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PALEONTOLOGY Copyright © 2020

The Authors, some
Molecular identification of fungi microfossils rights reserved;
exclusive licensee
in a Neoproterozoic shale rock American Association
for the Advancement
of Science. No claim to
S. Bonneville1*, F. Delpomdor2, A. Préat1, C. Chevalier3, T. Araki4, M. Kazemian4, original U.S. Government
A. Steele5, A. Schreiber6, R. Wirth6, L. G. Benning6,7 Works. Distributed
under a Creative
Precambrian fossils of fungi are sparse, and the knowledge of their early evolution and the role they played in Commons Attribution
the colonization of land surface are limited. Here, we report the discovery of fungi fossils in a 810 to 715 million NonCommercial
year old dolomitic shale from the Mbuji-Mayi Supergroup, Democratic Republic of Congo. Syngenetically pre- License 4.0 (CC BY-NC).
served in a transitional, subaerially exposed paleoenvironment, these carbonaceous filaments of ~5 m in width
exhibit low-frequency septation (pseudosepta) and high-angle branching that can form dense interconnected
mycelium-like structures. Using an array of microscopic (SEM, TEM, and confocal laser scanning fluorescence
microscopy) and spectroscopic techniques (Raman, FTIR, and XANES), we demonstrated the presence of vestigial
chitin in these fossil filaments and document the eukaryotic nature of their precursor. Based on those combined
evidences, these fossil filaments and mycelium-like structures are identified as remnants of fungal networks and

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represent the oldest, molecularly identified remains of Fungi.

INTRODUCTION like structures found in the Lakhanda group (1015 to 1025 Ma) (11),
Fungi are a eukaryotic kingdom that performs critical roles in the or even an older structure found in 2.4 billion–year–old basalt (12)
soil ecosystem (1). By forming vast microscopic filamentous networks were inferred to be of fungal nature, yet their conclusive attribution
(mycelium) in symbiosis with the roots of most plants (mycorrhiza), to Fungi remains problematic.
fungi can enhance rock weathering (2) and help the nutrient supply
of plants, particularly in young, poorly evolved soils. Because of these
abilities, ancestral fungi were crucial partners of the first phototrophs RESULTS
that colonized land surfaces (3). Although these associations are Geological setting
acknowledged as a requirement of terrestrial invasion, the timing Here, we report the presence of filamentous networks (Figs. 1 and 2
of this evolutionary transition is largely unknown (4). The Rhynie and fig. S1) attributed to Fungi in dolomitic shale rock of the Mbuji-­
cherts [407 million years (Ma) old] (5) with their superbly preserved Mayi Supergroup (MMS) in the Sankuru-Mbuji-Mayi-Lomami-Lovoy
fungi are considered a milestone of fungal fossil record and early (SMMLL) Basin, south-central of Democratic Republic of Congo
colonization of land. However, despite their Devonian age, fungal (13) (fig. S2). The filamentous fossils were identified in a thin section
remains in the Rhynie chert display a remarkable diversity (6), includ- cut in BIIc8 level (fig. S2) from a depth of 118.2 m in the Kanshi B13
ing members of Chytridiomycota, Blastocladiomycota, Glomeromycota, drillcore (sample BK457b, core 118/4). The fungal networks were
Mucoromycotina, and Ascomycota. Accordingly, molecular clock found embedded (Fig. 1F) in a homogenous mineral matrix preserved
studies have placed the divergence of the main groups of Fungi within as cylindric filaments, as clearly visible in Figs. 1C and 2. This
the Meso-Neoproterozoic (4). The terrestrialization of fungi dates “three-dimensional” (3D) preservation results from an early dolomitic
from sometime between the Ordovician (443 to 485 Ma) to <800 Ma, cementation [possibly due to schizohaline conditions (14)], which
while the earliest obligate biotrophic Glomeromycota fossils date hindered substantial compaction during burial (see fig. S3 and
from 455 to 460 Ma (4). The large uncertainty in the timing of fun- “Paleoenvironment and petrography of the BIIc subgroup” in the
gal evolution and their transition to land essentially stems from the Supplementary Materials for details). This BIIc8 dolomitic shale is a
scarcity and the ambiguous nature of the Precambrian fungi that member of the BII group of the MMS that dominantly consists of
are notoriously difficult to distinguish from prokaryotic remains. A shaley and stromatolitic dolostones with dolomitic shale interbeds.
number of Precambrian remains (e.g., Caudosphaera, Hurionospora, The lower BII group (i.e., BIIa-BIIb) accumulated on a marine
Shuiyousphaeridium, Horodyskia, and Tappania) (7–8), lichen-like outer-to-inner ramp, while the paleoenvironment of the upper BII
fossils (9) from ~ 600 Ma, as well as filament fragments and spores (BIIc-BIId; fig. S2) evolved periodically toward a coastal, lagoon,
from 0.9 billion to 1 billion years ago in Arctic Canada (10), mycelium-­ perennial lacustrine pond with the deposition of a well-preserved
fossil assemblage (15) and the development of cyanobacterial
1 mats and stratiform stromatolites (16). The transition to terrestrial
Biogéochimie et Modélisation du Système Terre, Département Géosciences,
Environnement et Société, Université Libre de Bruxelles, 50 Av. F. D. Roosevelt, paleoenvironments in the upper BIIc is further evidenced by (i) the
1050 Brussels, Belgium. 2Illinois State Geological Survey, University of Illinois at presence of coated pisoids and vadose/meteoric cements related to
Urbana-Champaign, 615 E. Peabody Dr., Champaign, IL 61820, USA. 3Center for subaerial exposures (fig. S3) and (ii) a depleted, uniform light rare
Microscopy and Molecular Imaging, Université Libre de Bruxelles, 12 rue des
professeurs Jeener et Brachet, Charleroi 6041, Belgium. 4Diamond Light Source,
earth elements concentrations reflecting riverine water and continental
Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UK. sediment inputs (17) (“Paleoenvironment and petrography of BIIc
5
Geophysical Laboratory, Carnegie Institution of Washington, 1530 P St NW, subgroup” in the Supplementary Materials). The MMS deposition
Washington, DC 20005, USA. 6German Research Centre for Geosciences, GFZ, time frame is constrained (13) with an Ar-Ar age of 882.2 ± 9 Ma on
Telegrafenberg, 14473 Potsdam, Germany. 7Department of Earth Sciences, Free
University of Berlin, 12249 Berlin, Germany. a dolerite sill at the BI/BII group contact (fig. S2). Within BIIc, the
*Corresponding author. Email: [email protected] 13C of carbonate rocks presents a large positive-to-negative excursion

