LETTERS

Organic-walled microfossils in 3.2-billion-year-oldshallow-marine siliciclastic depositsEmmanuelle J. Javaux1, Craig P. Marshall2 & Andrey Bekker3

Although the notion of an early origin and diversification of life onEarth during the Archaean eon has received increasing support ingeochemical, sedimentological and palaeontological evidence,ambiguities and controversies persist regarding the biogenicityand syngeneity of the record older than Late Archaean1–3. Non-biological processes are known to produce morphologies similarto some microfossils4,5, and hydrothermal fluids have the potentialto produce abiotic organic compounds with depleted carbonisotope values6, making it difficult to establish unambiguoustraces of life. Here we report the discovery of a population of large(up to about 300 mm in diameter) carbonaceous spheroidal micro-structures in Mesoarchaean shales and siltstones of the MoodiesGroup, South Africa, the Earth’s oldest siliciclastic alluvial to tidal-estuarine deposits7. These microstructures are interpreted asorganic-walled microfossils on the basis of petrographic and geo-chemical evidence for their endogenicity and syngeneity, theircarbonaceous composition, cellular morphology and ultrastruc-ture, occurrence in populations, taphonomic features of soft walldeformation, and the geological context plausible for life, as wellas a lack of abiotic explanation falsifying a biological origin. Theseare the oldest and largest Archaean organic-walled spheroidalmicrofossils reported so far. Our observations suggest that rela-tively large microorganisms cohabited with earlier reportedbenthic microbial mats8 in the photic zone of marginal marinesiliciclastic environments 3.2 billion years ago.

Until now, Archaean carbonaceous microstructures have beendescribed mostly from hydrothermal and sedimentary cherts1,2,5.Archaean siliciclastic lithologies have been largely overlooked by micro-palaeontologists, although they are routinely examined in Proterozoicsuccessions. Early microfossil reports9 from acid-macerated shales ofthe Fig Tree Group (Barberton Greenstone Belt (BGB), South Africa),which is about 3.3 Gyr old, have been deemed to be abiogenic10.Sedimentary structures interpreted as microbial mats, and also pre-served organic matter with negative carbon isotope values, have beendescribed from Archaean unsilicified11 siliciclastic successions, includ-ing the Moodies Group8, and from cherts12 of South Africa. Proterozoicshales and siltstones deposited in intertidal to deep-basinal marineenvironments are known to preserve organic-walled microfossils,sometimes at the ultrastructural level13,14; we therefore investigateda well-preserved Archaean siliciclastic succession within a well-constrained geological context of the BGB.

Here we report on a population of carbonaceous spheroidal micro-structures from bedded siltstones and shales of the MesoarchaeanMoodies Group, BGB. Our study of 55 samples from five short drillholes drilled from the underground levels 600 m below the surface inthe Agnes gold mine, Moodies Hills Block (Supplementary Fig. 1a),shows that the microstructures are relatively well preserved andcommon in several samples. They occur in the Clutha Formation, at

the base of the Moodies Group, in interlayered laminated grey shales,siltstones and wavy-laminated clay-rich and organic-matter-richlayers, possibly representing microbial mat structures. Flaser bedding,small-scale cross-bedding, and mud-draped current ripples wereobserved in drill core samples, polished slabs and thin sections (Sup-plementary Fig. 2). These sedimentary structures indicate depositionin shallow-water environments above the wave base.

The Moodies Group is the uppermost of three stratigraphic unitsthat comprise the Swaziland Supergroup in the BGB (SupplementaryFig. 1b). It consists of an up to 3.7-km-thick succession of alluvial toshallow-marine sandstones with subordinate conglomerates and mud-stones, as well as iron formation and volcanic rocks15. Deposition of theMoodies Group began shortly after 3,226 6 1 and 3,222 110/24 Myrago (age of an ignimbrite and porphyritic intrusion, respectively, at thetop of the underlying Fig Tree Group15,16) but before 3,207 6 2 Myr ago(age of a dacitic dyke cross-cutting the basal part of the MoodiesGroup15). The minimum age is also constrained by the 3,109 110/28-Myr-old Salisbury Kop Pluton that intruded the Moodies Groupin the eastern part of the greenstone belt16.

