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Organic-walled microfossils in 3.2-billion-year-old
shallow-marine siliciclastic deposits
Emmanuelle J. Javaux1, Craig P. Marshall2 & Andrey Bekker3

Although the notion of an early origin and diversification of life on the base of the Moodies Group, in interlayered laminated grey shales,
Earth during the Archaean eon has received increasing support in siltstones and wavy-laminated clay-rich and organic-matter-rich
geochemical, sedimentological and palaeontological evidence, layers, possibly representing microbial mat structures. Flaser bedding,
ambiguities and controversies persist regarding the biogenicity small-scale cross-bedding, and mud-draped current ripples were
and syngeneity of the record older than Late Archaean13. Non- observed in drill core samples, polished slabs and thin sections (Sup-
biological processes are known to produce morphologies similar plementary Fig. 2). These sedimentary structures indicate deposition
to some microfossils4,5, and hydrothermal fluids have the potential in shallow-water environments above the wave base.
to produce abiotic organic compounds with depleted carbon The Moodies Group is the uppermost of three stratigraphic units
isotope values6, making it difficult to establish unambiguous that comprise the Swaziland Supergroup in the BGB (Supplementary
traces of life. Here we report the discovery of a population of large Fig. 1b). It consists of an up to 3.7-km-thick succession of alluvial to
(up to about 300 mm in diameter) carbonaceous spheroidal micro- shallow-marine sandstones with subordinate conglomerates and mud-
structures in Mesoarchaean shales and siltstones of the Moodies stones, as well as iron formation and volcanic rocks15. Deposition of the
Group, South Africa, the Earths oldest siliciclastic alluvial to tidal- Moodies Group began shortly after 3,226 6 1 and 3,222 110/24 Myr
estuarine deposits7. These microstructures are interpreted as ago (age of an ignimbrite and porphyritic intrusion, respectively, at the
organic-walled microfossils on the basis of petrographic and geo- top of the underlying Fig Tree Group15,16) but before 3,207 6 2 Myr ago
chemical evidence for their endogenicity and syngeneity, their (age of a dacitic dyke cross-cutting the basal part of the Moodies
carbonaceous composition, cellular morphology and ultrastruc- Group15). The minimum age is also constrained by the 3,109 110/
ture, occurrence in populations, taphonomic features of soft wall 28-Myr-old Salisbury Kop Pluton that intruded the Moodies Group
deformation, and the geological context plausible for life, as well in the eastern part of the greenstone belt16.
as a lack of abiotic explanation falsifying a biological origin. These Disseminated particulate carbonaceous material occurs in most
are the oldest and largest Archaean organic-walled spheroidal samples taken from the five drill cores studied, but the carbonaceous
microfossils reported so far. Our observations suggest that rela- microstructures are present only in 22 of the 55 analysed samples and
tively large microorganisms cohabited with earlier reported within four of the five drill cores studied, and are abundant in only four
benthic microbial mats8 in the photic zone of marginal marine samples. The carbonaceous microstructures were observed in thin
siliciclastic environments 3.2 billion years ago. sections cut parallel (Fig. 1a, b) and perpendicular (Fig. 1c) to the
Until now, Archaean carbonaceous microstructures have been bedding. They are compressed parallel to the bedding, implying that
described mostly from hydrothermal and sedimentary cherts1,2,5. they were emplaced in the sediments before burial compaction. Their
Archaean siliciclastic lithologies have been largely overlooked by micro- black colour is similar to the colour of the surrounding particulate
palaeontologists, although they are routinely examined in Proterozoic organic matter disseminated as fine particles or larger irregular clots
successions. Early microfossil reports9 from acid-macerated shales of (Fig. 1a, b). Both observations indicate that the microstructures were
the Fig Tree Group (Barberton Greenstone Belt (BGB), South Africa), deposited with the sediments and are not derived from contamination
which is about 3.3 Gyr old, have been deemed to be abiogenic10. or artefacts of laboratory procedures. The studied samples are devoid
Sedimentary structures interpreted as microbial mats, and also pre- of hydrothermal veins, faults and fractures that could allow exogenous
served organic matter with negative carbon isotope values, have been material into the rock, and lack evidence of boring by endolithic micro-
described from Archaean unsilicified11 siliciclastic successions, includ- organisms. The microstructures are resistant to acids, indicating recal-
ing the Moodies Group8, and from cherts12 of South Africa. Proterozoic citrant organic composition. After acid maceration they conserve their
shales and siltstones deposited in intertidal to deep-basinal marine vesicle shape, demonstrating that they represent large microstructures
environments are known to preserve organic-walled microfossils, and are not formed by the agglomeration of fine organic particles. The
sometimes at the ultrastructural level13,14; we therefore investigated microstructures occur as populations of isolated unicells, not as
a well-preserved Archaean siliciclastic succession within a well- clusters or colonies. The minimum apparent diameter ranges from
constrained geological context of the BGB. 31.09 to 298.35 mm (n 5 98, mean 121.89 mm) with a mode between
Here we report on a population of carbonaceous spheroidal micro- 50 and 75 mm (Fig. 2). The microstructures have black and chagrinate
structures from bedded siltstones and shales of the Mesoarchaean (covered with very fine granules) structurally distinct walls, showing
Moodies Group, BGB. Our study of 55 samples from five short drill thin concentric or lanceolate folds, wrinkling and, sometimes, folding
holes drilled from the underground levels 600 m below the surface in over (Fig. 1af). Scanning electron microscope (SEM) imaging of
the Agnes gold mine, Moodies Hills Block (Supplementary Fig. 1a), the surface of carbonaceous microstructures shows a highly and irre-
shows that the microstructures are relatively well preserved and gularly wrinkled texture of degraded and collapsed organic walls
common in several samples. They occur in the Clutha Formation, at (Fig. 1gj), which we interpret here as a taphonomic feature rather
Department of Geology, University of Liege, 17 allee du 6 Aout B18, Liege 4000, Belgium. 2Department of Geology, University of Kansas, 1475 Jayhawk Boulevard, Lawrence, Kansas
66044, USA. 3Department of Geological Sciences, University of Manitoba, 125 Dysart Road (Wallace Building), Winnipeg, Manitoba R3T 2N2, Canada.

