ARTICLES PUBLISHED: 24 APRIL 2017 | VOLUME: 1 | ARTICLE NUMBER: 0141

Fungus-like mycelial fossils in 2.4-billion-year-old vesicular basalt

Stefan Bengtson1*, Birger Rasmussen2*, Magnus Ivarsson1, Janet Muhling2,​3, Curt Broman4, Federica Marone5, Marco Stampanoni5,​6 and Andrey Bekker7

Fungi have recently been found to comprise a significant part of the deep biosphere in oceanic sediments and crustal rocks. Fossils occupying fractures and pores in Phanerozoic volcanics indicate that this habitat is at least 400 million years old, but its origin may be considerably older. A 2.4-billion-year-old basalt from the Palaeoproterozoic Ongeluk Formation in South Africa contains filamentous fossils in vesicles and fractures. The filaments form mycelium-like structures growing from a basal film attached to the internal rock surfaces. Filaments branch and anastomose, touch and entangle each other. They are indistin- guishable from mycelial fossils found in similar deep-biosphere habitats in the Phanerozoic, where they are attributed to fungi on the basis of chemical and morphological similarities to living fungi. The Ongeluk fossils, however, are two to three times older than current age estimates of the fungal clade. Unless they represent an unknown branch of fungus-like organisms, the fossils imply that the fungal clade is considerably older than previously thought, and that fungal origin and early evolution may lie in the oceanic deep biosphere rather than on land. The Ongeluk discovery suggests that life has inhabited submarine volcanics for more than 2.4 billion years.

he deep biosphere, hidden beneath land and sea, represents a chlorite, K-feldspar, quartz and calcite, with accessory apatite and major portion of life’s habitats and biomass on Earth1. In spite Fe-Ti oxides. Amygdales and veins are present, characterized by Tof significant discoveries from scientific ocean drilling and chlorite and calcite representing mineral infills of original vesicles metagenomics, the deep biosphere remains largely uncharted and and fractures in the lavas. The spherical to subspherical amygda- its geological history almost entirely unknown. The deep habitats les are up to 1.5 mm in diameter (Figs 2 and 3). Most have rims are protected from most of the hazards of surface life, and the deep composed of masses of very fine-grained, brownish green chlo- environments would have been potentially available to life from the rite, chlorite 1. Thermometry of chlorite 1 yields metamorphic early stages of Earth’s history. Here, we report filamentous struc- temperatures in the range of 179–260 °C (Supplementary Fig. 1; tures preserved in carbonate- and chlorite-filled amygdales and Supplementary Discussion). Where filaments are present, they are fractures in basaltic lavas of the 2.4-Gyr-old Ongeluk Formation, defined by chlorite 1. No carbonaceous material has been detected South Africa. Their morphology, dimensions and striking similar- within the filaments (Supplementary Fig. 2a). Calcite typically forms ity to fungi in Phanerozoic volcanics2–7 indicate that they represent a cylindrical layer of constant thickness around the filaments; the fossilized fungus-like mycelial organisms. The observation that blocky arrangement of crystals in the calcite, without clear relation fungus-like organisms inhabited submarine basaltic lavas more than to filament morphology (Fig. 4e), suggests that the calcite has been 2.4 Gyr ago (Ga) suggests that this habitat was extremely conserva- recrystallized. Fine-grained chlorite 1 fills the space between the cal- tive across the Proterozoic and Phanerozoic eons, and raises ques- cite cylinders (Figs 2b,d,e,g and 4; Supplementary Figs 3a,b, 4 and 5). tions about the antiquity of fungi and the early history of eukaryotes. A second generation of chlorite, chlorite 2, coarser-grained and apple green, commonly intergrown with quartz and chalcopy- Geological setting rite, is present in some amygdales and fractures. Chlorite 2 is not The Ongeluk Formation is a 900-m-thick succession of basalts in pervasive but overprints chlorite 1, including filaments defined the Griquatown West Basin, South Africa. The lavas are regionally by chlorite 1 (Fig. 4d,e; Supplementary Fig. 4d). Thermometry extensive and comprise massive flows, pillow lavas and hyaloclas- of chlorite 2 gives metamorphic temperatures of 319–411 °C tites that extruded onto the seafloor around 2.4 Ga; the basalts have (Supplementary Figs 1 and 5; Supplementary Discussion). Its asso- undergone only very low-grade metamorphism (Supplementary ciation with chalcopyrite, occurrence in veins and otherwise non- Discussion). The fossiliferous sample (AG4) is a 25-cm-long ¼ core pervasive distribution suggest that the growth of chlorite 2 was derived from drill depth 21.79–22.04 m of the Agouron drill hole linked to hydrothermal fluids. GTF01, which penetrated the lower part of the Ongeluk Formation (Fig. 1). About 70 of the ~100 observed amygdales contain filaments. Filament structure and morphology The sample is a chlorite-altered basalt with a relict igneous The filaments extend from rims of chlorite 1 attached to amygdale texture consisting of pseudomorphs of pyroxene and plagioclase and fracture walls, and form a tangled network inside vesicles and (Supplementary Discussion). The groundmass consists of intergrown fractures in the rock (Figs 2–4; Supplementary Fig. 3). The density

