Fossilized anaerobic and possibly methanogenesis-fueling fungi identified deep within the Siljan impact structure, Sweden

The deep biosphere is the largest microbial habitat on Earth by volume and encompasses diverse, often severely energy-limited ecosystems. While research has focused largely on prokaryotes, microorganisms from all three domains of life are present and active in deep aquifers in Precambrian crystalline rocks. Eukaryotes, including fungi, have been detected at kilometer depths, though prokaryotes generally dominate with depth. Fungi have been found in deep present-day fracture waters and as ancient remnants in fractured igneous rocks, yet their ecological role and residence time in the deep igneous environment remain poorly understood. In continental igneous aquifers, anoxic conditions typically develop within tens of meters depth, implying that deep biosphere fungi must be adapted to anaerobic lifestyles. Obligate anaerobic fungi (notably Neocallimastigomycota) possess hydrogenosomes and produce H2, CO2, lactate, and formate as metabolic by-products, and are known to form syntrophic relationships with methanogens. It has been hypothesized that deep subsurface anaerobic fungi could produce H2 that fuels autotrophic prokaryotes (e.g., methanogens and sulfate reducers), potentially playing a key role in subsurface methane generation. However, in situ evidence for such consortia in the deep subsurface has been lacking. This study investigates the Siljan impact structure in Sweden for fossil fungi in anoxic fractured crystalline bedrock, aiming to identify fungal remains, constrain their age, and evaluate their potential syntrophic link to methanogenesis using mineral paragenesis, imaging, staining, biomarkers, and stable isotope analyses.

Prior work shows that the continental deep biosphere hosts substantial biomass and ancient microbial lineages adapted to subsurface conditions. Eukaryotes, including fungi, have been reported from deep aquifers, though their proportion declines with depth. Fossil and extant fungi have been documented in igneous and sedimentary contexts, including deep Swedish fractured rocks and Neoproterozoic and Phanerozoic deposits. Obligate anaerobic fungi (Neocallimastigomycota) are well characterized in herbivore rumens, where they harbor hydrogenosomes and produce H2, forming syntrophic associations with methanogens. Similar syntrophy has been proposed for deep subsurface settings, where fungi could degrade organic matter and supply H2 to autotrophic prokaryotes, potentially enhancing methane production compared to bacteria–methanogen consortia. Additionally, dimorphic fungi (yeast and hyphal phases) can adapt to diverse conditions, and facultative and obligate anaerobes have been found in various anoxic environments beyond animal guts. Biomarker studies indicate that sterol biosynthesis requires oxygen; under anoxia, some eukaryotes substitute sterols with tetrahymanol or hopanoids. Tetrahymanol and its diagenetic products (e.g., gammacerane, 30nor-gammacerane) serve as biomarkers for anaerobic eukaryotes. In the Siljan structure, microbial methane formation and hydrocarbon migration/biodegradation have been documented, providing a context to assess potential fungi–methanogen interactions and mineral isotopic signatures of methanogenesis.

Study site and sampling: The late Devonian Siljan impact structure (central Sweden) hosts down-faulted Ordovician–Devonian sediments overlying Paleoproterozoic crystalline rocks. Cored boreholes to ~700 m depth intersect fractured zones. One highly fractured, porous section in granite–rhyolite at 534–542 m depth in borehole CC1 was selected after screening fractures for fossilized microorganisms. Five fractures were sampled from this section (>125 m below the sedimentary contact). Open cavities with euhedral calcite, pyrite, quartz, and clay provided colonization space. Fractures were mechanically opened; exposed surfaces with carbonaceous material and mineral coatings were examined.

Imaging and mineralogical characterization: Scanning Electron Microscopy (SEM) and Environmental SEM (ESEM; low-vacuum, uncoated samples) with EDS were used to characterize morphology, mineralization (clay, calcite, pyrite), and elemental composition of filaments and associated materials. Synchrotron Radiation X-ray Tomographic Microscopy (SRXTM) on aliquots from mycelium-bearing fragments (TOMCAT beamline, 20–25 keV; 10×–20× objectives; voxel size 0.325–0.65 µm) visualized mineralized filaments enclosed within euhedral calcite overgrowths and 3D relationships among calcite, pyrite, and mycelium.

