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

Abstract

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.

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