Characterizing open ocean biodiversity at basin scales remains challenging due to sparse sampling and limited opportunities to validate satellite-derived estimates of plankton community composition. Through the Citizens of the Sea initiative, we deployed standardized environmental DNA (eDNA) sampling kits on 27 sailing vessels during the 2024 Pacific Rally circumnavigating between New Zealand, Tonga, and Fiji, generating 728 surface ocean eDNA samples across 1.5 million km2. Using three complementary metabarcoding markers (bacterial 16S rRNA, eukaryotic 18S rRNA and metazoan Cytochrome Oxidase I genes), and following rigorous bioinformatic quality control, we obtained 31,177 unique Amplicon Sequence Variants (ASVs), providing the first high-resolution plankton biodiversity assessment of this understudied region. After accounting for human-derived variation, all metabarcoding markers revealed clear latitudinal patterns in alpha diversity and community composition. ASV richness increased significantly with sea-surface temperature and community turnover was strongly correlated with gradients in temperature and chlorophyll-a. Comparisons between eDNA-inferred phytoplankton functional groups and satellite-based estimates showed moderate agreement for prokaryote-dominated communities (R2 = 0.27) but a weak correlation for eukaryotic phytoplankton (R2 < 0.15), reflecting functional-type-specific mismatches between molecular and remote sensing approaches. Our results reinforce that citizen science fleets can generate high-quality oceanographic eDNA datasets at scales unattainable by research expeditions, while demonstrating the strong potential of these initiatives to bridge molecular observations with satellite-derived assessments and improve our understanding of open ocean plankton biodiversity.
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