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Citation for the original published paper (version of record):
Bengtson, S., Ivarsson, M., Astolfo, A., Belivanova, V., Broman, C. et al. (2014)
Deep-biosphere consortium of fungi and prokaryotes in Eocene sub-seafloor basalts..
Geobiology, 12(6): 489-496
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Geobiology (2014), 12, 489–496
DOI: 10.1111/gbi.12100
Deep-biosphere consortium of fungi and prokaryotes in
Eocene subseafloor basalts
S. BENGTSON,1 M. IVARSSON,1 A. ASTOLFO,2 V. BELIVANOVA,1 C. BROMAN,3
F. MARONE2 AND M. STAMPANONI2,4
1
Department of Palaeobiology and Nordic Center for Earth Evolution, Swedish Museum of Natural History, Stockholm,
Sweden
2
Swiss Light Source, Paul Scherrer Institute, Villigen, Switzerland
3
Department of Geological Sciences, Stockholm University, Stockholm, Sweden
4
Institute for Biomedical Engineering, University and ETH Z€
urich, Z€
urich, Switzerland
ABSTRACT
The deep biosphere of the subseafloor crust is believed to contain a significant part of Earth’s biomass, but
because of the difficulties of directly observing the living organisms, its composition and ecology are poorly
known. We report here a consortium of fossilized prokaryotic and eukaryotic micro-organisms, occupying
cavities in deep-drilled vesicular basalt from the Emperor Seamounts, Pacific Ocean, 67.5 m below seafloor
(mbsf). Fungal hyphae provide the framework on which prokaryote-like organisms are suspended like cobwebs and iron-oxidizing bacteria form microstromatolites (Frutexites). The spatial inter-relationships show
that the organisms were living at the same time in an integrated fashion, suggesting symbiotic interdependence. The community is contemporaneous with secondary mineralizations of calcite partly filling the
cavities. The fungal hyphae frequently extend into the calcite, indicating that they were able to bore into
the substrate through mineral dissolution. A symbiotic relationship with chemoautotrophs, as inferred for
the observed consortium, may be a pre-requisite for the eukaryotic colonization of crustal rocks. Fossils
thus open a window to the extant as well as the ancient deep biosphere.
Received 4 May 2014; accepted 13 August 2014
Corresponding authors: S. Bengtson, Tel.: +46 8 5195 4220; e-mail: [email protected]
and M. Ivarsson, Tel.: +46 8 5195 4188; e-mail: [email protected]
INTRODUCTION
The oceanic crust makes up the largest potential habitat
for life on Earth (Schrenk et al., 2009). Yet little is known
about the abundance, diversity, and ecology of its biota.
Considering that deep-sea sediments are estimated to contain the Earth’s largest proportion of micro-organisms
(Parkes et al., 1994; Whitman et al., 1998), we should
expect a significant biosphere to be hosted in deep crustal
environments as well. Textural alterations of glassy basaltic
rocks and cryptoendoliths in basalts (Schumann et al.,
2004; Furnes et al., 2008; Ivarsson et al., 2008; Peckmann
et al., 2008; Eickmann et al., 2009), and genomic investigations of drill cores in basalts (Lever et al., 2013) suggest
ongoing microbial interactions, but direct observations of
the living micro-organisms are mostly beyond our reach,
and the biochemical pathways enabling life in these
extreme environments are largely unknown. Neither is it
known to what degree the subsurface environments are
sufficiently stable to allow the establishment of mutualistic
symbiotic relationships, important to sustain an efficient
circulation of nutrients. We report here the discovery of a
fossil microbial community consisting of eukaryotic heterotrophs (fungi) and prokaryotic putative chemoautotrophs
living in a spatially organized association deep in submarine
vesicular basalts.
