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 used single cells of two soil and two marine ciliate species to examine phenotypic and ribotypic traits and their relationships across lag, log, plateau, cystic stages and temperatures. We found that 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 the relative quantity of resting cysts in the protistan populations with total cellular rDNA and rRNA copy numbers. 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 as a key factor that slows cell division. Single-cell high throughput sequencing and analysis after correcting sequencing errors revealed multiple rDNA and rRNA variants per cell. Both encystment and temperature affected the number of rDNA and rRNA variants in several cases. The divergence of rDNA and rRNA sequence in a single cell ranged from 1% to 10%, depending on species. These findings have important implications for inferring cell-based biological traits (e.g., species richness, abundance and biomass, activity, and community structure) of protists using molecular approaches. ImportanceBased on phenotypic traits, traditional surveys usually characterize organismal richness, abundance, biomass, and growth potential to describe diversity, organization and function of protistan populations and communities. The ribosomal RNA gene (rDNA) and its transcripts have been widely used as molecular markers in ecological studies of protists. Nevertheless, the manner in which these molecules relate to cellular (organismal) and physiological traits remains poorly understood, which could lead to misinterpretations of protistan diversity and ecology. The current research highlights the dynamic nature of cellular rDNA and rRNA contents, which tightly couple with multiple phenotypic traits in ciliated protists. We demonstrate that quantity of resting cysts and maximum growth rate of a population can be theoretically estimated using ribotypic trait-based models. The intra-individual sequence polymorphisms of rDNA and rRNA can be influenced by encystment and temperature, which should be considered when interpreting species-level diversity and community structure of microbial eukaryotes.