On the nature of the earliest known lifeforms
- Max-Planck Institute for Biochemistry, Germany
- Chair of Ecological Microbiology, BayCeer, University of Bayreuth, Germany
- Earth and environmental sciences, Ludwig Maximilian University, Germany
- CNRS-Centre de Biophysique Moléculaire, France
- Advanced Light Microscopy Facility, European Molecular Biology Laboratory, Germany
- Key Laboratory of Agro-Ecological Processes in Subtropical Regions, Taoyuan Agroecosystem Research Station, Institute of Subtropical Agriculture, Chinese Academy of Sciences, China
- Department of Biosciences, Center for Electromicrobiology, Denmark
- Department of Botany I, Ludwig Maximilian University, Germany
Figures
Figure 1 with 6 supplements
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 Figure 1—figure supplement 1 and 2). 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, FM5-95 (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 and Packer, 1987; Delarue et al., 2020 ; Schopf and Packer, 1987; Delarue et al., 2020). Cyan arrows in images (E-H) point to cytoplasm sandwiched between large hollow vesicles. The arrow in the image I points to the dual membrane enclosing the microfossil. Morphologically similar images of EM-P cells are shown in Figure 1—figure supplement 3. 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 ICVs as shown in (D & E) (arrows) (also see Figure 1—figure supplements 4–6). Scale bars: (A-D) (0.5 µm) (E, K, & L) (2 µm), and 5 µm (F).
Figure 1—figure supplement 1
EM-P cells with intracellular vesicles (ICV).
Images (A-E) show STED microscope images of EM-P cells in different stages of intracellular vesicle formation. Cells in these images were stained with FM5-95 (membrane, white) and PicoGreen (DNA, red). Image (B) shows the first stage of vesicle formation by a process similar to endocytosis. Images (C-E) show a gradual increase in the number of intracellular vesicles. Image (E) shows the 3D-rendered STED microscope image of EM-P cells with hollow invaginations on the cell surface. Images (G-K) show TEM images of EM-P cells with intracellular vesicles. Arrows in these images point to the gradual collapse of ICVs, leading to the formation of spherical invaginations on the cell surface. Scale bars: 5 μm (A–E), 10 μm (F), and 250 nm (G–K).
Figure 1—figure supplement 2
Variation in the morphology of EM-P cells and intracellular vesicles.
Images (A-J) are STED microscopy images of EM-P cells with intracellular vesicles. These images show variations in the size and number of vacuoles observed within EM-P. Cells in these images were stained with FM5-95 (membrane, white) and PicoGreen (DNA, red). Scale bars: 5 μm (A–G) & 10 μm (H–J).
Figure 1—figure supplement 3
Morphological comparison of the Apex Chert microfossils with EM-P.
Images (A-D) show spherical microfossils with hollow intracellular compartments and two layers of the outer cell membrane (arrows) (originally published by Schopf and Packer, 1987; Schopf and Packer, 1987). Images (E & F) are morphologically analogous to EM-P cells with hollow intracellular compartments. Like microfossils, EM-P cells also appear to have a dual outer membrane due to the juxtaposition of vesicle membrane against cell membrane (arrows). Scale bar: 10 μm (E & F).
Figure 1—figure supplement 4
Morphological comparison between EM-P and the Strelley Pool Formation (SPF) microfossils.
Images (A-C) are SEM images of spherical microfossils reported from the SPF site (originally published by Delarue et al., 2020). Images (D & F) are 3D rendered STED microscope images of similar EM-P cells with hexagonal internal vacuoles. Image (E) is the individual Z-stack image of cells shown in image (D). Arrows in this image point to regions of cells with large and small polygonal vacuoles. Cells in images (D-F) were stained with membrane stain, FM5-95. A comparison of SPF microfossils with EM-P was also shown in Figure 1—figure supplements 5 and 6. Scale bar: 10 μm (D & F).
Figure 1—figure supplement 5
Morphological comparison between EM-P and Strelley Pool Formation SPF microfossils.
Images (A-C) are SEM images of spherical microfossils reported from the SPF site (originally published by Delarue et al., 2020; Delarue et al., 2020). Images (D & E) are SEM images of morphologically analogous EM-P cells with hexagonal internal vacuoles. Red and cyan arrows in all images point to either surface depressions or inward cell membrane bending in both SPF microfossils and EM-P cells. As shown in the figure, we assume these structures are formed due to invagination, complete lysis, or individual vacuole membranes, as shown in Figure 1—figure supplement 6D and E. Scale bar: 0.5 μm (E).
Figure 1—figure supplement 6
Morphological comparison between EM-P and SPF microfossils.
Images (A-C) are SEM images of SPF spherical microfossils with spherical/polygonal surface depressions (originally published by Delarue et al., 2020; Delarue et al., 2020). Images (D & E) are TEM images of morphologically analogous EM-P cells. Red arrows in these images point to surface depressions formed by the partial collapse of the vacuole membrane. Blue arrows in these images point to surface depressions formed by the complete rupture of the vacuole membrane. Scale bar: 200 nm (D & E).
Figure 2 with 5 supplements
Morphological comparison between the Mt. Goldsworthy microfossilsand 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). 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 Figure 2—figure supplements 1–5. Scale bars: 20 μm (A–E) & 20 μm (F–I).
Figure 2—figure supplement 1
Morphological comparison between EM-P and the Mt. Goldsworthy Formation microfossils.
Images (A-C) are the microfossils reported from the Mt. Goldsworthy locality in the Pilbara formations (originally published by Sugitani et al., 2009; Sugitani et al., 2013). Images (D-F) are morphologically analogous to EM-P cells. The highlighted regions in images (D-F) show in sequence the ICVs with tiny daughter cells, the lysis and release of these daughter cells into the surrounding media, the lysis of the vesicle membrane, and the release of a cluster of intracellular daughter cells. Similar lysis of cells, intracellular vesicles, and the clusters of daughter cells (highlighted region in C) can be seen in images (A-C). Scale bar: 10 μm.
Figure 2—figure supplement 2
Morphological comparison between EM-P and the Mt. Goldsworthy Formation microfossils.
Images (A & B) are Mt. Goldsworthy Formation microfossils, either with hollow intracellular vesicles (A, blue arrows) or spherical clumps of organic inclusions (B) (originally published by Lepot et al., 2013). Images C-F show morphologically analogous EM-P cells. Image (C) shows an intact and lysed EM-P cell. Image (D) shows a close-up view of the individual vesicle with daughter cells. Image (E&F) shows clumps of daughter cells formed after the lysis of vesicles. EM-P daughter cells exhibit all the morphological features of Mt. Goldsworthy microfossils, like the cluster of spherical daughter cells, daughter cells undergoing binary fission, and filamentous extensions. These cells also resemble the microfossils reported from SPF in their morphology (Appendix 1—figures 8 and 9). Scale bar: 10 μm (B–F).
Figure 2—figure supplement 3
Morphological comparison between EM-P and the Mt. Goldsworthy Formation microfossils.
Images (A & B) show the microfossils reported from Mt. Goldsworthy’s locality within SPF (originally published by Lepot et al., 2013). Images (C-F) show the TEM images of EM-P cells. Red arrows in these images point to the tiny globules reported from the Mt. Goldsworthy Formation and EM-P daughter cells. EM-P daughter cells and the globules reported from the Mt. Goldsworthy Formations exhibit similar morphological features like membrane overhangs. The green arrows in images (A & C) point to the EM-P cells with uneven distribution of intracellular organic carbon. The blue arrow in image (A) points to a spherical cell with hollow intracellular spaces. Similar EM-P cells were shown in Figure 1 & Appendix 1—figures 1–3. Scale bar: 500 nm (C–E) and 1 μm (F).