1Department of Palaeobiology and Nordic Center for Earth Evolution, Swedish Museum of Natural History, SE-10405 Stockholm, Sweden. 2Department of Applied Geology, Curtin University, Bentley, Western Australia 6102, Australia. 3School of Earth and Environment, The University of Western Australia, Perth, Western Australia 6009, Australia. 4Department of Geological Sciences, Stockholm University, SE-10691 Stockholm, Sweden. 5Swiss Light Source, Paul Scherrer Institute, CH-5232 Villigen, Switzerland. 6Institute for Biomedical Engineering, University and ETH Zürich, CH-8092 Zürich, Switzerland. 7Department of Earth Sciences, University of California, Riverside, California 92521, USA. *e-mail: [email protected]; [email protected]

NATURE ECOLOGY AND EVOLUTION 1, 0141 (2017) | DOI: 10.1038/s41559-017-0141 | www.nature.com/natecolevol 1 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. ARTICLES NATURE ECOLOGY AND EVOLUTION

24° E 28° E ab c ve Kaapvaal Transvaal 24° S c Yj Craton an South Africa Ca

26° S Griqualand Johannesburg 1 mm 100 µm10 µm West d e Chl1 f 28° E 100 km an Kathu Ca

28° S Black Ridge Thrust Vryburg GTF01 Chl1 10 µm 10 µm Prieska Kuruman e 30° S h 100 µm

Griqualand West Transvaal NE g Bushveld Complex Griquatown 50 km Ca Chl1 Upper Transvaal Upper Transvaal Ca Supergroup Supergroup Upper Transvaal Supergroup Kuruman and Griquatown Penge Iron Fm. Iron Formations Olifantshoek SW GTF01 Group Chl1 Campbellrand Subgroup Malmani Subgroup Prieska Postmasburg Schmidtsdrif Subgroup Wolkberg Group and Elim Groups 10 µm 100 µm Koegas Subgrp i jk SW NE ve bf bf Mooidraai Chl1 Formation Carbonate platform Hotazel Fm. Banded iron formation k bf Chl1 Ca 10 µm Ca hy Submarine basalt Diamictite Ongeluk l Ca 5 µm Formation Chl1 100 m GTF01 µ hy 50 µm Banded iron formation

