Prototaxites reinterpreted as mega-rhizomorphs, facilitating nutrient transport in early terrestrial ecosystems

Abstract

The enigmatic fossil Prototaxites found in successions ranging from the Middle Ordovician to the Upper Devonian was originally described as having conifer affinity. The current debate, however, suggests that they probably represent gigantic algal–fungal symbioses. Our re-investigation of permineralized Prototaxites specimens from two localities, the Heider quarry in Germany and the Bordeaux quarry in Canada, reveals striking anatomical similarities with modern fungal rhizomorphs Armillaria mellea. We analysed extant fungal rhizomorphs and fossil Prototaxites through light microscopy of their anatomy, Fourier transform infrared spectroscopy, X-ray microscopy, and Raman spectroscopy. Based on these comparisons, we interpret the Prototaxites as fungi. The detailed preservation of cell walls and possible organelles seen in transverse sections of Prototaxites reveal that fossilization initiated while the organism was alive, inhibiting the collapse of delicate cellular structures. Prototaxites has been interpreted to grow vertically by many previous workers. Here we propose an alternative view that Prototaxites represents a complex hyphal aggregation (rhizomorph) that may have grown horizontally similar to modern complex aggregated mycelial growth forms, such as cords and rhizomorphs. Their main function was possibly to redistribute water and nutrition from nutrient-rich to nutrient-poor areas facilitating the expansion for early land plant communities.

