Letter https://doi.org/10.

1038/s41586-019-1217-0

Early fungi from the Proterozoic era in Arctic

Canada
Corentin C. Loron1*, Camille François1, Robert H. Rainbird2, Elizabeth C. Turner3, Stephan Borensztajn4 &
Emmanuelle J. Javaux1*

Fungi are crucial components of modern ecosystems. They may have crown group Opisthokonta, which comprises metazoans, fungi and
had an important role in the colonization of land by eukaryotes, their protist relatives8,9.
and in the appearance and success of land plants and metazoans1–3. Several Precambrian microfossils have been tentatively interpreted
Nevertheless, fossils that can unambiguously be identified as fungi as fungi in the past fifty years10 (for further examples and specimen
are absent from the fossil record until the middle of the Palaeozoic names, see Supplementary Information). A few Proterozoic fossils—
era 4,5. Here we show, using morphological, ultrastructural notably, parts of the Ediacaran biota—have been compared to lichen-
and spectroscopic analyses, that multicellular organic-walled like organisms11. All of these previous studies have focused on broad
microfossils preserved in shale of the Grassy Bay Formation morphological comparisons; in isolation, this methodology cannot
(Shaler Supergroup, Arctic Canada), which dates to approximately fully support a specific affiliation with the fungi4. Nevertheless, when
1,010–890  million years ago, have a fungal affinity. These combined with the acetolysis resistance of chemically recalcitrant struc-
microfossils are more than half a billion years older than previously tures that have been tested in several fungal lineages12, the Palaeozoic
reported unambiguous occurrences of fungi, a date which is record4,5 indicates that fungi have a strong potential for preservation in
consistent with data from molecular clocks for the emergence of the geological record. To conclusively identify fungal or other eukary-
this clade6,7. In extending the fossil record of the fungi, this finding otic fossils, morphological evidence needs to be coupled with analyses
also pushes back the minimum date for the appearance of eukaryotic of the ultrastructure and chemical composition of microfossils13.

Fig. 1 | Microphotographs of O. giraldae

a b c d
specimens. a, Sketch of O. giraldae, displaying
Microfibrillary the main features of the microfossil.
wall structure b–h, Unornamented terminal sphere (spore).
Unornamented b–g, Transmitted light microscopy images that
terminal sphere
(spore)
show specimens with secondary branching at
Bilayered
wall structure
a right angle (b, d–g), with terminal spheres
connected together (c), with a bulbous
Bulbous connection (e) and with tertiary branching
connection
(d, f, g). Arrows show septate connections.
Right-angled e f g Details of sample and specimen numbers have
branching
filament
previously been published14. h, SEM image shows
with septa the compressed vesicle. Inset 1 shows the two
(hyphae) layers of the vesicle wall, with an amorphous
outer layer (AOL) and a fibrillary inner layer
(FIL). Inset 2 shows the intertwined microfibrils
of the microfibrillary wall structure. i, TEM
h 1 1 i images show the compressed vesicles of the
bilayer wall structure in ultra-thin section, with
FIL
magnification in insets 1 and 2. The outer layer
1 2 AOL Resin (OL), the inner layer (IL) and intracellular space
OL
IL (ICS) are indicated in both insets. Scale bars,
ICS 20 μm (h), 200 nm (h inset 1), 300 nm (i insets 1
and 2), 400 nm (h inset 2), 1,600 nm (i), 30 µm
Resin (b–g). Images b–g are modified from a previous
2 publication14.

2
2
Resin
OL
IL
1 OL

Resin

1
Early Life Traces & Evolution–Astrobiology Laboratory, UR Astrobiology, Geology Department, University of Liège, Liège, Belgium. 2Geological Survey of Canada, Ottawa, Ontario, Canada. 3Harquail
School of Earth Sciences, Laurentian University, Sudbury, Ontario, Canada. 4UMR 7154 CNRS, Institut de Physique du Globe de Paris, Paris, France. *e-mail: [email protected]; [email protected]

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 RESEARCH Letter

SEM images (secondary electron) also reveal the presence of locally

Deformation N–H
well-preserved and intertwined (approximately 15–20-nm thick)
O–H stretching

N–H stretching

C–H stretching

Saccharides
microfibrils, which make up the walls (Fig. 1h, Extended Data Fig. 2).
Ultrastructural analyses using transmission electron microscopy

