diversity (Δ+). We compared diversity patterns of fish assemblages formed only by native species against those of assemblages formed by native and non-native species (Scenario 1); then, we removed non-native species from their original assemblages and recalculated their diversity values to compare them with those of fish assemblages formed only by native species again (Scenario 2). We also investigated: (1) whether non-native’s fish assemblages are associated with land use, topographic and watercourse connectivity variables; (2 …