Fig. 4: Hypersalinity induces sheet dissociation and encystation.

a, The laboratory evaporation setup. b, The salinity over time in n = 12 splash pools (Fig. 3h and Extended Data Fig. 3) and in the laboratory (lab.) setup. Splash pools with less than three points below saturation are not shown. c, Quantification of salinity (top) and the fraction of cells in multicellular versus unicellular forms (bottom) during gradual evaporation. Data are mean ± s.d. n = 2 biological replicates (n = 11). d, Sheet dissociation and reformation during evaporation–rehydration in c. Top left, salinity. Top right, time. Scale bars, 20 μm. e, Cell division and aggregation restore multicellularity after rehydration. Time is shown as h:min. n = 2 biological replicates, n = 2 technical replicates each (n = 4). Scale bar, 25 μm. f–i, Morphological changes during evaporation. At 35 ppt (f), C. flexa cells display microvilli (green pseudocolour) and a flagellum (magenta). Inset: magnified view of the dashed square, showing the microvilli and flagellum. During evaporation (g–i), sheets dissociate and cells encyst, frequently lacking a collar (c) and a flagellum (fl) (g) but sometimes (h), although not always (i), displaying filopodia-like protrusions (pink arrowheads). Top right, salinity. Scale bars, 5 μm (f) and 2 μm (g–l). j–l, In flagellates (j), F-actin distributes in the collar (white arrowhead) and filopodia (fp). Filopodia-like protrusions (pink arrowheads) and an actin cortex (cx) are seen during early encystation (k, white arrow) and disappear in later cysts (l). Top right, salinity. m, The growth rate of cells at different salinities during evaporation. n, Schematic of dissociation–encystation. N:C ratio, nucleus-to-cytoplasm ratio. o, Desiccated sheets after rapid evaporation. Scale bar, 10 μm. p, The survival of sheets and cysts after desiccation. Data are mean ± s.d. n = 6 technical replicates. q, Sheets that have captured fluorescently labelled bacteria. Scale bar, 25 μm. r, Bacterial capture by colonies (n = 9) and unicellular flagellates (n = 9). For f–m, q and r, n = 3 biological replicates, n = 2 technical replicates each (n = 6). For m and r, the black circles show the mean values; the error bars show the s.d.; and the diamonds show the replicate means. P values in m, p and r were calculated using Mann–Whitney U-tests. The figure is related to Extended Data Figs. 5 and 6, Supplementary Figs. 5–8 and Supplementary Videos 17–19.