Re-evaluation of endolithic microfossils in pillow basalt of two Variscan orogens

Re-evaluation of endolithic microfossils in pillow basalt of two Variscan orogens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Re-evaluation of endolithic microfossils in pillow basalt of two Variscan orogens Dan Strömbäck, Jörn Peckmann, Sandra Siljeström, Ashley Krüger, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6512033/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract The presence of fossilised fungi within deep crustal rock formations has been established based on fossil evidence from 400 Ma continental crust and 81 Ma oceanic basaltic crust. Moreover, the Palaeoproterozoic Ongeluk Formation contains putative fungal remains reaching 2.4 Ga. The resulting gap of 2 billion years raises questions regarding the history of fungi in marine subsurface environments, in particular the lack of bona fide fossils in ophiolites, sections of layered basalts from mid-ocean ridges. Devonian examples of endolithic microorganisms preserved in marine pillow basalt stem from the Arnstein locality, Rheinisches Schiefergebirge, and the Kahlleite locality, Thüringer Wald, Germany, and have previously been found to contain filaments of microorganisms with uncertain biological affinity. The filamentous fossils were investigated using environmental scanning electron microscopy, Raman spectroscopy, confocal microscopy, widefield microscopy, and optical light microscopy. Energy dispersive spectroscopy analyses of several of the inferred microfossils revealed high carbon content and clay minerals, pointing to a mode of mineralization in association with organic matter and agreeing with a biological origin. Raman spectroscopy revealed that particularly iron oxide minerals are typified by carbon contents. Element compositions similar to younger mineralised fungal remains and morphologies resembling sporophores and hyphae agree with the interpretation of the Arnstein and Kahlleite fossils as marine fungi, shedding new light on many of the previously undetermined fossils and plausibly narrowing the fossil gap of oceanic deep subsurface fungi by at least 300 million years. ophiolite endolith fungus Devonian Rheinisches Schiefergebirge Thüringer Wald Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Below the surface of Earth resides the deep biosphere, the second largest biosphere on Earth (Orcutt et al., 2013; Goordial et al. 2021). Despite its size, little is known about its composition and modes of operation. Furthermore, most studies of the deep biosphere focused on deep-sea sediments, resulting in a lack of knowledge of microbial life in the oceanic and continental igneous crusts (Goordial et al. 2021). Research on microbial life in the deep oceanic crust began in the 1980s with the finding of potential bioalteration textures in volcanic glass (Ross & Fisher 1986). At this time, however, no mechanism was known to support such biological activity in subsurface environments. Volcanic rock of oceanic crust was considered to be a sterile environment after formation, requiring ingression of microorganisms from seawater to explain the presence of biogenic fossil-like structures (Bengtson et al. 2014). Rock dwelling organisms, including those that inhabit the inner space of the lithosphere, are termed “endoliths” (Ivarsson et al. 2021). These are further grouped by the kind of structural cavity they are found in. Euendoliths actively penetrate rock interiors and settle within these formations, chasmo- and cryptoendoliths inhabit already present cavities. Chasmoendoliths are defined by inhabiting fissures and cracks, whereas cryptoendoliths settle in structural cavities like vesicles. In suboceanic environments, cryptoendoliths are introduced into newly formed bedrock from circulating seawater via the cracks and vesicles that form in the rock as the magma cools and volatiles escape (Eickmann et al. 2009; Schmid-Beurmann et al. 2023). Microcracks connect vesicles and form paths throughout the rock, allowing microorganisms to disperse and colonize wide areas. Peckmann et al. (2008) and Eickmann et al. (2009) reported examples of cryptoendoliths living within Devonian pillow basalts of the Arnstein (Rheinisches Schiefergebirge) and Kahlleite (Thüringer Wald) localities in Germany. Fossils of proposed terrestrial fungi have been reported from the Devonian period, occurrences contemporaneous with the findings of Peckmann et al. (2008) and Eickmann et al (2009) (Ivarsson et al. 2013). Schumann et al. (2004) and Ivarsson (2012) have argued for the presence of fossilised fungi in rocks of the oceanic crust, the latter after re-evaluating what was previously considered to be prokaryotic traces. Bengtson et al. (2014) examined fungi and prokaryotic organisms in subseafloor basalts from the Eocene epoch. Body fossils of fungi are commonly represented by mycelial or mycelial-like networks, with anastomoses between branches, hyphae, hyphal tips, and septa (Ivarsson et al. 2020). Sporophores, the fruiting bodies of fungi, have also been identified (Ivarsson et al. 2018). Putative Palaeoproterozoic fossils, 2.4 Ga, resembling fungi have been found in the Ongeluk Large Igneous Province (LIP) of southern Africa (Bengtson et al. 2017). These findings however predate the earliest known fungi by one billion years. and their fungal affiliation is thus not confirmed. Evidence of fungi within the continental deep biosphere ranges from present to around 400 Ma (Ivarsson et al. 2012). In oceanic basalts, the known range is smaller, ranging from today to ca. 81 Ma (Ivarsson et al. 2012). If the fungus-like Ongeluk fossils would indeed represent crown group fungi, a gap of over two billion years would exist until the next observation of fungus-like mycelial networks. Herein, we revisit the Devonian cryptoendolithic microfossils described by Peckmann et al. (2008) and Eickmann et al. (2009). We re-evaluate the previous findings and provide a more in-depth analysis of the inferred biological affinity. Using microscopy and spectroscopy techniques including optical, and confocal microscopy and Raman spectroscopy the aim of this study is to constrain the biological affiliations of the filamentous fossils. The observed morphologies among the Devonian fossils suggest fungal affiliations. Finding such evidence of fungal life within the studied Devonian basalts extends the known maximum age of fungal life in the oceanic basalt by over 300 million years, not considering the problematic Palaeoproterozoic Ongeluk fossils. 