geobiology, 12(6): 489-496 bengtson, s., ivarsson, m., astolfo, a.,...

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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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Deep-biosphere consortium of fungi and prokaryotes inEocene subseafloor basaltsS . BENGTSON,1 M. IVARSSON,1 A. ASTOLFO,2 V . BELIVANOVA,1 C. BROMAN,3

F . MARONE2 AND M. STAMPANONI2 , 4

1Department of Palaeobiology and Nordic Center for Earth Evolution, Swedish Museum of Natural History, Stockholm,

Sweden2Swiss Light Source, Paul Scherrer Institute, Villigen, Switzerland3Department of Geological Sciences, Stockholm University, Stockholm, Sweden4Institute 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 cob-

webs 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 interdepen-

dence. 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 con-

tain 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 investi-

gations 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 hetero-

trophs (fungi) and prokaryotic putative chemoautotrophs

living in a spatially organized association deep in submarine

vesicular basalts.

Fossilized mycelial micro-organisms in subseafloor bas-

alts 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. 489This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution andreproduction in any medium, provided the original work is properly cited.

Geobiology (2014), 12, 489–496 DOI: 10.1111/gbi.12100

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 ques-

tionable whether abiotic sources or flow through basalt are

able to ensure a steady supply of accessible organic com-

pounds over geological time. The alternative is a biological

source of organic compounds provided by chemoautotro-

phic communities. At hydrothermal vents eukaryotes are

known to live in symbiotic relationships with chemoauto-

trophs (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 chemo-

autotrophs 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 submar-

ine 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 Nin-

toku 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, environ-

mental scanning electron microscopy (ESEM), energy dis-

persive 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 micro-

scope (ESEM) with a field emission gun (XL30 ESEM-

FEG) was used to analyze the minerals and fossils. The

ESEM was equipped with an Oxford X-act energy disper-

sive spectrometer (EDS), backscatter electron detector

(BSE), and a secondary electron detector (SE). The accel-

eration 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 ana-

lyzed in low vacuum to avoid charging effects at the sur-

face 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 X-

ray 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 inter-

est. 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 rear-

ranged into flat- and darkfield-corrected sinograms. Recon-

struction 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 tomogra-

phy 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 Avi-

zo software. With the 109 lens used, the resulting voxel

size was 0.65 lm.

Raman spectroscopy

The instrument used was a confocal laser Raman spectrom-

eter (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 incor-

porated 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.

490 S. BENGTSON et al.

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 soft-

ware.

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 Finni-

gan MAT 252 mass spectrometer was used. Based on these

measurements, the reproducibility was calculated to be bet-

ter 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

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 micro-

stromatolite. The assemblage co-occurs with secondary cal-

cite 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 sig-

nal without evidence of influence from biological processes,

which would be expected to produce more negative d13Cvalues (Table S1). There is no indication of recrystalliza-

tion that might have obliterated the original isotopic com-

position (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 net-

works as mycelia. These terms are typically applied to fungi

and actinobacteria and may be understood in a non-sys-

tematic, descriptive sense. However, Ivarsson et al. (2012)

demonstrated the fungal nature of similar thin hyphae in

A

C D

F

E

G H I

B

B

1 mm 100 µm

100 µm 100 µm

10 µm100 µm 10 µm

10 µm

100 µm

bf

bf

bf

bf

hy

hy

hy

hy

hy

hy

hy

cw

cw

cw cw

cw

cw

cw

ca

Fx

Fx

baba

ca

ca

ca

ca

my

Fig. 1 Fungal–prokaryote colony in vesicular

basalt, Koko Seamount, 67.5 m.b.s.f. (A) Light

micrograph of connected vesicles. (B) Close-

up 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.


Deep-biosphere consortium 491

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). Fre-

quently, it is cracked up and partly flaked off from the basal-

tic 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 con-

sists of a basal hematite layer with carbon, an upper hematite

layer, and a montmorillonite crust (Fig. S2). The montmo-

rillonite 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).

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–

D,E, S3). Thicker portions are expressed as hematite cylin-

ders 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).

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, sim-

ilar 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 fre-

quently form globular bodies, up to 60 lm in diameter,

within the calcite (Fig. 2D, Movie S1).

‘Cobwebs’

Sheets of montmorillonite between hyphae become trans-

parent in SRXTM and are seen to contain minute bodies,

CBA

ED

100 µm10 µm

10 µm

100 µm

100 µm

cwcw

ca

ca

ca

vo

vo

ba

chychy

chy

chy chy

chyba

bf

bf

bf

bf

bf

cw hy hy

hy

gl

gl

gl

gl

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).



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 cobweb-

like 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).

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 mate-

rial 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 lmin 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 sec-

ondary calcite (Fig. 3B), and in almost all of these

instances the apparent direction of growth is into the min-

eral (Fig. 3B; Movie S1), indicating that, like the void-fill-

ing hyphal networks, these structures grew into the

mineral, dissolving it along the way, rather than being dia-

genetically 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 & Coni-

glio, 1991; B€ohm & Brachert, 1993; Rodr�ıguez-Mart�ınez

et al., 2011). We will refer to them under this name.

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 inter-

face with the voids.

2 The thin hyphae emanate from the basal film, but only

in the voids, not in the calcite.

CB

A

D 100 µm

100 µm

100 µm

10 µm

hy

hy

hy

hy

ca

ca

ca

ca

ba

ba

bf

bf

vo

vo

vo

chy

Fx

Fx

Fx

cw

cw

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.



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, net-

works of coarse hyphae with large globules character-

ize the calcite.

4 The thin hyphae adapt themselves to the calcite sur-

faces 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, show-

ing 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 subse-

quent 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 (R�evillon 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 dis-

tance 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 termi-

nations (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 environ-

ments, 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 ‘cob-

webs’ 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 discus-

sion in the literature (reviewed by Rodr�ıguez-Mart�ınez

et al., 2011). Most interpretations have landed in a bacte-

rial 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 Fru-

texites to be bacterial in origin, for three main reasons:

1 Recent formation of Frutexites-like structures in sub-

terranean rocks has been shown to be associated with

the chemolithotrophic iron-oxidizing bacteria Gallio-

nella ferruginea (Rodr�ıguez-Mart�ınez 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



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 domi-

nated by crystal shapes and differ substantially from

the bacterially induced ones.

3 The Koko Seamount Frutexites are dominated by goe-

thite, 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.

Symbiotic relationships

We propose that three separate organisms lived simulta-

neously in physical contact in the voids in the Eocene basalt:

the fungi (basal film with protuberances and mycelial net-

work), 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 chemoau-

totrophic 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 limit-

ing in this environment even without the presence of fungi.

Symbiosis between prokaryotic and eukaryotic cells is a

globally important phenomenon that influences the physi-

ology, 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 habi-

tat 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.

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 bio-

topes within the crust . . .’. Today, by detailed studies of

the fossil record and with the aim of powerful 3D tech-

niques, we are able to visualize this hidden biosphere. Fos-

sil 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 his-

tory of the deep biosphere.

ACKNOWLEDGMENTS

We thank S. Ohlsson (Swedish Museum of Natural His-

tory) for laboratory assistance, M. Ahlbom and H. Sieg-

mund (Stockholm University) for assistance with ESEM/

EDS analysis and isotope analysis, respectively, S. Lindb-

lom (Stockholm University) for providing the samples, and

M. Rosing (University of Copenhagen) for valuable discus-

sion. We acknowledge funding from the Swedish Research

Council (Contracts No. 2010-3929 and 2012-4364), Dan-

ish 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 spectro-

scopic 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.



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