Aim  To investigate biogeographical patterns of the obligate freshwater fish order Characiformes. Location  South America. Methods  Parsimony analysis of endemicity, likelihood analysis of congruent geographical distribution, and partition Bremer support were used. Results  Areas of endemism are deduced from parsimony analysis of endemicity, and putative dispersal routes from a separate analysis of discordant patterns of distribution. Main conclusions  Our results demonstrate the occurrence of 11 major areas of endemism and support a preferential eastern–western differentiation of the characiforms in the Amazonian region, contrasting with the southern–northern differentiation of terrestrial organisms. The areas of endemism identified seem to be deeply influenced by the distribution of the emerged land during the 100‐m marine highstand that occurred during the late Miocene and allow us to hypothesize the existence of eight aquatic freshwater refuges at that time. The raw distribution of non‐endemic species supports nine patterns of species distribution across the 11 areas of endemism, two of which support a southern–northern differentiation in the eastern part of the Amazon. This result shows that the main channel of the Amazon limited dispersal between tributaries from each bank of the river. The levels of endemism further demonstrate that the aquatic freshwater refuges promoted allopatric speciation and later allowed the colonization of the lowlands. By contrast, the biogeographical pattern found in the western part of the Amazon is identified as a result of the Miocene Andean foreland dynamic and the uplift of the palaeoarches that promoted allopatric divergence across several sedimentary basins by the establishment of disconnected floodplains. The assessment of conflicting species distributions also shows the presence of seven putative dispersal routes between the Amazon, Orinoco and Paraná rivers. Our findings suggest that, rather than there being a single predominant process, the establishment of the modern South American freshwater fish biotas is the result of an interaction between marine incursions, uplift of the palaeoarches, and historical connections allowing cross‐drainage dispersal.

International audienceKnowledge of fish migration is a prerequisite to sustainable fisheries management and preservation, especially in large international river basins. In particular, understanding whether a migratory lifestyle is compulsory or facultative, and whether adults home to their natal geographic area is paramount to fully appraise disruptions of longitudinal connectivity resulting from damming.In the Amazon, the large migratory catfishes of the Brachyplatystoma genus are apex predators of considerable interest for fisheries. They are believed to use the entire length of the basin to perform their life cycle, with hypothesized homing behaviours. Here, we tested these hypotheses, using the emblematic B. rousseauxii as a model species.We sampled adults close to major breeding areas in the Amazon basin (upper Madeira and upper Amazonas) and assessed their lifetime movements by measuring variations in 87Sr/86Sr along transverse sections of their otoliths (ear stones) using laser ablation multicollector mass spectrometry (LA-MC-ICPMS).We demonstrate that larvae migrate downstream from the Andean piedmont to the lower Amazon, where they grow over a protracted period before migrating upstream as adults. Contrary to prevailing inferences, not all fish spend their nursery stages in the Amazon estuary. By contrast, the passage in the lower or central Amazon seems an obligate part of the life cycle. We further evidence that most adults home to their natal geographic area within the Madeira sub-basin. Such long-distance natal homing is exceptional in purely freshwater fishes.Synthesis and applications. By using otolith microchemistry, we were able to demonstrate a seemingly compulsory basin-wide migratory life cycle of large Amazonian catfishes. This makes them the organisms performing the longest migrations (>8000 km) in fresh waters. This exceptional life history is already jeopardized by two dams recently built in the Madeira River, which block a major migration route and access to a substantial part of their spawning grounds. Major impacts can be anticipated from the current and forthcoming hydroelectric development in the Amazon basin, not only on the populations and fisheries of this apex predator, but also on Amazonian food webs through trophic cascades