Figure 2—figure supplement 4
Mrphological comparison between EM-P and the Mt. Goldsworthy Formation microfossils.
Images (A & B) are the microfossils reported from the Mt. Goldsworthy Formation (Sugitani et al., 2007; Retallack et al., 2016). These images show hollow spherical cells associated with clusters of spherical structures. Images (E-J) are the phase-contrast microscope images of EM-P, morphologically similar to the Mt. Goldsworthy Formation microfossils. Images (D & E) are the time series images of the magnified region of image *(C). Images (G-J) are the time series images of the magnified region of image F. Time series images show the movement of spherical structures attached to the inner spheroid. Scale bars: 20 μm (A & B) and 10 μm (C & F).
Figure 2—figure supplement 5
Morphological comparison between EM-P and the Mt. Goldsworthy Formation microfossils.
Images (A-D) are the microfossils reported from the Mt. Goldsworthy Formation (Sugitani et al., 2007; Retallack et al., 2016). The black arrow in these images points to the deepening focal depths. The white arrow in image (C) points to the internal carbon-rich spheroidal structure. Images (E-I) are the phase-contrast microscope images of EM-P, morphologically similar to Mt. Goldsworthy formation microfossils. Arrows in these images point to a similar internal spheroidal structure observed in the Mt. Goldsworthy Formation microfossils. Scale bar: 20 μm (A–D) and 10 μm (E–I).
Figure 3
Morphological comparison of the Cleaverville microfossils with EM-P.
Images (A-E) are the microfossils reported from Cleaverville 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: 20 μm (F–K).
Figure 4
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., 2007; Sugitani et al., 2007). 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: 50 μm (A–C) & 10 μm (D).
Figure 5 with 9 supplements
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 (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 Figure 5—figure supplement 1). Images (E & F) are microfossils reported from the Sulphur Spring site (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 Figure 5—figure supplements 1–9, Video 17). Cells and membrane debris in these images were stained with the membrane stain FM5-95 (yellow). Scale bars: A (50 μm), (G & J) (20 μm).
Figure 5—figure supplement 1
Morphological comparison between the Sulphur Spring Formation microfossils and EM-P.
Images (A-C & G) are organic structures reported from the Sulphur Spring Formation (originally published by Duck et al., 2007). Images (D-F) are Phase-contrast (D & E) and TEM (E) images of morphologically analogous structures formed by EM-P. Images (D & E) show daughter cells attached to membrane debris. CM and V in (C & F) stand for Cell Membrane and Vacuole, respectively. The movement of daughter cells attached to membrane debris can be seen in Video 17. Image (H) shows the 3D reconstituted confocal image of folded membrane debris similar to the ones reported from the Sulphur Spring Formation. Scale bars: 10 μm (C & E) and 200 nm (D).
Figure 5—figure supplement 2
Morphological comparison between EM-P and the Mt. Goldsworthy microfossils.
Images (A, B & C) are organic structures reported from the Mt. Goldsworthy Formation (originally published by Sugitani et al., 2007; Retallack et al., 2016). Images (D & E, F & G,) and (H & I) (magnifier region of image-F, highlighted in cyan box) are anterior and posterior views of morphologically analogous membrane debris produced by EM-P cells. Both sets of images show a film-like membrane with clusters of hollow spherical attachments (cyan arrows). Images (H & I) show flexible film-like membrane debris of EM-P with similar spherical attachments. Scale bars: 50 μm (A) and 10 μm (D–G).
Figure 5—figure supplement 3
Morphological comparison between EM-P and the Mount Grant microfossils.
Images (A, B & C) are organic structures reported from the Mount Grant Formation (Sugitani et al., 2007; Retallack et al., 2016). Scale bar: 20 μm (A–C). Images (D & H) are morphologically analogous to EM-P’s membrane debris. Arrows in the images point to spherical inclusions attached to thread-like membrane debris. Membrane debris in images (G & H) should have formed by lysis and collapse of either individual or cluster of large spherical cells of EM-P with hexagonal vesicles (Figure 5—figure supplement 4). Scale bars: 10 μm (D–H).
Figure 5—figure supplement 4
Morphological comparison between EM-P and the SPF organic structures.
Images (A-D) are organic structures reported from the Mount Grant Formation (Sugitani et al., 2013; Schopf and Barghoorn, 1967). Scale bar: 20 μm (A–C). Images (E-G) are TEM images of morphologically analogous EM-P cells. Arrows in the images (B & F) point to spherical inclusions attached to thread-like membrane debris. Images (H-M) show the step-by-step transformation of EM-P cells into polygonal cell beds. Boxed regions within these images show the presence of a polygonal honeycomb-like structure within the cell debris. Scale bars: 1 μm (E–G) & 10 μm (H–M).
Figure 5—figure supplement 5
Morphological comparison of the SPF microfossils with EM-P.
Images (A & B) are organic structures reported from the SPF (Sugitani et al., 2013; Schopf and Barghoorn, 1967). Scale bars in images (A & B) are 20 μm & 50 μm, respectively. Images (C-D) are morphologically analogous to EM-P cells or their membrane debris (E). Dual-walled honeycomb-like structures could be seen in both images (A & C) (white arrows). Black arrows in images (A & D) point to a string of spherical daughter cells attached to the walls of the honeycomb. Boxed regions in images (B & E) show similarities between film-like debris reported from SPF and membrane debris observed in EM-P incubations. These structures have spherical daughter cells with a membrane wrapping (arrows). Scale bar: 10 μm (C&E) & 1 μm (D).
Figure 5—figure supplement 6
Morphological comparison between EM-P membrane debris and SPF membrane debris.
Images (A-C) are organic structures reported from SPF formation (originally published by Delarue et al., 2020; Delarue et al., 2020). These images show folded membrane-like structures. Images (D, E & F) are SEM, phase-contrast, and 3D-rendered STED images of morphologically analogous EM-P’s membrane debris. Arrows in images (B, C & E) point to the bending of the membrane at the edges in both SPF organic structures and EM-P. Arrows in image F point to spherical daughter cells of EM-P enclosed within the membrane debris. Scale bars: 2 μm (D), 10 μm (E) and 20 μm (F).
Figure 5—figure supplement 7
Morphological comparison between EM-P and the Farrel Quartzite film-like structures.
Images (A & B) are organic structures reported from the Farrel quartzite formation (Retallack et al., 2016; Kapteijn et al., 2022). Both images show a folded film-like membrane with spherical inclusions. Image (C) shows a wrinkled membrane with spherical cells attached, similar in structures to the Farrel Quartzite microfossils. Scale bar: 20 μm (C).
Figure 5—figure supplement 8
Morphological comparison between EM-P and the Moodies Group microfossils.
Images (A & B) are organic structures reported from the Moodies Group (Köhler and Heubeck, 2019) . Both images show a folded film-like membrane with spherical inclusions (black arrows). Image (C) shows a wrinkled membrane with spherical cells attached to it, similar in structures to the Moodies microfossils. Scale bar: 10 μm.
Figure 5—figure supplement 9
Morphological comparison of organic structures reported from the Dresser Formation with EM-P.
Images (A-E) show organic structures reported from the Dresser Formation (originally published by Wacey et al., 2018a; Wacey et al., 2018a). It shows wavy filamentous structures (blue arrows) with individual (red arrow) or aggregations (box) of hollow spherical inclusions. Image (B) is the membrane debris formed by lysis of EM-P, which is analogous in its morphology to organic structures reported from the Dresser Formation. Cells and membrane debris in image (F-H) were stained with FM5-95 (red) membrane stain. The boxed regions within (A, F, & G) show similar clusters of hollow spherical vacuoles. Scale bar: 10 μm (F & G).