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a b c

100 m 100 m 50 m

d e f

50 m 50 m 100 m

g i

50 m 10 m 20 m

h j

200 nm

50 m 2 m

m n

30 m

200 nm

Figure 1 | Carbonaceous microstructures in situ in thin sections and b), and concentric folds (d, g, h), wrinkling (i), lanceolate fold (e) and
extracted from the rock by acid maceration. Images were produced with a collapsing over (f), which are all typical taphonomic features of soft wall
transmitted light microscope (af), a backscattered environmental SEM deformation. SEM images show the highly folded, wrinkled and degraded
(gj) and a TEM (kn). Arrows point to spheroidal microstructures in texture of the wall (gj). TEM images show the compressed vesicle walls
section subparallel to the bedding (a, b), compressed microstructures in surrounding the cell lumen (arrowed) in semi-thin (k) and ultra-thin
section across the bedding (c), microstructures extracted from the rock by (n) sections and the homogeneous ultrastructure (l, m) of the roughly 160-
acid maceration (dn), disseminated organic particles (short arrows in nm-thick wall, torn and wrinkled in places (l).

than an ornamentation. SEMenergy-dispersive X-ray analyses show The taphonomic features of soft wall deformation, commonly
occasional disseminated arsenopyrite and other sulphide crystals on observed in Proterozoic and Phanerozoic organic-walled micro-
the walls of the microstructures. Transmission electron microscope fossils with well-accepted biogenicity, are due to their loss of turgor
(TEM) analyses of the wall ultrastructure show unambiguously that pressure and degradational collapse during decay17 before flattening
they represent flattened hollow organic-walled vesicles with the cell of the hosting shales and siltstones during compaction, and show
lumen visible between the compressed walls (Fig. 1k, n) rather than flexibility of the original organic wall. Another common feature with
large kerogen particles. The organic wall shows folding along its length Proterozoic fossiliferous siliciclastic rocks is the low total organic
(Fig. 1k, n) and seems disrupted in places because the 60-nm-thick carbon content ranging from 0.07 to 0.37wt%, with an average of
ultra-thin sectioning cut through highly wrinkled and degraded walls, 0.17wt% (n 5 22). Generally, Proterozoic shales with a high total
as observed in SEM images. Moreover, some small mineral grains were organic carbon content contain only particulate organic matter
ripped off during sectioning, as demonstrated by the presence of holes without structurally preserved walls, whereas shales with a low total
and, occasionally, pyrite cubes in the resin. The roughly 160-nm-thick organic carbon content (grey shales) may preserve, sometimes
wall appears torn and wrinkled in places, and has a homogeneous exquisitely, organic structures with cell walls13,17. Other important
ultrastructure (Fig. 1l, m). controls on the preservation potential of microorganisms are their
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25 residues containing microfossils (Supplementary Table 1). The data

