Mixotrophy emerges as an optimal strategy in mature waters of the Amazon River plume.

Phytoplankton, namely diatoms and cyanobacteria, combine photoautotrophy and the uptake of dissolved organic matter (osmotrophy) for a mixotrophic living. All other photosynthetic protists, except diatoms, are potentially phagotrophs, and currently classified as mixoplankton. This functional group occupies a unique position between autotrophs and heterotrophs in planktonic food webs, producing a greater carbon stock and higher-quality food for metazoans than phytoplankton do. However, field studies remain challenging due to the difficulty of distinguishing their sole activity in seston containing a mixture of all functional groups. During April/May 2018 and 2021, we examined seston using compound-specific stable nitrogen isotope analysis of amino acids to determine its trophic dynamics along the Amazon River plume. Based on the comparison of nitrogen isotopes in glutamic acid and alanine with phenylalanine, we found a dominance of mixotrophs in the Outer Plume Margin, a region of mature waters around 27 days old. Mixotrophy appears to be the optimal growth strategy in these heterogeneous margins as part of the succession of microalgae functional diversity along the plume. Our study highlights the urgent need to study mixotrophs and mixoplankton in situ within a multidisciplinary framework, pioneering the use of amino acid nitrogen isotopes in field research in this area.