Figure 6
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, FM5-95 (red), and imaged using a STED microscope. Images (G-J) are the microfossils reported from the SPF (originally published by Sugitani et al., 2007). Scale bars: (A-F) (10 μm), (G & H) (20 μm), and I (50 μm).
Figure 7
Morphological comparison of the Buck Reef Chert b-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, 2009). 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: 50 μm.
Figure 8 with 4 supplements
Morphological comparison between laminated structures reported from the Buck Reef Chert and structures formed by EM-P.
Image (A) shows laminated structures reported from the Buck Reef Chert (originally published by Tice, 2009). 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 Buck Reef Chert formation, filamentous membrane debris bifurcating to form spherical/lenticular gaps can be seen in several regions (Figure 8—figure supplements 1–4). 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, & Figure 8—figure supplement 3). Membranes were stained with Nile red, and imaging was done using an STED microscope. The scales: 50 μm.
Figure 8—figure supplement 1
Morphological comparison of the Moodies Group laminations with EM-P’s membrane debris.
Image (A) shows a-type laminations reported from the Moodies Group (originally published by Homann et al., 2018; Kazmierczak et al., 2009). Image B is a 3D-rendered confocal image of EM-P’s cell debris. Arrows in images (A & B) point to the lenticular gaps within the membrane debris. Scale bar: 20 μm (B).
Figure 8—figure supplement 2
Morphological comparison between laminated structures reported from the Moodies Group and structures formed by EM-P.
Images (A-C) are laminated structures reported from the Moodies Group (originally published by Hickman-Lewis and Westall, 2021; Hickman-Lewis and Westall, 2021). They show filamentous structures with lenticular gaps. 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, and some had a honeycomb pattern within them, indicating the presence of large spherical EM-P cells with intracellular vesicles (Figure 8, Figure 8—figure supplements 3 and 4). Membranes were stained with Nile red, and imaging was done using an STED microscope. Scale bar is 50 μm.
Figure 8—figure supplement 3
Lenticular membrane debris of EM-P.
Image (A) shows spherical EM-P cells covered in fabric-like membrane debris. Images (B-E) show an EM-P biofilm at different focal planes (bottom to top). The boxed region in image (D) shows the honeycomb pattern within the lenticular gap (also see Figure 8—figure supplement 4 & Video 21). Scale bar: 20 μm.
Figure 8—figure supplement 4
Lenticular membrane debris of EM-P.
The image shows the membrane debris produced by the lysis of EM-P cells. A honeycomb pattern within the lenticular structure suggests the presence of individual cells or EM-Ps vesicles during the time of their formation. (also see Video 21). Scale bar: 20 μm.
Appendix 1—figure 1
Morphological comparison between EM-P and the Farrel Quartzite microfossils.
Image (A) shows microfossils with hollow polygonal vacuoles from the Farrel Quartzite formation (originally published by Retallack et al., 2016; Kapteijn et al., 2022). Images (B-D) are images of morphological analogous EM-P cells with polygonal vacuoles. Cells in images (B-D) were stained with membrane stain, FM5-95. Polygonal vacuoles in these images could have formed by the confinement of many vacuoles within a cell, as shown in Figure 1—figure supplement 4F. The cytoplasm in these cells was restricted to narrow spaces between the vacuoles, as shown in Figure 1E and F. Scale bar: 10 μm (B) and 20 μm (C).
Appendix 1—figure 2
Morphological comparison between honeycomb structures reported from the Turee Creek Formations and EM-P.
Image (A) shows a spherical microfossil reported from the Turee Creek Formation (originally published by Barlow and Van Kranendonk, 2018; Sugitani et al., 2009). Images (B-D) are morphologically analogous to EM-P cells. Cells in these images were stained with membrane stain, FM5-95. Scale bar: 20 μm.
Appendix 1—figure 3
Morphological comparison between EM-P and the Fig Tree Formation microfossils.
Images (A-F) show spherical microfossils reported from Fig Tree Formation (originally published by Schopf and Barghoorn, 1967; Sugitani et al., 2007). Images (G-I) are morphologically analogous phase-contrast images of EM-P. The presence of regions with and without organic carbon can be seen in microfossil images A-E and EM-P cell shown in images (G & H). Distinctive spherical vacuoles devoid of organic carbon can be seen in images (B & C), (G & H). Image (F) shows hollow structures morphologically similar to hollow ICVs released by the lysis of EM-P cells (J & K). Scale bar: 10 μm (G–K).
Appendix 1—figure 4
Sequential stages of intracellular daughter cell formation.
Images (a-d) are TEM images of EM-P cells showing the formation of daughter cells into hollow ICVs. Arrows in image (A) point to the first step in daughter cell formation by a process resembling budding. Images (B-D) show the gradual growth of these buds into a string of daughter cells. Scale bar: 500 nm.
Appendix 1—figure 5
Lifecycle of EM-P cells reproducing via the formation of internal daughter cells.
Images (A-H) show phase-contrast images of EM-P reproducing by forming internal daughter cells. These images show a gradual increase in the volume of the ICV and a proportional decrease in the cytoplasmic volume of the cells. Images (I & J) show EM-P cells in their mid and late stationary growth phase. Insert in the image I show a magnified view of the highlighted cell. White arrows in image-J point to internal daughter cells. Scale bars: 10 μm.
Appendix 1—figure 6
Lifecycle of EM-P cells reproducing via the formation of internal daughter cells.
Images (A-H) show phase-contrast images of EM-P cells undergoing lysis and release of internal daughter cells. The highlighted region in image (B) and images (C-E) shows a cell undergoing lysis and cells in different stages of lysis. Arrows in image (B) point to thin strands of membrane debris formed during cell lysis. Arrows in images (F & G) point to clusters of daughter cells released by the lysis of EM-P cells. These cell clusters gradually dispersed, leading to the formation of individual daughter cells (H). TEM images of such clusters of interconnected daughter cells and individual daughter cells with membrane overhangs are shown in Appendix 1—figure 7F. Scale bar: 10 μm.
Appendix 1—figure 7
Morphological comparison between the SPF microfossils and EM-P.
Images (A-D) are hollow spherical SPF microfossils with internal inclusions (originally published by Sugitani et al., 2013; Schopf and Barghoorn, 1967). Images (D-F) are images of EM-P cells undergoing lysis (E) and dispersion (F) of intracellular vacuoles. Cyan arrows in images (D and E) point to the presence and absence of the outer cell membrane. Black arrows in images (C & F) point to filamentous structures interlinking spherical structures. Scale bar: 50 μm (A), 20 μm (B & C), 2 μm (D & E), and 5 μm (F).
Appendix 1—figure 8
Morphological comparison between EM-P and Strelley Pool Formation (SPF) microfossils.
Images (A-D) depict SPF microfossils with intracellular organic inclusions (originally published by Wacey et al., 2011). Image (E) shows morphologically analogous EM-P cells. Images (A-C) show chains and clumps of cells. A similar cluster of cells can be seen in image (E) (boxed regions). Image (D) shows spherical microfossils with intracellular spherical inclusions. Similar EM-P cells with intracellular daughter cells can be seen in image (E) (yellow arrows). Lysis and release of daughter cells can be seen in Appendix 1—figure 6 and Video 4.
Appendix 1—figure 9
Morphological comparison between EM-P and the Strelley Pool Formation (SPF) microfossils.