combined suggest that the presence of microfossils probably reflects
n = 98 environmental conditions that influenced the preservation of organic
fabrics rather than post-depositional contamination. Our carbon
Number of specimens

isotope data and total organic carbon contents agree well with the
15 previously published data for siliciclastic sediments of the Moodies
Group as well as all other units more than 3.0 Gyr old8,21 and are
consistent with a biological origin of the microstructures.
10 Simple organic molecules are readily synthesized by non-
biological processes in laboratory experiments or delivered by
5 meteorites, and they can self-assemble into vesicles22 with sizes
ranging from a few tens of nanometres up to 100 mm in diameter23.
However, it is uncertain whether these vesicles can form in nature,
0 whether they would be preserved in the rock record and whether they
0 50 100 150 200 250 300
Minimum diameter (m)
are acid-resistant. The necessary hydrothermal or low-ionic-strength
conditions22 clearly differ from the shallow-water marine siliciclastic
Figure 2 | Size distribution of carbonaceous spheroidal microstructures depositional environments of the studied stratigraphic level in the
preserved in the 3.2-Gyr-old shales and siltstones of the Moodies Group, Moodies Group (see Supplementary Information). Abiotic carbona-
Barberton Greenstone Belt, South Africa.
ceous vesicles may also form by other processes such as fluid migration
along microfractures in cherts24, around silicified mineral casts5 and
habitat, original cell composition and local taphonomic conditions around silica spheres formed in silica-saturated waters25. These struc-
during early diagenesis. tures might be preserved in the rock record as three-dimensional
The Raman first-order spectra (Fig. 3) demonstrate that these structures in carbonaceous cherts5, but they are not expected to form
microstructures are composed of a network of disordered sp2 car- two-dimensional, flattened, folded and acid-resistant carbonaceous
bonaceous material (see Supplementary Information). The line vesicles in fine-grained siliciclastic sediments. To our knowledge,
shape of the carbon first-order spectra is similar to that of the car- none of the known abiotic processes can account for the morpho-
bonaceous microstructures in situ and the surrounding particulate logical, ultrastructural and geochemical observations presented here.
organic matter as well as the carbonaceous microstructures extracted The oldest unambiguous organic-walled microfossils preserved in
from the rock (Fig. 3). The spectra indicate a metamorphic grade of unsilicified fine-grained siliciclastic sediments are of late Palaeo-
upper greenschist facies, which is consistent with the metamorphic proterozoic age (about 1.651.8 Gyr old14) and include relatively large
grade experienced by the host rocks during the tectonic events 3.13.2 vesicles (up to 238 mm) of unidentified microorganisms interpreted as
and 2.7 Gyr ago15,16,18, thereby supporting the syngeneity of the car- possible protists or cyanobacteria, and one eukaryotic taxon of orna-
bonaceous microstructures within the host rock. However, matura- mented vesicles. Early reports9 of globular-type A microfossils from
tion or metamorphism of almost all naturally occurring organic acid-macerated shales of the roughly 3.3-Gyr-old Fig Tree Group have
matter, whether biological or abiological (for example alkanes synthe- been questioned10. Large (up to 90 mm) Archaean carbonaceous
sized by FischerTropsch-type processes) in origin, give rise to similar spheroidal microstructures have been reported from the carbona-
thermally mature productscovalently crosslinked aromatic hydro- ceous cherts of the roughly 3.3-Gyr-old Kromberg Formation,
carbons and other aromatic subunits that become transformed and BGB26, but were reinterpreted as abiotic self-organized structures5
condensed through carbonization and graphitization19. Consequently, although associated hollow kerogenous filaments may be biogenic1.
Raman spectroscopy of overmature carbonaceous material cannot Similar-sized but more diverse carbonaceous spheroidal microstruc-
provide definitive evidence of its biogenicity by itself19,20. tures interpreted as probable microfossils27 are abundant in the lat-
Carbon isotope analyses of the bulk sample kerogen (n 5 22) erally extensive black chert beds of the roughly 2.97-Gyr-old Farrel
revealed negative d13C values ranging between 216.4% and Quartzite, Gorge Creek Group in the Mount GoldsworthyMount
228.3%, with an average of 222.4%. There is no difference in Grant area, Pilbara Craton, Western Australia. Carbonaceous micro-
carbon isotope values between samples with and without microfossils structures include possible microbial films with associated small
as well as between bulk samples with microfossils and their macerated spheres, large (1090-mm) hollow spheroids, and spindle-like struc-
tures. This assemblage was interpreted as a probable diverse microbial
D community flourishing in partly evaporitic basin with terrigenous
G sediment input27. The cell size of the microfossil population described
D2 in our study is up to 298 mm and larger than any other reported
Intensity (arbitrary units)