Amides
(TEM) show that the flattened microfossils are hollow, with a bilay-
ered wall that consists of an electron-dense thick inner layer and a thin
electron-tenuous outer layer (Fig. 1i).
This combination of complex morphology, right-angle branching,
multicellularity, bilayered wall ultrastructure, compositional recalci-
trance and relatively large size (not by itself a criterion) permits the
unambiguous placement of the microfossil O. giraldae among eukar-
yotes; together they indicate the presence of a complex cytoskeleton,
Microfossil 1 which is absent in prokaryotes19.
We performed Raman thermometry using Raman reflectance,
which is an effective method for analysing low metamorphic-grade
fossiliferous Proterozoic shale (see, for example, ref. 20 and refer-
ences therein). Average temperature estimates are approximately
195 °C (Supplementary Table 1). This low temperature is within the
diagenesis window (<200 °C), which confirms that the rocks are not
Chitosan standard
metamorphosed, consistent with the regional geology15,16 and with the
quality of microfossil preservation. Analyses of extracted and in situ
microfossils (in thin sections) produce similar estimates of temper-
ature, which—as has previously been documented20—indicates that
extraction by acid maceration does not affect Raman analyses. This
excellent preservation permits investigation of the molecular structure
of the fossil wall using Fourier-transform infrared (FTIR) spectroscopy
α-Chitin standard
analyses. O. giraldae and the other microfossils from the same sam-
ples all exhibit a low degree of thermal alteration, consistent with the
geological setting. This low degree of alteration—when combined
with the microfossils being preserved flattened parallel to the shale
laminations—demonstrates that the microfossils were deposited
together and contemporaneously with sedimentation (syngenicity),
4,000 3,000 2,000 1,000 650 rather than being a later contaminant.
Wavenumber (cm–1) To investigate the chemical composition of the wall, we performed
Fig. 2 | Spectra obtained with FTIR microspectroscopy. Representative FTIR spectroscopy on isolated microfossils. The spectra show absorp-
spectra of one specimen (extracted microfossil 1) are compared to tion bands that are typical of chitin (N-acetyl-d-glucosamine) and/
spectra of chitosan and α-chitin standards. Dark grey bands indicate the or chitosan (deacetyled d-glucosamine) (Fig. 2, Supplementary Table
typical absorption bands of chitin and chitosan. The presence of spectral 2 and ref. 21). The typical absorption bands of cellulose are absent22.
peaks for chitin and chitosan in O. giraldae supports its fungal affinity. The combination of the microfossil morphology, wall ultrastruc-
See Supplementary Table 2 for band assignments. Each measurement was ture and chemistry of these microfossils is consistent with a fungal
repeated three times with similar results.
affinity. The septate filaments with right-angled branching and ter-
minal spheres may be interpreted as hyphae with terminal spores,
Here we report abundant specimens of the organic-walled microfos- similar to the spore-bearing stages of many fungi4,7. The relatively
sil Ourasphaira giraldae14, which are preserved parallel to lamination large size of the microfossils and their gross morphology bear some
in shallow-water estuarine shale of the Grassy Bay Formation (Shaler resemblance to fungal sporangia, but sphere opening or disinte-
Supergroup) from the Brock Inlier in the Northwest Territories of gration—which would indicate sporangial cleavage—is absent. A
Canada15,16 (Extended Data Fig. 1). Uranium–lead dating of detrital polarized growth and osmotrophic nutrition are characteristic of the
zircon grains from the underlying Nelson Head Formation provide Chytridiomycota, Blastocladiomycota and Dikarya (Ascomycota and
a maximum possible depositional date of 1,013 ± 25 million years Basidiomycota)23. It is therefore possible that O. giraldae represents one
ago (Ma) for the Grassy Bay Formation. Rhenium–osmium dating of these clades. Septate hyphae are characteristic of the Dikarya, a few of
organic matter in black shale from the overlying Boot Inlet Formation the Zoopagomycota (such as the Kickxellales, in which the hyphae have
has yielded a date of 892 ± 13 Ma. These dates constrain the deposi- a distinctive helicoidal morphology) and of the late stages of spores in
tional date of the Grassy Bay Formation to between 1.0 and 0.9 billion the Mucoromycota, Chytridiomycota and Blastocladiomycota7,24,25.
years ago (Ga)17,18. Unlike in Dikarya, septa in the microfossil specimens are not regularly
The organic-walled microfossils consist of multicellular, branching, distributed along the hyphae; molecular clocks indicate a minimum
septate filaments with terminal spheres. Their resistance to the acid date of 452 Ma for divergence of Dikarya6, much later than the rocks
treatment used for mineral dissolution indicates a recalcitrant keroge- from which these fossils were obtained. These similarities and differ-
nous composition, as confirmed by Raman microspectroscopy. Spheres ences suggest that O. giraldae was probably in the stem Dikarya, or
are 33 to 80 μm in diameter (n = 27, M = 54.3, σ = 13.6) and are in a clade of the total fungi group that had some—but not all—of the
linked to the filaments (10 to 35 μm in length) by a single cylindrical or characteristics of Dikarya.
bulbous connection (Fig. 1a–g). Filament branching is right-angled, Fungal walls are bilayered, as has been documented with TEM4,25
and up to three orders of branching are present (Fig. 1a–g). The con- (Fig. 1i). The inner cell wall structure consists of intertwined microfi-
nections between the spheres and the filaments—and between first-, brils; these are locally well-preserved and visible in SEM images of the
second- and third-order branches—are septate (Fig. 1b–g). fossil material (Fig. 1h). The outer matrix—which is also visible in some
Transmitted-light and scanning electron microscopy (SEM) exam- of the fossil specimens (Fig. 1h, main panel and inset 1)—is composed
inations show smooth unornamented walls of filaments and spheres, mainly of chitin, glucan and proteins. In Dikarya, microfibrils are made
although taphonomic pitting and localized degradation are present. of α-chitin and fixed by β-glucan, whereas in Mucoromycota the chitin