2 Samples and methods 2.1 Samples The samples analysed in this study have previously been investigated by Peckmann et al. (2008) and Eickmann et al. (2009). Five thin sections from the Arnstein locality and three from the Kahlleite locality in Thuringia were studied herein (Table 1). Peckmann et al. (2008) collected vesicular pillow basalt from the Arnstein locality in the northeastern Rheinisches Schiefergebirge (Rhenish Massif). Vesicular pillow basalt from the Kahlleite locality of the eastern Thüringer Wald (Thuringian Forest) was collected by Eickmann et al. (2009). Some of the studied thin sections were partially stained with a mixture of potassium ferricyanide and alizarin red dissolved in 0.1% HCl to recognize carbonate minerals. Table 1: Samples collected by Peckmann et al. (2008) and Eickmann et al. (2009). Thin sections with asterisks were renamed for this report. Location Slide labels Thuringia TH-be-08-4 Thuringia TH-be-08-09A Thuringia TH-be-08-09D Arnstein Ar-03* Arnstein Ar-04* Arnstein Ar-05* Arnstein Ar-07* Arnstein Ar-08* 2.2 Methods 2.2.1 Microscopy The thin sections were studied with an Olympus BX51 optical light microscope. Images were captured using Olympus cellSens Dimension imaging software. Measurements of filament diameters for comparative analysis were made using the built-in line measuring tool in the software. 2.2.2 Environmental Scanning Electron Microscopy and EDS analysis Images of thin sections from the Arnstein and Kahlleite localities were produced with an XL30 environmental scanning electron microscope (ESEM) with an Oxford x-act energy dispersive spectrometer (EDS), a backscatter electron detector and a secondary electron detector attached. The acceleration potential was set to 20 kV, the microscope was calibrated using a cobalt standard, and analyses were recorded using AZTEC software to study elemental composition of the samples, either to create a spectrum of compositions using Point & ID for elemental peak analysis or to produce K series maps. 2.2.3 Confocal microscopy Images were acquired with epifluorescence using a ZEISS AxioObserver laser scanning confocal microscope 780 (LSM) with an LD C-Apochromat 63x/1.15 W Korr M27 objective at the Imaging Facility at Stockholm University. The immersion medium was water. An argon laser was used at a wavelength of 458 nm and a power of 25 W. The image channel wavelength was 488 nm. Gain #1 was set to 850, Gain #2 was set to 333. Both scanning modes were set to “frame”. Bits per pixel was set to 16. Beam splitter filter #1 was set to “MBS 488” and beam splitter filter #2 was set to “plate”. The image detection wavelength ranges were ca. 491.54 to 625.05. 2.2.4 Widefield microscopy Widefield images of sample Ar-04 were taken with a ZEISS AXIO Observer.Z1/7 inverted light microscope to provide a three-dimensional view. Micrographs were taken with the ZEISS ZEN 3.1 (blue edition) software using a Hamamatsu camera with a Plan-Apochromat 40x/0.95 Korr M27 objective. 2.2.5 Raman spectroscopy Raman data was collected of a thin section from the Kahlleite locality (Thuringia TH-be-08-09D) using a WITec Alpha 300 RAS system (Oxford Instruments, Ulm, Germany) that has been customised to incorporate confocal Raman spectroscopic imaging. The excitation source was a frequency-doubled solid-state YAG laser (532 nm) and a spectrometer diffraction grating of 600 L mm −1 was implemented (0-3600 cm -1 ). The measurements were done using a Zeiss LD EC Epiplan-Neofluar Dic 50x / 0.55 NA objective with the laser operating at 0.5-2 mW to minimize laser damage of refractory carbon and sensitive minerals such as iron oxides. No obvious changes were observed in either the optical image or spectra at different laser powers indicating no effect from the laser. Both Raman 2D images and single spectra were collected. Single spectra were collected with an integration time of 1 s per accumulation and 50 accumulations. Confocal Raman images (10x10 µm) were collected at double sampling rate (20 pixels per line and 20 pixels per image). Integration time per pixel was 1 s. 3 Results 3.1 Host rock and calcite matrix The ophiolites from Arnstein and Kahlleite localities consist of pillow basalt with ubiquitous veins and vesicles (Figure 1), the former pore space is filled with sparry calcite cement. The host rock differs in colour and appearance between the samples, but the secondary calcite matrix is similar, with differences mainly in the size of the equant crystals of calcite cement. The host rock in sample Ar-05 is light and homogenous with larger crystals embedded in the rock matrix (Figure 1a). The basalt in sample Ar-03 is darker with variations in colour and a more heterogeneous appearance (Figure 1b). Samples TH-be-08-4 and TH-be-08-09A (Figures 1c, d) are typified by dark basalt and abundant vesicles filled by light calcite cement. 