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Fig. 1. Textures and structures observed via light or scanning electron microscopy of a thin section from the fossiliferous dolomitic shale rock from
BIIc8 (MMS). (A) Composite image of light microscopy (LM) views of dark, nontranslucide, interconnected filaments forming part of a large mycelium-like
structure covering ~0.2 mm2 detailed in fig. S1 (as well as other mycelium-like structures). Note that the area shown in (B) was exposed to WGA-FITC in Fig. 2.
(C) Scanning electron microscopy micrograph illustrating the presence of cells (~20 to 25 m in length) regularly spaced by putative septa or pseudosepta
(white arrows) and showing putative anastomoses (red arrow).(D) LM image of a mycelium network of dark-colored filaments with Y- and T-branching (red
arrows); black arrow marks the position of an FIB foil #4984 cut across a filament in the thin section. Inset shows a portion of mycelial network composed of dark,
nontranslucide filaments branching at a right angle. (E) Histogram showing the width frequency distribution of filaments, with the solid line depicting the
median at 5.5 m (the dashed line as first and third quartiles at 4.5 and 6.7 m). (F) Encrusted aspect of these fungal networks and the absence of tunneling
patterns suggesting the syngenicity of these remains.

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angle branching and, possibly, anastomosing filaments (Figs. 1 and 2

and fig. S1), common features of fungal networks yet rare for pro-
karyotes. The width of filaments for extant and fossil fungi can easily
range from 2 to >20 m (19). Thus, the size of the fossil filaments
observed here fits well with fungal dimensions. However, size alone
cannot be a reliable criterion to distinguish fungal from bacterial
remains as prokaryotes also form filaments with large dimensions.
A previous study (15) of rocks from the BIe-BIIc6 interval reported
putative diversified prokaryotic and eukaryotic assemblages typical
for the Tonian period (1000 to 720 Ma) with abundant sphaeromorph
and acanthomorph acritarchs. In the BIIc8 unit, three putative
fungal species—Eomycetopsis septata, Eomycetopsis cylindrica, and
Eomycetopsis rugosa—were documented (20) (depth interval between
118.4 and 122.90 m, i.e., 20 cm above our fossiliferous rocks at 118.2 m).
They were described as cylindrical filaments aggregated in groups
and parenchyma-like masses with septate filaments. At a later stage,
those fossils first attributed to Fungi (20) were reassigned to tubular
sheaths of cyanobacteria (21). Thus, to unequivocally determine

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whether the filamentous networks that we describe here represent
Neoproterozoic fungal or cyanobacterial remains, an in-depth chemical
characterization is required.