Archaean sphaeromorphs, but comparable in size to the oldest un-

ambiguous organic-walled microfossils reported from the late Palaeo-
proterozoic era, extending their record in fine-grained siliciclastic
sediments by more than 1 Gyr. The apparent long gap in the siliciclas-
tic fossil record might be explained, at least in part, by the limited
number of micropalaeontological studies of organic-matter-poor
shales relative to those of the more conspicuous organic-matter-rich
black shales, cherts and carbonates of Archaean and Palaeoproterozoic
1,800 1,600 1,400 1,200 1,000 Extant microorganisms producing large organic-walled vesicles are
Raman shift (cm1) unknown among archaea28 but include protists and some bacteria. The
large bacterial cells include the symbiont Epulopiscium sp. (80 mm wide
Figure 3 | Raman microspectroscopy. First-order Raman spectrum of
carbonaceous microstructures (top trace) and disseminated particulate and 600 mm long), which lives in the nutrient-rich gut of tropical fish28,
carbonaceous material (bottom trace) in situ in thin section compared with and myxobacteria sporangioles (up to 200 mm in diameter), which
that of carbonaceous microstructures extracted by acid maceration (middle enclose myxospores and live predominantly in soil28. The sulphur
trace) (band assignments: D band, 1,355 cm21; G band, 1,590 cm21; D2 bacterium Thiomargarita namibiensis forms chains of unconnected
band, 1,620 cm21). mucus-sheathed spheroidal cells up to 750 mm in diameter, with up
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to 98% of the cytoplasm filled with vacuoles, and inhabits organic 514.5-nm line of a 5-W Ar1 laser (Stabilite 2017 laser; Spectra-Physics).
matter-rich and sulphidic sediments with changing redox conditions Carbon isotope analyses were performed by high-temperature oxidation in
in deep hydrothermal sites and coastal upwelling zones28. Large cya- helium flow with a Flash 1112 Elemental Analyser (Thermo Electron Inc.)
nobacteria include spheroidal sheaths 3060 mm in diameter enve- coupled to a Delta Plus XL (Thermo Electron Inc.) through a Conflo III. Data
were calibrated against the international standards L-SVEC and IAEA-CH6. The
loping small baeocyte cells29 and oval to sausage-shaped thick-walled
accuracy of d13C measurements was 0.1% (n 5 38).
cysts up to 100 mm long called akinetes28,29. The large organic-walled
microfossils reported here are unlikely to be related to the bacterial Full Methods and any associated references are available in the online version of
symbiont, to extant large sulphur bacteria or to myxobacteria, because the paper at
these organisms are not known to form recalcitrant biopolymers and
Received 12 June; accepted 15 December 2009.
live in completely different ecological niches from those in which the Published online 7 February 2010.
Moodies microfossils were deposited. Although extant protists may
have unornamented unilayered walls, a protistan affinity cannot be 1. Buick, R. in Paleobiology II (eds Briggs, D.E.G. & Crowther, P.R.) 1321 (Blackwell
Science, 2001).
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Moodies microfossils are ancestors of extant organisms rather than appearance of eukaryotes and cyanobacteria. Nature 455, 11011105 (2008).
members of extinct stem clades, their size, taphonomy, acid resistance 4. Garca-Ruiz, J. M. et al. Self-assembled silica-carbonate structures and detection
of ancient microfossils. Science 302, 11941197 (2003).
and habitat in the photic zone could suggest a cyanobacterial affinity, 5. Brasier, M. D. M. c. L. o. u. g. h. i. n. N., Green, O. & Wacey, D. A fresh look at the
although the Moodies microfossils are larger than any reported extant fossil evidence for early Archaean cellular life. Phil. Trans. R. Soc. B 361, 887902
or fossil cyanobacteria. Although the global increase in atmospheric (2006).
