Coupling between ribotypic and phenotypic traits of protists across life-cycle stages and temperatures

Relationships between ribotypic and phenotypic traits of protists across life-cycle stages remain largely unknown. Herein, we assessed the effect of life-cycle stage (lag, log, plateau, cyst) and temperature on quantity and polymorphisms of ribosomal (r)RNA gene (rDNA) and rRNA transcripts by using single cells of ciliate species. Colpoda inflata and C. steinii demonstrated allometric relationships between 18S rDNA copy number per cell (CNPC), cell volume (CV), and macronuclear volume across all life-cycle stages. Integrating previously reported data of Euplotes vannus and Strombidium sulcatum indicated taxon-dependent rDNA CNPC-CV functions. Ciliate and prokaryote data analysis revealed that the rRNA CNPC followed a unified power-law function, only if the rRNA-deficient resting cysts were not considered. Hence, a theoretical framework was proposed to estimate quantity of resting cysts of a protistan population. Using rDNA CNPC was a better predictor of growth rate at a given temperature than rRNA CNPC and CV, suggesting replication of redundant rDNA operons is a key factor that slows cell division. Single-cell high throughput sequencing revealed hundreds to thousands of rDNA and rRNA variants. The number of rDNA variants corresponded to the amount of cellular rDNA. Despite intra-individual divergence reaching up to 9%, operational taxonomic units (OTUs) clustered in a species-specific manner and independently of life-cycle stage or temperature. This indicates limited influence of said divergence on distributional pattern of OTU richness among samples.