3.2 Morphologies of microfossils All studied thin sections from the Arnstein locality contained filaments consisting of an inner strand, a sheath and in some cases an outer layer of iron oxide trapped in calcite (Figure 2a). EDS analyses of the samples reveal the elements Ca, Al, Si, K and Mg, common to clay minerals. A vesicle with several filaments in sample Ar-03 was previously described by Peckmann et al. (2008; their figures 2, 3). Another vesicle in sample Ar-03 with two separate generations of calcite was observed, with one containing several cross sections of filaments with outer layers consisting of ferric iron minerals and inner strands (Figure 3). Some filaments present in both localities contained the inner strand and the outer sheath but lacked the surrounding layer of iron minerals (Figure 4). These were smaller in size, 8.6-28.4 µm, compared to those with the secondary iron mineral layer, ca. 21.3-126.5 µm. Cross sections of the Ar-04 filaments show that the clear outer layers encase the inner strand fully (Figure 4), and some filaments were apparently branching. Filaments are overall less common in the Kahlleite rocks, but the different types of microstructures tend to occur together like in sample TH-be-08-09D (Figure 4c), whereas the different types of filaments tend to occur isolated in individual vesicles in the Arnstein samples. One instance of clustered filaments within a vesicle shows an irregular assemblage of filaments with internal segmentation in sample TH-be-08-09D (Figure 4c, d). In the filament rich vesicle of sample TH-be-08-09D, a structure made up of two filaments connected at a septum by a string was present (Figure 4d). Bulbous ends in filaments Filaments with bulbous ends were observed for both localities. One filament in sample Ar-05 revealed large bulbs at the end of a bulbous tail (Figure 5e). Sample Ar-04 contained several vesicles with sheathed filaments, including many with bulbous ends. To visualise these microstructures in detail, optical, brightfield and confocal microscopy were used. Images reveal the structures to be cohesive rather than separate, layered structures (Figure 5d). Similar structures were present in the samples of the Kahlleite locality, e.g. TH-be-08-09D. (Figure 5e). Confocal and widefield views of the samples show individual spherules embedded in the structures (Figs 5b, c). 3.3 Raman analysis Raman image scans of thin section TH-be-08-09D (Figure 7) show calcite (peaks at 157, 282 and 1087 cm -1 ; Figure 7c), while ferric iron minerals (hematite 225, 293, 409, 613, 660 and 1320 cm -1 ; Figure 7c) are associated with sheathed filamentous microstructures (cf. Faria et al. 1997 and 2007). In addition to the iron oxides, there is also a peak at 1560-1580 cm -1 that could potentially be assigned to the G-band of refractory carbon associated with the microstructures. Any D-band of refractory carbon (~1350 cm-1) is obscured by the strong peak produced by hematite at 1320 cm -1 . 4 Discussion Because a biological origin of a fossil-like microstructure is not always evident, it is necessary to have criteria for what can be interpreted as either a biological or an abiotic trace. Ivarsson et al. (2021) presented a flowchart where the most compelling evidence for biology, including syngenetic biomarkers (known only to occur during biological processes) and complex morphologies that are unlikely to result from abiotic processes, should be compared to abiotic signatures. Should a microstructure show compelling but inconclusive evidence for both biological and abiotic origin, it may be described as a dubiofossil (Ivarsson et al. 2021). Morphology is overall a less convincing character but may in some situations be the only available data (Ivarsson et al. 2021). Morphology alone is still able to provide unique data, i.e. the physical characters of a putative fossil. Problems may arise due to abiotic processes by chance producing microstructures that appear to be biological in origin (Ivarsson et al. 2021). In such a scenario it is helpful to know the taphonomic processes involved in fossilisation or diagenetic processes that might form a host rock. Morphology is most useful when combined with chemical analyses and should not be the sole argument for a microstructure being biotic in origin (Ivarsson et al. 2021). McMahon (2019) proposed an abiotic explanation of hyphae and hyphal networks pointing to irregularities in filaments and shows that in laboratory experiments, branches will grow out of a central origin and show a similar appearance to hyphal growth. This shows that abiotic growth of filaments can occur and that such processes may also explain the filaments present in the Arnstein and Kahlleite samples. However, the structures described in McMahon (2019) differ from those described herein, with the possible exception of the apparent hyphal tip in Fig. 4 e. The vesicular pillow basalts from Germany yielded single filaments eventually growing into bulbous shapes on the ends rather than the bulbs being the origin points with several points of branching. Furthermore, the central strands of the Devonian filaments do not consist of iron oxyhydroxide minerals but clay minerals consistent with clay remineralisation on an organic matrix (Fig. 2 ; cf. Konhauser & Urrutia 1999). Peckmann et al. (2008) and Eickmann et al. (2009) did not distinguish morphological or chemical characters among the fossilised structures diagnostic for biological affiliation, so resorted to labelling them as microfossils. They pointed towards several morphological traits in support of a biological origin, including the size range being similar to that of microorganisms, a distinct lack of variation, their positioning, and growth being independent of