Heterotrophic nanoflagellates are the chief agents of bacterivory in the aquatic microbial loop but remain underrepresented in culture collections and in genomic databases. We isolated and characterized a representative of the previously uncultured freshwater Cryptomonad Group 1 (CRY1a) lineage using a genome-streamlined, ultra-small and abundant microbe Planktophila versatilis as a prey and Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH) probe-based screening. This isolate, Tyrannomonas regina, is one of the most dominant ubiquitous heterotrophic cryptomonads in freshwaters. It is a small heterotrophic nanoflagellate (ca. 3-5 μm) and has the smallest genome of any cryptomonad sequenced thus far. The compact genome (ca. 69 Mb) revealed no traces of a photosynthetic lifestyle, consistent with its phylogenomic placement as a sister clade to cryptophytes that are characterized by the acquisition of a red-algal symbiont. Moreover, in comparison to its photosynthetic counterparts, its genome presents substantially lower repeat content and endogenous viral elements. Genomes of two giant viruses, Tyrannovirus reginensis GV1 and GV2, were also recovered from the same culture and represent a viral genus that has been described so far solely by metagenome-recovered genomes. Collectively, these findings provide insights into genomic ancestry and evolution, widespread ecological impact, and interactions of an elusive protist lineage and illustrate the advantages of culture-centric approaches towards unfolding complex tapestries of life in the microbial world.

Biology Centre of the Czech Academy of Sciences Institute of Hydrobiology Na Sádkách 7 37005 České Budějovice Czech Republic

Department of Biological Sciences and Bioengineering Inha University 22212 Incheon Republic of Korea

Faculty of Science University of South Bohemia 37005 České Budějovice Czech Republic

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