Images (A-C) show clusters of faint and dark spherical microfossils reported from SPF (originally published by Sugitani et al., 2015b; Barlow and Van Kranendonk, 2018). Images (D & E) show morphologically similar structures observed in EM-P incubations. Image A shows spherical clusters of spherical granules. Based on their morphological similarity, they likely represent the tiny intracellular daughter cells, as shown in D (highlighted region) and Appendix 1—figure 8. The thread-like structures in C (arrows) likely are the membrane debris often found associated with the release of daughter cells, as shown in (D & E) (arrows). Scale bars in images (D & E) are 10 μm.
Appendix 1—figure 10
Morphological comparison between EM-P and the Waterfall microfossils.
Images (A & B) are microfossils reported from the Waterfall locality within the Strelley Pool Formation (SPF) (originally published by Sugitani et al., 2015b; Barlow and Van Kranendonk, 2018). Images (C & D) show morphologically analogous EM-P cells. Arrows in images (C & D) point to spherical intracellular vacuoles within the cell. Image E shows sequential stages involved in the formation of structures shown in images (A & B). Sequential steps are indicated by numbers next to the arrows. Scale bars: 10 μm (C–E).
Appendix 1—figure 11
Morphological comparison between EM-P and Waterfall microfossils.
Images (A, B, & C) are microfossils reported from the waterfall locality within SPF (originally published by Sugitani et al., 2015b; Barlow and Van Kranendonk, 2018). Images (D-F) are phase-contrast images of morphologically analogous structures formed by EM-P. Red arrows in these images point to intracellular vacuoles with daughter cells of different sizes. Open red arrows point to a vacuole with a relatively large daughter cell. The white arrows in image (F) point to the ruptured region of the cell membrane and the release of the intracellular vesicles (ICVs). Scale bars: 10 μm (D–F).
Appendix 1—figure 12
Morphological comparison between honeycomb structures reported from the Turee Creek Formations and EM-P.
Images (A, B, & C) are microfossils reported from the Turee Creek locality within the Strelley Pool Formation (SPF) (originally published by Barlow and Van Kranendonk, 2018; Sugitani et al., 2009). Images (D-F) are phase-contrast images of morphologically analogous structures formed by EM-P. The boxed region in image (A) resembles the lysed cells of EM-P, shown in image (D). Arrows in image (B & E) point to the similar organic carbon inclusions (B) and morphologically analogous daughter cells (E) within a hollow spherical EM-P cell. Insert in images (B & E) show spherical cells with two intracellular vesicles or nearly equal volume. Scale bars: 10 μm (D–F).
Appendix 1—figure 13
The sequence of morphological transformations of EM-P leading to the formation of cells having the appearance of a thick cell wall.
Images (A-G) show the sequential morphological transformations of EM-P cells with one large central vacuole and multiple smaller vacuoles. Over the course of its growth, smaller vacuoles were squeezed between the cell membrane and the vacuole membrane (F & H), leading to the appearance of a porous cell wall. Images (F-G and H) are phase-contrast and TEM images of such EM-P cells. The image (I) shows a spherical microfossil reported from Dresser formation (originally published by Wacey et al., 2018a). Insert in the image-I shows the TEM image of the region indicated by the blue line. The porous nature of this region is similar to the EM-P cell shown in image (H). Scale bars: 10 μm (A–G) and 200 nm (H).
Appendix 1—figure 14
Morphological comparison of organic structures reported from the Dresser Formation with EM-P.
Images (A & B) are spherical microfossils reported from the Dresser formation (originally published by Wacey et al., 2018a). Images (C & D) are STED microscope images of morphologically analogous EM-P cells. Cells in these images were stained with FM5-95 (membrane, white) and PicoGreen (DNA, red). Images (G-I) are phase-contrast images of morphologically analogous EM-P cells. Closed arrows in these images point to the regions of the cell with the cytoplasm (organic carbon), and open arrows point to the hollow space created by the intracellular vesicles (ICVs). Scale bars 10 μm (C–I).
Appendix 1—figure 15
Morphological comparison of Dresser formation spherical microfossils with EM-P.
Images (A-D) were aggregations of spherical microfossils reported from Dresser formation (originally published by Wacey et al., 2018a). (E-I) are images of morphologically analogous structures observed from EM-P. Blue arrows in images (C & D) point to cells with discontinuous cell walls. Similar cell boundary features can be seen in EM-P (E–H). Sequential stages involved in forming such EM-P cells are shown in Appendix 1—figure 16. The red arrow in image (D) points to structures with organic carbon attached to the hollow spherical cells. Image-g showed morphologically analogous EM-P cells. Scale bar: 10 μm (E–I).
Appendix 1—figure 16
Sequential morphological transformation of EM-P cells with cytoplasm to hollow vacuoles.
Images (A-I) are phase-contrast images of EM-P. In sequence, they show formation (B) and an increase in the size of the intracellular vacuole (B–G). Over the course of its growth, cytoplasm from the parent cell is transferred into the daughter cells in the vacuole (barely visible tiny spherical structures in the vacuole), which leads to the gradual depletion of cytoplasmic volume and a gradual increase in vesicle volume. In their late growth stages, the presence of cytoplasm is restricted to the periphery of the cell as discontinuous patches (H & I). Scale bars: 10 μm.
Appendix 1—figure 17
Morphological comparison of the Dresser Formation spherical microfossils with EM-P.
Images (A-C) were aggregations of spherical microfossils reported from the Dresser Formation (originally published by Wacey et al., 2018a). Images (D &E) are TEM images of morphologically analogous EM-P cells. Image (A) shows hollow spherical aggregations that were devoid of organic carbon. Similar EM-P cells were shown in image (D). Red arrows in this image point to Y-shaped junctions with organic carbon. Such EM-P cells, over the course of their growth, underwent membrane rupture to release daughter cells (image (E), purple arrows). The process of lysis and dispersion of these cells is shown in Image (E). The morphology of these EM-P cells was very similar to the organic structures reported from the Dresser Formation (Image B). Like EM-P cells, daughter cells can also be seen nearby (red box). Image (C) shows an isolated spherical structure, with internal spherical inclusions with the presumed thick and uneven cell wall. Morphologically analogous EM-P cells can be seen in Image E (Cyan box). Scale bar: 250 nm (E).
Appendix 1—figure 18
Morphological comparison between EM-P and the Kromberg Formation microfossils.
Images (A & B) are microfossils reported from the Kromberg Formation (originally published by Kaźmierczak and Kremer, 2019; Wacey et al., 2011). Images (C, D, & E) are SEM and TEM images of morphologically analogous EM-P cells. Such EM-P cells were formed by deflation caused by lysis and release of daughter cells. SEM images show similar surface depressions in both the Kromberg microfossils and EM-P cells (arrows in A & C). Different stages of surface depression formation are shown in Figure 1—figure supplement 1G–K. Arrows in images (B & D) point to similar surface textures of microfossils and EM-P cells. Scale bars: 10 μm (A & B), 0.5 μm (C & D), and 250 nm (E).
Appendix 1—figure 19
Reproduction by budding and binary fission.
Images (A-E) are the phase-contrast images of EM-P reproducing by budding or binary fission. The arrows in the images point to different stages of EM-P reproduction. The arrows in images (B & C) point to the cells undergoing budding. Scale bars: 10 μm.
Appendix 1—figure 20
Morphological comparison between the North-Pole locality microfossils and EM-P.