Chitin staining: Individual filaments were hand-picked and stained with Calcofluor White; fluorescence microscopy assessed chitin presence. Positive staining in non-mineralized segments indicated chitin; negative reactions were attributed to degradation/mineralization.

Stable isotope microanalysis (SIMS): Calcite crystals intergrown with filaments were mounted in epoxy, polished, and imaged to identify growth zones (older Calcite-1 cores, younger Calcite-OG overgrowths). A Cameca IMS1280 microprobe (10 µm spot) measured δ13C across growth transects. Results were normalized for instrumental mass fractionation using a matrix-matched calcite reference (δ13C = −0.22 ± 0.11‰ V-PDB). Precision: ±0.3–0.5‰.

Organic geochemistry (GC–MS): Fungal material and mineral coatings (including some hyphae) were solvent-cleaned, ground, and sequentially extracted (dichloromethane/methanol; dichloromethane; hexane). Extracts were derivatized (BSTFA; TMCS/methanol) and analyzed by GC–MS (on-column injection; 80–325°C oven ramp; He carrier). Compound identification leveraged spectral libraries and literature data. Biomarker suites (hopanoids, 30nor-gammacerane, steranes), fatty acid distributions, and unresolved complex mixtures were assessed.

Geochronology linkage: The outermost calcite growth zone (Calcite-OG) that encloses filaments was previously dated by high-spatial-resolution U–Pb to 39.2 ± 1.4 Ma, providing an age constraint for fungal fossilization. This study leverages that published age to constrain timing of fungal occurrence.

Ancillary data: Previously reported gas isotopes in nearby boreholes (δ13CCH4 ~ −64 ± 2‰; δ13CCO2 ~ +5 to +9‰) and pyrite δ34S (−40 ± 1‰) from within the mycelium network were integrated into interpretations.

• Morphology and composition of filaments: Carbonaceous filamentous structures form mycelium-like networks protruding from biofilm-like coatings on fracture surfaces at 534–542 m depth. Filaments are ~10 ± 5 µm in diameter, extend >1 mm, display regular septa at ~50–60 µm, frequent branching, and tapered hyphal bridges at mineral contacts. Spherical bodies (5–15 µm) occur at hyphal tips and on surfaces; smaller spheres appear in budding pairs, larger as single bodies, consistent with yeast-like forms. Filaments are partly to completely mineralized by clay (smectite/montmorillonite-like) and carry micron-scale calcite precipitates; microcrystalline pyrite and late calcite occur within the mycelial network.
• Chitin detection: Calcofluor White staining was positive in parts of filaments, indicating chitin and supporting fungal affinity; non-reactive segments correspond to mineralized/degraded portions.
• Intergrowth with calcite and timing: Filaments are intergrown with and enveloped by euhedral calcite overgrowths (Calcite-OG) that post-date older calcite (Calcite-1). SRXTM shows filaments enclosed within calcite. The Calcite-OG has an age of 39.2 ± 1.4 Ma (Eocene), providing the first temporal constraint on fossilized fungi in continental igneous crust at this site.
• Stable carbon isotopes: SIMS transects reveal a shift from relatively 13C-light δ13C in Calcite-1 to 13C-heavy values in Calcite-OG that embeds the filaments. δ13Ccalcite in Calcite-OG reaches up to +8.1 ± 0.4‰ (median +6.0‰; n=14), consistent with precipitation from 13C-enriched DIC following microbial methanogenesis (carbonate reduction pathway). Older Calcite-1 lacks this enrichment.
• Sulfur isotopes: Pyrite within the mycelium network has very low δ34S (−40 ± 1‰; n=37), indicating microbial sulfate reduction.
• Biomarkers: Organic extracts are dominated by an unresolved complex mixture of degraded organic matter. Hopanoids (C27–C35) and 30nor-gammacerane are present; notably, steranes are absent. 30nor-gammacerane is significantly enriched in extracts from permineralized hyphae compared to mineral-dominated samples. Fatty acids C14–C22 (dominated by C16 and C18), minor unsaturated C16:1 and C18:1, and odd-chain/iso/anteiso C15 and C17 acids were detected in hyphae extracts.
• Environmental context: Methane-dominated gas was encountered during drilling; bitumen and seep oil of shale origin occur in fractures in both sedimentary cover and deeper crystalline rocks, indicating inputs of degradable organic matter into the fracture network. Collectively, these findings identify fossilized, chitin-bearing anaerobic fungi deep in fractured crystalline rock, temporally linked to 13C-enriched calcite formed during methanogenesis, and containing a biomarker (30nor-gammacerane) consistent with anaerobic eukaryotic lipid pathways.

