We compared environmental influences on the assemblages of stream fishes of contiguous but biogeographically distinct parts of the Upper Paraguay and Paraná basins in Brazil, aiming to describe the main distribution patterns of fish species in the headwater streams. We analyzed bimonthly samples in 10 streams in each basin from January to November 2004. Sixty fish species were collected, including 40 species in streams of the Paraguay Basin and 42 species in streams of the Paraná Basin. The species abundance rank did not differ between the basins. We found a clear differentiation between the Paraguay and Paraná stream fish assemblages. There were significant differences in species composition among streams, but no seasonal differences in species composition. Connectivity between streams explains the differences in species composition for the Paraguay streams, but not for the Paraná streams. Hydrological characteristics were the main factors determining species distribution in both basins, suggesting that the migratory capacity of each species and biogeographical barriers may act to filter the local species composition from the total species pool of colonizers.

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