(A-C) are TEM images of EM-P showing sequential stages of bud formation. Images (D-E) are phase-contrast images of EM-P cells that appear to be reproducing by budding or binary fission, depending on the size of the daughter cells. Images G-N show spherical microfossils reported from the North Pole locality (originally published by Buick, 1990; Kaźmierczak and Kremer, 2019). These microfossils resemble EM-P cells reproducing by budding. Arrows in images (A & G) point to pustular protuberances in EM-P and the North-Pole locality microfossils. Images (E, F, I & K) show EM-P cells and microfossils reproducing by binary fission. Image (D) and highlighted regions in images (E & F) (in white) show EM-P cells reproducing by the formation of multiple buds (also Figure 1B). Highlighted region in image (F) (in yellow) shows cells forming Y-junctions, similar to the microfossils shown in N. Scale bars: 500 nm (A & B), 10 μm (D–F), and 100 μm (G–N).
Appendix 1—figure 21
Comparison of EM-P with the Swartkoppie Formation microfossils.
Images (A-K) are the images of the Swartkoppie microfossils (originally published by Knoll and Barghoorn, 1977; Knoll and Barghoorn, 1977). Images (I-R) are images of EM-P exhibiting morphological similarities with the Swartkoppie microfossils. Image (A) shows cells with membrane overhangs (red box), cells in dyads (green box), and individual spherical cells (arrows). Morphologically analogous EM-P cells can be seen in the images mentioned below in the red and green boxes. Images (M, N, & O) are phase-contrast, SEM, and TEM images of cells with membrane extensions, like the microfossil cell shown within the red box. Images (P-R) show a sequence of stages involved in EM-P’s cell division. Images (B-F) and (G-K) show the Swartkoppie microfossils reproducing by binary division. Morphologically similar EM-P cells were shown in image (L) (white arrows). Numbers next to the arrows indicate different stages of cell division. Scale Bar: 10 μm (A), 0.5 μm (L & N), 0.25 μm (O, Q, & R) and 1 μm (M & P).
Appendix 1—figure 22
Comparison of EM-P with the Sheba Formation microfossils.
Images (A & B) show spherical microfossils reported from the Sheba Formation (originally published by Hickman-Lewis et al., 2018; Hickman-Lewis et al., 2019). Image (A) shows the aggregation of spherical microfossils within organic carbon clasts. Image (B) shows a magnified image of spherical cells with unevenly distributed cytoplasm. Images (C & G) are morphologically analogous EM-P cells. Like the Sheba Formation, microfossils' spherical EM-P cells formed filamentous overhangs (arrows in images (C-F), Video 16). EM-P cells were also noticed to have uneven cytoplasm (E) (Figure 2). The arrow in image A points to spindle-like cells with filamentous extensions that are morphologically similar to EM-P cells, as shown in image (C). Scale bars: 10 μm (C).
Appendix 1—figure 23
Comparison of EM-P with the Sheba Formation microfossils.
Images (A & B) show spherical microfossils reported from the Sheba Formation (originally published by Homann, 2019; Buick, 1990). Image (B) shows microfossils reproducing by what appears to be binary fission. Images (C-F) are the phase-contrast and STED microscope images of morphologically similar EM-P cells. Images (C-E) show spherical EM-P cells that are surrounded by membrane debris. Image (F) shows such debris composed of extracellular DNA (red). Scale bar: 10 mm.
Appendix 1—figure 24
Comparison of the Onverwacht group microfossils with EM-P.
Image (A) shows the Onverwacht microfossils (originally published by Walsh, 1992). Images (C & D) show sub-micrometer size cells EM-P, exhibiting morphological similarities with the Onvewacht microfossils. Images (D-I) show a cluster of EM-P cells organized in different configurations and interpretive drawings of morphologically similar Onverwacht microfossils (originally published by Walsh, 1992). Scale bars: (B & C) (5 μm).
Appendix 1—figure 25
Morphological comparison between EM-P and the Sulphur Spring Formation microfossils.
Images (A & B) are microfossils reported from the Sulphur Spring Formations (originally published by Wacey et al., 2014). Red arrows in these images point to filamentous structures with spherical inclusions. Images (C-F) are morphologically analogous to EM-P cells. Similar to images A & B, EM-P exhibited strings of spherical daughter cells. Arrows in these images point to the branching of the filaments. Scale bars: 10 μm (C–F).
Appendix 1—figure 26
Morphological comparison between EM-P and the Sulphur Spring Formation microfossils.
Images (A & B) are microfossils reported from Sulphur Spring Formations (originally published by Wacey et al., 2014). Red arrows in these images point to filamentous structures with spherical inclusions. The purple arrow in image (B) points to clusters of spherical organic structures from which filamentous structures appear to have originated. Yellow arrows point to the branching within the filamentous structures. Images (C & D) show morphologically similar EM-P cells. Images show spherical EM-P cells with filamentous extensions. Most filamentous extensions contain spherical daughter cells. Scale bars: 10 μm (B & C).
Appendix 1—figure 27
Morphological comparison between EM-P and the Sulphur Spring Formation microfossils.
Images (A-C) are microfossils reported from the Sulphur Spring Formations (originally published by Wacey et al., 2014). The boxed regions in image A show spherical pyrite-encrusted microfossils within hollow filaments. Images (B & C) show close-ups of spherical structures with wrinkly surfaces. Image (D) shows hollow filaments with spherical inclusions morphologically analogous to the Sulphur Spring Formation microfossils (Scale bar: 10 μm). Arrows in the image point to spherical daughter cells within the filaments. Insert at the bottom of the image shows an SEM image of such a cell with a wrinkled surface (Scale bar: 1 μm). The Insert image in the top right corner of image (D) shows a TEM image of such hollow filaments (Scale bar: 500 nm).
Appendix 1—figure 28
Morphological comparison between the Mt. Grant microfossils with EM-P.
Images (A & B) show microfossils reported from the Mt. Grant Formation (originally published by Sugitani et al., 2007; Retallack et al., 2016). Images (B-D) are morphologically analogous to EM-P daughter cells. Morphologically analogous EM-P cells shown in image (A) can be seen in motion in Video 14. Scale bars: 100 μm (A) & 10 μm (B–D).
Appendix 1—figure 29
Multilayered EM-P cells.
Images (A & B) show a top and lateral view of EM-P cells growing in multiple layers at the bottom of the chamber slide. Cells in the above images were stained with membrane stain, FM5-95. Scale bar: 20 μm (A).
Appendix 1—figure 30
Cell lysis and release of DNA observed in EM-P.
Images (A & B) show the lysis and release of cell constituents like DNA (red) into their surroundings. Image (B) shows the aggregation of cells within the biofilms with a considerable amount of extracellular DNA. Cells in these images were stained with FM5-95 (membrane, white) and PicoGreen (DNA, red). Scale bars: 10 μm.
Appendix 1—figure 31
Morphological comparison of the North Pole locality microfossils and EM-P.
Images (A & B) are the organic structures reported from the North-pole locality (originally published by Buick, 1990; Kaźmierczak and Kremer, 2019). Image (D) shows a similar aggregation of spherical EM-P cells observed in our study. Arrows in images (A & C) point to the membrane debris between the cell aggregates. Further comparison of North Pole locality microfossils and EM-P cells is shown in Appendix 1—figures 32 and 33. Scale bars: (A) (1 mm), (B) (100 μm), and (C) (20 μm).
Appendix 1—figure 32
Morphological comparison of the North Pole locality microfossils and EM-P.
Images (A-C) are the organic structures reported from the North Pole locality (originally published by Buick, 1990; Kaźmierczak and Kremer, 2019). They show organic mats composed of individual hollow spherical cells and large gaps within the mats (A & B). Images (D & E) are similar mat-like organic structures formed by EM-P. The highlighted regions in (D) show morphologically similar gaps observed in the North Pole locality and EM-P biofilms. Scale bars: (D & E) (10 μm).