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 Letter RESEARCH

O. giraldae

Opisthokonta
<1,013 ± 25 to >892 ± 13 Ma Fungi
Choanoflagellates

Metazoans

Amoebozoa
Rhizaria

Stramenopiles

Alveolata

Archaeplastida
Rhodophyta
Excavata

1,700 1,500 1,000 500 Ma

Fig. 3 | Simplified phylogenetic relationships of main eukaryotic occurrences (black dots) or molecular fossils (white dots). Details of dates,
supergroups. Diagram shows O. giraldae and its date range, and therefore fossils and geological occurrences can be found in the Supplementary
the minimum date for the most recent common ancestor of fungi and Information. Details about the relationships between the branches have
the metazoan–choanoflagellate clade. The minimum dates for the previously been published9.
appearance of each lineage are shown; these are based on either body fossil

in the microfibrils is replaced by chitosan21,25. Some of the life stages The later colonization of terrestrial settings by fungi may have
of pseudofungi (Oomycetes and Hyphochytridiomycetes) resemble preceded and aided the colonization of land by plants through myc-
the microfossils that we studied in terms of possessing local septa and orrhizal symbioses and through soil processing, which would have
branching, but have a low potential for preservation4. Although these provided ecological niches, improved the substrate, augmented nutrient
pseudofungi may have chitin in their wall components26, the major uptake and increased aboveground productivity1,2. As multidiscipli-
constituent of their walls is cellulose. Chitin synthesis may have evolved nary studies of Proterozoic fossil assemblages progress, we predict that
early in eukaryotes27, and FTIR spectroscopy has proven efficient more fossil fungi and other early eukaryotes will be discovered and will
in detecting chitin and chitosan at temperatures28 up to 280 °C. improve our understanding of the evolution of the early biosphere.
The presence of chitin and/or chitosan in our thermally immature
specimens is therefore consistent with a fungal affinity. Online content
Several multicellular micro- and macro-organisms (including Any methods, additional references, Nature Research reporting summaries, source
pseudofungi, some life stages of xanthophyte algae, and slime moulds) data, statements of data availability and associated accession codes are available at
are known to exhibit morphologies of ramified branching and terminal https://doi.org/10.1038/s41586-019-1217-0.
spheres that are broadly similar to the microfossils discussed here; how- Received: 23 January 2019; Accepted: 18 April 2019;
ever, the combination of morphology, wall ultrastructure, chitin and/or Published online xx xx xxxx.
chitosan chemistry, and the absence of cellulose shown by the micro-
fossils are most consistent with a fungal affinity4,7,25. Other organisms 1. Kenrick, P. & Crane, P. R. The origin and early evolution of plants on land. Nature
that produce chitin (arthropods, chrysoflagellates, diatoms and ciliates) 389, 33–39 (1997).
2. Jeffries, P., Gianinazzi, S., Perotto, S., Turnau, K. & Barea, J. M. The contribution
are morphologically very distinct from our specimens29. In addition, a of arbuscular mycorrhizal fungi in sustainable maintenance of plant health and
fungal affinity for these microfossils is consistent with molecular clock soil fertility. Biol. Fertil. Soils 37, 1–16 (2003).
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To date, the earliest uncontested fossil fungi are specimens from the (2017).
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fungi, metazoans and their protist relatives8,9 (Fig. 3). (2005).
As a consequence of their role in biological cycles (such as the deg- 11. Retallack, G. J. Ediacaran life on land. Nature 493, 89–92 (2013).
12. Graham, L. E., Trest, M. T. & Cook, M. E. Acetolysis resistance of modern fungi:
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they may have had similar roles in the Proterozoic era3. Fungi were 13. Marshall, C. P., Javaux, E. J., Knoll, A. H. & Walter, M. R. Combined micro-Fourier
transform infrared (FTIR) spectroscopy and micro-Raman spectroscopy of
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Organic-walled microfossils from the late Mesoproterozoic to early
some marine forms are known4,25. Because O. giraldae is preserved in Neoproterozoic lower Shaler Supergroup (Arctic Canada): diversity and
shallow-water estuarine shale of the Grassy Bay Formation, this fungus biostratigraphic significance. Precambr. Res. 321, 349–374 (2019).
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from land or marine niches. Grassy Bay, and Boot Inlet Formations in the Brock Inlier, Northwest Territories (NTS