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OPEN ACCESS | Article
Prototaxites reinterpreted as mega-rhizomorphs,
facilitating nutrient transport in early terrestrial
ecosystems
Vivi Vajda , Larissa Cavalcante, Kristoer Palmgren, Ashley Krüger , and Magnus Ivarsson
Department of Palaeobiology, Swedish Museum of Natural History, SE 104 05 Stockholm, Sweden
Corresponding authors: Vivi Vajda (email: vivi.vajda@nrm.se); Magnus Ivarsson (email: magnus.ivarsson@nrm.se)
Abstract
The enigmatic fossil Prototaxites found in successions ranging from the Middle Ordovician to the Upper Devonian was origi-
nally described as having conifer anity. The current debate, however, suggests that they probably represent gigantic algal–
fungal symbioses. Our re-investigation of permineralized Prototaxites specimens from two localities, the Heider quarry in Ger-
many and the Bordeaux quarry in Canada, reveals striking anatomical similarities with modern fungal rhizomorphs Armil-
laria mellea. We analysed extant fungal rhizomorphs and fossil Prototaxites through light microscopy of their anatomy, Fourier
transform infrared spectroscopy, X-ray microscopy, and Raman spectroscopy. Based on these comparisons, we interpret the
Prototaxites as fungi. The detailed preservation of cell walls and possible organelles seen in transverse sections of Prototaxites re-
veal that fossilization initiated while the organism was alive, inhibiting the collapse of delicate cellular structures. Prototaxites
has been interpreted to grow vertically by many previous workers. Here we propose an alternative view that Prototaxites repre-
sents a complex hyphal aggregation (rhizomorph) that may have grown horizontally similar to modern complex aggregated
mycelial growth forms, such as cords and rhizomorphs. Their main function was possibly to redistribute water and nutrition
from nutrient-rich to nutrient-poor areas facilitating the expansion for early land plant communities.
Key words: Prototaxites, Devonian, fungus, algae, lichen, hot-spring deposit
Introduction
The evolution of land plants and their colonization of
terrestrial environments are among the fundamental top-
ics within the field of geobiology (Edwards et al. 2014). The
rise of plants can be traced via fossil spores preserved in the
geological record by these early terrestrial plants. Although
traces of early land plants have been recorded from succes-
sions as old as the Ordovician (Rubinstein et al. 2011;Badawy
et al. 2014;Rubinstein and Vajda 2019;Leslie et al. 2021), a
major radiation occurred during the late Silurian and Early
Devonian, when plants developed more complex forms, in-
creased in abundance, and became well established on most
continents (Gray et al. 1974;Richardson and McGregor 1986;
Mehlqvist et al. 2012,2014,2015;Wellman et al. 2013,2022;
Leslie et al. 2021). This was also a time when other groups
of organisms developed in the terrestrial realm and left be-
hind peculiar fossil remains. One such organism was Pro-
totaxites loganii Dawson. This puzzling fossil represents an
organism that inhabited the late Silurian to Late Devonian
terrestrial landscape (420–370 Ma). It was by far the largest
land-living organism at the time, and possibly represented an
important carbon source for the early fauna that otherwise
only shared the eco-space with minute early land plants and
arthropods (Retallack 2001;Hagström and Mehlqvist 2012).
Indeed, Hueber (2001) identified the earliest records of ter-
restrial arthropod borings in a Prototaxites “log”. The archi-
tecture, systematic anities, and ecology of these organisms
have been debated intensely since their initial description by
Dawson (1859) who described it under the species name P. lo-
ganii owing to its superficial resemblance to a conifer trunk.
The fossils are remarkably robust, reaching tree size (up to
8 m long and 1 m in diameter), in some cases branching dis-
tally, and incorporating concentric layers of longitudinally
aligned, smooth, slender tubes interwoven with dierentially
thickened, banded tubes (Schweitzer 1983). Moreover, many
of the fossils show little compaction indicating a rigid archi-
tecture of the original organism, or a very rapid process of
permineralization.
The suggested anities of these fossils have, over the past
160 years, included the trunks of vascular plants (Dawson
1859), kelp-like aquatic algae (Schweitzer 1983), or rolled up
mats of liverworts (Graham et al. 2010). Hueber (2001) in-
terpreted Prototaxites as a giant sporomorph of basidiomy-
cote anities, whereas Retallack and Landing (2014) favoured
a provisional assignment to Glomeromycota.Honegger et al.
(2018) described P. loganii as a giant sporophore (basidioma)
and identified fertile Prototaxites taiti in Rhynie cherts. Boyce
et al. (2007) obtained variable δ13C isotope values from
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Prototaxites, suggesting heterotrophic nutrition on isotopi-
cally distinct substrates consistent with a fungal anity.
Most recent interpretations have converged on Prototaxites
being either a fungal fruiting body or large lichen (Selosse
2002;Nelsen and Boyce 2022). In summary, Nematophytales
(including Prototaxites) have no clear anities with extant
groups but probably constitute a fully extinct array of fungi
(Edwards and Axe 2012). Studies of Prototaxites have indicated
