Nuclear diversity in Charophyceae revealed by fluorescence staining and its implications for genome size estimation

We established a protocol for reliable nuclear visualization in Charophyceae, revealed diverse nuclear organization across cell types and species, and identified suitable cells for genome size estimation via flow cytometry. Charophyceae are multicellular green algae closely related to land plants and are established model systems for understanding plant evolution. Yet key cellular parameters like genome size remain poorly characterized. We combined fluorescence microscopy, transmission electron microscopy (TEM), and flow cytometry to characterize nuclear diversity across cell types and species of Characeae and to identify a cell type suitable for genome size estimation. Among three DNA-intercalating fluorochromes, propidium iodide labeled nuclei most reliably. Nuclear morphology varied widely across cell types: mononucleated cells were found in vegetative apical cells, the coronula of oogonia, and spermatogenous filaments of antheridia, whereas multinucleation predominated in other tissues, e.g., cortical cells, spine cells, stipulodes and rhizoids. Nuclei in Chara hispida showed a significant gradient in cross-sectional area along the thallus axis. In the apical internodes, nuclei were larger and more heterogeneous, whereas in the basal internodes they were smaller and more uniform, which is consistent with possible endopolyploidy. TEM confirmed the nuclear identity of crescent-shaped structures. Relatively large nuclei were found in rhizoids and spine cells. Only Sphaerochara canadensis showed an organized nuclei pattern. Whole-thallus preparations did not yield a defined nuclear peak by flow cytometry, but antheridia of Chara tomentosa produced a sharp peak, from which a genome size of 5.32 pg (1C) was estimated. The protocol established here provides a simple, reproducible framework for visualizing nuclei and estimating genome size in Charophyceae, and helps address longstanding questions in this group, such as the mechanisms and functions of multinucleation, the site of meiosis, and genome evolution.