Morphological comparison of the Apex Chert and the Strelley Pool Formation microfossils with EM-P.

Images A-D are TEM images of EM-P cells forming ICVs and intracellular daughter cells. The numbered arrows in these images point to different stages of ICV formation (see Fig. S1). Images E, F, K & L show TEM, SEM, and STED microscope images of EM-P cells with ICVs and surface depressions (black arrows). Cells in image F were stained with universal membrane stain, FMTM5-95 (red), and DNA stain, PicoGreen (green). Images G-J & M are spherical microfossils reported from the Apex Chert and the Strelley Pool Formation, respectively (originally published by Schopf et al., 1987 & Delarue et al., 2019)(34,35). Cyan arrows in images E-H point to cytoplasm sandwiched between large hollow vesicles. The arrow in the image I point to the dual membrane enclosing the microfossil. Morphologically similar images of EM-P cells are shown in Fig. S3. Black arrows in images K-M point to surface depressions in both EM-P and the Strelley Pool Formation microfossils, possibly formed by the rupture of ICV’s as shown in D & E (arrows) (also see Fig. S4-S6). Scale bars: A-D (0.5µm) E, K & L (2 µm), and 5 µm (F).

Morphological comparison between the Mt. Goldsworthy microfossils and EM-P.

Images A-E show the process of cell lysis and release of intracellular vesicles in EM-P. Image A shows an intact cell with intracellular vesicles. Images B-E show lysis and gradual dispersion of these vesicles. Insert in image D shows enlarged images of individual ICVs. Images F-I show spherical microfossils reported from the Mt. Goldsworthy formation (originally published by Sugitani et al., 2009)(39). The arrow in this image, A & F, points to a cell surrounded by an intact membrane. The black arrow in these images points to filamentous extensions connecting individual vesicles. The boxed region in images D & I highlights a similar discontinuous distribution of organic carbon in ICVs and microfossils. Also see Fig. S13-S17. Scale bars: 20μm (A-E) & 20μm (F-I).

Morphological comparison of the Cleaverville microfossils with EM-P:

Images A-E are the microfossils reported from Cleverville formation (originally reported by Ueno et al., 2006). Images F-K are the EM-P cells that are morphologically analogous to the Cleverville Formation microfossils. Open arrows in images A, B, F & G point to the membrane tethers connecting the spherical cells within the filamentous extensions. Red arrows in the images point to the cells that have a similar distribution of organic carbon within the cells. Boxed and magnified regions in images B, F & G highlight the arrangement of cells in the filaments in pairs. The boxed region in image H highlights the cluster of hollow vesicles in EM-P incubations similar to the hollow organic structures in the Cleverville Formation, as shown in image c. Images D, E, and I-J show spherical cells that were largely hollow with organic carbon (cytoplasm) restricted to discontinuous patches at the periphery of the cell. Scale bars: 20μm (F-K).

Morphological comparison between EM-P and the Mt. Goldsworthy microfossils.

Images A, B & C are organic structures reported from the Mt. Goldsworthy Formation (Sugitani et al., 2009)(41). Image D shows morphologically analogous film-like membrane debris observed in EM-P incubations. Arrows in images A-D point to either clusters or individual spherical structures attached to these film-like structures. Scale bar: 50μm (A-C) & 10μm (D).

Morphological comparison of the Mt. Goldsworthy and the Sulphur Spring microfossils with EM-P.

Image A-C are microfossils reported from the Mt. Goldsworthy Formation (37)(Sugitani et al., 2007). Image D is the 3D-rendered STED microscope images of morphologically analogous membrane debris of EM-P cell with attached daughter cells (highlighted region) (also see Fig. S40). Images E & F are microfossils reported from the Sulphur Spring site (57)(Duck et al., 2007), showing spherical structures attached to membrane debris. Images G & J are the morphologically analogous structures observed in EM-P incubations. Images H & I show the magnified regions of G & J showing spherical EM-P daughter cells attached to membrane debris (also see Fig. S40-S47, Movie 17). Cells and membrane debris in these images were stained with the membrane stain FMTM5-95 (yellow). Scale bars: A (50μm), G & J (20μm).

Sequential steps involved in the formation of honeycomb-shaped mats:

Images A-C show single EM-P cells that gradually transformed from spherical cells with intracellular vesicles into honeycomb-like structures. Images D-E show a similar transformation of biofilms composed of individual spherical cells into honeycomb-like structures. Cells in these images are stained with membrane stain, FMTM5-95 (red), and imaged using a STED microscope. Images G-J are the microfossils reported from the SPF (originally published by Sugitani et al., 2007)(37). Scale bars: A-F (10μm), G & H (20μm), and I (50μm).

Morphological comparison of the Buch Reef Chert

β-laminations with EM-P’s membrane debris. Image A shows a 3D-rendered image of EM-P’s membrane debris. Cells in the image are stained with membrane stain Nile red and imaged using a STED microscope. Images B & C show β-type laminations reported from Buch Reef Chert (originally published by Tice et al., 2009)(59). The boxed region in image-a highlights the membrane-forming rolled-up structures containing spherical daughter cells, as described in the case of BRC organic structures. Scale bars: 50μm.

Morphological comparison between laminated structures reported from the Moodies Group and structures formed by EM-P.

Images A & B are laminated structures reported from the Moodies Group (originally published by Homann et al., 2015)(61). They show filamentous structures with lenticular gaps. These gaps were filled with quartz granules. Image C is a 3D-rendered confocal image of EM-P’s membrane debris. Filamentous membrane debris bifurcating, forming spherical\lenticular gaps, can be seen in several regions. Some spherical/lenticular gaps were hollow (box), and some had a honeycomb pattern within them (arrow), indicating the presence of large spherical EM-P cells with intracellular vesicles (D). 3D rendered STED microscope images of individual spherical/lenticular gaps with and without EM-P cell debris were shown in Fig. S75 & S76, and S77. Membranes were stained with Nile red, and imaging was done using a STED microscope. The scales: 50μm.