Fig. 1: C. flexa sheets can form clonally but display aggregative features.
From: Clonal-aggregative multicellularity tuned by salinity in a choanoflagellate
a, Choanoflagellates (turquoise) are the sister group to animals (Metazoa). The phylogenetic relationships depicted are based on previous studies2,12. Polytomies indicate uncertain relationships. b, Bright-field image of a C. flexa sheet in its relaxed conformation (shown in the schematic). Inset: magnified view of the dashed square showing flagella (magenta pseudocolour) and collar–collar contacts between cells (green pseudocolour). Scale bars, 20 μm (main image) and 10 µm (inset). n = 3 biological replicates, n = 56 sheets. c, Diagnostic features of a choanoflagellate. C. flexa cells within a sheet are linked by their collars (pink arrow). d, Three-dimensional reconstruction of a confocal z stack of a fixed sheet (inverted conformation, shown in the schematic), with cell bodies stained with a membrane/cytoplasmic dye (FM 1-43FX, magenta) and collars stained with an F-actin dye (phalloidin-rhodamine, white). n = 7 biological replicates, n = 30 sheets. Scale bar, 20 μm. e, Stills from a bright-field timelapse analysis of sheet formation by serial cell division from a single cell (white arrowhead). After each division, sister cells remain connected by collar–collar contacts (black arrow). Note that cells retract their flagellum during division. Time is shown as h:min. n = 3 biological replicates. Scale bar, 10 μm. f, The cell lineage tree as a function of time in e shows asynchronous divisions during colony formation (approximately every 8–10 h). g, Stills from a bright-field timelapse analysis of a medium-sized sheet expanding by cell division (orange, pink, blue and yellow pseudocolour) and by aggregation (white arrow, green pseudocolour). Time is shown as h:min. n = 2 biological replicates, with n = 2 technical replicates each (n = 4). Scale bar, 25 μm. h, Schematics of the mechanisms that establish multicellularity in C. flexa: colonies can form and grow by cell division but might also expand by aggregation. Sister cells adhere to each other through collar–collar contacts (pink arrowhead). The figure is related to Supplementary Fig. 1 and Supplementary Videos 1–3.