Makganyene Figure 2 | Ongeluk vesicular basalt with filamentous fossils, petrographic Koegas Subgrp thin sections. a, Basalt with vesicles frequently connected by veins; Griquatown Formation Carbonate platform Swedish Museum of Natural History X6129. b,c, Anastomosing network; X6130. d,e, Vesicle with broom structure; note distinction between calcite Kamden Member Kuruman (light) and chlorite (dark) cement; X6131. f, Anastomosis; X6132. g, Broom Formation structure in fracture (same specimen as in Fig. 4); X6133. h, Broom; X6134. Klein Naute i, Vesicle connected to vein filled with calcite (light) and chlorite (dark) Nauga Slope and Sandstone cement; X6135. j–l, Basal film and marginal network; X6136. Panels a–i Formation basinal show transmitted light images; panels j–l show ESEM images produced in backscatter mode. Lettered frames indicate position of enlargements in Schmidtsdrif Shale other panels. an, anastomosis; bf, basal film; Ca, calcite; Chl1, chlorite 1; hy, hypha; ve, vein; Yj, Y-junction. Figure 1 | Geological map and stratigraphic section of the Griqualand West sub-basin, showing the location of Agouron drill hole GTF01 (28° 49 39.7 S, 23° 07 24.1 E). The fossiliferous sample is from the ′ ′′ ′ ′′ Branchings at acute angles, Y-junctions, are common among lower part of the Ongeluk Formation (drill depth 21.79 m). Fm., formation; the free filaments (Figs 2c and 3f,g). T-junctions also occur subgrp, subgroup. Modified from ref. 53, Geological Society of America. (Fig. 3g), although considerably less frequently. Filaments with different orientation commonly touch and entangle each other of the filamentous network typically decreases towards the centre of (Fig. 3f,i), and crossing filaments sometimes seem to merge the cavities (Fig. 2b,d,j, 3a,e and 4b; Supplementary Fig. 3a,b). The seamlessly. Where none of the filaments change direction, chlorite rim represents an uneven basal film consisting of a jumbled the crossing is interpreted as coincidental (Fig. 2l). This phe- mass with little space remaining between filaments (Figs 2j,k and 3e; nomenon of taphonomic/diagenetic filament merging makes Supplementary Fig. 3). Scanning electron microscopy (SEM)/ it sometimes difficult to identify true branching, where a single back-scattered electron (BSE)/wavelength-dispersive X-ray spec- filament is split into two. When Y-junctions on the same appar- troscopy (WDS) images confirm that the structure and compo- ently branching filament point in opposite directions (Fig. 3c), sition are identical between filaments and basal film (Fig. 2j,k; one or both junctions may represent false branching; this can Supplementary Figs 4 and 5). also be indicated by the filament being thicker, or even appear- Filaments are 2–12 μ​m wide; the width is usually constant within ing doubled, below a Y-junction. There are, however, a number a filament. No internal septa have been identified, but original inter- of cases where the morphology of the junction leaves little nal structure is not preserved (Fig. 2k,l). The filaments typically doubt of true branching (Figs 2c and 3f,g). In particular, where form straight or curved sections, rarely with irregular wiggly parts. successive Y-branching takes place from a stem of constant diam- Filaments frequently form loops of different diameter, from about eter, the branching is real and not due to bundling of separate 10 μ​m (Fig. 3h) to 80 μ​m or more (Fig. 3i). filaments (Fig. 3f,g).