that this organism had a lifestyle adapted to a terrestrial en-
vironment, and although an upright growth habit has been
proposed for Prototaxites by many previous authors, convinc-
ing evidence for this is meagre (Graham et al. 2010;Retallack
2022).
Here we explore the morphological and chemical similari-
ties between extant fungal rhizomorphs and Prototaxites spec-
imens derived from two Northern Hemisphere localities. We
assess the depositional environment of the Early Devonian
ecosystem in which these giant organisms lived and discuss
their influence on the early land plant communities.
Materials
Fossil material
The studied fossil material is represented by two Early De-
vonian Prototaxites associations, one from the Heider quarry
(50◦5700N, 007◦1800 E), Germany, and the other from
the Bordeaux quarry (48◦0248N, 066◦4657 E), Canada. The
specimens are part of the paleobotanical collections hosted
at the Swedish Museum of Natural History (NRM). The mate-
rial from the Heider quarry was originally collected by Prof.
H.-J. Schweitzer and described in 1983 as Prototaxites cf. lo-
ganii (Dawson). The Heider quarry belongs to the Wahnbach-
Schichten of the Rheinische Schiefergebirge (shale moun-
tains) and hosts one of the most important Early Devonian
fossil assemblages. The successions are near-shore marine
and contain not only a rich fish and eurypterid fauna but also
a range of transported early land plant remains (Gross 1933).
The Bordeaux quarry is located on the southern shores
of the Gaspé on Chaleur Bay and the river mouth of Res-
tigouche River in Quebec, Canada. The fossils were collected
from the Campbellton Formation, which has been dated to
an Early Devonian, Emsian age (Kennedy et al. 2013). The as-
semblage was collected by Dawson in 1881 when visiting the
area. The successions represent flood plain deposits interca-
lated with coarser transported material, in which the Proto-
taxites were found. The Campbellton Formation is overlying
the Val d’Amour Formation of Pragian age, which contains
abundant volcanoclastic deposits (Kennedy et al. 2013).
Extant fungal isolates
Three extant fungal isolates T4 (Armillaria mellea), T8 (Postia
rennyi), and T9 (Fomitopsis pinicola) of saprophytic fungi from
the Tyresta National Reserve, Stockholm, Sweden, sampled in
September 2021, were used in the study. The samples were
collected from fungal fruiting bodies on tree trunks (Figs.
1A–1C). Within 24 h, the samples were placed in Petri dishes
with a growth medium (Sigma–Aldrich’s potato glucose agar),
and spiked with cloramphenicol (0.05 g L−1)at25◦C. The
plates were checked every other day, and when fungal growth
was observed, samples were transferred to a new Petri dish
and left to incubate. These steps were repeated until isolates
were obtained. All three extant fungi were used for Fourier
transform infrared (FTIR) analysis for compositional compar-
ison with the fossils and for calculating the R3/2 branching
index. The A. mellea isolate forming morphologically distinct
rhizomorphs was used as a morphological comparison to the
fossil material (Figs. 1D–1F).
Methods
Light and fluorescence microscopy combined
with staining
The fungi and fossil samples were studied using a stereomi-
croscope (Olympus SZX2-ILLT) and an Olympus BX51 micro-
scope with an X-cite Series 120Q fluorescence light source.
The fungi were stained with CalcoFlourWhite (BioTium), a
dye that binds to chitin. Before staining, the samples were
treated with sterile gloves and forceps to reduce the intro-
duction of foreign fluorescent particles.
ESEM
An XL30 environmental scanning electron microscope
(ESEM) with a field emission gun (XL30 ESEM-FEG) was used
to analyse the fossil material and the rhizomorphs. The ESEM
was equipped with an Oxford x-act energy dispersive spec-
trometer, a backscatter electron detector, and a secondary
electron detector. The acceleration voltage was 20 kV. The
instrument was calibrated with a cobalt standard. Peak and
element analyses, together with element mapping, were
recorded using the accompanying AZTEC software.
Raman confocal microspectroscopy
Raman confocal microspectroscopy was used to identify
mineral species and the presence of organic matter in the
samples as well as characterizing peak temperatures for
the fossils. Raman spectra were collected at the Depart-
ment of Materials and Environmental Chemistry (MMK)——
Stockholm University (SU) using a Horiba LabRAM HR 800
Raman spectrometer equipped with an air-cooled double-
frequency Nd:YAG laser operating at 532 nm. All Prototaxites
samples were mounted on glass slides and exposed to a laser
power of 5 mW through an objective lens with 50×magnifi-
cation (NA 0.42). A diraction grating with 600 grooves mm−1
was used to resolve the spectra. Acquisition time was set be-
tween 2 and 4 s, with 30–50 scans accumulated from 100 to
3400 cm−1with a spectral resolution of 2 cm−1for obtain-
ing the spectra. Five to ten spectra were taken from dierent
parts of each sample.
Spectra postprocessing was performed with Origin 2021
(OriginLab Corp., Northampton, MA, USA), and a linear base-
line was created for baseline correction, using fixed points
at the shoulders of each visible band. Band deconvolution in
the range 1000–1800 cm−1was performed using an iterative
procedure with Multiple Peak Fit, using a Gaussian function,
until fit converged with R2≥0.997.