Chitin staining
One of the major constituents of fungal cell wall is chitin, a biopolymer
of  1–4–bonded N-acetylglucosamine, which is abundant in fungi
and more generally in animal taxa (i.e., ciliates, arthropods, chryso-
phytes, and diatoms), yet absent in prokaryotic organisms such as
Actinomycetes (which can also form mycelium-like structures).
Hence, chitin alone cannot be used as a unique defining trait of fungi;
however, the chitin-producing organisms listed above are morpho-
logically distinct from our fossils in that they do not form mycelial
networks, and the presence of chitin within our filaments would be
a strong argument for a fungal affinity. Chitin can be preserved
throughout geological time. It has been detected in insects in an
Fig. 2. Confocal laser scanning fluorescence microscopy using WGA-FITC of Oligocene shale (22) and in fungi preserved in Cretaceous amber
mycelium-like structure illustrated in Fig. 1. Overview image in (A) shows the (23), and it was even found in a 505-Ma-old marine sponge from
absence of large natural autofluorescence (i.e., without WGA-FITC) of the thin the Burgess shale (24). Those discoveries support the idea that the
section. In contrast, with WGA-FITC labeling, the same area exhibits filamentous, main control of chitin preservation is not age but rather the environ-
mycelium-like structure visible in (B) (top section). (C to F) High-resolution confocal ment, with organic matter–rich sediment being ideal for preserva-
fluorescence views of the mycelium-like structures. The WGA-FITC binds specifically tion (22). Here, we document the presence of chitin in the fossil
on the cell wall of the fossil filaments, which appears cylindrical (as evidenced by a filaments by staining with wheat germ agglutinin conjugated with
rounded cross section of filaments). Several septa (s) are also stained. Note in (C)
fluorescein isothyocyanate (WGA-FITC), a highly specific dye of
that the arrows highlight septa present in putative anastomosing filaments (a)
illustrated in Fig. 1A (arrows; images are mirrored as confocal microscope is inverted).
N-acetylglucosamine trimers (19). WGA-FITC has been widely
Insets in (F) illustrate septa (ps) with perforation or “bulged,” which resembles used to discriminate between fungal hyphae and filamentous pro-
pseudosepta (39). Scale bars, 10 m (C to F). karyotes in soils, sediments, and rocks, as well as in cyanobacteria-­
fungus endosymbiosis (i.e., Geosiphon-Nostoc) and has also been
applied as a fungal marker in Eocene basalt (19) and in Cretaceous
(±7‰) coeval to the Bitter Springs 13C anomaly at ca. 810 Ma, while amber (23). We used confocal laser scanning fluorescence micros-
the age of the overlaying Kabele-Kabenga conglomerate is estimat- copy and observed that the WGA-FITC binds to extensive portions
ed at 715 Ma (18) (fig. S2). Therefore, the estimated age of the fossil- of the mycelial structure (Fig. 2). In the mycelium, WGA-FITC was
iferous dolomitic shales from BIIc8 is between ca. 810 and 715 Ma. detected in the cell wall of filaments that appear cylindrical (rounded
cross section of a filament in Fig. 2C), suggesting a preservation in
Microfossil morphology three dimensions of the remains. A few septa were stained as well
We document dark, nontranslucide, cylindrical filaments typically (Fig. 2, C to F) and, on some instances, what appear to be “incomplete
between 3.5 and 11.5 m in width (Figs. 1 and 2 and fig. S1), extending septa” (pseudosepta) that do not fully separate adjacent cells (Figs. 2C
over several hundreds of micrometers in length (>1 mm of cumu- and insets of 2F). Confocal views also allow observing some putative
lated length in Fig. 1D). These filaments sometimes evolve into anastomosis of fungal filaments (Fig. 2C). In contrast to our observations
dense interconnected networks of ~500 m in diameter (Fig. 1B). In in filamentous networks, staining was negative when WGA-FITC
these mycelium-like structures, filaments exhibit multiple-order, high-­ was exposed to inclusions of “nonfungal” organic matter in BIIc6 shale,

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Fig. 3. Confocal raman spectroscopy of the filamentous network illustrated in Fig. 1A-B. (A) Reflected LM of a filamentous network in Fig. 1A. (B) G band intensity
map of the filament shown in (A). a.u., arbitrary units. (C) Representative spectra showing the D and G bands analyzed (marked “D” at ~1350 cm−1 and “G” at ~1600 cm−1
on the spectra) from the areas marked by crosses in (B). The coloration of the spectra corresponds to the color of the crosses seen in (B). (D) G band peak center map
from the same area (i.e., B) that shows significant variations in the G band peak center across the filamentous network. (E) Representative Raman spectrum illustrating the
deconvolution in D1, D3, D4, D5, and G bands used to calculate diagenetic peak temperature and the determination of the parameter I(1350)/I(1600). The thermal matu-
ration of the fossilized filament was derived according to the FWHM-D1 geothermometer relationship in (26).

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indicating that WGA-FITC maintained its high-specificity binding aliphatic signal with peaks at 2960 cm−1 (vas CH3), 2920 cm−1 (vas CH2),
when exposed to ancient, mature organic matter (fig. S4). Considering 2875 cm−1 (vs CH2), and 2850 cm−1 (vs CH3), and at lower wave
the fungus-like morphology, the colocalization/binding of the WGA- numbers 1458 cm−1, 1408 cm−1 (as and s in CH2), and, finally,
FITC with/to cell wall/septa and its specific affinity for chitin, we 727 cm−1, the latter being indicative of (CH2)n polymethylenic chains
assert that the staining can be used as a clear indicator of chitinous with n > 4. As kerogen matures, aromaticity tends to increase relative
remnants. Simultaneously, the detection of vestigial chitin in these to the aliphatic character with fully aromatic, pure graphite being
filamentous fossils is a strong indication of their fungal origin. the ultimate end member of this process. Here, the transformation
into graphite is far from complete as the aliphatic vibrations are still
Organic matter ultrastructure in fossil filament preeminent. As for the formation of those long aliphatic chains, it
Raman microspectroscopy on filaments (Fig. 3) revealed two broad has been extensively studied in fossil and thermally matured cuticle
peaks at ~1350 cm−1 (Fig. 3D) and at ~1600 cm−1 (Fig. 3G) typically of arthropods (22), which are made of a similar protein-chitin complex
associated with low structural order, amorphous carbonaceous matter than fungal cell wall. With time, this structural assemblage is altered
(25). Deconvolution of those two spectral features provided D1, D3, by the breaking of ester, amide, and glycosidic bonds, hence pro-
D4, and G bands (Fig. 3E), which are characteristic of organic matter ducing free aliphatic groups and unsaturated carbon that then
that experienced advanced diagenesis or low-grade metamorphism polymerize into long polymethylenic chains (32, 33). Aliphatic
(26). We calculated the peak temperature of the fossil filament (and molecules associated with vestigial chitin would then create a hydro-
of the dolomitic shale organic matter away from mycelium/filament phobic, protective barrier against microbial or aqueous thermolytic
structure) to a range between 150° and 250°C (Fig. 3), which is also degradation and may explain the excellent preservation of these

