Dependence of genome size and copy number of rRNA gene on cell volume in dinoflagellates

Dinoflagellates are an ecologically important group of protists in aquatic environment and have evolved many unusual and enigmatic genomic features such as immense genome sizes, high repeated genes, and a large portion of hydroxymethyluracil in DNA. Although previous studies have observed positive correlations between the large subunit (LSU) rRNA gene copy number and genome size of a variety of eukaryotic organisms (e.g. higher plants and animals), or between cell volume and LSU rRNA gene copy number, and/or between genome size and cell size, which suggests a possible co-evolution among these three features in different lineages of life, it remains an open question regarding the relationships among these three parameters in dinoflagellates. For the first time, we estimated the copy numbers of the LSU rRNA gene, the genome sizes, and cell volumes within a broad range of dinoflagellates (covering 15 species of 11 genera) using single-cell qPCR-based assay (determining LSU rRNA gene copy number), FlowCAM (cell volume measurement), and ultraviolet spectrophotometry (genome size estimation). The measured copy number of LSU rRNA gene ranged from 398 ± 184 (Prorocentrum minimum) to 152,078 ± 33,555 copies•cell⁻¹ (Alexandrium pacificum), while the genome size and the cell volume ranged from 5.6 ± 0.2 (Karlodinium veneficum) to 853 ± 19.9 pg•cell⁻¹ (Pseliodinium pirum), and from 1,070 ± 225 (Kar. veneficum) to 168,474 ± 124,180 μm³ (Ps. pirum), respectively. Together with the three parameters measured in literature, there are significant positive linear correlations between LSU rRNA gene copy numbers and genome sizes, cell volumes and LSU rRNA gene copy numbers, and between genome sizes and cell volumes via comparisons of multi-model regression analyses, suggesting a dependence of genome size and rRNA gene copy number on the cell volumes of dinoflagellates. Validation of the measurement methods was conducted via comparisons between reported data in the literature and that predicted using the linear equations we obtained, and between genome size measured by flow cytometry (FCM) and ultraviolet spectrophotometry (Nanodrop). These results provide insightful understandings of dinoflagellate evolution in terms of the relationships among genomes, gene copy number, and cell volume, and of rRNA gene-based studies in intra-populational and intra-individual genetic diversity, taxonomy, and diversity assessment in the environment of dinoflagellates. The results also provide a dataset useful for reads calibration in environmental metabarcoding studies of dinoflagellates and selection of candidate species for whole genome sequencing.