Oligotrophic ocean gyre ecosystems may be expanding due to rising global temperatures [1] [2] [3] [4] [5] . Models predicting carbon flow through these changing ecosystems require accurate descriptions of phytoplankton communities and their metabolic activities [6] . We therefore measured distributions and activities of cyanobacteria and small photosynthetic eukaryotes throughout the euphotic zone on a zonal transect through the South Pacific Ocean, focusing on the ultraoligotrophic waters of

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... e South Pacific Gyre (SPG). Bulk rates of CO 2 fixation were low (0.1 µmol C l −1 d −1 ) but pervasive throughout both the surface mixed-layer (upper 150 m), as well as the deep chlorophyll a maximum of the core SPG. Chloroplast 16S rRNA metabarcoding, and single-cell 13 CO 2 uptake experiments demonstrated niche differentiation among the small eukaryotes and picocyanobacteria. Prochlorococcus abundances, activity, and growth were more closely associated with the rims of the gyre. Small, fast-growing, photosynthetic eukaryotes, likely related to the Pelagophyceae, characterized the deep chlorophyll a maximum. In contrast, a slower growing population of photosynthetic eukaryotes, likely comprised of Dictyochophyceae and Chrysophyceae, dominated the mixed layer that contributed 65-88% of the areal CO 2 fixation within the core SPG. Small photosynthetic eukaryotes may thus play an underappreciated role in CO 2 fixation in the surface mixed-layer waters of ultraoligotrophic ecosystems.