The evolution of the eukaryotic cell paved the way for the emergence of all complex life on Earth. Despite its significance, the environmental context of early eukaryote evolution is largely unknown1,2. Here we use the geological record to reconstruct the habitats of the oldest known fossil eukaryotes, approximately 1.75–1.4 billion years old. Our integrated palaeontological, sedimentological and geochemical analyses show that although fossil eukaryotes are found in samples deposited in a range of environments from coastal to offshore, they are almost entirely restricted to those from settings with oxygenated bottom waters. This distribution suggests these organisms were aerobes (obligate, facultative and/or microaerophilic) and, given their size and morphological complexity, probably possessed mitochondria. Furthermore, their near absence from otherwise fossiliferous anoxic samples suggests a benthic habit, as planktonic eukaryotes would be expected to be present in both oxic and anoxic samples. We propose that eukaryotes were largely restricted to oxic benthic habitats for much of the Proterozoic eon, only expanding into planktonic habitats during the Neoproterozoic era (1–0.54 billion years ago). This late ecological expansion could account for the mismatch between the appearance of eukaryotic body fossils and molecular biomarkers3 and explain the stepwise increase in eukaryote diversity during the Neoproterozoic era4.