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habitat of the Moodies microfossils might suggest either early evolu- Archean life: microbial mats in Earths oldest siliciclatic tidal deposits (3.2 Ga
Moodies Group, South Africa). Geology 34, 253256 (2006).
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Archean. J. Afr. Earth Sci. 33, 427436 (2001).
Regardless of their biological affinities, these large microorganisms, if
11. Noffke, N., Beukes, N., Bower, D., Hazen, R. M. & Swift, D. J. P. An actualistic
crown group ancestors, must have either used or maintained local perspective into Archean worlds(cyano-)bacterially induced sedimentary
redox gradients in shallow-marine environments. If this is indeed so, structures in the siliciclastic Nhlazatse Section, 2.9 Ga Pongola Supergroup, South
geochemical studies of these shallow-water environments might recog- Africa. Geobiology 6, 520 (2008).
nize local redox cycling almost 0.80.9 Gyr before the global increase in 12. Tice, M. M. & Lowe, D. R. Photosynthetic microbial mats in the 3.416-Myr-old
ocean. Nature 431, 549552 (2004).
atmospheric oxygen30. 13. Javaux, E. J., Knoll, A. H. & Walter, M. R. TEM evidence for eukaryotic diversity in
The Moodies Group provides an unusual window into the ecology mid-Proterozoic oceans. Geobiology 2, 121132 (2004).
of Mesoarchaean ocean, demonstrating the early evolution of a 14. Knoll, A. H., Javaux, E. J., Hewitt, D. & Cohen, P. Eukaryotic organisms in
moderately diverse ecosystem in the photic zone of marginal marine Proterozoic oceans. Phil. Trans. R. Soc. B 361, 10231038 (2006).
siliciclastic environments, in which large recalcitrant organic-walled uni- 15. Heubeck, C. & Lowe, D. R. Depositional and tectonic setting of the Archean
Moodies Group, Barberton Greenstone Belt, South Africa. Precambr. Res. 68,
cells or colonial envelopes lived contemporaneously with earlier reported 257290 (1994).
benthic microbial mats. As palaeontological studies of Archaean shales 16. Kamo, S. L. & Davis, D. W. Reassessment of Archean foredeep sedimentation
move forwards, we predict that focus on organic-matter-lean shales will related to crustal shortening: a reinterpretation of the Barberton Mountain Land,
yield significant discoveries, improving our understanding of the early South Africa, based on UPb dating. Tectonics 13, 167192 (1994).
evolution of the biosphere. 17. Butterfield, N. J., Knoll, A. H. & Swett, N. Paleobiology of the Neoproterozoic
Svanbergfjellet formation, Spitsbergen. Fossils Strata 34, 184 (1994).
18. Tice, M. M., Bostick, B. C. & Lowe, D. R. Thermal history of the 3.53.2 Ga
METHODS SUMMARY Onverwacht and Fig Tree Groups, Barberton greenstone belt, South Africa,
Thin sections were cut from rock samples perpendicular and parallel to the inferred by Raman microspectroscopy of carbonaceous material. Geology 32,
bedding. Optical microscopy was performed with a Zeiss Axioimager micro- 3740 (2004).
scope equipped with an Axiocam MRc5. About 25 g of each sample was demi- 19. Marshall, C. P. et al. Structural characterization of kerogen in 3.4 Ga Archaean
neralized by treatment with HF/HCl followed by settling and decanting. Part of cherts from the Pilbara Craton, Western Australia. Precambr. Res. 155, 123
the residue was mounted on microscope slides. Single microfossils were hand-
20. Pasteris, J. D. & Wopencka, B. Necessary, but not sufficient: Raman identification
picked under an inverted microscope by using a micropipette, then deposited of disordered carbon as a signature of ancient life. Astrobiology 3, 727738
uncoated on an aluminium stub and imaged by backscattered electron micro- (2003).
scopy with a Philips ESEM (Environmental Scanning Electron microscope) 21. Strauss, H. & Moore, T. B. in The Proterozoic Biosphere: a Multidisciplinary Study
LX30 FEG (field emission gun) at 15 kV. Single microfossils were embedded in (eds Schopf, J.W. & Klein, C.) 709798 (Cambridge Univ. Press, 1992).