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a b c Biogenicity and syngenicity an fb Crucial to the interpretation of the filaments are the issues of bio- an genicity and syngenicity: do the filaments represent biological organisms and when did they form relative to the age of the rock? 10 µm 10 µm Filamentous fabrics are not uncommon in basaltic rocks, although most reported cases refer to tunnelling in volcanic glass and its e d alteration products8. Both biogenic and abiogenic mechanisms may be responsible for such tunnels, and distinguishing between the two 9–12 100 µm1br 0 µm causes is difficult and controversial . A number of observations clearly indicate, however, that the Ongeluk structures were formed e br f as filaments in voids, not as tunnels in minerals: Yj tf tf • Although tunnels may take on a variety of shapes, including branching and dendritic ones13, several features of the Ongeluk Yj structures are incompatible with tunnels. The frequent fusing of adjacent filaments (Fig. 3f,i), resulting in false branching (Fig. 3c), implies that they are physical entities often touching and entan- 10 µm Yj 10 µm gling each other. This is consistent with flexible filaments in a void, gij bp but not with tunnelling in rock. Similarly, the recurring cases of anastomosis (Figs 2 and 3) are difficult to reconcile with tunnels. Yj • The morphology of the filaments and the mineral paragenetic Yj sequence in the fractures (Fig. 4) are identical to those of the Tj 10 µm bp adjacent vesicles, implying that the vesicles, like the fractures, started out as voids and underwent the same history of coloniza- h lo tf lo tion and paragenesis. • A number of different spherical or globular structures are found bp 14 10 µm 10 µm 10 µm in volcanic and subvolcanic rocks . They may be formed as gas bubbles in the magma (vesicles), as radial growth of crys- Figure 3 | Ongeluk vesicle with filamentous fossils, SRXTM surface/ tals (spherulites), or as the result of immiscibility of compo- volume renderings; Swedish Museum of Natural History X6137. nent magmatic fluids (varioles). Vesicles usually become filled a, Section through complete vesicle; frame indicates region depicted in e. by secondary minerals formed at low temperatures, forming b,c, Anastomoses and false branching. d,e, Brooms. f,g, Y-junctions, amygdales. The Ongeluk spherical structures are filled with T-junctions and touching filaments. h,i, Loops and touching filaments. minerals (mainly calcite and chlorite) characteristic of amyg- j, Bulbous protrusions. an, anastomosis; bf, basal film; bp, bulbous dales; they show neither spherulitic structure nor magmatic protrusion; br, broom; fb, false branching; lo, loop; tf, touching filaments; composition, and so may confidently be interpreted as having Tj, T-junction; Yj, Y-junction. begun as gas bubbles (Supplementary Discussion). • The Ongeluk filaments fulfil established criteria15 distinguish- ing cryptoendoliths (cavity-dwellers) and chasmoendoliths Anastomoses, where a branched-off filament meets and merges (fracture-dwellers) from euendoliths (rock-borers) and abiotic with another, occur with some frequency (Figs 2c,f and 3b,c). As with processes forming microtunnels in rock (Supplementary Discus- branching, it may be difficult to distinguish coincidental coming- sion). They show pre-metamorphic growth into fluid-filled cavi- together of independent filaments from true anastomoses, but ties, curvilinear and branching forms with circular cross-section the frequency of apparent anastomoses with consistent morphol- and non-uniform diameter, and preservation in clays with or ogy (for example, Fig. 3b,c) indicates that the phenomenon is real. without organic matter in carbonate-filled vesicles; all listed as Nonetheless, anastomoses do not dominate the filament tangles to characters typical of crypto- and chasmoendoliths15. the extent that they form interlocking networks. The filaments sometimes carry bulbous protrusions, 5–10 μ​m The authors of a previous study16 reported a variety of structures in diameter. These tend to congregate on the basal parts of fila- interpreted as ambient inclusion trails in an Archaean pyroclastic tuff. ments and on basal films, and be more rare on distal parts of Their ‘type 1 microtubes’ show a compositional similarity with the filaments (Fig. 3j). Ongeluk structures: both have chloritic cores surrounded by calcite. A recurring feature is a bundle of filaments giving off diverging They differ from the latter, however, in being straight and very regular. branches to form a broom-like structure, here termed ‘broom’, that A commonly stated criterion for biogenicity of microfossils is the extends from the basal film or from the substrate (Figs 2e,g,h and 3d,e). presence of original organic carbon in the structures; this has even In some vesicles, there are brooms consisting of tens of diverging been cited as a necessary criterion12,17. However, organic carbon filaments, some with their bases apparently attached to the vesicle is seldom preserved in environments of highly oxidized minerals, wall and some produced by branching (Supplementary Fig. 3c,d). such as calcite or hematite; organically preserved microfossils are The basalt is permeated by veins that are frequently seen to con- predominantly found under preservational conditions of low per- nect to the spherical/subspherical vesicles (Fig. 2a,i; Supplementary meability and reactivity, as in cherts18. As the absence of organic Video). The veins, down to 5 μ​m in width, are filled with chlorite carbon in a fossil is seldom reported in the literature, the lack of and calcite similar to that which fills the vesicles. One large vein, such carbon is frequently overlooked. It is, however, a common >​2.2 mm long and >​0.2 mm wide, comprises a zone of densely condition18. For example, we have investigated well-preserved iron- intertwined filaments that occurs between the basalt wall rock oxidizing bacteria in a Quaternary microbialite where filaments and the centre of the vein. Filaments adjacent to the margin of are encrusted with hematite, and Raman spectroscopy failed to the vein are commonly truncated by chlorite sheets and veinlets reveal any organic carbon signal19. The lack of detectable carbona- (Fig. 4), representing a later stage of chlorite growth (chlorite 2; ceous matter in the Ongeluk filaments is thus not a valid argument Supplementary Discussion). against their biogenicity.

