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Photosynthesis in eukaryotes first arose through phagocytotic processes wherein an engulfed cyanobacterium was not digested, but instead became a permanent organelle. Other photosynthetic lineages then arose when eukaryotic cells engulfed other already photosynthetic eukaryotic cells. Some of the resulting lineages subsequently lost their ability for phagocytosis, while many others maintained the ability to do both processes. These mixotrophic taxa have more complicated ecological roles, in that they are both primary producers and consumers that can shift more towards producing the organic matter that forms the base of aquatic food chains, or towards respiring and releasing CO 2 . We still have much to learn about which taxa are predatory mixotrophs as well as about the physiological consequences of this lifestyle, in part, because much of the diversity of unicellular eukaryotes in aquatic ecosystems remains uncultured. Here, we discuss existing methods for studying predatory mixotrophs, their individual biases, and how single-cell approaches can enhance knowledge of these important taxa. The question remains what the gold standard should be for assigning a mixotrophic status to ill-characterized or uncultured taxa—a status that dictates how organisms are incorporated into carbon cycle models and how their ecosystem roles may shift in future lakes and oceans. This article is part of a discussion meeting issue ‘Single cell ecology’.