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97-A, D). Geological Survey of Canada Open File 8394 (Canada Geological 30. Taylor, J. W. & Berbee, M. L. Dating divergences in the fungal tree of life: review
Survey, Natural Resources Canada, 2018). and new analyses. Mycologia 98, 838–849 (2006).
17. van Acken, D., Thomson, D., Rainbird, R. H. & Creaser, R. A. Constraining the 31. Javaux, E. J. & Knoll, A. H. Micropaleontology of the lower Mesoproterozoic
depositional history of the Neoproterozoic Shaler Supergroup, Amundsen Roper Group, Australia, and implications for early eukaryotic evolution.
Basin, NW Canada: rhenium–osmium dating of black shales from the Wynniatt J. Paleontol. 91, 199–229 (2017).
and Boot Inlet Formations. Precambr. Res. 236, 124–131 (2013).
18. Rainbird, R. H. et al. Zircon provenance data record the lateral extent of Acknowledgements This research was supported by the Agouron Institute,
pancontinental, early Neoproterozoic rivers and erosional unroofing history of the FRS-FNRS-FWO EOS ET-Home grant 30442502 and the ERC Stg ELiTE
the Grenville orogen. Geol. Soc. Am. Bull. 129, 1408–1423 (2017). FP7/308074. We thank M. Giraldo, M.-C. Sforna, Y. Cornet and S. Smeets
19. Javaux, E. J., Knoll, A. H. & Walter, M. Recognizing and interpreting the fossils of (University of Liège) for technical support and the Geological Survey of
early eukaryotes. Orig. Life Evol. Biosph. 33, 75–94 (2003). Canada’s Geomapping for Energy and Minerals Program for fieldwork logistics.
20. Baludikay, B. K. et al. Raman microspectroscopy, bitumen reflectance and illite
crystallinity scale: comparison of different geothermometry methods on Reviewer information Nature thanks Linda Graham and the other anonymous
fossiliferous Proterozoic sedimentary basins (DR Congo, Mauritania and reviewer(s) for their contribution to the peer review of this work.
Australia). Int. J. Coal Geol. 191, 80–94 (2018).
21. Mohaček-Grošev, V., Božac, R. & Puppels, G. J. Vibrational spectroscopic Author contributions C.C.L. and E.J.J. conceived the study and interpreted the
characterization of wild growing mushrooms and toadstools. Spectrochim. Acta data. C.F. and C.C.L performed the Raman and FTIR analyses. C.C.L., C.F. and
A 57, 2815–2829 (2001). S.B. performed the TEM and SEM sample preparation and observations. R.H.R.
22. Kačuráková, M., Capek, P., Sasinková, V., Wellner, N. & Ebringerová, A. FT-IR study and E.C.T. sampled the rocks and collected the geological data. C.C.L. and E.J.J.
of plant cell wall model compounds: pectic polysaccharides and wrote the paper with contribution from all the authors. E.J.J. supervised the
hemicelluloses. Carbohydr. Polym. 43, 195–203 (2000). project.
23. Riquelme, M. & Sánchez-León, E. The Spitzenkörper: a choreographer of fungal
growth and morphogenesis. Curr. Opin. Microbiol. 20, 27–33 (2014). Competing interests The authors declare no competing interests.
24. Spatafora, J. W. et al. A phylum-level phylogenetic classification of zygomycete
fungi based on genome-scale data. Mycologia 108, 1028–1046 (2016). Additional information
25. Webster, J. & Weber, R. Introduction to Fungi (Cambridge Univ. Press, Extended data is available for this paper at https://doi.org/10.1038/s41586-
Cambridge, 2007). 019-1217-0.
26. Mélida, H., Sandoval-Sierra, J. V., Diéguez-Uribeondo, J. & Bulone, V. Analyses of Supplementary information is available for this paper at https://doi.org/
extracellular carbohydrates in oomycetes unveil the existence of three different 10.1038/s41586-019-1217-0.
cell wall types. Eukaryot. Cell 12, 194–203 (2013). Reprints and permissions information is available at http://www.nature.com/
27. Richards, T. A., Leonard, G. & Wideman, J. G. What defines the “kingdom” fungi? reprints.
Microbiol. Spectr. 5, FUNK-0044-2017 (2017). Correspondence and requests for materials should be addressed to C.C.L. or
28. Wanjun, T., Cunxin, W. & Donghua, C. Kinetic studies on the pyrolysis of chitin E.J.J.
and chitosan. Polym. Degrad. Stabil. 87, 389–394 (2005). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional
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34) (ed. Gupta, N. S.) 1–34 (Springer Science and Business Media, New York,
2010). © The Author(s), under exclusive licence to Springer Nature Limited 2019