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in line with the Raman-derived temperature calculated for asphaltite organic remains (32, 34). In our case, even though the aliphatic
inclusions in various BIIc dolostone (27). Note that the dissolution signal is strong, the degradation of this protein-chitin complex into
of fungal hypha and chitin was shown both to start at much higher aliphatic compounds appears incomplete as we found sharp vibra-
temperature ~250° to 300°C and to complete at 380° to 390°C (28). tions characteristic of amide at 3286 and 3070 cm−1 and especially at
Here, considering the peak temperature experienced by the fossil 1651 and 1540 cm−1, which are assigned respectively to amide A
filaments, it is likely that they retain some characteristics or struc- (v N─H), amide B and amide I (v C═O), amide II ( N─H). As for
tural compounds (e.g., chitin) that can be diagnostic of the original carbohydrates (usually between 950 and 1200 cm−1), they are depleted;
microorganism. however, we were still able to detect a pyranose peak (v C─C and vas
Recently, the Raman 1350 to 1600 cm−1 intensity ratio—I(1350)/­ C─O) at 1170 cm−1. The fact that, in addition to the aromatic and
I(1600)—which was previously used to estimate the structural order aliphatic vibrations, amide and pyranose peaks are present is a
of carbonaceous matter (29), was combined with the Fourier transform testimony of the exceptional preservation of those filamentous re-
infrared (FTIR) branching index of aliphatic chains, R3/2 (derived mains. So far, the oldest fossil exhibiting organic remains other than
from the 2800- to 3000-cm−1 spectral regions). The idea is to use the kerogens was a Vauxia gracilenta sponge (505 Ma), in which vestigial
I(1350)/I(1600) versus R3/2 trends to discriminate between pro- chitin was identified (24). Our SR-FTIR data for the fossil filament
karyotic and eukaryotic fossils (29–31). Eukaryotic cells differ widely are remarkably close to those reported for V. gracilenta (fig. S5 and
from the prokaryotic ones in terms of their cell structure and chemical table S1 for the respective peak positions), which supports the notion
compositions (i.e., different membranes, cell wall, and organelles), that vestiges of chitin are present (in line with the positive WGA-
which result in different taphonomic behaviors and distinctive FITC staining). SR-FTIR spectra of the fossil filament differ, how-
I(1350)/I(1600) versus R3/2 trends (29, 30). Synchrotron radiation– ever, from pure chitin (fig. S5). This is to be expected, because the
based (SR)–micro-FTIR (FTIR) spectra (Fig. 4) acquired on the precursor organic matter (i.e., fungal cell walls) of the fossil filaments
fossil filaments revealed a strong aliphatic signal with an average contained not only chitin but also large amounts of phospholipids,
branching index R3/2 of 0.34 ± 0.1. This rather low R3/2 value indicates proteins, and glucans. The latter together with the carbohydrate
the presence of long straight-chain aliphatic molecules and fits reason- moieties of chitin were likely preferentially degraded, and thus, it
ably well with known R3/2 values for extant eukaryotic groups (31) appears logical to find carbohydrate bands to be almost absent in
including fungi (Fig. 5). When compared with I(1350)/I(1600) ver- the SR-FTIR spectra of the fossil filament (apart from pyranose
sus R3/2 signatures of known fossils of eukaryotic and prokaryotic vibrations at 1171 cm−1; Fig. 4 and fig. S5). With respect to amide I
microorganisms, our fossilized filaments with a I(1350)/I(1600) and II vibrations, there is a good match with those of pure chitin—
ratio of ~0.6 ± 0.06 fall well in the eukaryotic trend and clearly differ usually 1650 to 1660 cm−1 and 1550 to 1560 cm−1, respectively.
from a prokaryotic signature (Fig. 5). This combined Raman-FTIR Together, those SR-FTIR data revealed that the fossil filament is
signature suggests a eukaryotic precursor for the organic matter of made of partially kerogenized organic matter, which exhibits vibra-
the fossil filament. This result, together with the morphology, dimen- tion bands consistent with vestigial “chitin,” hence supporting our
sions, and the presence of chitin remnants, further supports the idea previous positive WGA-FTIC staining essay.
of a fungal origin for those fossil filaments.
Carbon and nitrogen speciation
Chemical characterization and molecular signature Ultrathin sections of filaments were sampled perpendicularly across
Beyond the R3/2 ratio, the SR-FTIR data also revealed additional the filament by focused ion beam (FIB) milling (see Fig. 1D for
important information about the functional groups present in the localization and fig. S6 for detailed views) and analyzed by x-ray
fossil filaments (Fig. 4; table S1 for detailed assignments). First, the absorption near-edge structure (XANES) spectroscopy at the carbon
kerogenization of the fossil filaments is evidenced by aromatic and nitrogen K-edge. Synchrotron XANES analyses is an excellent
bands at 1597 cm−1 (v C═C), 3070 cm−1 (v C─H), and 1270 cm−1 nondestructive and direct method for quantifying speciation and
(C─O─C in aromatic ether/phenol groups) and by the strong bonding in organic molecules in organic-rich fossils. Recently, XANES