cleavage planes as well as morphological structures pointing to cell division or differentiation. Filament size and growth direction were also arguments for their biological affiliation. Furthermore, they argued for the observed septa as evidence of biology and that they may agree with a fungal affiliation. The filaments described herein are identical to fossilised hyphae described from similar environments in previous studies (Ivarsson et al. 2020). Both the internal structure with sheaths and the mycelial like appearance are in accordance with fossil fungi rather than prokaryotic remains (Ivarsson et al. 2020). Iron-oxidising bacteria commonly mineralize as homogenous iron oxides, sometimes with secondary, irregular growth. Conversely, fossilised hyphae from fungi usually appear as more complex ultrastructures. Thus, our interpretation is that the filaments represent fossilised fungal hyphae. In the context of fungi, the hyphae with bulbous ends are identical to fungal sporophores (Webster & Weber 2007). The seamless transition from filamentous hyphae to a terminal bulb with a slightly irregular surface are all characteristic for fungal sporophores. In Fig. 5 b, it is possible that the contents visible are remnants of unreleased spores. While the size ranges partly coincide, there are clear size differences between the filaments with an outer layer of a ferric iron mineral and the filaments without (Fig. 6 ), but EDS analysis revealed a similar composition of clay minerals (Fig. 2 ). Filaments with the outer layer of ferric iron minerals were more commonly found as a single type of filament within a vesicle and would commonly take up a lot of space within the vesicle (Fig. 2 ); large clusters with abundant microstructures without iron minerals occur but are less common than filaments with iron mineral coatings. Further studies might reveal new information about these apparently distinctive morphologies. Our re-evaluation of the studied samples suggests that some of the filamentous microstructures represent sheathed hyphae in mycelial networks, similar to the fossils presented in Ivarsson et al. (2020), some surrounded by a layer of a ferric mineral. We did not, however, find anastomoses in these samples. The Raman image scans of the Thuringian hyphae (Fig. 7 c) reveal potential refractory carbon together with hematite. Iron oxide minerals have a high affinity to carbon (Ivarsson et al. 2015), yet microfossils consisting of iron minerals from oceanic basalt are not necessarily associated with carbon (Qu et al. 2023). Some samples were difficult to analyse with ESEM due to being carbon coated, and the G bands from the Raman analysis are consistent with refractory carbon (Zhao et al. 2011). However, because this carbon is present throughout the scanned filaments and not found outside of them, it is likely remaining organic carbon from the organisms rather than remnants of a carbon coating. EDS analysis suggested a clay-like phase, but this could not be confirmed by Raman spectroscopy. Thus, it is likely a clay-phase that is poorly crystalline. Biogenicity is also supported by the presence of a sporophore-like structure (Fig. 5 ), with the confocal view confirming that the observed detail represents one cohesive structure and not an accumulation of unrelated fossilised structures. The widefield image in Fig. 5 shows spheres within these microstructures that are separate from the body of the terminal bulbs, consistent with the presence of spores. The possible identification of spores within the sporophore-like structures supports the assignment of the filamentous Arnstein and Kahlleite microfossils to fungi. The presence of these spheres in association with sporophore-like structures agrees with the interpretation of the microstructures as fossilised fungi. The leftmost sporophore in Fig. 5 a appears ruptured and likely contained spores that were released at some point, and at least one spherule is present outside of it. Figure 5 f shows a morphology in a Kahlleite sample resembling a sporophore, similar to the ones in the Arnstein samples. Regrettably, only few filaments exist that show all of each of these characteristics. The chemically studied filaments in Kahlleite have not yet shown up alongside sporophores. The presence of sporophore-like microstructures in both the Arnstein and Kahlleite samples from two different mountain ranges indicates that vesicular basalt provided a common subseafloor habitat for fungi in the Devonian period. The structure shown in Fig. 4 d resembles a dolipore septum, such as the one presented in Lima-Zaloumis et al. (2022). In their study, this type of microstructure has been suggested to represent evidence of basidiomycetes. The findings presented in this paper suggest that a suboceanic, basaltic bedrock could have hosted fungi as early as the Devonian period. Further research into the biological affinity of life within the basaltic oceanic crust should include investigations of traces of fossil life within ophiolites, for instance either with confocal microscopy or X-ray tomography as well as isotope analysis. 5 Conclusions Filamentous microstructures within vesicles of Devonian pillow basalt from the Rheinisches Schiefergebirge and the Thüringer Wald, previously reported by Peckmann et al. (2008) and Eickmann et al. (2009), were revisited herein. Our re-evaluation of the microstructures as well as identification of new microstructures suggests that many of them plausibly represent fungi, in one case basidiomycetes specifically. While morphology as a criterion sometimes comes with ambiguities, several of the identified microstructures have not yet been shown to result from abiotic processes. Some experiments with abiotic growth result in suspiciously biomorphic structures, however none of which resembles the microstructures observed in the Devonian pillow basalts. These findings support further investigations of submarine basalt as a hub for well-preserved microfossils. Furthermore, our findings reinforce the idea to target basaltic bedrock in extraterrestrial missions such as the search for life on Mars. Declarations Competing interests: The authors did not receive support from any organization for the submitted work. Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Dan Strömbäck, Sandra Siljeström, and Magnus Ivarsson. The first draft of the manuscript was written by Dan Strömbäck and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. 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Physica B: Condensed Matter 406:3876–3884. https://doi.org/10.1016/j.physb.2011.07.016 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major Revision 30 Dec, 2025 Reviewers agreed at journal 09 May, 2025 Reviewers invited by journal 08 May, 2025 Editor assigned by journal 24 Apr, 2025 First submitted to journal 23 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6512033","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":453964029,"identity":"a708e97b-badd-425f-8282-1fbab914e1cc","order_by":0,"name":"Dan Strömbäck","email":"","orcid":"","institution":"Swedish Museum of Natural History: Naturhistoriska riksmuseet","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Strömbäck","suffix":""},{"id":453964030,"identity":"5c9e7657-4c51-4309-a73b-25f90d80c7c3","order_by":1,"name":"Jörn Peckmann","email":"","orcid":"","institution":"University of Hamburg: Universitat Hamburg","correspondingAuthor":false,"prefix":"","firstName":"Jörn","middleName":"","lastName":"Peckmann","suffix":""},{"id":453964031,"identity":"6fe5b236-2ff3-46a4-ab76-1b7a6265d971","order_by":2,"name":"Sandra Siljeström","email":"","orcid":"","institution":"RISE Research Institutes of Sweden AB","correspondingAuthor":false,"prefix":"","firstName":"Sandra","middleName":"","lastName":"Siljeström","suffix":""},{"id":453964032,"identity":"7ca51ddb-ca15-4a5b-97fe-a844675712c5","order_by":3,"name":"Ashley Krüger","email":"","orcid":"","institution":"Swedish Museum of Natural History: Naturhistoriska riksmuseet","correspondingAuthor":false,"prefix":"","firstName":"Ashley","middleName":"","lastName":"Krüger","suffix":""},{"id":453964033,"identity":"6f2a4e8a-5ed0-42c6-b6a9-3869e17d9d8e","order_by":4,"name":"Magnus Ivarsson","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYBADHhhDjnQtxqRbl9hASIVu+/Frj3n+MMgYHD/7+OOPijvp26VPJzD+qMCtxexMTrkxbxsDj8GZdDNpnjPPcnf25W5g5jmDR8uBnDRp3gYGHsmGNDZmxrbDuRvO8G4AMvBoOf8mTRroMB7J/mfMH3+2HU43AGph/PkPj5Yb6cekedgYePgl0hgkeNsOJ4C0MPDiCQSzG2/YJOe2SQC1PGMD+uWwIchhh3mO4XNY+jOJN39s7Nn405iBIXZYHmjLxoc/anBrAUaiAZCQQBU7gE8DAwP7A/zyo2AUjIJRMAoAAkpNZvpmSKwAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-4548-1560","institution":"Swedish Museum of Natural History: Naturhistoriska riksmuseet","correspondingAuthor":true,"prefix":"","firstName":"Magnus","middleName":"","lastName":"Ivarsson","suffix":""}],"badges":[],"createdAt":"2025-04-23 11:15:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6512033/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6512033/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82648096,"identity":"f6eaf746-17ca-4722-a1b1-810029e546fd","added_by":"auto","created_at":"2025-05-13 16:33:55","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":840786,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of thin sections showing host basalt and secondary calcite veins and vesicles. a. Ar05. b. Ar-03. c. TH-be-08-4. d. TH-be-08-09A. ‘Cc’ = Calcite, ‘Bas’ = Basalt\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6512033/v1/39c45dcffe224a2aa8b913d2.jpeg"},{"id":82647756,"identity":"50ca6863-ebad-4f7a-a419-c0b97f89ba84","added_by":"auto","created_at":"2025-05-13 16:25:55","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":869476,"visible":true,"origin":"","legend":"\u003cp\u003eDetail of slide Ar-03; the same detail has previously been described by Peckmann et al. (2008). a. Optical microscope image of filamentous microstructures. The yellowish layers surrounding the filaments are ferric clays. b. ESEM image, rotated ca. 90° clockwise compared to a. c. On the left: EDS layered image. On the right: Individual elements. e. ESEM image of filament without a secondary ferric layer. White crosses indicate where spectra in f were obtained. f. Spectrum graphs indicate a presence of clay minerals containing Al, K, Mg, and Si.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6512033/v1/311f1a033e974478c45a987b.jpeg"},{"id":82648098,"identity":"9524ce0c-c09f-4bfc-8746-07388f19dc72","added_by":"auto","created_at":"2025-05-13 16:33:55","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":812450,"visible":true,"origin":"","legend":"\u003cp\u003eTwo generations of calcite within a vesicle in sample Ar-03. Inner strands are present inside the circular shapes containing iron oxides and clay minerals. ‘Cc’ = Calcite, ‘Bas’ = Basalt\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6512033/v1/268f4f8e08de4675609c07c0.jpeg"},{"id":82647769,"identity":"3a780b3a-fcd5-4956-88d8-2c621c3151a1","added_by":"auto","created_at":"2025-05-13 16:25:55","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1043923,"visible":true,"origin":"","legend":"\u003cp\u003eFilaments in calcite matrices in Arnstein and Kahlleite samples. Inner strands are surrounded by a sheath along the entire filament. Circular morphologies are cross sections through these filaments. a. Ar-07. b. TH-be-08-4. Starts of branching visible as bumps along the sides. c. TH-be-08-09D. White