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 Letter RESEARCH

Methods FTIR microspectroscopy. The analyses were performed with a Hyperion 2000
Sample preparation. The shale sample 15RAT-021A1 (Grassy Bay Formation, Bruker microscope coupled to a Tensor 27 FTIR spectrometer at the Early Life
Shaler Supergroup, Canada) was cut into thin sections parallel to lamination for Traces and Evolution–Astrobiology Laboratory. The specimens were pipetted out
the Raman analyses. A fraction of the sample was demineralized by treatment of Milli-Q water and deposited on ZnSe plates. Data were collected with a con-
with HF and HCl, using a previously published method32, without centrifuga- ventional Globar source equipped with 15× objective (NA = 0.4) and a liquid-ni-
tion to minimize mechanical shocks. A part of the residue was mounted on micro- trogen-cooled MCT-A detector. Background was collected on a ZnSe plate free
scopic slides. Single microfossils were hand-picked under an inverted microscope of sample, by combining 32 accumulations. Thirty-two scans were accumulated
with a micropipette, and deposited on a glass slide and ZnSe disc for spectroscopic in transmission mode from each specimen with a spectral range that spanned
analyses. between 650 cm−1 and 4,000 cm−1, with a resolution of 4 cm−1. The spectra were
Optical microscopy. Optical microscopy was performed on a Zeiss Axio imager treated, atmospheric water and CO2 were removed and the baseline corrected,
microscope equipped with an Axiocam MRc5. Measurements were taken using using the software ‘Opus 8.0’. Standards for chitin (shrimp shells, Sigma, C7170-
the software AxioVision. 100G) and chitosan (shrimp shells, ≥ 75%; Sigma-Aldrich, C3646-10G) were each
SEM. SEM analyses were performed using an Auriga 40 Field Emission Gun deposited on ZnSe plates. Spectra were acquired with the same parameters as those
Scanning Electron Microscope (FEG SEM) Zeiss, at the electron microscopy used for the microfossil analyses. Band assignments of the spectra are based on
platform of the Institut de Physique du Globe de Paris (Platform PARI). The previous studies21,36–39 (see also Supplementary Table 2).
images were taken using the Secondary Electron Detector InLens and/or Everhart Reporting summary. Further information on research design is available in
Thornley at 15-keV accelerating voltage, with a beam current at 800 pA and a the Nature Research Reporting Summary linked to this paper.
working distance between 2.5 and 7.5 mm. Analyses were performed on speci-
mens deposited on glass slides, and Au-coated with a Quorum Q150 ES metallizer. Data availability
TEM. Isolated microfossils were embedded in agar, dehydrated in a series of eth- All processed data are included in Supplementary Tables 1 and 2. Raw data are
anol solutions and then infiltrated successively with propylene oxide and ethanol, available from the corresponding authors upon reasonable request. Microfossil
propylene oxide and epoxy resin, and pure epoxy resin. Samples were polymerized specimens are accessible at the Early Life Traces and Evolution–Astrobiology
in an oven at 60 °C for at least 12 h. The resin blocks were cut into 1-mm-thick Laboratory.
(semi-thin) and 50-nm-thick (ultra-thin) sections with a diamond knife. Sections