Fossilized mycelial micro-organisms in subseafloor basalts have been interpreted as remnants of fungi (Schumann
et al., 2004; Reitner et al., 2006; Peckmann et al., 2008;
Eickmann et al., 2009; Ivarsson, 2012; Ivarsson et al.,
2012, 2013). The ability of fungi to live in such extreme
environments is corroborated by discoveries of viable yeast
© 2014 The Authors. Geobiology Published by John Wiley & Sons Ltd.
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reproduction in any medium, provided the original work is properly cited.
489
490
S. BENGTSON et al.
cells deep in continental crust (Ekendahl et al., 2003) and
of active fungal genes in deep-sea sediments at various
depths below the seafloor (Orsi et al., 2013a,b). These
occurrences raise several questions regarding metabolism
and ecology of fungal communities in subseafloor basalts.
All known fungal species are heterotrophs, and it is questionable whether abiotic sources or flow through basalt are
able to ensure a steady supply of accessible organic compounds over geological time. The alternative is a biological
source of organic compounds provided by chemoautotrophic communities. At hydrothermal vents eukaryotes are
known to live in symbiotic relationships with chemoautotrophs (Cavanaugh et al., 2006), and fungi are known
from such environments (Connell et al., 2009). Fungi are
notable for entering into symbiotic relationships, although
such a relationship with chemosynthesizers has not yet
been demonstrated in living forms. Symbiosis with chemoautotrophs would enable fungi to populate and thrive in
subseafloor settings and expand their ecological niches.
MATERIAL AND METHODS
The Emperor Seamounts in the Pacific Ocean is a submarine chain of seamounts formed by hotspot volcanism
between ~81 and ~43 Ma. The seamounts stretch for
about 5000 km in a north–south trend. At ~43 Ma, the
direction changes to a northwest–southeast trend, and the
Hawaiian Islands continue to the Recent. The ages
increase, in a classical hotspot fashion, away from the active
hotspot in the southeast called Loihi Seamount.
During Ocean Drilling Program (ODP) Leg 197, three
seamounts belonging to the Emperor Seamounts were
drilled: Detroit Seamount (~81 Ma), Nintoku Seamount
(~56 Ma), and Koko Seamount (~48 Ma). Sites 1203 and
1204 were drilled at Detroit Seamount, Site 1205 at Nintoku Seamount, and Site 1206 at Koko Seamount (Tarduno
et al., 2002). The material described in this study is from
sample ODP 197-1206A- 4R-2, 0, Koko Seamount, from a
depth of 67.5 m below seafloor (Ivarsson et al., 2013).
Areas with fossils were localized with light microscopy in
drill core pieces. Selected areas were reduced to cubes of
~1 9 1 cm in diameter by sawing. The reduced pieces
were further investigated with optical microscopy, environmental scanning electron microscopy (ESEM), energy dispersive spectrometry (EDS), synchrotron radiation X-ray
tomographic microscopy (SRXTM), Raman spectroscopy,
and stable isotope mass spectrometry. The investigated
material is deposited in the Palaeobiology collections of
the Swedish Museum of Natural History (X5311).
Optical microscopy
Light microscopy and imaging were performed with a Nikon
SMZ1500 microscope fitted with a Nikon D80 camera.
Environmental scanning electron microscopy
A Philips XL30 environmental scanning electron microscope (ESEM) with a field emission gun (XL30 ESEMFEG) was used to analyze the minerals and fossils. The
ESEM was equipped with an Oxford X-act energy dispersive spectrometer (EDS), backscatter electron detector
(BSE), and a secondary electron detector (SE). The acceleration voltage was 20 or 15 kV depending on the nature
of the sample, and the instrument was calibrated with a
cobalt standard. Peak and element analyses were made
using INCA Suite 4.11 software. The samples were analyzed in low vacuum to avoid charging effects at the surface and thus were not coated, with neither C nor Au, to
enable EDS analyzes of C.
Synchrotron radiation X-ray tomographic microscopy
(SRXTM)
The samples were mounted on a 3 mm-wide brass peg.