Disseminated particulate carbonaceous material occurs in mostsamples taken from the five drill cores studied, but the carbonaceousmicrostructures are present only in 22 of the 55 analysed samples andwithin four of the five drill cores studied, and are abundant in only foursamples. The carbonaceous microstructures were observed in thinsections cut parallel (Fig. 1a, b) and perpendicular (Fig. 1c) to thebedding. They are compressed parallel to the bedding, implying thatthey were emplaced in the sediments before burial compaction. Theirblack colour is similar to the colour of the surrounding particulateorganic matter disseminated as fine particles or larger irregular clots(Fig. 1a, b). Both observations indicate that the microstructures weredeposited with the sediments and are not derived from contaminationor artefacts of laboratory procedures. The studied samples are devoidof hydrothermal veins, faults and fractures that could allow exogenousmaterial into the rock, and lack evidence of boring by endolithic micro-organisms. The microstructures are resistant to acids, indicating recal-citrant organic composition. After acid maceration they conserve theirvesicle shape, demonstrating that they represent large microstructuresand are not formed by the agglomeration of fine organic particles. Themicrostructures occur as populations of isolated unicells, not asclusters or colonies. The minimum apparent diameter ranges from31.09 to 298.35mm (n 5 98, mean 121.89mm) with a mode between50 and 75mm (Fig. 2). The microstructures have black and chagrinate(covered with very fine granules) structurally distinct walls, showingthin concentric or lanceolate folds, wrinkling and, sometimes, foldingover (Fig. 1a–f). Scanning electron microscope (SEM) imaging ofthe surface of carbonaceous microstructures shows a highly and irre-gularly wrinkled texture of degraded and collapsed organic walls(Fig. 1g–j), which we interpret here as a taphonomic feature rather

1Department of Geology, University of Liege, 17 allee du 6 Aout B18, Liege 4000, Belgium. 2Department of Geology, University of Kansas, 1475 Jayhawk Boulevard, Lawrence, Kansas66044, USA. 3Department of Geological Sciences, University of Manitoba, 125 Dysart Road (Wallace Building), Winnipeg, Manitoba R3T 2N2, Canada.

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than an ornamentation. SEM–energy-dispersive X-ray analyses showoccasional disseminated arsenopyrite and other sulphide crystals onthe walls of the microstructures. Transmission electron microscope(TEM) analyses of the wall ultrastructure show unambiguously thatthey represent flattened hollow organic-walled vesicles with the celllumen visible between the compressed walls (Fig. 1k, n) rather thanlarge kerogen particles. The organic wall shows folding along its length(Fig. 1k, n) and seems disrupted in places because the 60-nm-thickultra-thin sectioning cut through highly wrinkled and degraded walls,as observed in SEM images. Moreover, some small mineral grains wereripped off during sectioning, as demonstrated by the presence of holesand, occasionally, pyrite cubes in the resin. The roughly 160-nm-thickwall appears torn and wrinkled in places, and has a homogeneousultrastructure (Fig. 1l, m).

The taphonomic features of soft wall deformation, commonlyobserved in Proterozoic and Phanerozoic organic-walled micro-fossils with well-accepted biogenicity, are due to their loss of turgorpressure and degradational collapse during decay17 before flatteningof the hosting shales and siltstones during compaction, and showflexibility of the original organic wall. Another common feature withProterozoic fossiliferous siliciclastic rocks is the low total organiccarbon content ranging from 0.07 to 0.37wt%, with an average of0.17wt% (n 5 22). Generally, Proterozoic shales with a high totalorganic carbon content contain only particulate organic matterwithout structurally preserved walls, whereas shales with a low totalorganic carbon content (‘grey shales’) may preserve, sometimesexquisitely, organic structures with cell walls13,17. Other importantcontrols on the preservation potential of microorganisms are their

100 µm 100 µm 50 µm

50 µm 50 µm 100 µm

50 µm 10 µm

2 µm50 µm

20 µm

d e f

g

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i

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l

m n

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Figure 1 | Carbonaceous microstructures in situ in thin sections andextracted from the rock by acid maceration. Images were produced with atransmitted light microscope (a–f), a backscattered environmental SEM(g–j) and a TEM (k–n). Arrows point to spheroidal microstructures insection subparallel to the bedding (a, b), compressed microstructures insection across the bedding (c), microstructures extracted from the rock byacid maceration (d–n), disseminated organic particles (short arrows in

b), and concentric folds (d, g, h), wrinkling (i), lanceolate fold (e) andcollapsing over (f), which are all typical taphonomic features of soft walldeformation. SEM images show the highly folded, wrinkled and degradedtexture of the wall (g–j). TEM images show the compressed vesicle wallssurrounding the cell lumen (arrowed) in semi-thin (k) and ultra-thin(n) sections and the homogeneous ultrastructure (l, m) of the roughly 160-nm-thick wall, torn and wrinkled in places (l).