circle indicates zoom-in area in d. d. Zoomed in area from c.Arrow points at a segmentation. On the left-hand side, a string-like filament connects two larger segments. e. Multiple ends of sheathed filaments in Ar-08. ‘Cc’ = Calcite\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6512033/v1/bd1dd815fda3477f232523c2.jpeg"},{"id":82648696,"identity":"497cd770-11ee-4696-95a5-df32099a8d76","added_by":"auto","created_at":"2025-05-13 16:41:55","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1011760,"visible":true,"origin":"","legend":"\u003cp\u003eBulbous morphologies at the ends of filaments in Arnstein and Kahlleite samples. Contents visible within the structures. a. Ar-04. b. Widefield image of structure attached to a vesicle wall in Ar-04. Spherules visible inside the bulbous end. c. Confocal image of the right-most structure in 6a. Spherules visible within the structure. Arrow points towards a spherule. d. Brightfield view of the structure in 6a shows that the visible morphology is one coherent structure. e. Optical light microscopy view of structure in Ar-05. In this instance, the tail also appears somewhat bulbous. f. Structure growing from basalt wall in TH-be-08-09D\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6512033/v1/ec7f6d2239e0d4c867843f8f.jpeg"},{"id":82648695,"identity":"0f4d0cbd-16b2-4a28-8ecf-08462d4d257c","added_by":"auto","created_at":"2025-05-13 16:41:55","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":112928,"visible":true,"origin":"","legend":"\u003cp\u003eDiameter comparison between filaments with and without secondary ferric layers. For the latter filaments, only the diameter excluding the secondary mineral growth is considered. 36 measurements make up the range of diameters in the category without a secondary layer, ten samples make up the range in the category of filaments with a mineral coating\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6512033/v1/026d548be7d69092558703c8.jpeg"},{"id":82647759,"identity":"c77bf9e1-09f5-40f4-a23b-dee5de69560b","added_by":"auto","created_at":"2025-05-13 16:25:55","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":184582,"visible":true,"origin":"","legend":"\u003cp\u003ea. Micrograph of sample TH-be-08-09D. The white square indicates the area of a Raman image scan. b. Raman image scan of filament showing hematite (blue) and calcite (red). c. Spectra showing hematite and potential organics (top) and calcite (bottom)\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6512033/v1/ba88d475cbc3c9e21fb8ef6b.jpeg"},{"id":82649164,"identity":"a647e6f2-aadf-49ed-a73e-0cef2f2a7ed9","added_by":"auto","created_at":"2025-05-13 16:49:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5368808,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6512033/v1/f87fd155-5068-4c26-acf3-f0cd95b5adb7.pdf"}],"financialInterests":"","formattedTitle":"Re-evaluation of endolithic microfossils in pillow basalt of two Variscan orogens","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eBelow the surface of Earth resides the deep biosphere, the second largest biosphere on Earth (Orcutt et al., 2013; Goordial et al. 2021). Despite its size, little is known about its composition and modes of operation. Furthermore, most studies of the deep biosphere focused on deep-sea sediments, resulting in a lack of knowledge of microbial life in the oceanic and continental igneous crusts (Goordial et al. 2021). Research on microbial life in the deep oceanic crust began in the 1980s with the finding of potential bioalteration textures in volcanic glass (Ross \u0026amp; Fisher 1986). At this time, however, no mechanism was known to support such biological activity in subsurface environments. Volcanic rock of oceanic crust was considered to be a sterile environment after formation, requiring ingression of microorganisms from seawater to explain the presence of biogenic fossil-like structures (Bengtson et al. 2014).\u003c/p\u003e \u003cp\u003eRock dwelling organisms, including those that inhabit the inner space of the lithosphere, are termed \u0026ldquo;endoliths\u0026rdquo; (Ivarsson et al. 2021). These are further grouped by the kind of structural cavity they are found in. Euendoliths actively penetrate rock interiors and settle within these formations, chasmo- and cryptoendoliths inhabit already present cavities. Chasmoendoliths are defined by inhabiting fissures and cracks, whereas cryptoendoliths settle in structural cavities like vesicles. In suboceanic environments, cryptoendoliths are introduced into newly formed bedrock from circulating seawater via the cracks and vesicles that form in the rock as the magma cools and volatiles escape (Eickmann et al. 2009; Schmid-Beurmann et al. 2023). Microcracks connect vesicles and form paths throughout the rock, allowing microorganisms to disperse and colonize wide areas.\u003c/p\u003e \u003cp\u003ePeckmann et al. (2008) and Eickmann et al. (2009) reported examples of cryptoendoliths living within Devonian pillow basalts of the Arnstein (Rheinisches Schiefergebirge) and Kahlleite (Th\u0026uuml;ringer Wald) localities in Germany. Fossils of proposed terrestrial fungi have been reported from the Devonian period, occurrences contemporaneous with the findings of Peckmann et al. (2008) and Eickmann et al (2009) (Ivarsson et al. 2013). Schumann et al. (2004) and Ivarsson (2012) have argued for the presence of fossilised fungi in rocks of the oceanic crust, the latter after re-evaluating what was previously considered to be prokaryotic traces. Bengtson et al. (2014) examined fungi and prokaryotic organisms in subseafloor basalts from the Eocene epoch. Body fossils of fungi are commonly represented by mycelial or mycelial-like networks, with anastomoses between branches, hyphae, hyphal tips, and septa (Ivarsson et al. 2020). Sporophores, the fruiting bodies of fungi, have also been identified (Ivarsson et al. 2018). Putative Palaeoproterozoic fossils, 2.4 Ga, resembling fungi have been found in the Ongeluk Large Igneous Province (LIP) of southern Africa (Bengtson et al. 2017). These findings however predate the earliest known fungi by one billion years. and their fungal affiliation is thus not confirmed.