SRXTM was performed at the TOMCAT beamline at the
Swiss Light Source at the Paul Scherrer Institute. The Xray energy was set to 41 keV for maximum penetration.
First, for each sample, a fast overview scan (not shown) at
low magnification but capturing the entire object was
acquired to identify the coordinates of the regions of interest. With this information, selected 1.6 9 1.6 9 1.6 mm3
volumes within the larger 1 9 1 9 1 cm3 specimens were
then imaged at higher resolution (local tomography). For
the results presented here, 1501 projections were acquired
equiangularly over 180°, online post-processed and rearranged into flat- and darkfield-corrected sinograms. Reconstruction was performed on a Linux PC farm using highly
optimized routines based on the Fourier Transform
method (Marone & Stampanoni, 2012). To mitigate
image artifacts occurring as a consequence of the strong
incompleteness of these datasets captured in local tomography geometry and strongly affecting the quality of the
results, corrected sinograms were constant padded prior to
reconstruction (Marone et al., 2010). Slice data derived
from the scans were then analyzed and rendered using Avizo software. With the 109 lens used, the resulting voxel
size was 0.65 lm.
Raman spectroscopy
The instrument used was a confocal laser Raman spectrometer (LabRAM HR 800; Horiba Jobin Yvon, Villeneuve
d’Ascq, France), equipped with a multichannel air-cooled
charge-coupled device detector. Excitation was provided by
an Ar-ion laser (k = 514 nm) source. The laser power at
the sample surface was 8 mW. The instrument was incorporated with an Olympus BX51 microscope. The laser
beam was focused through an 809 objective with a
© 2014 The Authors. Geobiology Published by John Wiley & Sons Ltd.
Deep-biosphere consortium
491
some of the vugs, the basaltic walls are entirely covered by
fossil films, whereas in some the fossils form isolated
patches. The assemblage consists of a basal film from which
filaments arise to form mycelium-like networks, which in
turn are overgrown by cobweb-like structures and microstromatolite. The assemblage co-occurs with secondary calcite in the voids. Stable isotope measurements of the
calcite, with d13C ranging between 1.0 and 1.5& and
d18O ranging between 2.7 and 3.6&, give a seawater signal without evidence of influence from biological processes,
which would be expected to produce more negative d13C
values (Table S1). There is no indication of recrystallization that might have obliterated the original isotopic composition (Walter et al., 2007). The fossils are mineralized
by iron oxides and montmorillonite (Figs S1–S4). The
montmorillonite is not visible in the SRXTM images,
resulting in apparent morphological differences between
SEM (Fig. 1C) and SRXTM (Fig. 1D) renderings.
We will refer to the filaments as hyphae and to the networks as mycelia. These terms are typically applied to fungi
and actinobacteria and may be understood in a non-systematic, descriptive sense. However, Ivarsson et al. (2012)
demonstrated the fungal nature of similar thin hyphae in
working distance of 8 mm; the resulting analyzed spot size
was about 1 lm. The spectral resolution was ~0.3 cm 1.
The accuracy of the instrument was controlled by repeated
use of a silicon wafer with a Raman line at 520.7 cm 1.
The Raman spectra were obtained with LabSpec 5 software.
Stable isotopes
For stable isotope analysis, calcite crystals were sampled
from six different vesicles with varying amounts of fossils
present. 0.2 mg carbonate was flushed with helium gas in
a septum-seal glass vial. 100 ll of 99% H3PO4 was added
to each sample for reacting to CO2. For the analyses of
stable isotope signatures, a GasbenchII coupled to a Finnigan MAT 252 mass spectrometer was used. Based on these
measurements, the reproducibility was calculated to be better than 0.07& for d13C and 0.15& for d18O.