Appendix 1—figure 33
Morphological comparison of the North Pole locality microfossils and EM-P.
Images (A-C) are the organic structures reported from the North-pole locality (originally published by Buick, 1990; Kaźmierczak and Kremer, 2019). Images (D & E) show phase-contrast images of EM-P cells. Image (D) shows an aggregation of hollow EM-P vesicles with filamentous structures. The method of their formation is shown in Videos 5–9. Scale bars: (D & E) (10 μm).
Appendix 1—figure 34
Morphological comparison of North Pole locality microfossils and EM-P.
Images (A-C) are the organic structures reported from the North-Pole locality (originally published by Buick, 1990; Kaźmierczak and Kremer, 2019). Images (D & E) show phase-contrast images of EM-P cells. Image (D) shows an aggregation of hollow EM-P vesicles with filamentous structures. The method of their formation is shown in Videos 5–9. Scale bars: (D & E) (10 μm).
Appendix 1—figure 35
Morphological comparison of the North Pole locality microfossils and EM-P.
Images (A-C) are the organic structures reported from the North Pole locality (originally published by Buick, 1990; Kaźmierczak and Kremer, 2019). Images (D-G) show phase-contrast images of EM-P cells. Image (D) shows an aggregation of hollow EM-P vesicles with filamentous structures. The method of their formation is shown in Videos 5–9. Arrows in images point to the similarities in the filamentous structure from both north-pole formation and EM-P. Scale bars: (D & E) (10 μm).
Appendix 1—figure 36
Morphological comparison of Strelley Pool Formation (SPF) microfossils with EM-P.
Images (A-C) are the organic structures reported from the SPF (originally published by Sugitani et al., 2010);. Images (D & E) show phase-contrast images of EM-P cells. Highlighted regions in images (D & E) show EM-P cells forming hexagonal structures, similar to SPF microfossils. Scale bar: (D & E) (20 μm).
Appendix 1—figure 37
A morphological comparison between organic structures was reported from the Nuga Formation and honeycomb structures of EM-P.
Images (A & B) are honeycomb-shaped organic structures reported from the Nauga Formation (originally published by Kazmierczak et al., 2009; Ueno et al., 2006). Arrows in (A) point to the magnified image. Arrows in (B) point to the honeycomb structures within the organic structures. Image (D) shows morphologically analogous structures formed by EM-Ps. Cells in images (D-F) were stained with membrane stain, FM5-95 (yellow). Boxed regions within Image (C) show polygonal structures similar in their morphology to organic structures shown in Image (B). The scale bar: 20 μm (C).
Appendix 1—figure 38
Morphological comparison between organic structures reported from the Buck Reef Chert (BRC) Formation and honeycomb structures of EM-P.
Image (A) is a honeycomb-shaped organic structure reported from the BRC (originally published by Tice, 2009; Tice and Lowe, 2004). Images (B & C) are anterior and posterior views of morphologically analogous EM-P’s membrane debris. Cells in images (B-D) are stained with membrane stain, FM5-95 (yellow). Image (D) is the magnified region of the biofilm showing honeycomb structures. Images (E & F) are phase-contrast images of large spherical EM-P cells with polygonal vacuoles undergoing clumping to form large honeycomb-shaped mats. Scale bars: (B & C) (100 μm) and (E & F) (10 μm).
Appendix 1—figure 39
Morphological comparison between organic structures reported from the BRC Formation and honeycomb structures of EM-P.
Images (A & B) show the bifurcating or very fine strands of carbonaceous matter within the BRC formation (originally reported by Tice and Lowe, 2006; Schopf et al., 2017). Image (C) is the morphologically similar structure formed by EM-P. The subsequent disintegration of these honeycomb structures into strands of filamentous membrane is shown in Appendix 1—figure 40B and C. Scale bars: (C) (20 μm).
Appendix 1—figure 40
Morphological comparison of honeycomb structures reported from the Turee Creek Formations and EM-P.
Image (A) shows a tangled network of microfossil structures reported from the Turee Creek Formation (originally published by Barlow and Van Kranendonk, 2018; Sugitani et al., 2009). Images (B & C) are morphologically analogous structures formed by EM-P. Cells in these images are stained with membrane stain, FM5-95, and imaged using a STED microscope. Boxed regions and arrows in image (A) highlight the spherical cells closely associated with honeycomb-shaped organic structures (Appendix 1—figures 36 and 39C). Arrows in (B & C) point to spherical daughter cells attached to membrane debris. Based on the morphological resemblance, we propose that the Turee Creek organic structures were leftover membrane debris of EM-P-like cells rather than an entangled network of filamentous microfossils. Scale bars: (A) (2 mm), (B & C) (50 μm).
Appendix 1—figure 41
Morphological comparison of organic structures reported from the Moodies Group with EM-P.
Images (A-C) are the microbial mats reported from the Moodies Group. Honeycomb-like polygonal structures (Gamper et al., 2012; Westall et al., 2006). Images (D-G) are morphologically analogous structures observed in EM-P incubations. Images (D & E) show the encrusted surface and interior of the biofilm. The hexagonal structures underneath the surface can be seen in image (E). Image (F) shows the individual EM-P cells that constitute the biofilm. Image (G) shows the deeper layers of the biofilm with a distinctive honeycomb pattern. Scale bars: (D-G) (20 μm).
Appendix 1—figure 42
Morphological comparison between honeycomb structures of EM-P organic structures reported from Strelley Pool Formation (SPF).
Images (A-C) are honeycomb-shaped organic structures reported from SPF (originally published by Schopf et al., 2017; Gamper et al., 2012). The circled region in Image (B) points to flattened honeycomb structures. Images (D & E) show morphologically analogous honeycomb-like structures observed in EM-Ps incubations. Cells in images (D-F) were stained with membrane stain, FM5-95. The scale bar: 20 μm (D).
Appendix 1—figure 43
Membrane debris formation.
Images (A-E) show different stages involved in forming fabric-like membrane debris formation. Image (A) shows a biofilm with stacks of spherical EM-P cells. Image (B) shows the membrane debris formed during the cell lysis. Image (C) shows the gradual accumulation and increase in the surface area of membrane debris. Image (D) shows the top view of the biofilm with sheets of membrane debris growing out of the biofilm surface. A later stage of the biofilm largely engulfed in the membrane debris is shown in Appendix 1—figure 45. Image (E) is the lateral view of the EM-P biofilm shown in (D). Cells and membrane debris in these images were stained with membrane stain, FM5-95, and imaged using a STED microscope. Scale bars: 20 μm.
Appendix 1—figure 44
Cell lysis and formation of membrane debris within multilayered EM-P cells.
Images (A & B) show a top and lateral view of EM-P cells growing in multiple layers at the bottom of the chamber slide. Cells in the above images were stained with membrane stain, FM5-95. The boxed region in image (A) shows the membrane debris formed from the lysis of cells. Scale bar: 20 μm (A).
Appendix 1—figure 45
Membrane debris of EM-P.
The image shows membrane debris of EM-P forming a layer over a mat of multilayered cells. Based on the morphological similarities with laminated structures, we presume such structures were formed by a similar process. The lateral view of the image, along with its morphological comparison with α-type laminations reported from BRC, was shown in Appendix 1—figure 46. Membranes were stained with Nile red and were imaged using a confocal microscope. Scale bar: 100 μm.
Appendix 1—figure 46
Morphological comparison between the BRC laminations and membrane debris of EM-P.
