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Heavy-ion beams have been widely utilized as a novel and effective mutagen for mutation breeding in diverse species, including algae, but a preferred mutant cannot be easily obtained without a suitable large-scale screening method. We devised a unique, convenient, and effective method for screening mutants of Haematococcus pluvialis to isolate a strain resistant to environmental stress with low white fluorescence, i.e., a robust strain. Haematococcus was irradiated with heavy-ion beams of carbon ions, argon ions, and iron ions at various doses, after which approximately 10,000 surviving colonies were inoculated into 96-well plates, cultured for approximately 2 weeks, and then left to dry in a refrigerator for 3–12 months without a lid. In these unattended 96-well plates, cells in approximately one-third of the wells died and became white, and the remaining wells were approximately evenly split between red and green. The robustness of wild-type and mutant strains isolated from red and green wells was compared under severe environmental-stress conditions (125 µmol photons m−2 s−1, continuous light period, 45 mM sodium acetate). In the wild-type strain, most cells died, and 93.9% of cells emitted white autofluorescence. In contrast, few G4 cells emitted white autofluorescence, indicating a survival rate of 91.8%. Strains with excellent carotenoid production, such as G7 and R1, showed greater robustness compared to wild-type strains.

The evolution of dioecy from hermaphroditism allows for avoidance of self-pollination, and its evolutionary background has been investigated both experimentally and theoretically since it was first proposed by Darwin. To reproduce this evolution, we screened hermaphroditic mutants of Silene latifolia using heavy-ion beam or γ-ray irradiation and characterized the phenotypes of their floral organs. Our scatterplots indicate severe deviations from the trade-off relationships between pollen and ovule numbers and between seed and germinated pollen numbers in hermaphroditic mutant S. latifolia. These deviations presumably led to promotion of dioecy from the ancestral state of S. latifolia. To infer the likely flower phenotypic characteristics of the ancestral plant of S. latifolia before evolving dioecy, the flowers of Silene viscosa, a naturally hermaphroditic plant related to S. latifolia were also characterized. S. viscosa exhibits both spatial separation of stamens from pistils within the flower (reverse herkogamy) and temporal separation of stamen and pistil maturation (dichogamy), raising the question of whether hermaphroditic mutant S. latifolia, which is thought to be the ancestral state, would possess these functions. We show that two hermaphroditic mutants of the dioecious plant S. latifolia exhibit signs of protogyny (reverse dichogamy) and approach herkogamy, as pistils were constantly longer than stamens. These findings illustrate the evolution of dioecy from hermaphroditism as a self-pollination avoidance mechanism and to balance the investments into male and female functions.