RESULTS
The microbial assemblage investigated here occurs in open
or partly open vugs in the vesicular basalt (Fig. 1A). In
1 mm
A
B
100 µm
hy
Fx
bf
cw
ca
ca
ba
hy
B
ba
Fig. 1 Fungal–prokaryote colony in vesicular
basalt, Koko Seamount, 67.5 m.b.s.f. (A) Light
micrograph of connected vesicles. (B) Closeup of mycelium in A. Broken hyphae show
brown hematite core inside light crust of
montmorillonite. (C) ESEM micrograph of
mycelium and ‘cobwebs’. (D) SRXTM
isosurface rendering of the same region as in
C, bringing out the thin ferruginous cores
rather than the thick montmorillonite crust. (E)
SRXTM combined volume and surface
rendering of mycelium and ‘cobwebs’ at
calcite–void interface. (F) SRXTM isosurface
rendering of dog-tooth calcite enveloped, and
partly penetrated, by hyphae. (G, H) SRXTM
volume renderings of hyphae and ‘cobwebs’.
Arrows point to examples of alignment of the
minute bodies. (I) ESEM micrograph of
hyphae, ‘cobwebs’, and Frutexites. Hyphae
and
‘cobwebs’
are
encrusted
with
montmorillonite, Frutexites is not (cf. B).
Legend (for all figures): ba, basalt; bf, basal
film; ca, calcite; chy, coarse hypha; cw,
‘cobweb’; Fx, Frutexites; gl, globular feature;
hy, hypha; my, mycelium; vo, void.
cw
my
C
D
cw
cw
E
cw
hy
ca
hy
hy
ca
bf
bf
100 µm
100 µm
F
100 µm
100 µm
G
H
10 µm
I
Fx
10 µm
bf
10 µm
hy
ca
cw
cw
© 2014 The Authors. Geobiology Published by John Wiley & Sons Ltd.
hy
492
S. BENGTSON et al.
D,E, S3). Thicker portions are expressed as hematite cylinders with a less-dense (less X-ray attenuating) core (Fig. 3A),
whereas thinner ones appear as single hematite strands with
no resolvable core (Movie S1). Near the basal film, the
hyphae are mostly slender; further from the film they
thicken, branch, and anastomose to form a mycelium
(Fig. 1B–F) and frequently penetrate the secondary calcite
(Figs 1E, 3D, Movie S1). Where they meet calcite surfaces
they either (i) creep along the surface of the mineral, (ii)
enter crevices in the mineral, or (iii) penetrate the mineral,
forming deep galleries (Fig. 1E,F, Movie S1).
equivalent rocks, and the new data reported in the present
paper are consistent with a fungal affinity of the structures.
Basal film
The basal film covers the basalt interfaces with the voids and
with the secondary calcite but is nowhere seen to extend
over the surfaces of the calcite (Fig. 2A,E, Movie S1). Frequently, it is cracked up and partly flaked off from the basaltic substrate, leaving a space beneath (Figs 1A, 2A–C, 3A).
Such detachment occurs both in the voids and in the calcite
(Fig. 2A, upper and lower part, respectively). The film consists of a basal hematite layer with carbon, an upper hematite
layer, and a montmorillonite crust (Fig. S2). The montmorillonite and upper hematite layers peter out where the film
faces the calcite, leaving only the hematite–carbon layer.
Morphologically, there is also a distinct difference between
the parts of the films that are covered by calcite and those
that are exposed to the voids. In the exposed parts, thin
hyphae 2–10 lm in diameter emerge from the film to form
mycelia filling up part of the voids and entering the calcite
through its interface with the voids. These thin hyphae are
missing where the basal film is covered by calcite (Movie
S1). In both the exposed and calcite-covered parts, the
surface of the film is smooth or forms hemispherical to
cylindrical protrusions (Fig. 2C) that may extend into a
mycelium-like network with coarse hyphae (Fig. 2E).