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Fig. 1. Saprophytic fungi from the Tyresta National Reserve, Stockholm, Sweden, sampled in September 2021 used to extract
fungal isolates (A) T4 (Armillaria mellea), (B) T8 (Postia rennyi), and (C) T9 (Fomitopsis pinicola).(D) Isolate in Petri dish. Note the
predominant growth as rhizomorph and (E) detailed image of one rhizomorph. (F) Microphotograph of a cut rhizomorph
showing the hollow interior.

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FTIR microscopy
An FTIR microscope was used to characterize the miner-
alogical and organic content of the fossils, and used as ba-
sis for calculating the R3/2 value that reflects the lipidic com-
position of cellular membranes among dierent organisms,
thus enabling determination of biological anity (Igisuetal.
2009). FTIR spectra were recorded at MMK——SU, on a Varian
670-IR microscope equipped with a motorized x–y–zstage and
a mercury cadmium telluride detector cooled with liquid ni-
trogen. The spectra were collected in both transmission and
attenuated total reflectance (ATR) modes with a diamond ATR
element. The infrared transmission spectra of the thin sec-
tion from the Bordeaux quarry Prototaxites and extant fungi
isolates were recorded with a 100 μm×100 μm aperture
and using a CaF2window. The infrared spectra in ATR mode
were taken directly from a selected darker area of the Heider
quarry Prototaxites fossil sample. For each spectrum, 256 scans
were accumulated at a spectral resolution of 4 cm−1, and they
were recorded in the frequency range of 600–4000 cm−1.Fi-
nally, spectra from three to five locations in each sample were
taken, and IR background spectra were taken before each
sample. Spectra processing and further data analyses were
performed with Origin 2021 (OriginLab Corp.). No baseline
correction was necessary.
3D XRM
One cubic centimetre of the Prototaxites sample (Je-Sch0263)
was cut for scanning using a 3D X-ray microscope (XRM). Scan-
ning was carried out at the Stockholm University Brain Imag-
ing Centre (SUBIC) using the ZEISS Xradia Versa 520 X-ray mi-
croscope. The sample was placed in a 14 mL Falcon round bot-
tom polypropylene test tube packed with polystyrene pack-
ing material to keep the sample stable during scanning. The
following scan parameters were used: 140 kV at 10 W with
a 4 s exposure time. A total of 1601 projections were taken
in full 360◦rotation. A 4×objective was used to image a
pixel size of 1.6 μm. Scanning was completed using Xradia
Scout & Zoom software. The reconstructed .ti stack was im-
ported and visualized using Object Research Systems (ORS)
(Montreal, Quebec, Canada) Dragonfly software using a non-
commercial license.
Results and interpretation
Modern rhizomorph morphology
Here we present the results for the fossil Prototaxites,us-
ing modern fungus as an analogue. We compare and con-
trast results from other studies with our own observations
of morphological and anatomical similarities between Pro-
totaxites and modern rhizomorphs. At subsampling, the iso-
lated fungus in T4 produced rhizomorphs ∼1mmwideand
3–5 cm long (Figs. 1D–1F). Rhizomorphs were visualized by
ESEM (Figs. 2A–2C) and staining with CalcoFlourWhite using
fluorescence microscopy (Figs. 2D and 2E). Each rhizomorph
consists of a bundle of thousands of parallel-fused hyphae
forming a tissue-like substrate at the margins surrounding a
central cavity (Figs. 1D and 1E). The tubular elements are sev-
eral centimetres long and 1–8 μm in diameter. The internal
architecture of the studied modern rhizomorphs is character-
ized by a peripheral zone of parallel-bundled hyphae fused
into a tissue-like substrate that surrounds a central cavity.
Prototaxites morphology
The tubular elements (7–10 μm in diameter) of Prototax-
ites are clearly visible (Figs. 3A–3D) and occur in bundles
divided by growth increments. The density, diameter, and
wall thickness of the tubular structures and the width of the
growth increments vary between specimens. The tubes are
“spaghetti-like” in longitudinal section, unbranched, and
aseptate (Fig. 3D).
The Prototaxites specimen from the Heider quarry has tubu-
lar structures that occur as alternating light and dark bands
in cross section (Fig. 3A). The lighter bands consist of tubu-
lar structures with diameters up to ∼50 μm, while the darker
bands consist of tubular structures with diameters of ∼10 μm
(Figs. 3B–3D). According to the ESEM (Fig. 5) and Raman spec-
troscopy (Fig. 6), the lighter bands consist of quartz, while
the darker bands consist of organic matter alternated with
quartz.
The Prototaxites specimens from the Bordeaux quarry ex-
hibit growth increments (Figs. 4A and 4B). Well-preserved
tubular structures are found throughout (Figs. 4B–4D). In
cross section, these are circular or near-circular with diame-
ters up to ∼10 μm(Figs. 4E and 4F), also consisting of quartz.
ESEM imaging reveals the lighter bands to be more homo-
geneous relative to the darker bands (Figs. 5A–5G). The dark
bands are somewhat heterogeneous with abundant carbona-
ceous matter in between the quartz. Element mapping high-
lights abundant C in the bands between the quartz, and oc-
casional traces of Ca (Figs. 5E and 5F).
In the centre of most tubes, a dark circular nuclei-like
structure, 1–2 μm in diameter, is seen in cross section (Figs.
3E–3G). These structures are detected in most tubes when
analysing the fossil in cross section and we interpret them
as contracted, fossilized cell content (organelles).
Raman spectroscopy
Overall, the Raman spectra of the Prototaxites fossils show
the presence of bands in the first- and second-order regions
of the Raman spectra (Figs. 6A and 6B), characteristic of car-
bonaceous material (Kouketsu et al. 2014;Qu et al. 2015,
2018;Henry et al. 2019;Bonneville et al. 2020). The first-
order Raman spectral region contains two main bands (1100–
1800 cm−1), whereas the second-order region is character-
ized by several overlapping bands (2400–3500 cm−1), previ-
ously interpreted as overtones and inelastic scattering of the
bands present in the first-order region (Henry et al. 2019). Ad-
ditionally, bands at 125, 202, and 461 cm−1(Fig. 6B) are char-
acteristic of quartz minerals (Chukanov and Vigasina 2020).
As expected, the carbonaceous material occurs mainly in
the darker regions of the Heider quarry fossil (Fig. 6D). In
the first-order region of the spectra, the graphite (G) band is
commonly attributed to in-plane vibrations of carbon atoms
and is observed at ∼1600 cm−1(Qu et al. 2018;Henryetal.
2019;Bonneville et al. 2020). The disordered (D) band occurs
at ∼1350 cm−1, and corresponds to ring breathing mode in
disordered carbonaceous material (Henry et al. 2019).

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Fig. 2. Photomicrographs and ESEM images of modern rhizomorph from sample T4. (A) ESEM image of a rhizomorph showing
the longitudinal growth direction of the hyphae. (B) Cross section through rhizomorph. The central cavity has collapsed due
to vacuum in the ESEM chamber. (C) Close-up of the cross section. Note the varying diameter of the tubular structures. (D, E)
Selected parts of rhizomorphs stained with ClacoFlourWhite and studied using fluorescence microscopy. (D) Tip of rhizomorph.
(E) Note the longitudinal direction of the hyphae.

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Fig. 3. Micrographs of Prototaxites, sample JE-Sch0252 from Heider quarry. (A) Overview of a thin section of Prototaxites in trans-
verse section; note the shift between the more organic-rich dark bands and quartz-dominated light bands. Cell size/crystallinity
increases with age from left to right. Darker bands get darker with age as well. (B, C) Micrograph, close-up of tubes in trans-
verse section. (D) Micrograph with close-up of the tubular structures, longitudinal section. Light band surrounded by two
darker bands. Cell diameter is larger in the bright band compared with dark bands. The possible cell nuclei are more com-
monly visible in the darker bands. Red arrows point to nuclei in bright bands. (E) Micrograph of Prototaxites cross section,
boundary between light and dark bands. Note the presence of dark specks, possible cell content. (F, G) Possible cell content
(arrows) preserved in the quartz-rich bright bands. (H) A large Prototaxites specimen Je-Sch2318 from Heider quarry.

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Fig. 4. Bordeaux quarry specimens of Prototaxites.(A,B)Prototaxites specimen S004496 displaying growth increments. (C, D) Mi-
crograph with close-up of the tubular structures, longitudinal section of specimen S04496-04 and S04496-05. (E, F) Micrograph,
close-up of tubes in transverse section of specimen S082540.