\u003c/p\u003e \u003cp\u003eEvidence of fungi within the continental deep biosphere ranges from present to around 400 Ma (Ivarsson et al. 2012). In oceanic basalts, the known range is smaller, ranging from today to ca. 81 Ma (Ivarsson et al. 2012). If the fungus-like Ongeluk fossils would indeed represent crown group fungi, a gap of over two billion years would exist until the next observation of fungus-like mycelial networks. Herein, we revisit the Devonian cryptoendolithic microfossils described by Peckmann et al. (2008) and Eickmann et al. (2009). We re-evaluate the previous findings and provide a more in-depth analysis of the inferred biological affinity. Using microscopy and spectroscopy techniques including optical, and confocal microscopy and Raman spectroscopy the aim of this study is to constrain the biological affiliations of the filamentous fossils. The observed morphologies among the Devonian fossils suggest fungal affiliations. Finding such evidence of fungal life within the studied Devonian basalts extends the known maximum age of fungal life in the oceanic basalt by over 300\u0026nbsp;million years, not considering the problematic Palaeoproterozoic Ongeluk fossils.\u003c/p\u003e"},{"header":"2 Samples and methods","content":"\u003ch2\u003e2.1 Samples\u003c/h2\u003e\n\u003cp\u003eThe samples analysed in this study have previously been investigated by Peckmann et al. (2008) and Eickmann et al. (2009). Five thin sections from the Arnstein locality and three from the Kahlleite locality in Thuringia were studied herein (Table 1).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ePeckmann et al. (2008) collected vesicular pillow basalt from the Arnstein locality in the northeastern Rheinisches Schiefergebirge (Rhenish Massif). Vesicular pillow basalt from the Kahlleite locality of the eastern Th\u0026uuml;ringer Wald (Thuringian Forest) was collected by Eickmann et al. (2009). Some of the studied thin sections were partially stained with a mixture of potassium ferricyanide and alizarin red dissolved in 0.1% HCl to recognize carbonate minerals.\u003c/p\u003e\n\u003cp\u003eTable 1: Samples collected by Peckmann et al. (2008) and Eickmann et al. (2009). Thin sections with asterisks were renamed for this report.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eLocation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eSlide labels\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eThuringia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eTH-be-08-4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eThuringia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eTH-be-08-09A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eThuringia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eTH-be-08-09D\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eArnstein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eAr-03*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eArnstein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eAr-04*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eArnstein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eAr-05*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eArnstein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eAr-07*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eArnstein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eAr-08*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003ch2\u003e2.2 Methods\u003c/h2\u003e\n\u003ch3 id=\"_Toc12862\"\u003e2.2.1 \u0026nbsp; \u0026nbsp;Microscopy\u003c/h3\u003e\n\u003cp\u003eThe thin sections were studied with an Olympus BX51 optical light microscope. Images were captured using Olympus cellSens Dimension imaging software. Measurements of filament diameters for comparative analysis were made using the built-in line measuring tool in the software.\u003c/p\u003e\n\u003ch3 id=\"_Toc12863\"\u003e2.2.2 \u0026nbsp; \u0026nbsp;Environmental Scanning Electron Microscopy and EDS analysis\u003c/h3\u003e\n\u003cp\u003eImages of thin sections from the Arnstein and Kahlleite localities were produced with an XL30 environmental scanning electron microscope (ESEM) with an Oxford x-act energy dispersive spectrometer (EDS), a backscatter electron detector and a secondary electron detector attached. The acceleration potential was set to 20 kV, the microscope was calibrated using a cobalt standard, and analyses were recorded using AZTEC software to study elemental composition of the samples, either to create a spectrum of compositions using Point \u0026amp; ID for elemental peak analysis or to produce K series maps.\u003c/p\u003e\n\u003ch3 id=\"_Toc12864\"\u003e2.2.3 \u0026nbsp; \u0026nbsp;Confocal microscopy\u003c/h3\u003e\n\u003cp\u003eImages were acquired with epifluorescence using a ZEISS AxioObserver laser scanning confocal microscope 780 (LSM) with an LD C-Apochromat 63x/1.15 W Korr M27 objective at the Imaging Facility at Stockholm University. The immersion medium was water. An argon laser was used at a wavelength of 458 nm and a power of 25 W. The image channel wavelength was 488 nm. Gain #1 was set to 850, Gain #2 was set to 333. Both scanning modes were set to \u0026ldquo;frame\u0026rdquo;. Bits per pixel was set to 16. Beam splitter filter #1 was set to \u0026ldquo;MBS 488\u0026rdquo; and beam splitter filter #2 was set to \u0026ldquo;plate\u0026rdquo;. The image detection wavelength ranges were ca. 491.54 to 625.05.