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of fungal hypha from the lichen Parmelia saxatalis (Fig. 6B). When

compared with other modern fungal species, the C- and N-XANES
of our fossil filaments exhibit the same trend than when it is com-
pared with chitin, i.e., more intense aromatic/olefinic and imine/
ketone/phenolic peaks (at 285.1/398.8 to 399.9 eV and 286 to 287.2 eV)
and reduced amide/carboxylic peaks (at 288.6 eV/401.3 eV). Equivalent
C- and N-XANES trends were observed in a recent experimental
study (36) simulating burial-induced maturation of various modern
microorganisms during advanced diagenesis. This difference in
C-XANES is particularly marked for Aspergillus fumigatus, but also
visible C-XANES of Paxillus involutus and for P. saxatalis although
to a lesser extent (Fig. 6A). Those two fungal species exhibit significant
aromatic/olefinic peaks and generally have C-XANES spectra close
to one of our fossil filaments with common absorption peaks
centered at ~285, 286.7, and 288.6 eV. As an element of comparison,
cyanobacteria Gloeobacter violaceus have distinctive C- and N-XANES
(25) with sharp amide peaks at 288.1 and 401.3 eV, a moderate aromatic
Fig. 4. Synchrotron-FTIR (average over 31 10 by 10–m areas) of fragments peak at 285 eV, and a shoulder for aldehydes at 289.4 eV, which differ

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of fossilized filaments illustrated in Fig. 1E. Assignments of absorption bands markedly from fungal and fossil filament XANES spectra (Fig. 6).
and vibration modes ( = deformation;  = stretching; s = symmetric; as = asymmetric)
Although not directly diagnostic, the fossil filaments exhibit C- and
are indicated in parentheses (see table S1 for full details). Note the strong contributions
of amide functional groups (at 1650, 1540, and 3286 cm−1) and aliphatic CH2 and
N-XANES spectral features consistent with the presence of ves-
CH3 moieties in the 2800 to 3000 cm−1 region, which is also evidenced by the presence tigial chitin and also share some similarities with modern fungal
of 727 cm−1 peak indicating long (CH2)n chains with n ≥ 4. (Inset) Detail of the 2800 to species.
3000 cm−1 spectral region used to calculate the branching index (i.e., R3/2—the
relative ratio of CH3 to CH2 carbon molecules) based on the intensity of their respective Syngeneticity of the fossil filaments
asymmetric stretching. The question naturally arises as to when these filamentous networks
formed relative to their host rocks. We did not detect any living fungal
cells or fresh fungal organic matter, which precludes recent contamina-
was used to detect chitin remnants in V. gracilenta (24) and to show tion. Rather, we showed that filamentous organic matter has undergone
that organic matter compositional heterogeneity (e.g., prokaryotic partial kerogenization as a result of advanced diagenesis, which suggests
versus eukaryotic microorganism) can be maintained throughout burial over geological time scales. We also found that all organic
pressure and temperature typically encountered during diagenesis matter found in the BI-BII groups of MMS, i.e., asphaltite (27),
(up to 250°C and 250 bars). Here, through deconvolution of our dolomitic shale organic matter (in the vicinity of filamentous net-
C-XANES (Fig. 6A and fig. S7), in the fossil filament we detected two works), and fossil assemblages (37) including our fossil filaments,
characteristic peaks of -chitin at 285.1 eV (C═C 1 s-* transition in exhibit the same range of Raman peak temperature of 150° to 250°C,
aromatic carbon) and at 288.6 eV (1 s-* transition of amide carbonyl indicating a common thermal exposure. Further, in several ultrathin
and C─N bonds). Compared with pure chitin, the strengthening of FIB sections, we detected paragenetic jarosite [KFe3+3(OH)6(SO4)2],
the aromatic carbon peak (285.1 eV) and the occurrence of two quartz around the filament (fig. S6). Alunite [KAl3(SO4)2(OH)6] was
additional features in the filament spectra (286.7 eV C═O/C═N also identified within fossil filaments (not shown). Those phases are
bonds and 287.5 eV for aliphatic carbon) are consistent with the likely related to evaporite-leaching fluid circulation induced by the
SR-FTIR results and illustrate the incomplete microbial degrada- Lufilian orogen (~560 to 480 Ma) (38). This important compres-
tion, the subsequent dehydration/heating processes of the advanced sional event is known to have been the cause of large sulfur mineral-
diagenesis, and the resulting kerogenization of the fossil filament. ization events in the Katangan Basin (southeast of the SMMLL
For instance, the presence of a ketone peak (286.7 eV C═O/C═N; Basin) and also that such mineralization was related to thermo-
Fig. 6 and fig. S7) indicates the dehydration/heating of polysaccharide chemical (280° to 390°C) sulfate reduction of evaporite-leaching
moieties in chitin and glucans of the fungal cell wall (33). The selec- fluid (38). This upward fluid migration in the adjacent SMMLL
tive degradation and the ensuing burial experienced by the fossil Basin is evidenced by anhydrite (CaSO4) in faults in the BIIb/d as
filament are also evidenced by the low levels of N in the fossil fila- well as by gypsum in BIe2 (16). This fluid circulation would explain
ments: C:N ratio varies from 50 to 199 versus 5 to 30 in fresh fungi. the occurrence of jarosite/alunite as well as the thermal maturation
Still, our N-XANES data confirmed the presence of amydil N with a (150° to 250°C) of the fossil filaments. Thus, on the basis of paragenetic
peak at 401.3 eV that corresponds to the main spectral feature of mineralization alone, a minimum Cambrian-Ordovician age can be
chitin (32) on the nitrogen K-edge (Fig. 6B). Other peaks are also inferred. Furthermore, the fungal filaments were not restricted to
present in the N-XANES, at 398.7 and 399.7 eV, which are associated cracks or voids and not related to tunneling—a known feature of
with pyridine and its derivatives (35). Those compounds were pre- fungal endolithic colonization. In addition, a previous study (20)
viously detected in Carboniferous and Silurian arthropod cuticles that sampled in close vicinity to our dolomitic shale in the BIIc8
(32) and were shown to form when chitinous tissues were subjected, level also described similar cylindrical, septated filaments (obtained
experimentally, to temperatures and pressures typical of advanced as palynomorph via acidic maceration). Thus, we consider the fossil
diagenesis (34). Those pyridine peaks (398.7 and 399.7 eV) in addi- filament described here as syngenetic, with the host dolomitic shale
tion to the amide peak (401.3 eV) were also found in the N-XANES rock formed ca. 810 to 715 Ma ago.