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Fig. 5. ESEM micrographs of Prototaxites, sample JE-Sch0252 from Heider quarry. (A) Overview showing the distribution of
bands seen in ESEM. Bands that appear dark in optical microscopy appear more heterogeneous and with more inclusions of
carbonaceous matter in between the quartz crystals when using ESEM (top and bottom sides of dashed lines). Light bands are
more homogeneous (in between dashed lines). (B) Composite image of element mapping. (C) Si K series elemental mapping.
(D) O K series elemental mapping. (E) Ca K series elemental mapping. (F) C K series elemental mapping. (G) S K series elemental
mapping.
Deconvolution of the two bands in this region showed the
presence of the D1 and G bands, together with the follow-
ing additional bands: D2, D4, D5, and D6 (Fig. 7A;Henryetal.
2019), which indicates that the fossil reached low- to medium-
grade thermal maturity (Kouketsu et al. 2014;Bonneville et
al. 2020). Using the FWHM-D1 relationship determined by
Kouketsu et al. (2014), the peak temperature experienced by
the fossil was calculated to be 269 ±14 ◦C.
The dierence in the Raman intensity between the darker
regions from which the spectra were acquired suggests that
the amount of organic matter varies within these regions.
The fringes are artefacts, most likely caused by internal re-
flections of the fossil and (or) glass slides. In contrast, the
spectra of the brighter parts of the fossil (Fig. 6E) did not
show bands corresponding to carbonaceous materials. In-
stead, these areas are characterized by a stronger band at
∼460 cm−1and a medium band at ∼200 cm−1bands, both
corresponding to vibrational modes of quartz.
The Bordeaux quarry fossil is entirely composed of dark
carbonaceous matter. The deconvolution of the Raman bands
in the first-order region shows the presence of the two main
bands D1 and G and three additional bands D3, D4, and
D5 (Fig. 7B), indicating that this fossil consists mainly of
low-grade carbonaceous material. Again, using the FWHM-
D1, the peak-metamorphic temperature was calculated to be
128 ±10 ◦C.
Finally, Raman 1350–1600 cm−1intensity ratios (I(1350)/I
(1600)) were previously used to estimate the structural order
of carbonaceous matter (Kouketsu et al. 2014;Qu et al. 2015).
In our study, this ratio was calculated considering only the
intensity of the two main bands, regardless of the resultant
deconvolution bands. As a result, the fossils from the Heider
and Bordeaux quarries have intensity ratios of 0.62 ±0.03 and
0.72 ±0.01, respectively, indicating the presence of organic
matter with lower structural order.
FTIR
Infrared spectra were acquired for Prototaxites from both
localities (Fig. 8). For the Heider quarry Prototaxites,nobands
corresponding to organic matter were observed. The bands at

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Fig. 6. Confocal Raman spectra acquired from Prototaxites, sample JE-Sch0252 from Heider quarry. (A) Representative Raman
spectrum of the carbonaceous matter in the fossils, with characteristic bands in the first- and second-order regions. The spec-
trum also indicates the presence of quartz mineral. (B) Two main bands from carbonaceous matter in the first-order region, the
disordered band (D) at 1357 cm−1and the graphite band (G) at 1605 cm−1. Characteristic quartz bands also appear at 461, 202,
and 125 cm−1. (C) Thin section of Heider quarry Prototaxites. The darker areas 1–5 correspond to the areas from which Raman
spectra were collected for analysis. The Raman spectra from brighter areas i–iv were also acquired for comparison. (D) Raman
spectra 1800–300 cm−1of the areas 1–5, indicating the presence of carbonaceous matter and quartz. This contrasts with the
spectra of areas i–iv (E), which did not show bands of carbonaceous matter, presenting only bands at ∼460 and ∼200 cm−1
characteristic of vibrational modes of quartz.