agar, dehydrated in a series of ethanol solutions, and then infiltrated successively 22. Deamer, D., Singaram, S., Rajamani, S., Kompanichenko, V. & Guggenheim, S. Self-
with propylene oxide/ethanol, propylene oxide/epoxy resin, and pure epoxy assembly processes in the prebiotic environment. Phil. Trans. R. Soc. B 361,
resin. Samples were polymerized in an oven at 60 uC for at least 12 h. After 18091818 (2006).
verification of proper orientation of microfossils, resin blocks were trimmed 23. Akashi, K.-I., Miyata, H., Itoh, H. & Kinosita, K. Jr. Preparation of giant liposomes in
and cut into 1-mm-thick semi-thin and 50-nm-thick ultra-thin sections with a physiological conditions and their characterization under an optical microscope.
Biophys. J. 71, 32423250 (1996).
diamond knife. Sections were put on copper grid and imaged with a JEM100SX
24. Van Zuilen, M. A., Chaussidon, M., Rollion-Bard, C. & Marty, B. Carbonaceous
at 80 kV. No staining was used because previous studies13 showed this to be
cherts of the Barberton Greenstone Belt, South Africa: isotopic, chemical and
unnecessary and it can be a source of artefacts. Raman spectra were collected structural characteristics of individual microstructures. Geochim. Cosmochim.
on thin sections and on isolated microfossils were collected on glass slides. Acta 71, 655669 (2007).
Spectra were recorded from two to ten different points in each sample to check 25. Jones, B. & Renaut, R. W. Microstructural changes accompanying the opal-A to
for the representative nature of the spectra, with a Renishaw inVia Reflex Raman opal-CT transition: new evidence from the siliceous sinters of Geysir, Haukadalur,
Microprobe using a Peltier-cooled charge-coupled device detector and the Iceland. Sedimentology 54, 921948 (2007).
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26. Walsh, M. M. Microfossils and possible microfossils from the early Archean analyses; J. Robertson for information on geology of the Agnes Gold Mine; and
Onverwacht Group, Barberton Mountain Land, South Africa. Precambr. Res. 54, A. H. Knoll for comments on an earlier version of the manuscript. The study was
271292 (1992). supported by a University of Liege Impulsion Grant (CFRA0805) and a University
27. Sugitani, K. et al. Diverse microstructures from Archaean chert from the Mount of Liege grant (RCFRA0036-J) to E.J., a National Science Foundation grant
GoldsworthyMount Grant area, Pilbara Craton, Western Australia: microfossils, (EAR-937 05-45484), a NASA Astrobiology Institute award (NNA04CC09A), a
dubiofossils, or pseudofossils? Precambr. Res. 158, 228262 (2007). Natural Sciences and Engineering Research Council of Canada 938 Discovery grant
28. Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.-H. & Stackebrandts, E. (eds) in to A.B., and Australian Research Council funding to C.M.
The Prokaryotes 3rd edn, Vol. 6, 11561163; Vol. 7, 31115 (Springer, 2006).
29. Waterbury, J. B. & Stanier, R. Y. Patterns of growth and development in Author Contributions E.J. and A.B. conceived the study and wrote the paper. E.J.
Pleurocapsalean cyanobacteria. Microbiol. Rev. 42, 244 (1978). discovered the microfossils and performed the microscopic, SEM and TEM
30. Bekker, A. et al. Dating the rise of atmospheric oxygen. Nature 427, 117120 analyses, and interpreted the data. C.M. performed the Raman analyses and
(2004). interpreted and wrote the results. A.B. conducted field work and carbon isotope
analyses and interpreted the sedimentary structures. All authors commented on
Supplementary Information is linked to the online version of the paper at the manuscript.
Author Information Reprints and permissions information is available at
Acknowledgements We thank M. Giraldo, J. Laval and N. Decloux for sample The authors declare no competing financial interests.
preparation; P. Compere for TEM imaging; C. Henrist for environmental SEM Correspondence and requests for materials should be addressed to E.J.
imaging and energy-dispersive X-ray analyses; T. Prokopiuk for carbon isotope (

