Understanding of trophic structure and flow of energy within river systems is essential for informed management of these ecosystems and conservation of native fish fauna and fisheries resources. Food resources used by riverine fish depend on productivity within the main stem and adjacent floodplains, terrestrial inputs, and longitudinal and lateral hydrologic connectivity. Trophic ecology of puye (Galaxias maculatus Jenyns, 1842) in Chilean river systems remains poorly understood and studies that combine short‐term (stomach content) and long‐term (stable isotope analyses) analyses have not been performed to date. In this study, we assessed spatial variability in diet of puye based on both stomach content and analyses of stable isotopes of carbon and nitrogen in 10 river systems of southern Chile (including main stem, headwater lakes, and estuaries). Fish in the main stems of more northern rivers, with predictable flow regimes, were characterised by the most diverse diets, broad niche space, and high proportion of terrestrial prey items and zooplankton in diet. Fish collected in lakes were characterised by narrow isotopic niche space and diet dominated by zooplankton. Diet of G. maculatus in Chilean river systems is associated with characteristics of riverine habitats within each river system (lentic, lotic, and estuarine) and hydrologic connectivity among them. Indeed, connectivity with floodplain habitats and direct supply from headwater lakes appeared to be primary drivers of fish diet in systems where these are present. These supplies maintain fish populations of good condition and allow freshwater recruitment. Therefore, maintaining seasonal flow dynamics and connectivity is essential to preserve the natural function of these river systems and conserve native fish populations.

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