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Fig. 7. Representative Raman spectra illustrating the deconvolution of the D and G bands for the Prototaxites fossils from both
Heider and Bordeaux quarries. (A) Deconvolution into D1, D2, D4, D5, D6, and G for the Raman spectra acquired of the Heider
quarry fossils. (B) Deconvolution into D1, D4, D5, D6, and G for the Raman spectra acquired of the Bordeaux quarry fossils.
In both cases, the FWHM-D1 was used for calculating the peak temperature experienced by the fossils, using the relationship
determined by Kouketsu et al. (2014).
1087, 798, 695, and 460 cm−1indicate the presence of mainly
organosilicon compounds, more specifically siloxanes, with
Si–O–Si, Si–O–C, and Si–CH3groups (Socrates 2001;Benning
et al. 2004;Launer and Arkles 2013).
For the Bordeaux Prototaxites sample, the 2950, 2925, and
2870 cm−1bands correspond to the asymmetric stretch of
methyl (vas CH3) and asymmetric and symmetric stretches
of methylene (vas CH2and vsCH2). The bands at wavenum-
bers lower than 2500 cm−1(1990, 1870, 1790, 1680, 1620,
1520, and 1490 cm−1) correspond to Si–O vibrations of quartz
(Igisu et al. 2009;Qu et al. 2015), which masked the other sig-
nals from the organic matter——hampering a direct compar-
ison of composition between the spectra of Prototaxites fos-
sils and extant fungi (Fig. S1). As an alternative, the bands
in the 3000–2800 cm−1can be used as follows: FTIR bands
corresponding to CH3and CH2asymmetric stretches in the
spectral region 2800–3000 cm−1were previously used to cal-
culate the branching index of aliphatic chains R3/2 (Igisuetal.
2009). For the extant fungi isolates T4, T8, and T9, the R3/2 val-
ues obtained were 0.90 ±0.05 (n=4), 0.950 ±0.002 (n=4),
and 0.90 ±0.02 (n=5), respectively. The value obtained for
the more well-preserved fossil Prototaxites from the Bordeaux
quarry was 0.97 ±0.01 (n=9), fitting the values for the extant
fungi analysed. The high values (>0.5) observed for both the
fossil and extant organisms indicate the prevalence of methyl
groups over methylene-chain structure, i.e., highly branched
aliphatic compounds.
Discussion
The enigmatic fossil Prototaxites loganii Dawson encoun-
tered from Silurian to Upper Devonian successions was in
the early days of its discovery interpreted as a conifer trunk,
and although that hypothesis has been disregarded (Retallack
and Landing 2014;Honegger et al. 2018, and references
therein), there is still an ongoing debate concerning their
anity with groups such as red and brown algae, liverworts,
fungi, and lichens put forward as the most possible parent
organisms.
Our geochemical and morphological analyses of the enig-
matic fossil Prototaxites loganii Dawson provide some new re-
sults.
The morphological and anatomical similarities between
the studied Prototaxites and modern rhizomorphs are striking;
both consist of tubular structures of similar length and diam-
eter, fused together into a tissue-like material. Rhizomorphs
are among the most complex organs produced by fungi, and
are the result of a coordinated growth of millions of bun-
dled hyphae. They are root-like structures constituted by a
series of dierentiated tissues, each with a distinctive hyphal
type, orientation, size, and function. The dense tissue-like pe-
ripheral zones of the rhizomorphs correspond to the dark
bands of the Heider quarry Prototaxites and are characterized
by abundant carbonaceous material and poorly crystalline
quartz, whereas the hollow interior of the rhizomorphs cor-
responds to the lighter bands of Prototaxites mineralized by
quartz and lacking carbonaceous material. The lighter parts
of Prototaxites likely represent earlier hollow vessels of the
massive structures that were mineralized by quartz upon
fossilization. The circular centralized structure within some
crystalline cell walls from the Heider quarry sample may rep-
resent cell wall decay products enclosed by quartz precipi-
tated from fluids supersaturated with silica during rapid per-
mineralization, which is common in permineralized cell lu-
mens. Fossilized fungi commonly display a central strand
throughout hyphae, which could be diagenetic owing to
shrinkage of the cell cytoplasm during the fossilization pro-
cess (Ivarsson et al. 2012). We contend that Prototaxites thus
represents giant fossil versions of modern rhizomorphs and

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Can. J. Microbiol. 69: 17–31 (2023) | dx.doi.org/10.1139/cjm-2021-0358 27
Fig. 8. Representative FTIR spectra acquired for Prototaxites fossils from both Bordeaux and Heider quarries. Upper figure: The
IR spectrum was acquired in the transmission mode from a thin section of the Bordeaux quarry fossil, and it is characterized by
aliphatic bands between 3000 and 2800 cm−1and bands corresponding to Si–O vibrational modes of quartz. The box between
3050 and 2750 cm−1was enlarged to highlight the aliphatic bands at 2950 and 2925 cm−1, which correspond to asymmetric
stretching modes of methyl and methylene, respectively. Their intensity was used to calculate the R3/2 ratio to determine the
carbon chain branching. Lower figure: The IR spectrum on the bottom was acquired from the Heider quarry fossil using an
ATR mode. Bands characteristic of organosilicon compounds were obtained, possibly indicating elemental substitution by
incorporation of silicon atoms from the environment during the fossilization process.
that the internal structure and morphology indicate that the
fossils represent a complex growth structure based on fused
hyphae. The Raman results for the Prototaxites sample from
the Bordeaux quarry reveal——based on the fact that the G
and D2 bands are merged into a single band——that this fos-
sil is composed mainly of low-grade organic matter. In con-
trast, the Heider quarry fossil presents a clear separation
between the G and D2 bands. The presence of both bands
has previously been recognized as an indication that the fos-
sil experienced temperatures high enough to partially trans-
form the organic matter into anthracite (Straka and Sýko-
rová 2018), which agrees with the dierent thermal matu-
ration temperatures calculated for each fossil of 128 ±10
and 269 ±14 ◦C, respectively. This could also explain the
results obtained by FTIR for the Heider quarry fossil, which
did not show any remains of aliphatic vibrations containing,
e.g., carbonyl or alkene groups. The spectra showed the pres-
ence of organosilicon compounds, suggesting that this fossil
might have undergone elemental substitution, incorporating
silicon atoms from the hydrothermal environment around
it during its permineralization process. This is further sup-
ported by the lack of contrast in the XRM data. Although the
data do show some small inclusions within the host rock,
the imaging of Prototaxites itself was unsuccessful. This is
likely due to the lack of contrast between the mycelia struc-
tures, surrounding fossilized organic material, and the host
rock.
The Prototaxites sample from Bordeaux quarry, on the other
hand, showed remains of aliphatic compounds, facilitating
the calculation of its R3/2 ratio (0.97 ±0.01). The R3/2 val-
ues reflect the lipidic composition of the membrane cells
of the dierent organisms, possible to tell apart due to the
distinct lipids’ structure (Igisu et al. 2009). Previous stud-
ies on thermal alteration of aliphatic C–H bonds during
simulated diagenesis on extant organisms showed that the
change in both aliphatic CH2and CH3bonds is delayed in
the presence of silica (Igisu et al. 2018). Igisu et al. (2018) also
showed that the R3/2 values of the altered organism did not