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habitat, original cell composition and local taphonomic conditionsduring early diagenesis.

The Raman first-order spectra (Fig. 3) demonstrate that thesemicrostructures are composed of a network of disordered sp2 car-bonaceous material (see Supplementary Information). The lineshape of the carbon first-order spectra is similar to that of the car-bonaceous microstructures in situ and the surrounding particulateorganic matter as well as the carbonaceous microstructures extractedfrom the rock (Fig. 3). The spectra indicate a metamorphic grade ofupper greenschist facies, which is consistent with the metamorphicgrade experienced by the host rocks during the tectonic events 3.1–3.2and 2.7 Gyr ago15,16,18, thereby supporting the syngeneity of the car-bonaceous microstructures within the host rock. However, matura-tion or metamorphism of almost all naturally occurring organicmatter, whether biological or abiological (for example alkanes synthe-sized by Fischer–Tropsch-type processes) in origin, give rise to similarthermally mature products—covalently crosslinked aromatic hydro-carbons and other aromatic subunits that become transformed andcondensed through carbonization and graphitization19. Consequently,Raman spectroscopy of overmature carbonaceous material cannotprovide definitive evidence of its biogenicity by itself19,20.

Carbon isotope analyses of the bulk sample kerogen (n 5 22)revealed negative d13C values ranging between 216.4% and228.3%, with an average of 222.4%. There is no difference incarbon isotope values between samples with and without microfossilsas well as between bulk samples with microfossils and their macerated

residues containing microfossils (Supplementary Table 1). The datacombined suggest that the presence of microfossils probably reflectsenvironmental conditions that influenced the preservation of organicfabrics rather than post-depositional contamination. Our carbonisotope data and total organic carbon contents agree well with thepreviously published data for siliciclastic sediments of the MoodiesGroup as well as all other units more than 3.0 Gyr old8,21 and areconsistent with a biological origin of the microstructures.

Simple organic molecules are readily synthesized by non-biological processes in laboratory experiments or delivered bymeteorites, and they can self-assemble into vesicles22 with sizesranging from a few tens of nanometres up to 100 mm in diameter23.However, it is uncertain whether these vesicles can form in nature,whether they would be preserved in the rock record and whether theyare acid-resistant. The necessary hydrothermal or low-ionic-strengthconditions22 clearly differ from the shallow-water marine siliciclasticdepositional environments of the studied stratigraphic level in theMoodies Group (see Supplementary Information). Abiotic carbona-ceous vesicles may also form by other processes such as fluid migrationalong microfractures in cherts24, around silicified mineral casts5 andaround silica spheres formed in silica-saturated waters25. These struc-tures might be preserved in the rock record as three-dimensionalstructures in carbonaceous cherts5, but they are not expected to formtwo-dimensional, flattened, folded and acid-resistant carbonaceousvesicles in fine-grained siliciclastic sediments. To our knowledge,none of the known abiotic processes can account for the morpho-logical, ultrastructural and geochemical observations presented here.

The oldest unambiguous organic-walled microfossils preserved inunsilicified fine-grained siliciclastic sediments are of late Palaeo-proterozoic age (about 1.65–1.8 Gyr old14) and include relatively largevesicles (up to 238mm) of unidentified microorganisms interpreted aspossible protists or cyanobacteria, and one eukaryotic taxon of orna-mented vesicles. Early reports9 of ‘‘globular-type A microfossils’’ fromacid-macerated shales of the roughly 3.3-Gyr-old Fig Tree Group havebeen questioned10. Large (up to 90mm) Archaean carbonaceousspheroidal microstructures have been reported from the carbona-ceous cherts of the roughly 3.3-Gyr-old Kromberg Formation,BGB26, but were reinterpreted as abiotic self-organized structures5

