Single Cell Fluorescence Action Spectra for the Targeted Study and Isolation of Phytoplankton Within Complex Assemblages

Discerning the diversity, abundance, and functional role of distinct phytoplankton groups is essential to the study of aquatic systems. The diversity and ecological roles of phytoplankton are still understudied relative to their importance in the ocean system. Here, we present a new flow cytometry method that uses up to five excitation colors to determine the relative fluorescence action spectra of phytoplankton within complex assemblages, thus leveraging the precise and high-throughput capabilities of flow cytometry and the unique combinations of photosynethetic pigments in phylogenetically related groups of phytoplankton. We tested the method on cultivated Synechococcus of known pigment composition and genotype then applied the method to a natural phytoplankton assemblage from several coastal and open ocean environments. We determined the relative fluorescence action spectra of up to 8 distinct phytoplankton populations. By coupling multi-laser flow cytometry to cell sorting we demonstrated that natural phytoplankton populations with similar relative fluorescence action spectra belonged to the same taxonomic classes based on 18S rRNA gene phylogeny. Based on these results, we suggest that multi-laser flow cytometry could be applied to enumeration and isolation of Class-level genotypically distinct phytoplankton groups for improved genome assembly from metagenomes, metabolic analysis including nutrient uptake, and targeted isolation for cultivation.