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28 Can. J. Microbiol. 69: 17–31 (2023) | dx.doi.org/10.1139/cjm-2021-0358
Fig. 9. Comparison between the R3/2 and I(1350)/I(1600) values for the Prototaxites fossils, eukaryotic and prokaryotic organisms.
(A) R3/2 ratio comparison of obtained values for extant fungi and the Prototaxites fossils (this study), and previously published
values for extant plants, algae, bacteria, and cyanobacteria (Igisu et al. 2009).(B)I(1350)/I(1600) versus R3/2 values of Prototaxites
fossil (purple; this study) and previously published values for fossil eukaryotes (green area; Qu et al. 2015) and fossil prokaryotes
(red area; Igisu et al. 2009;Qu et al. 2015).
Fig. 10. Illustration of the authors’ palaeoenvironmental and morphological interpretation of Prototaxites thriving in a Devo-
nian landscape. It is likely that Prototaxites explored land by foraging through sediment, soil, and aquatic environments.
significantly change when compared to the living organism,
having at most slightly increased. Analogously, despite un-
dergoing diagenesis, the calculated R3/2 value of fossil Pro-
totaxites (0.97 ±0.01) is likely to have remained close to
the living organism, due to a delay caused by the pres-
ence of silica. When considering the ratio calculated for the
extant fungi analysed in this study (average 0.915 ±0.008)
and the values found in the literature for extant plants and