although associated hollow kerogenous filaments may be biogenic1.Similar-sized but more diverse carbonaceous spheroidal microstruc-tures interpreted as probable microfossils27 are abundant in the lat-erally extensive black chert beds of the roughly 2.97-Gyr-old FarrelQuartzite, Gorge Creek Group in the Mount Goldsworthy–MountGrant area, Pilbara Craton, Western Australia. Carbonaceous micro-structures include possible microbial films with associated smallspheres, large (10–90-mm) hollow spheroids, and spindle-like struc-tures. This assemblage was interpreted as a probable diverse microbialcommunity flourishing in partly evaporitic basin with terrigenoussediment input27. The cell size of the microfossil population describedin our study is up to 298mm and larger than any other reportedArchaean 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 siliciclasticsediments by more than 1 Gyr. The apparent long gap in the siliciclas-tic fossil record might be explained, at least in part, by the limitednumber of micropalaeontological studies of organic-matter-poorshales relative to those of the more conspicuous organic-matter-richblack shales, cherts and carbonates of Archaean and Palaeoproterozoicage.

Extant microorganisms producing large organic-walled vesicles areunknown among archaea28 but include protists and some bacteria. Thelarge bacterial cells include the symbiont Epulopiscium sp. (80mm wideand 600mm long), which lives in the nutrient-rich gut of tropical fish28,and myxobacteria sporangioles (up to 200mm in diameter), whichenclose myxospores and live predominantly in soil28. The sulphurbacterium Thiomargarita namibiensis forms chains of unconnectedmucus-sheathed spheroidal cells up to 750mm in diameter, with up

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0 50 100 150 200 2500

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300Minimum diameter (µm)

Figure 2 | Size distribution of carbonaceous spheroidal microstructurespreserved in the 3.2-Gyr-old shales and siltstones of the Moodies Group,Barberton Greenstone Belt, South Africa.

1,800 1,600 1,400 1,200 1,000

Inte

nsity

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itrar

y un

its)

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Raman shift (cm–1)

Figure 3 | Raman microspectroscopy. First-order Raman spectrum ofcarbonaceous microstructures (top trace) and disseminated particulatecarbonaceous material (bottom trace) in situ in thin section compared withthat of carbonaceous microstructures extracted by acid maceration (middletrace) (band assignments: D band, 1,355 cm21; G band, 1,590 cm21; D2band, 1,620 cm21).

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to 98% of the cytoplasm filled with vacuoles, and inhabits organicmatter-rich and sulphidic sediments with changing redox conditionsin deep hydrothermal sites and coastal upwelling zones28. Large cya-nobacteria include spheroidal sheaths 30–60mm in diameter enve-loping small baeocyte cells29 and oval to sausage-shaped thick-walledcysts up to 100mm long called akinetes28,29. The large organic-walledmicrofossils reported here are unlikely to be related to the bacterialsymbiont, to extant large sulphur bacteria or to myxobacteria, becausethese organisms are not known to form recalcitrant biopolymers andlive in completely different ecological niches from those in which theMoodies microfossils were deposited. Although extant protists mayhave unornamented unilayered walls, a protistan affinity cannot bedefinitely inferred in the absence of wall ornamentation or complexwall ultrastructure, the only diagnostic criteria indicative of a eukar-yotic affinity for acid-resistant organic-walled microfossils13. If theMoodies microfossils are ancestors of extant organisms rather thanmembers of extinct stem clades, their size, taphonomy, acid resistanceand habitat in the photic zone could suggest a cyanobacterial affinity,although the Moodies microfossils are larger than any reported extantor fossil cyanobacteria. Although the global increase in atmosphericoxygen happened later, between 2.45 and 2.32 Gyr ago30, cyanobacteriamight have had anoxygenic photosynthetic ancestors or might havebeen producing oxygen in microenvironments well before their plane-tary impact on the atmospheric redox state. Because diffusion into andwithin prokaryotic cells restricts their maximum size to a few micro-metres in nutrient-poor environments, the large size, taphonomy, andhabitat of the Moodies microfossils might suggest either early evolu-tion of a compartmentalized eukaryotic cytoplasm or colonial enve-lopes of cyanobacteria, or extinct prokaryotes with an unknownmetabolism. At this point, the absence of definitive diagnostic featuresdoes not unambiguously relate the microfossils to a crown group.Regardless of their biological affinities, these large microorganisms, ifcrown group ancestors, must have either used or maintained localredox gradients in shallow-marine environments. If this is indeed so,geochemical studies of these shallow-water environments might recog-nize local redox cycling almost 0.8–0.9 Gyr before the global increase inatmospheric oxygen30.