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METHODS silicon wafer (111). A surface laser power of 1.01.5 mW was used to minimize
Scanning electron microscopy. The microstructures were deposited uncoated laser-induced heating of the samples. An accumulation time of 30 s and ten scans
on an aluminium stub and imaged by backscattered electron microscopy at the were used, which gave an adequate signal-to-noise ratio for the spectra. The scan
microscopy facility of the University of Liege with a Philips ESEM LX30 FEG at ranges were 8001800 cm21 in the carbon first-order region.
15 kV. Carbon isotope analyses. Carbon isotope analyses of organic matter were per-
Transmission electron microscopy. The microstructures were embedded in formed at the Department of Geological Sciences, University of Saskatchewan.
agar, dehydrated in a series of ethanol solutions, and then infiltrated with a Carbonate-bearing samples were acidified stepwise in 10%, 20% and 40% HCl to
mixture of propylene oxide/ethanol, followed by propylene oxide/epoxy resin, remove carbonate materials completely. The residue was then homogenized and
and then pure epoxy resin. Samples were then polymerized in an oven at 60 uC loaded into tin capsules. Stable isotope values were obtained with a Flash 1112
for at least 12 h. After verification of the proper orientation of individual micro- Elemental Analyser (Thermo Electron Inc.) coupled to a Delta Plus XL (Thermo
structures, resin blocks were trimmed and cut into 50-nm-thick ultra-thin sec- Electron Inc.) through a Conflo III. Samples were dropped under helium into an
tions with a diamond knife. No staining was used, because this had been shown oxidation furnace at 1,000 uC that was packed with chromium(VI) oxide and
to be unnecessary in previous studies13 and can be a source of artefacts. Sections silvered cobaltic/cobaltous oxide (used to remove any halogens). Organic
were put on a copper grid and imaged at the microscopy facility of the University materials were oxidized to carbon dioxide, various nitrogen-bearing gases and
of Liege with a JEM100SX at 80 KV. Negatives were scanned to obtain images. water. This gas mixture was then passed through a reduction furnace at 680 uC
Note that the TEM image on Fig. 1n is a photomontage of three images to obtain packed with elemental copper to reduce all nitrogen-bearing compounds to pure
an image of the whole microfossil at high resolution. nitrogen gas. The resulting gases were then passed through a water trap to
Raman microspectroscopy. Raman spectra were collected on thin sections, and eliminate moisture, and then a gas chromatography column at 50 uC to separate
isolated microfossils on glass slides. Because spectral features used to infer the the carbon dioxide for analysis in the mass spectrometer. Carbon isotope ratios
degree of crystallinity of disordered sp2 carbons vary depending on the orienta- are corrected for blank and 17O contribution and reported in % notation relative
tion of the crystallites to the exciting laser beam, spectra were recorded from two to the V-PDB scale. Carbon isotope data are calibrated against the international
to ten different points in each sample to check for the representative nature of the standards L-SVEC (d13C 5 246.6% V-PDB) and IAEA-CH6 (d13C 5 210.45%
spectra. The Raman spectra were acquired on a Renishaw inVia Reflex Raman V-PDB). IAEA-CH7, an intermediate international standard, gave the following
Microprobe with a Peltier-cooled charge-coupled device detector. The collection results: d13C 5 232.14 6 0.03% V-PDB (n 5 12), which compares well with its
optics is based on a Leica DM LM microscope. A refractive glass 503 objective accepted value of 232.15 6 0.10% V-PDB. The accuracy of data was monitored
lens was used to focus the laser on a 2-mm spot to collect the backscattered through routine analyses of in-house standards that were stringently calibrated
radiation. The 514.5-nm line of a 5-W Ar1 laser (Stabilite 2017 laser; Spectra- against the IAEA standards mentioned above. The accuracy of d13C measure-
Physics) oriented normal to the sample was used to excite the sample. The ments was 0.1% (n 5 38); weight-percentage C measurements had an accuracy
instrument was calibrated against the Raman signal of Si at 520 cm21 with a of between 1% and 0.5% (n 5 10).

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