Phytoplankton, the foundational organisms in ocean food webs, have been little studied in the Indonesian Throughflow region of the eastern Indian Ocean, the spawning area of Southern Bluefin Tuna. Here, we assess phytoplankton abundance, biomass, size structure, pigment composition, taxonomic diversity and percent functional mixotrophs of that region based on complementary approaches of flow cytometry, microscopy, taxon-specific pigments and rRNA gene sequencing. During summer (January–February) 2022, the region was characterized by warm (up to 30.5 °C), stratified, oligotrophic (nitrogen-limited) waters, with integrated euphotic zone (EZ) chlorophyll a (CHLa) of 13 mg m−2. EZ mean CHLa was low in the upper layer (85 ng L−1) and 3.8 times higher (320 ng L−1) at the pronounced deep CHLa maximum. EZ-integrated phytoplankton carbon averaged 1229 mg C m−2. Prochlorococcus dominated throughout the EZ, but eukaryotic carbon biomass was ∼4-times greater in the lower than upper EZ, along with a distinct community. In the upper EZ, haptophytes, dinoflagellates and prasinophycean taxa without prasinoxanthin contributed most to monovinyl chlorophyll a (MV-CHLa). In the more diverse lower EZ, haptophytes, dinoflagellates, prasinophycean taxa with prasinoxanthin, pelagophytes, and cryptophytes were the main contributors to MV-CHLa. Diatoms were a minor part of the community. A higher percentage of the upper EZ community showed mixotrophy (35–84%) relative to the lower EZ (30–51%). Nitrogen-fixing organisms (as symbionts of diatoms and free-living cyanobacteria taxa) were ubiquitous, but low in abundance. Overall, community characteristics were similar to those at the Hawaii Ocean Time-series site and the central Gulf of Mexico.
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