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Fig. 5. Combined FTIR and Raman biosignatures of fungi microfossils. (A) Plots of the FTIR parameter R3/2 versus Raman parameter I(1350)/I(1600) of fossilized fil-
ament from MMS (red) and comparison to eukaryotic (green) and prokaryotic (blue) fossils from previous studies (29–31). Within the Rhynie chert plant fossils, several
regions were measured including cell wall, epiderm, protoplasm, and extracellular carbon. Our fossilized filaments fall close to the protoplasm of plants. The colored areas
provide possible discrimination between eukaryote and prokaryote fossils based on their respective organic matter R3/2 and I(1350)/I(1600). The average values with SDs
are plotted. (B) Box-and-whisker plot of the R3/2 ratio of a range of modern organisms spanning across the three domains of life [data for eukaryotes, prokaryotes, and Archaea
are from (46, 47)]. R3/2 ratio quantifies the extent of branching and length of aliphatic moieties of the precursor membrane and can be used as a domain-specific proxy. As
fossilization proceeds, it seems that the R3/2 values tend to decline (47), which also seem to be the case here for fungi. *Note that “Extant fungi” values were calculated from
the FTIR spectra (48–51) of Rhizopus sp., Motierella alpina, Mucor circinelloides, Paxillus involutus, Aspergillus nidulans, Neurospora sp., Penicillium glabrum, and Umbelopsis isabellina.

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Ordovician (460 to 55 Ma). Considering that our study merged sev-

eral complementary, in situ, and spatially resolved molecular iden-
tifications by confocal Raman (Fig. 3), synchrotron-FTIR (Figs. 4
and 5 and fig. S5 and table S1), staining (Fig. 2), and XANES (Fig. 6),
we contend that the identification of chitin biosignatures in our sam-
ples is, by far, more comprehensive than the evidence reported (10)
recently, where only FTIR was used. In our study, confocal laser
scanning fluorescence microscopy was instrumental in demonstrating
the presence of vestigial chitin and the occurrence of key fungal fea-
tures such as septa and anastomosing filaments in those mycelium-­
like structures. Taking advantage of the unique sensitivity and spatial
resolution of synchrotron-based FTIR, XANES, and Raman
spectroscopy, the present study revealed that the molecular bio-
signature of the filamentous fossils has been exceptionally preserved
despite their 715- to 810-Ma-long geological history. We believe
that with the early cementation and burial in a shale rock rela-
tively rich in organic matter (presumably preventing oxidative
degradation) and a rather low peak-temperature (150° to 250°C),