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a c form spores, about 1 μ​m in diameter, on the mycelium, some- 23 b 1 mm times in sporangia 5–20 μ​m in size . Actinobacteria have a wide distribution in aquatic and terrestrial habitats, including various b extreme environments24. Like actinobacteria, fungi are widely distributed in terrestrial c and aquatic habitats, and they have recently been shown to be com- 5,25–29 Fracture centre mon inhabitants of deep marine sediments and crustal rocks . Hyphae in fungal mycelia vary in width between 2 and 27 μ​m30. Anastomoses are prevalent31 and the mycelia typically form net- d works of interconnected hyphae. Fungal spores are larger than those of actinobacteria, typically around 5 μ​m. Chl2 Chl1 Fungi have recently been found to play a leading role in the Phanerozoic subsurface biota through the discoveries of fossilized Filamentous zone fungal mycelia in vesicles in Devonian, Eocene and Quaternary submarine volcanics2–7,28. These fungi may form symbiotic assem- blages with prokaryotes32,33. 100 µm Basalt 100 µm The oomycetes were previously thought to be fungi, but 25 µm molecular systematics now places them close to the photosyn- de 34 Ca Ca thetic stramenopiles . Anastomoses between hyphae occasion- ally occur, but as a form of conjugation, not a mechanism to form Chl2 interlocking networks35. Chl2 When compared with modern mycelial organisms, the Ongeluk fossils in hyphal dimensions, network architecture and mode of life Chl1 Chl1 seem most consistent with fungi. If the 5–10 μ​m bulbous protru- sions are spores, those too agree with fungal but not actinobacterial Ca Ca dimensions. Ongeluk anastomoses closely mimic those in modern fungi (compare our Figs 2c,f and 3b,c with anastomoses in ref. 36, Figure 4 | Calcite- and chlorite-filled fracture with filamentous fossils in Fig. 1). Other features of the Ongeluk fossils, such as the basal Ongeluk vesicular basalt, petrographic thin section; Swedish Museum film and the tendency of filaments to protrude from it as brooms, of Natural History X6133. a, Overview of fracture, which is truncated are consistent with fungal mycelial morphology (for example, the along centre by edge of section. b–e, Fracture filling divided into central mycelial cords developed by many fungi under conditions of star- 37 zone and peripheral filamentous zone, parted by a band of chlorite 2; note vation ). The growth habit of the Ongeluk filaments in basaltic truncation of filaments by chlorite 2 band. Different intensity of calcite vesicles is morphologically almost identical to that seen in fungi in 2–7,32,33 interference colours in filamentous zone (e) indicates blocky distribution Phanerozoic volcanics (Supplementary Fig. 8) . The examples 3,4 of calcite crystals, not related to filament morphology; chloritic filaments from Devonian pillow lavas are particularly significant because are too thin to reveal interference colours of chlorite 1 (black arrow). Panels they show preservational features similar to those in the Ongeluk a–d show plane-polarized transmitted light images; the image in panel e vesicles, with mineral encrustations of the filaments (Supplementary was produced using crossed nicols. Lettered frames indicate position of Fig. 8a–d). In the Devonian occurrences, however, the encrusting enlargements in other panels. Ca, calcite; Chl1, chlorite 1; Chl2, chlorite 2. minerals include illite and glauconite as well as chamosite (chlorite). Although on the basis of morphology we cannot exclude the possibility that the Ongeluk fossils represent a separate branch With regard to syngenicity, the organisms must have invaded of fungus-like organisms, the similarities with fungi in the corre- the Ongeluk lavas while the vesicles and fracture-controlled poros- sponding Phanerozoic settings are striking. The presence of fungi ity were still open to the water column, a window that probably in early Palaeoproterozoic submarine volcanic rocks would, how- closed after ca. 10 Myr following the eruption of the lavas20. In any ever, overturn current concepts on the timing and circumstances of case, they should not be younger than ca. 2.06 Ga, at which time fungal origin and evolution. There is a strong consensus that fungi chloritization would have taken place (Supplementary Discussion). and nucleariids comprise the sister group of holozoans within the Supplementary Fig. 6 depicts the proposed formation sequence from clade Opisthokonta38–40, and the time of divergence of the two sister invasion of the organism through diagenesis and metamorphism. branches is commonly estimated to lie within the Mesoproterozoic Raman spectroscopy indicates the presence of carbonaceous or earliest Neoproterozoic41–47. The last common ancestor of crown- material in the Ongeluk host basalt that has not been subjected to group fungi is considered to have been non-filamentous, with temperatures higher than about 200 ±​ 30 °C; carbonaceous mate- flagellated spores, aquatic, but probably non-marine39. Under this rial in the Ongeluk basal sandstone and the underlying Makganyene scenario, marine and deep-biosphere fungi might represent migrated diamictite yields temperatures of around 370 °C (Supplementary terrestrial taxa, consistent with the predominance in marine and Fig. 7; Supplementary Discussion). The origin of the carbon is deep-biosphere environments of advanced forms5,7,26,27,29,48. Fungi unknown; it shows no affinity to the filaments (Supplementary Fig. 2). living in 2.4-Gyr-old submarine basalts, however, would imply that the fungal clade is considerably older than previously thought, and Biology of the filaments that fungal origin and early evolution may lie in the oceanic deep Filamentous growth is a recurring characteristic in many multicel- biosphere rather than on land. lular prokaryotes and algae, but mycelial networks consisting of Estimates of node ages from molecular clocks rely on calibra- branching filaments are known mainly from three modern groups tion against the fossil record. Whereas the Phanerozoic fossil of organisms: actinobacteria, fungi and the fungus-like eukary- record is sufficiently reliable to yield useful calibration points49, otic oomycetes. Mycelium-forming actinobacteria produce radi- the Proterozoic record is notoriously spotty, and interpretations of ating networks of branching filaments, 0.15–1.5 μ​m in diameter. Proterozoic fossils are frequently controversial (for example, alleged Anastomoses are generally absent21; occasional reports of anastomo- Proterozoic fungi46). Ages of Proterozoic nodes are therefore typi- ses in Streptomyces have not been confirmed22. Many actinobacteria cally based on extrapolations from Phanerozoic calibration points.

