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1. Flood-pulsed wetlands make vital contributions to local and global biodiversity. However, the patterns and controls of spatial and temporal variation in aquatic biodiversity in flood-pulsed wetlands are not well understood. We analysed the relationship between variation in hydrological regime and the patterns of aquatic biodiversity in a large pristine flood-pulsed wetland, the Okavango Delta, Botswana. 2. Surveys of water chemistry, diatoms and macroinvertebrates were conducted over the seasonal phases of the flood pulse. Hydrological variables of flood frequency and hydroperiod class were collated from 16 years of satellite images. Multivariate regression trees and generalised least squares regression were used to determine the chief controls of community composition and taxon richness. 3. Hydroperiod class, phase of the flood and conductivity explained 32% and 43% of the variation in diatom and invertebrate taxon richness, respectively. There was a negative relationship between hydroperiod class and invertebrate taxon richness on the rising, peak and receding flood, whereas at low flood there was no significant relationship. Multivariate regression tree analysis revealed hydroperiod class, phase of the flood and conductivity as the dominant forces shaping invertebrate and diatom community composition. 4. Seasonal and spatial variation in hydrological conditions are the principal drivers of variation in aquatic biodiversity in flood-pulsed wetlands. In pristine flood-pulsed wetlands, increased productivity caused by the arrival of the flood waters appears to override disturbance and connectivity in shaping taxon richness and community composition. Thus, these data suggest that the maintenance of a rich mosaic of habitats covering a broad range of hydroperiod is the key to preserving aquatic biodiversity and natural ecosystem function in flood-pulsed wetlands. [ABSTRACT FROM AUTHOR]