Material collection and culture
We collected a mature female gametophyte at Botofurinai, Muroran, Hokkaido, Japan (42°31′N, 140°98′E), in May 2013. Gametophytes of M. angicava release biflagellate gametes during spring at low tides (Togashi and Cox 2001). Gametes released from the gametophyte were separated from contaminants via their positive phototaxis (Togashi et al. 1999). The gametes were cultured under conditions described by Tatewaki (1969) with PES (Provasoli’s enriched sea-water) medium (Provasoli 1968) in culture chambers (LH-220S; NK System, Osaka, Japan). The gametes parthenogenetically developed into sporophytes at 14 °C under long-day conditions (14 h light:10 h dark cycle) created by cool white fluorescent lamps with an intensity of approximately 15 μmol photons/m2/s. The sporophytes released zoospores that developed into gametophytes at 12 °C under short-day conditions (10 h light:14 h dark cycle) created by cool white fluorescent lamps with an intensity of approximately 2.5 μmol photons/m2/s. The gametophytes were cultured at 10 °C under long-day conditions (14 h light:10 h dark cycle) created by cool white fluorescent lamps with an intensity of approximately 35 μmol photons/m2/s.
Observations of somatic cell divisions
We fixed and decolored gametophytes 120 min, 360 min and 600 min after the onset of the light period and every 60 min after the onset of the dark period with a 3:1 ethanol:acetic acid solution at room temperature for 72 h. For more detailed examination of the timing of mitosis, we fixed and decolored the gametophytes every 10 min from 30 to 220 min after the onset of the dark period with a 3:1 ethanol:acetic acid solution. The fixed specimens were dealcoholized with distilled water for 15 min, stained with 5 µg/ml 4′-6-diamidino-2-phenylindole (DAPI) in VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA, USA) for 10 min and softly pressed with a glass coverslip. We observed the specimens using an IX81 fluorescent inverted microscope (Olympus, Tokyo, Japan) with a CCD camera (Olympus) and a Disk-Spinning Unit for Confocal Imaging (DSU). In this study, we defined dividing cells as cells with condensed chromosomes, because it is difficult to distinguish between nondividing cells and cells undergoing mitosis that lack condensed chromosomes (e.g., prophase cells). We measured the frequencies of dividing cells in a microscopic field of 5791 µm2 (n = 10 fields per measurement).
Mitotic inhibitor treatments
From 60 to 180 min after the onset of the dark period, including the time when the cells had actively divided (see "Results" for more details), we treated the cells of young, growing M. angicava gametophytes with colchicine (0.05%, 0.1% or 0.5%), 8-hydroxyquinoline (2 mM), APM (1 µM, 5 µM, 10 µM or 50 µM), griseofulvin (1 µM, 5 µM, 10 µM or 50 µM) and oryzalin (1 µM, 5 µM, 10 µM or 50 µM) in PES medium in the culture chamber. Stock solutions of APM, griseofulvin and oryzalin were prepared with dimethylsulfoxide at 1 mM. They were diluted with PES medium. We then fixed and decolored them with a 3:1 ethanol:acetic acid solution. The specimens were dealcoholized with distilled water for 15 min, stained with 5 µg/ml DAPI in VECTASHIELD mounting medium for 10 min and softly pressed with a glass coverslip. We observed the specimens using an IX81 fluorescent inverted microscope. We measured the frequencies of dividing cells in a microscopic field of 5791 µm2 (n = 10 fields per measurement). The results were statistically analyzed with a 0.05 significance level with the Mann–Whitney U test using R version 3.2.3 (R Core Team 2015).
Three-dimensional observation of chromosomes
We obtained cross-sectional images of chromosomes stained with 5 µg/ml DAPI every 0.1 µm using an IX81 fluorescence microscope with a DSU and constructed three-dimensional images using MetaMorph software (Molecular Devices, Tokyo, Japan). We counted the number of chromosomes based on three-dimensional images.