Natural ponds in the Brazilian Cerrado harbor high biodiversity but are still poorly studied, especially their microbial assemblage. The characterization of the microbial community in aquatic environments is fundamental for understanding its functioning, particularly under the increasing pressure posed by land conversion and climate change. Here, we aim to characterize the structure (abundance, richness, and diversity) and composition of the Bacteria and Archaea in the sediment of two natural ponds belonging to different basins that primarily differ in size and depth in the Cerrado. Sediment samples were collected in the dry and rainy seasons and the transition periods between both. The structure and composition of Bacteria and Archaea were assessed by 16S rRNA gene pyrosequencing. We identified 45 bacterial and four archaeal groups. Proteobacteria and Acidobacteria dominated the bacterial community, while Euryarchaeota and Thaumarchaeota dominated the archaeal community. Seasonal fluctuations in the relative abundance of microbial taxa were observed, but pond characteristics were more determinant to community composition differences. Microbial communities are highly diverse, and local variability could partially explain the microbial structure's main differences. Functional predictions based in 16S rRNA gene accessed with Tax4Fun indicated an enriched abundance of predicted methane metabolism in the deeper pond, where higher abundance of methanogenic archaea Methanocella, Methanosaeta, and Methanomicrobiaceae was detected. Our dataset encompasses the more comprehensive survey of prokaryotic microbes in Cerrado's aquatic environments. Here, we present basic and essential information about composition and diversity, for initial insights into the ecology of Bacteria and Archaea in these environments.

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