Image (A) shows α-type laminations reported from the BRC (originally published by Tice, 2009; Tice and Lowe, 2004). Two filamentous layers with hollow spaces between them can be seen in image (A). The hollow space in between is filled with filamentous membrane debris and spherical inclusions. Images (B-E) show lateral sections of EM-P membrane debris, as shown in Appendix 1—figure 45. Open and closed arrows in all the images point to the top layer of the membrane enclosure and the bottom cell layer, respectively. The hollow space in between is filled with membrane debris and spherical cells. We presume that the lysis of these cells could have produced membrane debris like the ones observed in image (A). In support of this presumption, membrane debris similar to β-type laminations (indicated by the red box) was observed in EM-P batch cultures (Appendix 1—figures 51 and 54). Also, see Video 19. Scale bars: 5 mm (A) and 100 μm (E).
Appendix 1—figure 47
Membrane debris of EM-P.
The image shows EM-P cells covered in membrane debris. Images (B & C) are the magnified regions of image (A). Arrows in (C) point to the naturally formed lenticular gaps within the membrane debris. Scale bar: 100 μm (A).
Appendix 1—figure 48
Membrane debris of EM-P.
The image shows EM-P covered in membrane debris. Image (B) shows the magnified regions of image (A). Images (D & E) are the anterior and the posterior view of the membrane debris with cells. These images highlight the fabric-like texture of the membrane debris. Cells in these images were stained with FM5-95 membrane, white in (A & B) and gray in (C-E) and PicoGreen (DNA, red in A & B and cyan in C-E). Scale bar: 50 μm (A & C).
Appendix 1—figure 49
Morphological comparison between EM-P and the Chinaman Creek microfossils.
Images (A & B) are organic structures reported from the Chinaman Creek (Brasier et al., 2005; Tice, 2009). Image (C) is a composite image of morphologically analogous EM-P structures. Images (D-F) are magnified regions of image (C). All the images show spherical structures with or devoid of organic carbon enclosed in a film-like structure. Scale bar: 10 μm (C).
Appendix 1—figure 50
Morphological comparison of EM-P Membrane debris with laminated structures.
Images (A & B) show bifurcating laminated structures reported from the Moodies Group (originally published by Homann et al., 2015; Brasier et al., 2005). Image (C) is a 3D-rendered confocal image of image of morphological analogous membrane debris formed by EM-P cells. White arrows in B point to hollow lenticular regions within the laminations, similar to the one observed in EM-P. Arrows in images (A & C) point to the bifurcation of membrane debris. Membranes were stained with Nile red, and imaging was done using a point scanning microscope. Scale bar: 50 μm (C).
Appendix 1—figure 51
Sequential stages involved in the formation of β-laminations.
Images (A-E) shows the steps involved in the sequential transformation of membrane debris to β-type laminations. Image (A-C) shows the aggregation of vacuoles formed from the lysis of large spherical EM-P cells. Image (D) shows collapsed membrane debris formed by lateral compression or deflation of such aggregations after the release of daughter cells. The arrow in image (E) points to the individual membrane layers within the debris. Scale bars:10 μm.
Appendix 1—figure 52
Sequential stages involved in the formation of β-laminations.
Images (A-C) show the steps involved in the sequential transformation of membrane debris to β-type laminations. Image (A) shows the aggregation of vacuoles formed from the lysis of large spherical EM-P cells. Image (B) shows collapsed membrane debris formed by lateral compression or deflation of such aggregations after the release of daughter cells. Cyan and yellow arrows point to membrane debris and compressed membrane debris, respectively. Inset in image (B) is the magnified region of compressed membrane debris. Image-c shows structures similar to β-type laminations. Scale bars:10 μm.
Appendix 1—figure 53
Morphological comparison of the BRC β-laminations with EM-P’s membrane debris.
Images (A & B) show β-type laminations reported from the BRC (originally published by Tice and Lowe, 2004; Duck et al., 2007). These laminations are described as rolled-up or bundled filamentous structures. Image (C) shows morphologically analogous membrane debris formed by EM-P. Insets in image (C) are magnified regions of the debris showing bundled-up individual filamentous structures. Scale bars: 10 μm (C).
Appendix 1—figure 54
Morphological comparison of the BRC β-laminations with EM-P’s membrane debris.
Images (A & B) show β-type laminations reported from the BRC (originally published by Tice and Lowe, 2004; Duck et al., 2007). Image (C) shows an STED image of morphologically analogous membrane debris formed by EM-P. Spherical daughter cells (evidenced by the presence of DNA) are still attached to the membrane debris, which can be seen in the image. In the image, the membrane was stained with FM5-95 (red), and DNA was stained with PicoGreen (green). Scale bar: 10 μm (C).
Appendix 1—figure 55
Morphological comparison of the Moodies Group laminations with EM-P’s membrane debris.
Image (A-C) shows laminations reported from the Moodies Group (originally published by Homann et al., 2018; Kazmierczak et al., 2009). Image (D) is a 3D-rendered confocal image of EM-P’s membrane debris exhibiting hollow lenticular gaps (Video 20). Images (E-G) show the hollow lenticular structure at different Z-axis positions. Membranes were stained with Nile red, and imaging was done using an STED microscope. Scale bar: 50 μm.
Appendix 1—figure 56
Morphological comparison of the Moodies Group laminations with EM-P’s membrane debris.
Image (A) show β-type laminations reported from the Moodies Group (originally published by Homann et al., 2018; Kazmierczak et al., 2009). Laminations from the Moodies Group were shown to form raised filamentous structures (A). Arrows in the image point to spherical structures within the laminations. Images (B-D) show 3D-rendered confocal images of morphologically analogous EM-P’s membrane debris. Image (B) shows filamentous membrane debris of EM-P, that rose above layers of spherical cells. Images (C & D) are close-up images of raised filamentous membrane debris of EM-P from different viewing angles. Arrows in these images point to spherical cells attached to the membrane debris. Image (E) is a non-3D rendered image showing individual membrane layers within the debris, with spherical inclusions attached. Scale bar: 20 μm (B).
Appendix 1—figure 57
Swirl-like structures of EM-P’s membrane debris.
Images (A-F) show a 3D-rendered STED image of membrane debris observed in late-growth stages of EM-P (1–2 month-old). This debris typically formed raised mound-like structures. Images (A & B) show the top view of such structures, and image C shows the side view. Images (D-F) are images of such mounds at different depths (bottom to top). The formation of swirls can be seen in images (E & F). In the image, membrane debris is stained with Nile red. A morphological comparison of these structures with Archaean laminations is shown in Appendix 1—figure 58. Scale bar: 50 μm (A).
Appendix 1—figure 58
Morphological comparison of the Moodies Group laminations with EM-P’s membrane debris.
Images (A-F) shows raised mound-like structures reported from the Moodies Group (Homann et al., 2018; Kazmierczak et al., 2009). Image (C) shows the top view of such mounds. These mounds were shown to have filamentous extensions at their peaks. Images (G-J) are morphologically analogous structures formed by EM-P. In these images, the membrane debris is stained with Nile red. Images (I & J) show the top and side views of these raised mounds (depth coloring). Scale bar: 50 μm (G).
Appendix 1—figure 59
Morphological comparison of laminations reported from the BRC with EM-P’s membrane debris.
Images (A & B) show β-type laminations reported from the BRC (originally published by Tice, 2009; Tice and Lowe, 2004). Images (E & F) are frontal and lateral view of morphologically analogous structures formed by EM-P. Arrows in these images point to helical structures observed in microfossils and EM-P. Star signs in images (B, C, & E) point to regions showing the filamentous nature of the membrane debris. In the image, the membrane was stained with Nile red. Scale bars: 50 µm (E).
Appendix 1—figure 60
Chemical nature of EM-P’s crust.