Coarse hyphae
The protrusions emanating from the basal film are about
20–40 lm in width. Mostly they are hemispherical in
shape and form a botryoidal texture on the surface of the
film (Fig. 2C, lower part), but occasionally they protrude
as coarse hyphae (‘chy’ in Fig. 2). Where the film is
exposed to the void these are usually short, up to about
100 lm, and sometimes have a globular termination
(Fig. 2A–C), about twice the diameter of the supporting
hypha. Where the calcite abuts the film, the coarse hyphae
may grow to produce a mycelial network within the calcite
(Fig. 2E, 3B). These coarse hyphae have a dense core, similar to the hyphae of the main mycelial network, but the
main volume consists of a thick outer sheath, slightly less
X-ray attenuating than the surrounding calcite. They frequently form globular bodies, up to 60 lm in diameter,
within the calcite (Fig. 2D, Movie S1).
Hyphae
The thin hyphae emerging from the exposed basal film are
2–10 lm in diameter and up to several millimeters in length
(Figs 1, 2, 3A,D). They consist of a dense X-ray-attenuating
central part of hematite with small amounts of carbon,
encrusted by X-ray-transparent montmorillonite (Figs 1B–
A
100 µm
vo
cw
cw
Sheets of montmorillonite between hyphae become transparent in SRXTM and are seen to contain minute bodies,
10 µm
hy
gl
chy
hy
B
‘Cobwebs’
100 µm
C
gl
chy
cw
chy
gl
hy
bf
bf
bf
ba
D
10 µm
E
ca
gl
bf
ca
chy
chy
ca
ba
chy
vo
bf
100 µm
Fig. 2 Fungal–prokaryote colony, features of basal film. (A) SRXTM slice through region with basal film over basalt meeting both void and calcite. (B) SRXTM
volume rendering of part of the region represented by A (globular feature ‘gl’ is the same object in both). (C) SRXTM isosurface rendering of basal film with
sporophore-like object. (D) SRXTM slices (two slices at 90° angle to each other, intersection marked by vertical line) through coarse hypha connected with
large globular feature. (E) SRXTM slice showing basal film over basalt under calcite and coarse hyphae in calcite. Legend as in Fig. 1. SRXTM slices negative
(lighter tones represent higher X-ray attenuation).
© 2014 The Authors. Geobiology Published by John Wiley & Sons Ltd.
Deep-biosphere consortium
1–5 lm in size, commonly aligned in strings (Figs 1G,H,
3D; S1). These strings are suspended within planes
determined by the supporting hyphae, forming cobweblike structures (Figs 1B–I, 2A–C, 3A,D). The ‘cobwebs’
only occur where the supporting hyphae grow in the voids
and in crevices in the invaded crystals, not where the
hyphae form galleries in the crystals (Fig. 1E). Element
analyses by EDS of less-heavily encrusted strings indicate
that the bodies are preserved in iron oxides (Fig. S1).
A
cw
493
100 µm
vo
ba
hy
hy
Fx
Frutexites
Closely associated with the hyphae and ‘cobwebs’ is another
type of structure: fractally branching, cauliflower-like bodies
with a distinct direction of growth (Figs 1B,I, 3). The material is strongly X-ray-attenuating owing to a mainly goethite
composition with local transitions to poorly crystallized
hematite (Fig. S4). The major branches may reach 100 lm
in width and more than a millimeter in length. The internal
structure shows evidence of lamination suggesting successive
layers of growth (Fig. 3C). In some instances, the initial part
is attached to a hypha from the void-filling mycelial network
(Fig. 3D, coarse arrows), and occasionally hyphae are
encrusted by later growth stages (Fig. 3A).
The cauliflower-like bodies frequently penetrate the secondary calcite (Fig. 3B), and in almost all of these
instances the apparent direction of growth is into the mineral (Fig. 3B; Movie S1), indicating that, like the void-filling hyphal networks, these structures grew into the
mineral, dissolving it along the way, rather than being diagenetically embedded by the mineral. The growth direction
into the calcite may be explained by the fact that growth
starts on a substrate in the void, such as mycelial hyphae.