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Can. J. Microbiol. 69: 17–31 (2023) | dx.doi.org/10.1139/cjm-2021-0358 29
bacteria/cyanobacteria (0.41 ±0.09 and 0.69 ±0.03, respec-
tively; Igisu et al. 2009;Bonneville et al. 2020), the similarity
between Prototaxites and fungi is further supported (Fig. 9A).
Moreover, a combination between the I(1350)/I(1600) and
the branching index of aliphatic chains R3/2 was recently used
to dierentiate prokaryotes and eukaryotes, as their cellular
content and structure vary suciently, resulting in a dier-
ent trend between the two groups (Igisu et al. 2009;Qu et al.
2015,2018;Bonneville et al. 2020). When considering both
R3/2 and I(1350)/I(1600) mentioned for the Bordeaux quarry
Prototaxites, and comparing our results with the I(1350)/I
(1600) versus R3/2 signatures of previously reported eukary-
otic (plants from Rhynie chert) and prokaryotic organisms
(cyanobacteria from Bitter Springs and a stromatolite from
Wumishan) (Igisu et al. 2009;Qu et al. 2015,2018), the Proto-
taxites fossil falls clearly outside the range of prokaryotes, be-
ing closer to the eukaryotic range (Fig. 9B). Prototaxites,how-
ever, falls also outside the field indicated for eukaryotes in
Fig. 9B, since the latter is based only on data presently avail-
able for plants. A broader study of eukaryotic organisms, es-
pecially fungal I(1350)/I(1600) versus R3/2 signatures, could
in the future provide more detailed knowledge on Prototax-
ites anity within fungi.
Based on the morphology of Prototaxites and the FTIR data
collected, and combined with results from previous studies
(Boyce et al. 2007), we conclude that the Prototaxites speci-
mens of both Heider quarry and Bordeaux quarry most likely
are fungi. These fungi represent extinct giant forms of vegeta-
tive growth structures, similar to modern complex mycelial
growth forms, such as cords and rhizomorphs. Owing to their
similarity to tree trunks, it has been assumed that Prototax-
ites grew vertically. However, there are no conclusive results
that support this assumption. Furthermore, we were unable
to locate any mention of in situ vertical stumps in the pre-
vious literature, and the variations in diameter from stump
to tip seen in most illustrations cannot be corroborated by
publications except one, where the measured dierence in
diameter from base to tip is just a few decimetres (Hueber
2001). Considering our new data, it is possible that Prototax-
ites had a more varied spatial growth habit and that it was
just as likely to grow horizontally as it was to grow verti-
cally. The functions of both mycelia and rhizomorphs are
both explorational and for translocation of nutrients, wa-
ter, and oxygen from nutrient-rich to nutrient-poor environ-
ments (Yafetto 2018), thus enhancing the survival of fungi in
nutrient-limited settings. It is likely that Prototaxites explored
land by foraging through sediment, soil, and aquatic environ-
ments. It likely grew underground and, on the surface, per-
haps even in the air, but strictly vertical growth on an other-
wise barren or little-vegetated landscape seems unlikely (Fig.
10).
The proximity to areas with volcanic activity and hot
springs might explain the preservation by silica, which is also
supported by the occurrence of coarse-grained volcaniclastic
rocks dated to the early Pragian to early Emsian (Kennedy
et al. 2013). Hydrothermal silica-oversaturated fluids proba-
bly impregnated the Prototaxites rhizomorphs leading to ex-
ceedingly rapid permineralization. This may be the reason
for the high grade of preservation and the maintenance of
the original anatomy down to cellular level. Similar exam-
ples of rapid permineralization preserving exceptionally del-
icate features, such as nuclei and starch grains of osmunda-
ceous ferns and fungal hyphae, are known from hydrother-
mally influenced deposits of southern Sweden (Bomfleur et
al. 2014;McLoughlin and Bomfleur 2016). It is likely that
geothermal springs and associated groundwaters oversatu-
rated by silica could have been of importance for the survival
of these giant fungal growth structures. If local hot springs
acted as havens of nutrients and accessible bioavailable car-
bohydrates in the form of dead or living plants in an other-
wise plant-scarce environment, the growth of large rhizomor-
phic structures could translocate necessary nutrients from
one area to another. Fungi may have mined nutrient-rich ar-
eas for minerals and organic matter to enable growth further
away.
The ability to grow large translocation structures was prob-
ably an advantage for surviving in the sparsely vegetated
Early Devonian ecosystems where nutritional sources were
more scattered than in modern landscapes.
During the Early Devonian, terrestrial vegetation remained
sparse and was represented essentially by herbaceous plants
lacking secondary wood (Gerrienne et al. 2011) but evidence
of fungal–plant interactions extend back to the Early Devo-
nian, Rhynie chert (Strullu-Derrien et al. 2015,2018).
At that time, the population of living plants and accumula-
tions of dead plant matter available for fungal decomposition
was meagre comparable to our modern world.
However, local areas of flourishing vegetation were prob-
ably sucient to support the massive growth structures of
Prototaxites, and perhaps their size enabled necessary water
and nutritional translocations from nutrient-rich to nutrient-
poor areas. In modern rhizomorphs, the function of the cen-
tral cavity is mostly for translocating oxygen. To grow such a
massive fungal structure as Prototaxites, high amounts of oxy-
gen would have been required, suggesting that the core re-
gion of Prototaxites supplied the tissues with oxygen in the
dysaerobic subsurface to form an early terrestrial aeration
system. Thus, Prototaxites may have played a pivotal role in
Early Devonian ecosystems by connecting nodes of vegeta-
tion, aerating subsurface tissues, and translocating key nutri-
ents, through soils of an otherwise sparsely vegetated planet.
Article information
History dates
Received: 16 November 2021
Revised: 22 September 2022
Accepted: 20 October 2022
Accepted manuscript online: 13 December 2022
Version of record online: 13 December 2022
Copyright
© 2022 The Author(s). This work is licensed under a Creative
Commons Attribution 4.0 International License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduc-
tion in any medium, provided the original author(s) and
source are credited.

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30 Can. J. Microbiol. 69: 17–31 (2023) | dx.doi.org/10.1139/cjm-2021-0358
Data availability
The specimens are part of the paleobotanical collections
hosted at the Swedish Museum of Natural History (NRM). Data
generated or analysed during this study are available from
the corresponding author upon reasonable request.
Author information
Author ORCIDs
Vivi Vajda https://orcid.org/0000-0003-2987-5559
Ashley Krüger https://orcid.org/0000-0003-1196-8693
Author notes
This paper is part of a collection entitled Astrobiology.
Author contributions
Vivi Vajda: conceptualization, data curation, formal analy-
sis, funding acquisition, investigation, methodology, project
administration, writing – original draft, writing – review &
editing; Larissa Lopes Cavalcante: data curation, formal anal-
ysis, investigation, methodology, validation, writing – orig-
inal draft, writing – review & editing; Kristoer Palmgren:
formal analysis, investigation, methodology, writing – review
& editing; Ashley Krüger: data curation methodology, visual-
ization, writing – original draft, writing – review & editing;
Magnus Ivarsson: conceptualization, formal analysis, inves-
tigation, methodology, project administration, supervision,
writing – original draft, writing – review & editing.
Competing interests
The authors declare there are no competing interests.
Funding information
This research was supported by the Swedish Research Coun-
cil grant VR 2019-4061 (V.V.) and the Knut and Alice Wallen-
berg Foundation grant KAW 2020.0145 (V.V.). XRM scanning
data acquisition was supported by a grant to the Stockholm
University Brain Imaging Centre (SU FV-5.1.2-1035-15).
Supplementary material
Supplementary data are available with the article at https:
//doi.org/10.1139/cjm-2021-0358.
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