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 intracellular vesicles (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)(35,36). 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)(41). 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 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)(43). 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 (39)(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 (59)(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)(39). Scale bars: A-F (10μm), G & H (20μm), and I (50μm).

Morphological comparison of the Buck 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 Buck Reef Chert (originally published by Tice et al., 2009)(61). 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.

Image A shows laminated structures reported from the Moodies Group (originally published by Homann et al., 2015)(63). They show parallel layers of organic carbon with lenticular gaps. Together with the quartz, these lenticular gaps consist of clumps of organic carbon. Image B is a 3D-rendered confocal image of analogous membrane debris formed by EM-P. Images C & D are the magnified regions of C. Like Moodies formation, filamentous membrane debris bifurcating to form spherical\lenticular gaps can be seen in several regions (S75, S76, & S77). Some spherical/lenticular gaps were hollow, and some had an organic structure within them, even exhibiting a honeycomb pattern (arrow), suggesting the presence of large spherical EM-P cells with intracellular vesicles (D, & S77). Membranes were stained with Nile red, and imaging was done using a STED microscope. The scales: 50μm.