These fractally branching bodies are morphologically
identical to the microstromatolitic structures known as
Frutexites (Maslov, 1960), characteristic of cryptic habitats
and hydrothermal vents through the Proterozoic and
Phanerozoic (Walter & Awramik, 1979; Myrow & Coniglio, 1991; B€
ohm & Brachert, 1993; Rodrıguez-Martınez
et al., 2011). We will refer to them under this name.
bf
ca
B
100 µm C
Fx
chy
vo
ca
D
hy
100 µm
vo
cw
Fx
ca
ca
DISCUSSION
Temporal relationships between the fossil structures
To understand the assemblage of fossils in the vesicles, it is
first necessary to establish their temporal relationships
between each other and to the secondary calcite in the voids.
The following observations are significant in this regard:
1 The basal film covers the basalt interfaces with both
voids and calcite, but it never covers the calcite interface with the voids.
2 The thin hyphae emanate from the basal film, but only
in the voids, not in the calcite.
© 2014 The Authors. Geobiology Published by John Wiley & Sons Ltd.
10 µm
hy
ba
bf
Fig. 3 Fungal–prokaryote colony, features of Frutexites microstromatolites.
(A) SRXTM slice through Frutexites with associated hyphae and ‘cobwebs’.
(B) SRXTM slice through Frutexites partly within calcite. (C) SRXTM volume
rendering of Frutexites, sectioned to show internal lamination. (D) SRXTM
volume rendering (red/cyan stereo anaglyph) of Frutexites with associated
hyphae, basal film, and ‘cobwebs’, showing mutual relationships among
organisms and with surrounding basalt, calcite, and void. Coarse arrows point
to basal attachment of Frutexites to fungal hyphae. Legend as in Fig. 1.
494
S. BENGTSON et al.
3 There are further morphological differences in the film
at the two types of interface: shorter coarse hyphae
with globular terminations occur in the voids, networks of coarse hyphae with large globules characterize the calcite.
4 The thin hyphae adapt themselves to the calcite surfaces but can also bore into the calcite.
5 The ‘cobwebs’ are suspended on the thin hyphae in
the voids and where the hyphae rest in crevices in the
calcite, but they never occur on the hyphae making
boring galleries into the calcite.
6 Frutexites is attached to hyphae in the voids and often
penetrates the calcite but is not seen to displace
hyphae when growing into voids.
7 Even when forming a dense mycelium, hyphae do not
crowd around Frutexites or become engulfed by it.
These observations suggest that the basal film became
established on the basalt surfaces before the growth of the
calcite. It may have become partly smothered by the calcite,
but continued to grow and form a coarse hyphal network.
Where not covered by calcite it produced short protrusions
ending in bulbous terminations as well as thinner hyphae
forming a mycelium. These hyphae mainly grew in the voids,
but could also penetrate the calcite and form a network
within it. The ‘cobwebs’ suspended themselves on the
hyphae in the voids and followed the live hyphae as far as
they could into crevices in the calcite but not where the
hyphae bored into the calcite. Frutexites attached itself on
the hyphae in the voids and grew to considerable volume
but without disturbing or engulfing the hyphal network.
Thus, we propose that the organisms lived at the same
time, during the precipitation of the calcite. The hyphae
grew and penetrated the mineral after it was formed, rather
than having become embedded in growing crystals. These
observations provide a time constraint for the calcite, showing that it was formed after the film was laid down in the
voids but prior to the development of complex, void-filling,
calcite-boring mycelia. Together with the montmorillonite
preservation, this indicates that the colonization and subsequent rapid fossilization occurred in an aquatic environment
of ambient or higher temperatures, which coincides with the
late stages of seawater–rock interactions. The basalts were
subject to seawater alteration during 1–2 million years while
the sediment pile built up and finally isolated the volcanic
section (Revillon et al., 2007; Ivarsson et al., 2013), which
provides the time window for the colonization to occur.