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the fossil filaments have benefited from a very favorable pres-
ervation context over time scales. This exceptional preservation
made possible a whole range of chemical characterizations.
Although the width of the filament (Fig. 1E) is consistent with
either fungal or filamentous prokaryotic precursors, we could infer
that these remains had most likely a eukaryotic origin by combining
SR-FTIR with Raman spectral signatures (Fig. 5). We further
showed that despite a kerogenization process evidenced by intense
aromatic signals and a low C:N ratio (50–199), the fossil filaments
exhibit SR-FTIR, C-XANES, and N-XANES spectra sharing sig-
nificant similarities with modern fungal species and chitinous fossil
such as V. gracilenta and that they are not similar to typical pro-
karyotic microbes. We could even detect amide functional groups
(C-XANES absorption at 288.2 eV, N-XANES at 401.3 eV, and FTIR
bands at 1651 and 1540 cm−1) that were likely to be involved in the
cell wall protein-chitin complex synthesized by the once living fungi—
hence confirming our positive WGA-FITC staining of vestigial
Fig. 6. X-ray absorption near edge spectroscopy at the C and N K-edge performed chitin by confocal laser scanning fluorescence microscopy. On
on FIB foil 4984. (A) Compilation of C-XANES spectra for a range of modern fungi the basis of the combined evidence of presence of vestigial chitin,
(Paxillus involutus and Aspergillus fumigatus), fungal hypha in lichen (Parmelia saxatalis), eukaryotic nature, syngenecity, size, and morphology, the filaments
cyanobacteria (Gloeobacter violaceus) (36), -chitin, and fossil filament (FIB foil #4984; and mycelium-like structures are identified as the fossilized remnants
fig. S7 for detailed views). The colored bands denote the energy range for which of fungal networks.
various C functional groups are expected (see table S2). For the fossilized filament,
In our view, the observed septation of the hypha does not indicate a
the main peaks are centered at 285.1 eV (aromatic C═C), 286.7 (ketone and phenol
C), and a shoulder in amide and carboxylic energy bands (288 to 288.7 eV). A complete
dikaryan affinity, which appears unlikely considering the Neoproterozoic
deconvolution of the C-edge for fossilized filament was performed (fig. S7). Note that age of the remains and the much later timing of the Dikarya evolution
for P. involutus, the XANES was acquired on the FIB section at the interface between (Basidiomycota and Ascomycota). Instead, the low frequency of the
fungi and biotite; the double peak between 297 and 300 eV are, thus, absorption septation and also the presence of incomplete septa (pseudosepta)
peaks for potassium. (B) N-XANES spectra of fossilized filament, fungal hypha in in the fossil filaments resemble more Blastocladiomycota hypha (39).
Parmelia saxatalis, and -chitin (32). The fossilized filament exhibits spectral features at This basal phylum regroups parasitic or saprotroph fungal species
398.7 (imine), at 399.8 eV related to pyridine (35), and a shoulder at 401.3 eV, which and is found in both aquatic and terrestrial environments. Hence,
corresponds well with the main spectral feature of the -chitin (N 1 s-3p/* transition considering the very shallow, periodically subaerially exposed, lacustrine
in amide). Note that all those features are visible in the spectra of P. saxatalis. nature of the upper BIIc paleoenvironment where the fungal remains
were discovered, one may hypothesize that those fungi were in asso-
DISCUSSION ciation with (either parasitic or symbiotic) or decomposers of an
We report the presence of ancient fungal filaments and mycelium-­ early photosynthetic community in transition toward terrestrial life
like structures preserved in a Neoproterozoic dolomitic shale (ca. 810 (40). Ephemeral ponds, with their frequent wet-drying cycles (e.g.,
and 715 Ma). This discovery proves definitively that filaments, found along the shores), might have been favorable environments for
earlier in (20), which were later reassessed as cyanobacterial tubular physical interactions between fungi and algae. Those early associa-
sheaths, were in fact remains of fungal origin. Hence, this finding tions with photosynthetic organisms were presumably restricted to
extends the record of fungi fossils by more than 250 Ma with respect free living, and possibly lichenized, green algae or cyanobacteria (9),
to previously reported oldest fungi fossil dating from the mid-­ which may have represented a primitive form of cryptogamic cover

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SCIENCE ADVANCES | RESEARCH ARTICLE

(41). Although no such symbiotic relationships have been observed A cosmic ray reduction routine was used to reduce the effects
directly here, it is worth noting that the upper BIIc shale beds, in of stray radiation on Raman images, as was image thresholding to
addition to fungal remains, exhibit an abundant and diverse assem- reject isolated bright pixels. Fluorescence effects were inhibited by
blage of microfossils including 11 eukaryotic, acantomorph, presum- the use of specific peak fitting in place of spectral area sums and by
ably algal species (15). Hence, our study represents the oldest, the confocal optics used in the instrument. The effects of interfering
documented fungi to date and pushes well into the Neoproterozoic peaks were removed by phase masking routines based on multiple
the possibility that fungi helped to colonize land surface, almost peak fits as compared with standardized mineral spectra. This prod­
300 Ma before the first evidence of land plants. Overall, our discoveries uces an average spectrum over the number of pixels chosen in the
also lend support to previous assumptions regarding the role of fungi area of interest. Peak center maps were produced by using a Gaussian
in land colonization and, by extension, on the evolution of Earth’s fit to the G band and then confirmed by taking representative spectra
biogeochemical cycles (40, 42). Neoproterozoic soil development from the range of peak centers as seen in Fig. 3.
driven by early terrestrial biota has generated increasing amounts of
pedogenic clay minerals that helped to stabilize organic matter and Confocal laser scanning fluorescence microscopy
enhanced carbon burial, which, in turn, contributed to the stepwise The incubation with WGA-FITC lasted for 1 hour. The concentra-
oxygenation of the Late Precambrian. tion of the WGA-FITC in the incubation medium was 80 g ml−1
WGA-FITC in 10 mM phosphate buffer at pH 7.8. After incubation,
the samples were thoroughly washed with MilliQ water. Confocal
MATERIALS AND METHODS fluorescence imaging was realized on a Zeiss LSM-7 10 confocal