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Irrespective of the formidable molecular clock problems, the exis- X-ray energy was set to 15 keV for petrographic thin sections and 28 keV for tence of fungi near the beginning of the Proterozoic, before or at sawn-out 2 mm pillars. Objectives ×​4, ×​10 and ×​20 were used, for a voxel size of the very early stage of the Great Oxidation Event, would raise issues 1.625 μ​m, 0.65 μ​m and 0.325 μ​m, respectively. Pillars were first scanned in total at low magnification to identify fossiliferous vesicles later to be scanned at higher about the existence of other major eukaryote branches at the time. magnifications. For the results presented here, 1,501 projections were acquired Whether or not the Palaeoproterozoic Ongeluk fossils represent equiangularly over 180°, online post-processed and rearranged into flat- and fungi, the occurrence of remarkably similar fossils in Phanerozoic darkfield-corrected sinograms. Reconstruction was performed on a Linux PC farm vesicular basalts (Supplementary Fig. 8)2–7,32 suggests that this envi- using highly optimized routines based on the Fourier transform method52. Slice ronment has been extremely stable for billions of years. Locally and data derived from the scans were analysed and rendered using Avizo software. regionally, an environment such as that provided by the Ongeluk Data availability. The illustrated material is deposited at the Swedish Museum of lavas may be short-lived, however, and whether colonizing biota Natural History, Stockholm. The datasets generated and/or analysed during the under such conditions was preferentially supplied from the sea- current study are available from the corresponding author (S.B.) on request. water or from the subsurface environments is an open question. 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6 NATURE ECOLOGY AND EVOLUTION 1, 0141 (2017) | DOI: 10.1038/s41559-017-0141 | www.nature.com/natecolevol © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.