Nature of the organisms
Previous work has established thin mycelial structures from
these rocks as fungal hyphae based on characteristic fungal
morphologies like repetitive septa, anastomoses between
branches, a central strand, and the presence of chitin,
which together suggest affinity to the Dikarya (Ivarsson
et al., 2012, 2013). Because the hyphae seem to emanate
from the basal film and become wider with increasing distance from the film, we tentatively interpret the film and
its coarser protrusions to belong to the same organism as
the hyphae. The short coarse hyphae with globular terminations (Fig. 2A–C) may represent sporophores, but the
lack of internal structures does not allow determination of
which, if any, dikaryan sporophore type is represented. The
larger globular features connected to coarse hyphae within
the calcite may also be related to reproduction, but as
these hyphae appear to have been actively boring into the
calcite (see below), a function to propagate spores into the
surrounding medium seems less likely.
The regular alignment in strings of the minute bodies in
the ‘cobwebs’ indicates that the bodies are not accidentally
trapped particles but belong to an organized structure.
The size, morphology, and organization of the ‘cobwebs’
are concordant with prokaryotic cells, and the organized
association between the ‘cobwebs’ and the fungal hyphae
suggests a symbiotic relationship between the two. The
organization and placement of the ‘cobwebs’ would be
consistent with micro-organisms that utilize elements or
compounds dissolved or in suspension. The ‘cobweb’
structure is similar to that of the sulfur-oxidizing archaea
Pyrodictium (Rieger et al., 1995) and Euryarchaeon SM1
(Moissl et al., 2003), frequent in hydrothermal environments, as well as sulfur-oxidizing bacteria living on sulfidic
sediments (Fenchel & Glud, 1998; Thar & K€
uhl, 2001).
Lack of S in EDS analysis speaks against the presence of
sulfur oxidizers, however. As the putative cells in the ‘cobwebs’ consist of iron oxides, there is a possibility that the
organisms mediated iron oxidation metabolically, but the
composition might also be due to diagenesis, as suggested
by the similar composition of the fungal hyphae. The lack
of sulfides and the presence of iron oxides in the samples
exclude strictly anaerobic conditions.
The nature of Frutexites has been the subject of discussion in the literature (reviewed by Rodrıguez-Martınez
et al., 2011). Most interpretations have landed in a bacterial origin, but non-biogenic chemical precipitation has also
been entertained as an alternative. This question reflects
the well-known problem of how to distinguish biogenic
stromatolites from non-biogenic precipitates (Grotzinger
& Rothman, 1996). We interpret the Koko Seamount Frutexites to be bacterial in origin, for three main reasons:
1 Recent formation of Frutexites-like structures in subterranean rocks has been shown to be associated with
the chemolithotrophic iron-oxidizing bacteria Gallionella ferruginea (Rodrıguez-Martınez et al., 2011).
2 Chafetz & Guidry (1999), studying precipitates in
hydrothermal environments, found a morphological
and structural continuum from bacterially mediated
‘shrubs’ to abiotically precipitated dendrites. The
© 2014 The Authors. Geobiology Published by John Wiley & Sons Ltd.
Deep-biosphere consortium
structures at the biological end of the spectrum are
morphologically identical to Frutexites and include
goethite-rich structures with well-preserved bacterial
fossils. ‘Shrubs’ at the non-biogenic end are dominated by crystal shapes and differ substantially from
the bacterially induced ones.
3 The Koko Seamount Frutexites are dominated by goethite, whereas the basal films and hyphae attributed to
fungi mainly consist of hematite of likely diagenetic origin.
We therefore interpret the Koko Seamount Frutexites as
ferruginous microstromatolites formed by iron-oxidizing
bacteria.