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The dolomitic shale sample (BK457b, 118.2 m, core 118/4) from BIIc8 microscope using both Zeiss ×2 0/0 8 PlanApochromat and Zeiss
was cut to produce three petrographic, noncovered thin sections, of ×63/1.4 PlanApochromat objectives and with specific excitation
which one showed five areas where mycelia were apparent. The sections using a 488-nm Argon ion laser. Fluorescence detection was realized
were studied using light microscopy, scanning electron microscopy using a 500 to 550 bandpass emission filter and the Zeiss Airyscan
(SEM), chitin staining with WGA-FITC with fluorescence micros- GaAsP detector to increase signal-to-noise ratio and resolution.
copy, and SR-FTIR. Furthermore, nine FIB foils were sampled to Images were acquired using the Zeiss ZenBlack software (Zeiss,
be analyzed by synchrotron–XANES spectroscopy and by analytical Oberkochen, Germany). Fluorescence images of the BK233 sample
transmission electron microscopy (TEM). were done on a Zeiss AxioObserver Z1 widefield microscope using
a Zeiss ×20/0.8 PlanApochromat objective and a 493-nm light-emitting
Scanning electron microscopy diode excitation. Detection was done on a Hamamatsu Flash4.0
The images in Fig. 1 and figs. S1 and S6A were obtained with an sCMOS camera with a 1.5-s exposure time. Image adjustments (i.e.,
Ultra55 Field Emission Gun-SEM (Zeiss), operated at 2.00-kV brightness and contrast) and Z projections were done using Fiji soft-
accelerating voltage. The samples were uncoated. ware (43).

Confocal Raman imaging spectroscopy Synchrotron radiation–FTIR

Raman spectra and images were collected using a Witec -Scanning The FTIR measurements were all done at the beamline B22 at
Near-Field Optical Microscope customized to incorporate confocal Diamond Light Source (UK). All measurements were conducted in
Raman spectroscopic imaging. The excitation source was a frequency-­ a transmission mode with the samples—fragments of filamentous
doubled solid-state yttrium-aluminum-garnet (YAG) laser (532 nm) network—manually deposited on CaF2 slides. We used the Hyperion
operating between 0.3- and 1-mW output power (dependent on 3000 SR-FTIR setup to analyze our sample with the 36× objective.
objective), as measured at the sample using a laser power meter. Usually, 512 or 1024 scans were accumulated at a resolution of
Objective lenses used included a ×100 long working distance (LWD) 4 cm−1 between 500 and 4000 cm−1 (later cut between 1000 and
and a ×20 LWD with a 50-m optical fiber acting as the confocal 3500 cm−1) and generally with a 10 × 10–m aperture. Spectra were
pin hole. Spectra were collected on a Peltier-cooled Andor EMCCD then baseline corrected using the concave rubber band routine in
(electron multiplying charge-coupled device) chip after passing OPUS Spectroscopy software (Bruker).
through an f/4 300-mm focal length imaging spectrometer typically
using a 600 lines/mm grating. The lateral resolution of the instrument Ion milling by FIB
was as small as 360 nm in air when using the ×100 LWD objective, Nine ultrathin foils, typically 15 x 7 x 0.15 m, were sampled from
with a focal plane depth of ~800 nm. various filamentous networks using an FIB single-beam instrument
Typically, 2D imaging and single spectra modes were used (FEI FIB 200 TEM) at the German Research Centre for Geosciences
during this study. Single-spectrum mode allowed the acquisition of Potsdam following the procedure described in (44). This ion milling
a spectrum from a single spot on the target. Average spectra were procedure maintained textural and chemical integrity even in the
produced typically using integration times of 30 s per accumulation case of sensitive materials. Milling was conducted at low-gallium ion
and 10 accumulations to allow verification of weak spectral fea- currents to minimize gallium implantation or redeposition of the
tures. Target areas were identified on the thin section in reflected sputtered materials on the sample surface and to prevent significant
light. The height and width of the field of interest within the light changes in the speciation of complex carbon-based polymers.
microscopy image were then measured and divided by the lateral
resolution of the lens being used to give the number of pixels per X-ray absorption near-edge structure spectroscopy
line. The instrument then took a Raman spectrum (0 to 3600 cm−1 All XANES measurements were all done at the I08-SXM beamline
using the 600 lines/mm grating) at each pixel using an integration at the Diamond Light Source (UK). Collection of XANES spectra at
time of between 1 s per pixel. the C (from 280 to 310 eV) and N (from 395 to 420 eV) K-edges

Bonneville et al., Sci. Adv. 2020; 6 : eaax7599 22 January 2020 9 of 11

SCIENCE ADVANCES | RESEARCH ARTICLE

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Molecular identification of fungi microfossils in a Neoproterozoic shale rock
S. Bonneville, F. Delpomdor, A. Préat, C. Chevalier, T. Araki, M. Kazemian, A. Steele, A. Schreiber, R. Wirth and L. G. Benning

Sci Adv 6 (4), eaax7599.

DOI: 10.1126/sciadv.aax7599

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