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Protists play key roles in aquatic food webs as primary producers, predators, nutrient recyclers, and symbionts. However, a comprehensive view of protist diversity in freshwaters has been challenged by the immense environmental heterogeneity among lakes worldwide. We assessed protist diversity in the surface waters of 366 freshwater lakes across a north temperate to subarctic range covering nearly 8.4 million km² of Canada. Sampled lakes represented broad gradients in size, trophic state, and watershed land use. Hypereutrophic lakes contained the least diverse and most distinct protist communities relative to nutrient-poor lakes. Greater taxonomic variation among eutrophic lakes was mainly a product of heterotroph and mixotroph diversity, whereas phototroph assemblages were more similar under high-nutrient conditions. Overall, local physicochemical factors, particularly ion and nutrient concentrations …