495
A decade ago Baross et al. (2004) pointed out that even
after 40 years of scientifically coordinated drilling in the
oceanic basement ‘. . . so far, we have not been able to
visualize the microbial communities in their specific biotopes within the crust . . .’. Today, by detailed studies of
the fossil record and with the aim of powerful 3D techniques, we are able to visualize this hidden biosphere. Fossil data provide an essential tool to understand the ecology
and diversity of subseafloor biotopes that we cannot yet
observe in vivo, as well as a window onto the ancient history of the deep biosphere.
ACKNOWLEDGMENTS
Symbiotic relationships
We propose that three separate organisms lived simultaneously in physical contact in the voids in the Eocene basalt:
the fungi (basal film with protuberances and mycelial network), the prokaryotic cells suspended like cobwebs
between the fungal hyphae, and the iron-oxidizing bacteria
forming the microstromatolites. From a metabolic point of
view, we suggest a symbiosis between chemoautotrophic
prokaryotes and heterotrophic fungi in the deep crustal
environment would be functional. The role of the chemoautotrophic symbionts in such an arrangement could be to
utilize inorganic carbon such as CO2 and through oxidation
of reduced compounds transform it into organic molecules
accessible to the eukaryotic host. The fungal production of
CO2 could be utilized by the chemosynthetic community,
although it is not clear whether CO2 would have been limiting in this environment even without the presence of fungi.
Symbiosis between prokaryotic and eukaryotic cells is a
globally important phenomenon that influences the physiology, ecology, and evolution of virtually every organism
on Earth. Eukaryotic hosts expand their ecological niches
through symbiosis with metabolically diverse bacteria and
archaea (Stewart et al., 2005). Establishment of sustainable
eukaryotic colonies in a challenging environment such as
that of subseafloor basalts would probably be impossible
without chemosynthetic symbionts serving as sources of
accessible metabolites like carbohydrates.
When the basaltic vesicles were formed in molten
magma, they were sterile. A pre-requisite for colonization
by prokaryotes and fungi and for preservation of the habitat
is that the vesicles are connected to the overlying seawater
column and circulating brines by fractures, veins, pores, or
chains of adjacent vesicles. The club-shaped sporophore-like
structures of the fungi (Fig. 2) probably represent a means
of ensuring that propagules are released into the circulating
water to enable their distribution within the available habitat of connected vesicles. In view of the likely sporadic
interconnectedness of the deep habitats in crustal rocks, a
considerable diversity of biotopes is to be expected.
© 2014 The Authors. Geobiology Published by John Wiley & Sons Ltd.
We thank S. Ohlsson (Swedish Museum of Natural History) for laboratory assistance, M. Ahlbom and H. Siegmund (Stockholm University) for assistance with ESEM/
EDS analysis and isotope analysis, respectively, S. Lindblom (Stockholm University) for providing the samples, and
M. Rosing (University of Copenhagen) for valuable discussion. We acknowledge funding from the Swedish Research
Council (Contracts No. 2010-3929 and 2012-4364), Danish National Research Foundation (DNRF53), and Paul
Scherrer Institute (20130185).
AUTHOR CONTRIBUTIONS
M.I. and S.B. conducted the design and analyses and wrote
the paper. M.I. prepared the samples and acquired ESEM/
EDS data. S.B., A.A., V.B., F.M., and M.S. performed
SRXTM measurements. C.B. acquired Raman spectroscopic data. S.B. prepared light micrographs and
tomography renderings.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the
online version of this article:
Fig. S1 EDS spectra of a non-encrusted part of a ‘cobweb’ structure with
accompanying ESEM image.
Fig. S2 Raman spectra of three layers on a cavity wall.
Fig. S3 Raman spectra of a hypha core and a hypha sheath.
Fig. S4 Raman spectra of Frutexites microstromatolites on fungal hyphae.
Table S1. d13C and d18O values for six different euhedral calcite crystals
from three different vugs.
Movie S1. Sequence of orthoslices through SRXTM voxel stack.
© 2014 The Authors. Geobiology Published by John Wiley & Sons Ltd.