The Moodies Group provides an unusual window into the ecologyof Mesoarchaean ocean, demonstrating the early evolution of amoderately diverse ecosystem in the photic zone of marginal marinesiliciclastic environments, in which large recalcitrant organic-walled uni-cells or colonial envelopes lived contemporaneously with earlier reportedbenthic microbial mats. As palaeontological studies of Archaean shalesmove forwards, we predict that focus on organic-matter-lean shales willyield significant discoveries, improving our understanding of the earlyevolution of the biosphere.

METHODS SUMMARY

Thin sections were cut from rock samples perpendicular and parallel to the

bedding. Optical microscopy was performed with a Zeiss Axioimager micro-

scope equipped with an Axiocam MRc5. About 25 g of each sample was demi-

neralized by treatment with HF/HCl followed by settling and decanting. Part of

the residue was mounted on microscope slides. Single microfossils were hand-

picked under an inverted microscope by using a micropipette, then deposited

uncoated on an aluminium stub and imaged by backscattered electron micro-

scopy with a Philips ESEM (Environmental Scanning Electron microscope)

LX30 FEG (field emission gun) at 15 kV. Single microfossils were embedded in

agar, dehydrated in a series of ethanol solutions, and then infiltrated successively

with propylene oxide/ethanol, propylene oxide/epoxy resin, and pure epoxy

resin. Samples were polymerized in an oven at 60 uC for at least 12 h. After

verification of proper orientation of microfossils, resin blocks were trimmed

and cut into 1-mm-thick semi-thin and 50-nm-thick ultra-thin sections with a

diamond knife. Sections were put on copper grid and imaged with a JEM100SX

at 80 kV. No staining was used because previous studies13 showed this to be

unnecessary and it can be a source of artefacts. Raman spectra were collected

on thin sections and on isolated microfossils were collected on glass slides.

Spectra were recorded from two to ten different points in each sample to check

for the representative nature of the spectra, with a Renishaw inVia Reflex Raman

Microprobe using a Peltier-cooled charge-coupled device detector and the

514.5-nm line of a 5-W Ar1 laser (Stabilite 2017 laser; Spectra-Physics).

Carbon isotope analyses were performed by high-temperature oxidation in

helium flow with a Flash 1112 Elemental Analyser (Thermo Electron Inc.)

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

accuracy of d13C measurements was 0.1% (n 5 38).

Full Methods and any associated references are available in the online version ofthe paper at www.nature.com/nature.

Received 12 June; accepted 15 December 2009.Published online 7 February 2010.

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3. Rasmussen, B., Fletcher, I. R., Brocks, J. J. & Kilburn, M. R. Reassessing the firstappearance of eukaryotes and cyanobacteria. Nature 455, 1101–1105 (2008).

4. Garcıa-Ruiz, J. M. et al. Self-assembled silica-carbonate structures and detectionof ancient microfossils. Science 302, 1194–1197 (2003).

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16. Kamo, S. L. & Davis, D. W. Reassessment of Archean foredeep sedimentationrelated to crustal shortening: a reinterpretation of the Barberton Mountain Land,South Africa, based on U–Pb dating. Tectonics 13, 167–192 (1994).

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26. Walsh, M. M. Microfossils and possible microfossils from the early ArcheanOnverwacht Group, Barberton Mountain Land, South Africa. Precambr. Res. 54,271–292 (1992).

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Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements We thank M. Giraldo, J. Laval and N. Decloux for samplepreparation; P. Compere for TEM imaging; C. Henrist for environmental SEMimaging and energy-dispersive X-ray analyses; T. Prokopiuk for carbon isotope

analyses; J. Robertson for information on geology of the Agnes Gold Mine; andA. H. Knoll for comments on an earlier version of the manuscript. The study wassupported by a University of Liege Impulsion Grant (CFRA0805) and a Universityof Liege grant (RCFRA0036-J) to E.J., a National Science Foundation grant(EAR-937 05-45484), a NASA Astrobiology Institute award (NNA04CC09A), aNatural Sciences and Engineering Research Council of Canada 938 Discovery grantto A.B., and Australian Research Council funding to C.M.

Author Contributions E.J. and A.B. conceived the study and wrote the paper. E.J.discovered the microfossils and performed the microscopic, SEM and TEManalyses, and interpreted the data. C.M. performed the Raman analyses andinterpreted and wrote the results. A.B. conducted field work and carbon isotopeanalyses and interpreted the sedimentary structures. All authors commented onthe manuscript.

Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.Correspondence and requests for materials should be addressed to E.J.(ej.javaux@ulg.ac.be).

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938Macmillan Publishers Limited. All rights reserved©2010

METHODSScanning electron microscopy. The microstructures were deposited uncoated

on an aluminium stub and imaged by backscattered electron microscopy at the

microscopy facility of the University of Liege with a Philips ESEM LX30 FEG at

15 kV.

Transmission electron microscopy. The microstructures were embedded in

agar, dehydrated in a series of ethanol solutions, and then infiltrated with a

mixture of propylene oxide/ethanol, followed by propylene oxide/epoxy resin,

and then pure epoxy resin. Samples were then polymerized in an oven at 60 uCfor at least 12 h. After verification of the proper orientation of individual micro-structures, resin blocks were trimmed and cut into 50-nm-thick ultra-thin sec-

tions with a diamond knife. No staining was used, because this had been shown

to be unnecessary in previous studies13 and can be a source of artefacts. Sections

were put on a copper grid and imaged at the microscopy facility of the University

of Liege with a JEM100SX at 80 KV. Negatives were scanned to obtain images.

Note that the TEM image on Fig. 1n is a photomontage of three images to obtain

an image of the whole microfossil at high resolution.

Raman microspectroscopy. Raman spectra were collected on thin sections, and

isolated microfossils on glass slides. Because spectral features used to infer the

degree of crystallinity of disordered sp2 carbons vary depending on the orienta-

tion of the crystallites to the exciting laser beam, spectra were recorded from two

to ten different points in each sample to check for the representative nature of the

spectra. The Raman spectra were acquired on a Renishaw inVia Reflex Raman

Microprobe with a Peltier-cooled charge-coupled device detector. The collection

optics is based on a Leica DM LM microscope. A refractive glass 503 objective

lens was used to focus the laser on a 2-mm spot to collect the backscattered

radiation. The 514.5-nm line of a 5-W Ar1 laser (Stabilite 2017 laser; Spectra-

Physics) oriented normal to the sample was used to excite the sample. Theinstrument was calibrated against the Raman signal of Si at 520 cm21 with a

silicon wafer (111). A surface laser power of 1.0–1.5 mW was used to minimize

laser-induced heating of the samples. An accumulation time of 30 s and ten scans

were used, which gave an adequate signal-to-noise ratio for the spectra. The scan

ranges were 800–1800 cm21 in the carbon first-order region.

Carbon isotope analyses. Carbon isotope analyses of organic matter were per-

formed at the Department of Geological Sciences, University of Saskatchewan.

Carbonate-bearing samples were acidified stepwise in 10%, 20% and 40% HCl to

remove carbonate materials completely. The residue was then homogenized and

loaded into tin capsules. Stable isotope values were obtained with a Flash 1112

Elemental Analyser (Thermo Electron Inc.) coupled to a Delta Plus XL (Thermo

Electron Inc.) through a Conflo III. Samples were dropped under helium into an

oxidation furnace at 1,000 uC that was packed with chromium(VI) oxide and

silvered cobaltic/cobaltous oxide (used to remove any halogens). Organic

materials were oxidized to carbon dioxide, various nitrogen-bearing gases and

water. This gas mixture was then passed through a reduction furnace at 680 uCpacked with elemental copper to reduce all nitrogen-bearing compounds to pure

nitrogen gas. The resulting gases were then passed through a water trap to

eliminate moisture, and then a gas chromatography column at 50 uC to separate

the carbon dioxide for analysis in the mass spectrometer. Carbon isotope ratios

are corrected for blank and 17O contribution and reported in% notation relative

to the V-PDB scale. Carbon isotope data are calibrated against the international

standards L-SVEC (d13C 5 246.6%V-PDB) and IAEA-CH6 (d13C 5 210.45%V-PDB). IAEA-CH7, an intermediate international standard, gave the following

results: d13C 5 232.14 6 0.03% V-PDB (n 5 12), which compares well with its

accepted value of 232.15 6 0.10% V-PDB. The accuracy of data was monitored

through routine analyses of in-house standards that were stringently calibrated

against the IAEA standards mentioned above. The accuracy of d13C measure-

ments was 0.1% (n 5 38); weight-percentage C measurements had an accuracy

of between 1% and 0.5% (n 5 10).

doi:10.1038/nature08793

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