were put on copper grid with Formvar, and imaged at 80 keV with a transmission 32. Grey, K. A Modified Palynological Preparation Technique for the Extraction of Large
electron microscope JEM100SX. Neoproterozoic Acanthomorph Acritarchs and Other Acid-Soluble Microfossils.
Raman microspectroscopy. Raman spectra were collected on polished thin sec- (Geological Survey of Western Australian, Department of Minerals and Energy,
Perth, 1999).
tions and on isolated microfossils using a Renishaw INVIA Raman microspec-
33. Sforna, M. C., Van Zuilen, M. A. & Philippot, P. Structural characterization by
trometer, at the ‘Early Life Traces and Evolution–Astrobiology Laboratory’ Raman hyperspectral mapping of organic carbon in the 3.46 billion-year-old
(UR Astrobiology, Geology Department, University of Liège) (see Supplementary Apex chert, Western Australia. Geochim. Cosmochim. Acta 124, 18–33 (2014).
Information). Raman analyses were done using an Ar-ion, 40-mW monochro- 34. Liu, D. H. et al. Sample maturation calculated using Raman spectroscopic
matic 514-nm laser source. Laser excitation was focused through a 50× objective parameters for solid organics: methodology and geological applications. Chin.
to obtain a 1–2-μm spot size. The Raman spectrum of each point (around 20 Sci. Bull. 58, 1285–1298 (2013).
35. Sauerer, B., Craddock, P. R., AlJohani, M. D., Alsamadony, K. L. & Abdallah, W.
points were measured for each specimen) was acquired in static mode (fixed at a Fast and accurate shale maturity determination by Raman spectroscopy
wavenumber of 1,150 cm−1) for 1 × 1-s running time. This enabled the acquisi- measurement with minimal sample preparation. Int. J. Coal Geol. 173, 150–157
tion of Raman spectra with a 2,000-cm−1 detection range and a 4-cm−1 spectral (2017).
resolution. To process the data, we used the software ‘Renishaw Wire 4.1’. The 36. Paulino, A. T., Simionato, J. I., Garcia, J. C. & Nozaki, J. Characterization of
baseline subtraction protocol was performed on a truncated spectrum between chitosan and chitin produced from silkworm crysalides. Carbohydr. Polym. 64,
98–103 (2006).
1,000 and 1,800 cm−1, with a third-order polynomial fit. Data processing followed 37. Movasaghi, Z., Rehman, S. & Rehman, D. I. Fourier transform infrared (FTIR)
a previously published protocol33. Thermal maturity of carbonaceous material was spectroscopy of biological tissues. Appl. Spectrosc. Rev. 43, 134–179 (2008).
assessed following a previously published protocol20, using the Raman reflectance 38. Michell, A. J. & Scurfield, G. Composition of extracted fungal cell walls as
parameter (RmcR0%), which is an equivalent of vitrinite reflectance (vR0%)34,35. indicated by infrared spectroscopy. Arch. Biochem. Biophys. 120, 628–637
The RmcR0% parameter uses the positions (ω) of the D1 and G peaks, and is (1967).
39. Bahmed, K., Quilès, F., Bonaly, R. & Coulon, J. Fluorescence and infrared
defined as RmcR0% ≡ vR0 eq% = 0.0537 × (ωG − ωD1) − 11.21 (see ref. 34).
spectrometric study of cell walls from Candida, Kluyveromyces, Rhodotorula and
The Raman reflectance method appears to be a robust tool for evaluating the Schizosaccharomyces yeasts in relation with their chemical composition.
thermal maturity of carbonaceous material from Proterozoic rocks20. Biomacromolecules 4, 1763–1772 (2003).