The filament morphology (diameter, septation, branching, hyphal bridges), chitin staining, and clay mineralization are consistent with fungal hyphae rather than prokaryotes. Spherical bodies likely represent yeast-like cells, suggesting dimorphic fungal life cycles adapted to oligotrophic, anoxic fractures where hyphae explore for nutrients and yeast forms disperse via fluids. Preservation by clay mineralization explains the integrity of the mycelial network. The strictly anoxic setting below the redox front and presence of unaltered sulfides indicate anaerobic growth. The absence of steranes and detection of 30nor-gammacerane enriched in hyphae extracts point to tetrahymanol-derived biomarkers, consistent with anaerobic eukaryotic adaptations substituting sterols under oxygen limitation. The 13C-enriched calcite overgrowth intergrown with hyphae indicates in situ methanogenesis contemporaneous with fungal activity, supported by regional gas isotope data (13C-light CH4; 13C-heavy CO2). The most parsimonious interpretation is that anaerobic fungi degraded organic matter (e.g., infiltrated bitumen or prokaryotic biomass), producing H2 and other metabolites that fueled autotrophic methanogens (CO2 reduction pathway). Microbial sulfate reduction is also recorded by 34S-depleted pyrite within the mycelium, implying dynamic redox and electron-acceptor availability; sulfate reducers likely preceded or alternated with methanogens due to competition for H2. Although direct body fossils or specific biomarkers of methanogens were not detected in the fossilized biofilm, the spatial-temporal association between fungi and 13C-enriched calcite supports a syntrophic link. These results suggest anaerobic fungi can act as overlooked H2 providers that sustain autotrophic methanogenesis in crystalline basement aquifers, with implications for subsurface methane budgets and potential greenhouse gas emissions if mobilized. Impact-induced fracturing and organic matter ingress in structures like Siljan may have facilitated deep eukaryotic colonization and intermittently expanded subsurface ecological niches through geologic time.

This study documents fossilized, chitin-bearing fungal hyphae and yeast-like forms at ~540 m depth within fractures of the Siljan impact structure’s crystalline basement. Filaments are intergrown with Eocene calcite overgrowths that are 13C-enriched up to +8.1‰, indicating contemporaneous microbial methanogenesis. Biomarker evidence (30nor-gammacerane) suggests anaerobic eukaryotic lipid pathways consistent with sterol substitution under anoxia. Together, these lines of evidence imply that anaerobic fungi degraded organic matter and likely produced H2 that fueled autotrophic methanogens, establishing a putative syntrophic relationship in the deep igneous crust. This provides the first temporal constraint on fossil fungi in continental igneous crust and highlights fungi as potentially widespread decomposers and H2 providers in the deep biosphere. Future research should: (1) pursue enrichment, isolation, and co-culturing of native subsurface fungi with methanogens to directly test syntrophy and quantify H2 fluxes; (2) expand surveys of impact structures and crystalline aquifers for fungal fossils and tetrahymanol-derived biomarkers; (3) integrate isotopic, biomarker, and mineral paragenesis studies to constrain spatiotemporal dynamics of methanogenesis and sulfate reduction; and (4) assess the contribution of fungal-mediated processes to subsurface methane accumulations and potential atmospheric release.

• Active community analysis was not possible due to ongoing gas production tests, preventing direct detection of living methanogens or fungi in fracture waters.
• The relative contribution of fungal-derived versus prokaryotic-derived H2 to methanogenesis cannot be quantified in this fossil system.
• Interpretation of yeast-like spheres is based on morphology without molecular confirmation.
• Absence of detectable methanogen body fossils or diagnostic archaeal biomarkers in the fossil biofilm limits direct evidence for syntrophy; inferences rely on spatial association and isotopic signatures.
• Findings are from a single fractured interval within one borehole; broader spatial representativeness remains to be established.