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

The deep biosphere, encompassing diverse environments and ecosystems, is Earth's largest microbial habitat by volume. Severely energy-limited deep biosphere ecosystems, inhabited by ancient microbial lineages adapted to subsurface conditions, are of significant interest. Understanding these life forms and processes has implications for life in extreme environments, evolution on Earth, and the search for extraterrestrial life. While research has focused on prokaryotes, recent studies reveal microorganisms from all three domains of life are abundant and active in deep aquifers within Precambrian crystalline rocks, which comprise the largest volume of the continental crust. Eukaryotes, including fungi, have been detected at several kilometers depth, but the proportion generally decreases with increasing depth. Fungi have been detected in deep fracture waters and ancient fungal remains found in fractured igneous rock in Sweden. However, their ecological role and duration in this environment remain largely unresolved. The transition to anoxic conditions typically occurs within the upper tens of meters of continental igneous aquifers, suggesting the majority of deep biosphere microorganisms, including fungi, have adapted to anaerobic lifestyles. Studies of anaerobic fungi, primarily from the phylum *Neocallimastigomycota* found in herbivore rumens, reveal metabolic models involving hydrogenosomes (producing H₂, CO₂, lactate, and formate) and symbiotic relationships with prokaryotes such as methanogens. A similar syntrophic consortium has been proposed for the deep biosphere, where fungi might produce H₂ utilized by autotrophic prokaryotes, like sulfate reducers. Consortia of anaerobic fungi and methanogens could potentially enhance methane production from various organic matter sources. However, in situ evidence from the deep subsurface is lacking. This study investigates the late Devonian Siljan impact structure in Sweden for fossil fungi in anoxic environments using imaging techniques, staining, biomarkers, stable isotopes, and organic molecular remains of secondary minerals to link the fungi to prokaryotic metabolisms, such as methanogenesis. The Siljan impact structure, with its methane-dominated gas and organic material from shale origin, presents an ideal site for exploring ancient fungi-methanogen relationships.

Previous research has highlighted the existence of microbial life, primarily prokaryotic, in the deep subsurface biosphere. Studies have shown the presence of various microorganisms in energy-limited environments within the Earth's crust, including both extant and fossilized communities. However, the role of eukaryotes, especially fungi, in these deep subsurface ecosystems has been less explored. While some studies have reported the presence of extant fungi in deep groundwater, the evidence for ancient, fossilized fungi has been limited and often debated. This study builds upon previous findings of fungal remains in the Siljan impact structure, providing further insights into the nature and metabolic activities of these organisms in anoxic environments. The study also draws upon existing knowledge of anaerobic fungi from other environments, such as the rumens of herbivores, to inform the interpretation of the fossilized remains. The understanding of anaerobic fungal metabolisms, particularly their role in hydrogen production and their syntrophic relationships with methanogens, is crucial to contextualizing the findings of this research.

The study focused on the Siljan impact structure in Sweden, specifically analyzing samples from a drill core (CC1) at a depth of 534-542 m, within fractured granite-rhyolite. The samples contained filamentous carbonaceous material, calcite, pyrite, and clay minerals. Various analytical techniques were employed to characterize the samples and the fossilized fungal remains. These included: 1. **Microscopy:** Environmental Scanning Electron Microscopy (ESEM) and Scanning Electron Microscopy (SEM) were used to examine the morphology and microstructure of the carbonaceous filaments. Backscattered ESEM images provided information on elemental composition. 2. **Staining:** Calcofluor White staining was used to confirm the presence of chitin, a characteristic component of fungal cell walls. 3. **X-ray Tomography:** Synchrotron radiation X-ray tomographic microscopy (SRXTM) was used to visualize the three-dimensional structure of the filaments and their relationship with surrounding minerals. 4. **Stable Isotope Analysis:** Secondary Ion Mass Spectrometry (SIMS) microanalysis was employed to determine the stable carbon isotope (δ¹³C) ratios of calcite crystals associated with the fungal hyphae. This provided insights into the potential involvement of methanogenesis in the formation of the calcite. 5. **Biomarker Analysis:** Gas chromatography-mass spectrometry (GC-MS) was used to analyze organic extracts from the samples, identifying biomarkers such as 30nor-gammacerane (a degradation product of tetrahymanol, indicating anaerobic conditions) and various hopanoids. The absence of steranes was also noted. The combination of these techniques allowed for detailed characterization of the fossilized fungal remains, their mineralogical context, and their potential involvement in anaerobic microbial processes.