N A t U r e | www.nature.com/nature
 RESEARCH Letter

Extended Data Fig. 1 | Location of the study area in northwestern 121° 44′ 17′′ W). The stratigraphic column and geology have previously
Canada, highlighting the Brock Inlier. The white cross indicates been published14–16. The map is modified after a previous publication14.
the location at which the samples were extracted (68° 55′ 42′′ N,
 Letter RESEARCH

Extended Data Fig. 2 | Additional SEM images. a, b, O. giraldae (whole specimen) with right-angled branching hyphae (indicated with arrows).
d–g, Detailed images of microfibrils on the surface of the specimen shown in Fig. 1h.
 RESEARCH Letter

Extended Data Fig. 3 | Additional spectra of O. giraldae, showing a

more-advanced state of degradation. The typical peaks of chitin and
chitosan are present, but at a lower intensity than in the standard. The
region of the saccharides (wavenumber of 1,200–800 cm−1), which
is necessary for polymer recognition, is very weak in intensity. Each
measurement was repeated three times with similar results.
 nature research | reporting summary
Corresponding author(s): Corentin C. Loron
Last updated by author(s): Apr 12, 2019

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All processed data are included in Supplementary Material Table I and II. Raw data are available from the corresponding author upon reasonable request.
Microfossils specimens are accessible in the collections of the “Early Life Traces and Evolution” Laboratory, Geology department, University of Liège, Belgium.
October 2018

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nature research | reporting summary

Ecological, evolutionary & environmental sciences study design
All studies must disclose on these points even when the disclosure is negative.
Study description morphological, ultrastructural and microchemical analyses of organic-walled microfossils

Research sample A fossil population of Ourasphaera giraldae from one bed of Grassy Bay Formation, Shaler Supergroup, Arctic Canada. The sample
was chosen for its very good preservation and the abondance of microfossil specimens.

Sampling strategy We selected the best and complete specimens from the Ourasphaera giraldae population.

Data collection C.C.L. and E.J.J. conceived the study and interpreted the data. C.F. and C.C.L performed the RAMAN and FTIR analyses. C.C.L., C.F. and
S.B. performed the TEM/SEM sample preparation and observations. R.H.R and E.C.T. sampled the rocks and collected the geological
data. C.C.L and E.J.J. wrote the paper with contribution of all the authors. E.J.J. supervised the project.

Timing and spatial scale Analyses of microfossils were conducted during the year 2018

Data exclusions no data have been excluded

Reproducibility 27 microfossils were measured, 6 specimens were prepared for TEM, 5 specimens for SEM, 2 specimens for RAMAN (in thin section
and extracted) and 3 specimens were prepared for FTIR microspectroscopy

Randomization This is not relevant for our study because the studied microfossils represent one taxon

Blinding This is not relevant for our study because the studied microfossils represent one taxon

Did the study involve field work? Yes No

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Palaeontology
Specimen provenance Fraction of rock sample from one bed of Grassy Bay Formation, Shaler Supergroup, Arctic Canada, collected by R.H.R and E.C.T in
Canada with the support the Geological Survey of Canada, stored in the collection of the “Early Life Traces and Evolution”
Laboratory”, UR Astrobiology, Geology Department of the University of Liège, Belgium

Specimen deposition the microscopic slides are stored in the collection of the “Early Life Traces and Evolution” Laboratory”, UR Astrobiology, Geology
Department of the University of Liège, Belgium

Dating methods No new dates are provided

Tick this box to confirm that the raw and calibrated dates are available in the paper or in Supplementary Information.
October 2018