Image (A) shows a 30-month-old EM-P culture at the bottom of the bottle. Over the course of incubation, EM-P biofilm transformed into a brittle wafer due to salt encrustation. Images (B & C) show SEM images of EM-P. Scale bars in these images are 2 µm. The elemental composition of the solidified mat is shown in (D) (determined by SEM-EDX).
Appendix 1—figure 61
Morphological comparison of the Kromberg microfossils with EM-P.
Images (A & B) show mineral-encrusted microbial mats (A) and spherical microfossils (B) within these encrusted mats reported from the Kromberg Formation (originally published by Westall et al., 2001; Homann et al., 2015). Scale bars in images (A & B) are 20 µm. (C & D) show SEM images of morphologically analogous salt-encrusted EM-P cells after 30 months of incubation. The elemental composition of such microbial mats and individual cell morphologies are shown in Appendix 1—figure 60. Scale bars in images (C & D) are 2 µm.
Appendix 1—figure 62
Morphological comparison of the North Pole microfossils with EM-P.
Images (A-C) show stellate-shaped structures with filamentous projections reported from the North Pole locality (originally published by Buick, 1990; Kaźmierczak and Kremer, 2019). Scale bars: 100 µm. (D-K) are phase-contrast images of morphologically analogous EM-P cells. The stellar-shaped structure could have been the biologically induced mineral formed on the surface of the cells, as shown in Appendix 1—figure 60. The filamentous structures were the daughter cells growing out of the crust when encrusted cells were transferred into fresh media. Arrows in images (C, E, & F) point to a filamentous string of daughter cells extending out of cell clumps (Video 22). Images (D-K) also show salt-encrusted individuals or cells in dyads undergoing binary fission. Scale bars: 10 µm.
Appendix 1—figure 63
Morphological comparison of the Strelley Pool Formation (SPF) microfossils with EM-P.
Image A shows stellate microfossils with filamentous projections reported from the SPF (originally published by Sugitani et al., 2013; Schopf and Barghoorn, 1967). Scale bar: 200 µm. Images B-E are spinning-disk confocal images of morphologically analogous EM-P cells. Cells in these images are stained with membrane stain, FM5-95 (red), and PicoGreen (DNA, green). Like microfossils, EM-P cells revived from the above-described crust (Appendix 1—figure 62) formed filamentous daughter cells from below the stellate structures. Scale bars: 10 µm.
Appendix 1—figure 64
Association of salt with EM-P.
Images (A & B) are SEM images of EM-P. Image (A) shows EM-P cells forming membrane invaginations that resemble an endocytosis vesicle. These images EM-P with salt crystals within the membrane vesicles (closed arrows). Scale bars: 2 µm.
Appendix 1—figure 65
Morphological comparison of volcanic pumice with honeycomb-like structures observed in EM-P incubations.
Images (A & B) are the images of volcanic pumice (World Wide Web). Images (C-F) are the images of EM-P biofilm. Scale bars: 20 µm (C) and 10 µm (D–F).
Videos
Video 1
Video shows an EM-P cell with an intracellular daughter cell within its vesicle.
Scale bar: 5 μm.
Video 2
Videos show a gradual increase in the number of daughter cells within EM-Ps intracellular vesicles and a corresponding decrease in the volume of the cell cytoplasm.
Scale bar: 5 μm.
Video 3
Movies show a gradual increase in the number of daughter cells within EM-Ps intracellular vesicles and a corresponding decrease in the volume of the cell cytoplasm.
Scale bar: 5mm.
Video 4
Movies show a gradual increase in the number of daughter cells within EM-Ps intracellular vesicles and a corresponding decrease in the volume of the cell cytoplasm.
Scale bar: 5mm.
Video 5
Videos show sequential stages involved in EM-P cell lysis and the release of intracellular vacuoles containing daughter cells.
Videos also show the formation of cell debris during the release of ICVs. Scale bar: 5 μm.
Video 6
Movie show EM-P cell lysis and the release of intracellular vacuoles containing daughter cells.
Movie also show the formation of cell debris during the release of ICVs. Movies 6-9 show the sequential stages involved in the lysis and the release of intracellular vacuoles containing daughter cells. Scale bar: 5mm.
Video 7
Movie show EM-P cell lysis and the release of intracellular vacuoles containing daughter cells.
Movie also show the formation of cell debris during the release of ICVs (long strings of membrane debris). Movies 6-9 show the sequential stages involved in the lysis and the release of intracellular vacuoles containing daughter cells. Scale bar: 5mm.
Video 8
Movie show EM-P cell lysis and the release of intracellular vacuoles containing daughter cells.
Movie also show the formation of cell debris during the release of ICVs (long strings of membrane debris). Movies 6-9 show the sequential stages involved in the lysis and the release of intracellular vacuoles containing daughter cells. Scale bar: 5mm.
Video 9
Movie show EM-P cell lysis and the release of intracellular vacuoles containing daughter cells.
Movie also show the formation of cell debris during the release of ICVs (long strings of membrane debris). Movies 6-9 show the sequential stages involved in the lysis and the release of intracellular vacuoles containing daughter cells. Scale bar: 5mm.
Video 10
The video shows cell debris of EM-P cells with tiny daughter cells attached to them.
Scale bar: 5 μm.
Video 11
Movie along with 10-12 show the sequential stages involved in the formation such individual daughter cells and their subsequent transformation into a string-of-daughter cells.
Scale bar: 5mm.
Video 12
Movie along with 10-12 show the sequential stages involved in the formation such individual daughter cells and their subsequent transformation into a string-of-daughter cells.
Scale bar: 5mm.
Video 13
Movie along with movies 14 & 15 show sequential stages involved in the detachment and fragmentation of these strings of daughter cells into individual daughter cells.
Scale bar: 5mm.
Video 14
Movie along with movies 13 & 15 show sequential stages involved in the detachment and fragmentation of these strings of daughter cells into individual daughter cells.
Scale bar: 5mm.
Video 15
Movie along with movies 13 & 14 show sequential stages involved in the detachment and fragmentation of these strings of daughter cells into individual daughter cells.
Scale bar: 5mm.
Video 16
Movie show the constant movement of the daughter cells; such surface undulations should have provided the kinetic energy required for the fragmentation of strings of daughter cells into individual daughter cells.
Scale bar: 10mm.
Video 17
The video shows cell debris of EM-P cells with tiny daughter cells attached to them.
Scale bar: 5 μm.
Video 18
The video shows cell debris being formed within the biofilm.
The lateral view of the biofilm shows the membrane debris being pushed out of the biofilm. Cells in this movie are stained with membrane stain FM5-95 and imaged using a STED microscope.
Video 19
The video shows a layer of EM-P cells and membrane debris that was formed over this layer.
Several spherical cells can be seen attached to the membrane debris and in the free space between the cell layer and the wavy membrane. Membrane debris was often noticed to have lenticular gaps. Cells in this movie are stained with membrane stain FM5-95 and imaged using a STED microscope.
Video 20
The video shows layered membrane debris of EM-P forming hollow lenticular structures.
Such structures should have formed by random folding of membrane debris, as no indication of trapped cells was observed in these structures. Scale bar: 20 μm.
Video 21
The video shows solidified EM-P biofilm.
Hollow lenticular structures could be seen within the layers of membrane debris. Such structures should have formed honeycomb patterns within these lenticular gaps suggesting the presence of one’s intact EM-P cells within these structures. Scale bar: 20 μm.
Video 22
The video shows a string of EM-P cells growing out of the stellar-shaped salt-encrusted cells.
Scale bar: 10 μm.
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On the nature of the earliest known lifeforms
eLife 13:RP98637.
https://doi.org/10.7554/eLife.98637.3
