Linking Protistan Trophic Indicators to Phylogenetic Identity: From Single-Cell Grazing Evidence to Biogeographic Context.

Protists form the foundation of aquatic food webs and drive global nutrient cycles, yet distinguishing which species photosynthesize, graze or do both remains a major challenge because most are uncultivable and community surveys seldom resolve species-level traits. We developed a field grazing-scPCR framework that integrates short-term grazing assays, single-cell microscopy and 18S rRNA sequencing to link morphology, fluorescence-based trophic indicators, ingestion evidence and phylogenetic identity in 21 individually isolated protistan cells spanning freshwater to oceanic ecosystems. Using a conservative, phylogeny-informed classification, this approach confirmed constitutive mixotrophs (Cryptomonas curvata, Poterioochromonas malhamensis), identified a candidate non-constitutive mixotroph within Katablepharidaceae, and showed that prey-derived fluorescence can overestimate mixotrophy in natural assemblages. Two C. curvata isolates exhibited contrasting states, an active grazer with plastid autofluorescence and a non-grazing, aflagellate cyst retaining plastid fluorescence, highlighting the limits of single-time-point assays. Linking single-cell observations to MetaPR2 and Tara Oceans exact-match records further placed trophically characterized taxa in a broader biogeographic context. This framework advances species-level resolution of protistan trophic diversity in nature while underscoring the need to interpret fluorescence and ingestion signals in phylogenetic and ecological context.