The study identified fossilized fungal hyphae at a depth of 540 m within the fractured bedrock of the Siljan impact structure. The hyphae exhibited characteristics consistent with fungi, including their size, septation, branching patterns, and the presence of chitin confirmed through Calcofluor White staining. The filaments showed a tapered fashion at the mineral surface, similar to hyphal bridges observed in other fungal hyphae. Septa were observed at regular intervals (around 50-60 µm), and the filaments formed mycelium-like networks. Mineralization, primarily by clay minerals, was observed to varying degrees. Spherical structures resembling budding yeast cells were also identified. The close association of the hyphae with calcite overgrowths showing a strong ¹³C-enrichment (δ¹³C values up to +8.1‰) suggests a link to methanogenesis, with the fungi potentially producing H₂ that fueled the autotrophic methanogens. This ¹³C enrichment was notably absent in older calcite formations, suggesting a temporal relationship between fungal growth and methanogenesis onset. The detection of 30nor-gammacerane, a degradation product of tetrahymanol, further supports the anaerobic conditions under which the fungi lived and potentially points to a unique survival strategy, substituting sterols with tetrahymanol in the absence of oxygen. U-Pb dating of the calcite associated with the hyphae yielded an age of 39.2 ± 1.4 Ma. The study also detected pyrite with isotopically light δ³⁴S values (−40 ± 1‰), indicating the co-occurrence of microbial sulfate reduction. The absence of steranes and presence of 30nor-gammacerane support the inference that this fungus lived in an environment where sterol production was impossible and an alternative pathway was employed.

The findings provide compelling evidence for the existence of anaerobic fungi in the deep continental biosphere, extending back at least to the Eocene epoch. The strong ¹³C enrichment of the calcite associated with the fungi strongly suggests a link to methanogenesis, with the fungi likely playing a crucial role in providing H₂, a key electron donor for this process. The discovery of 30nor-gammacerane supports the interpretation that these fungi were living under strictly anaerobic conditions. This work challenges the long-held assumption that fungi are primarily aerobic organisms and highlights their potential importance in deep subsurface ecosystems. Furthermore, the presence of both hyphae and yeast-like structures suggests the fungi exhibited dimorphism, a trait possibly advantageous in the oligotrophic deep subsurface. The findings also have implications for our understanding of global biogeochemical cycles, particularly the production of methane, a potent greenhouse gas. Further research is necessary to fully understand the extent and significance of fungal contribution to methane production in the deep biosphere.

This study provides the first direct evidence of fossilized anaerobic fungi in the continental deep biosphere, dating back to the Eocene epoch. The strong isotopic evidence linking the fungi to methanogenesis highlights a novel, potentially widespread ecological role for anaerobic fungi in this vast, largely unexplored habitat. This discovery opens exciting avenues for future research into the diversity, metabolism, and ecological significance of anaerobic fungi in deep subsurface ecosystems and their contribution to global biogeochemical cycles. Further research using in vitro co-cultures of deep biosphere fungi and methanogens would help to further delineate the nature of this syntrophic relationship and the extent of fungal contributions to H2 production.

The study primarily relies on morphological and isotopic analyses of fossilized remains. The absence of intact biological molecules limits definitive taxonomic identification of the fungi and prevents detailed metabolic reconstructions. The interpretation of the spherical structures as yeast cells is based solely on morphology. The lack of active microbial communities' sampling in the water prevents the confirmation of active methanogenic communities' existence in the immediate vicinity of the fungal remains. The significance of fungal versus prokaryotic-derived H₂ for methanogenesis cannot be definitively assessed with the available fossil data.