\u003c/p\u003e\n\u003ch3 id=\"_Toc12865\"\u003e2.2.4 \u0026nbsp; \u0026nbsp;Widefield microscopy\u003c/h3\u003e\n\u003cp\u003eWidefield images of sample Ar-04 were taken with a ZEISS AXIO Observer.Z1/7 inverted light microscope to provide a three-dimensional view. Micrographs were taken with the ZEISS ZEN 3.1 (blue edition) software using a Hamamatsu camera with a Plan-Apochromat 40x/0.95 Korr M27 objective.\u0026nbsp;\u003c/p\u003e\n\u003ch3 id=\"_Toc12866\"\u003e2.2.5 \u0026nbsp; \u0026nbsp;Raman spectroscopy\u003c/h3\u003e\n\u003cp\u003eRaman data was collected of a thin section from the Kahlleite locality (Thuringia TH-be-08-09D) using a WITec Alpha 300 RAS system (Oxford Instruments, Ulm, Germany) that has been customised to incorporate confocal Raman spectroscopic imaging. The excitation source was a frequency-doubled solid-state YAG laser (532 nm) and a spectrometer diffraction grating of 600 L mm\u003csup\u003e\u0026minus;1\u003c/sup\u003e was implemented (0-3600 cm\u003csup\u003e-1\u003c/sup\u003e). The measurements were done using a Zeiss LD EC Epiplan-Neofluar Dic 50x / 0.55 NA objective with the laser operating at 0.5-2 mW to minimize laser damage of refractory carbon and sensitive minerals such as iron oxides. No obvious changes were observed in either the optical image or spectra at different laser powers indicating no effect from the laser. Both Raman 2D images and single spectra were collected. Single spectra were collected with an integration time of 1 s per accumulation and 50 accumulations. Confocal Raman images (10x10 \u0026micro;m) were collected at double sampling rate (20 pixels per line and 20 pixels per image). Integration time per pixel was 1 s.\u003c/p\u003e"},{"header":"3 Results","content":"\u003ch2\u003e3.1 Host rock and calcite matrix\u003c/h2\u003e\n\u003cp\u003eThe ophiolites from Arnstein and Kahlleite localities consist of pillow basalt with ubiquitous veins and vesicles (Figure 1), the former pore space is filled with sparry calcite cement. The host rock differs in colour and appearance between the samples, but the secondary calcite matrix is similar, with differences mainly in the size of the equant crystals of calcite cement. The host rock in sample Ar-05 is light and homogenous with larger crystals embedded in the rock matrix (Figure 1a). The basalt in sample Ar-03 is darker with variations in colour and a more heterogeneous appearance (Figure 1b). Samples TH-be-08-4 and TH-be-08-09A (Figures 1c, d) are typified by dark basalt and abundant vesicles filled by light calcite cement.\u003c/p\u003e\n\u003ch2 id=\"_Toc12869\"\u003e3.2 Morphologies of microfossils\u003c/h2\u003e\n\u003cp\u003eAll studied thin sections from the Arnstein locality contained filaments consisting of an inner strand, a sheath and in some cases an outer layer of iron oxide trapped in calcite (Figure 2a). EDS analyses of the samples reveal the elements Ca, Al, Si, K and Mg, common to clay minerals. A vesicle with several filaments in sample Ar-03 was previously described by Peckmann et al. (2008; their figures 2, 3). Another vesicle in sample Ar-03 with two separate generations of calcite was observed, with one containing several cross sections of filaments with outer layers consisting of ferric iron minerals and inner strands (Figure 3).\u003c/p\u003e\n\u003cp\u003eSome filaments present in both localities contained the inner strand and the outer sheath but lacked the surrounding layer of iron minerals (Figure 4). These were smaller in size, 8.6-28.4 \u0026micro;m, compared to those with the secondary iron mineral layer, ca. 21.3-126.5 \u0026micro;m. Cross sections of the Ar-04 filaments show that the clear outer layers encase the inner strand fully (Figure 4), and some filaments were apparently branching.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFilaments are overall less common in the Kahlleite rocks, but the different types of microstructures tend to occur together like in sample TH-be-08-09D (Figure 4c), whereas the different types of filaments tend to occur isolated in individual vesicles in the Arnstein samples. One instance of clustered filaments within a vesicle shows an irregular assemblage of filaments with internal segmentation in sample TH-be-08-09D (Figure 4c, d). In the filament rich vesicle of sample TH-be-08-09D, a structure made up of two filaments connected at a septum by a string was present (Figure 4d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBulbous ends in filaments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFilaments with bulbous ends were observed for both localities. One filament in sample Ar-05 revealed large bulbs at the end of a bulbous tail (Figure 5e). Sample Ar-04 contained several vesicles with sheathed filaments, including many with bulbous ends. To visualise these microstructures in detail, optical, brightfield and confocal microscopy were used. Images reveal the structures to be cohesive rather than separate, layered structures (Figure 5d). Similar structures were present in the samples of the Kahlleite locality, e.g. TH-be-08-09D. (Figure 5e). Confocal and widefield views of the samples show individual spherules embedded in the structures (Figs 5b, c).\u003c/p\u003e\n\u003ch2 id=\"_Toc12870\"\u003e3.3 Raman analysis\u003c/h2\u003e\n\u003cp\u003eRaman image scans of thin section TH-be-08-09D (Figure 7) show calcite (peaks at 157, 282 and 1087 cm\u003csup\u003e-1\u003c/sup\u003e; Figure 7c), while ferric iron minerals (hematite 225, 293, 409, 613, 660 and 1320 cm\u003csup\u003e-1\u003c/sup\u003e; Figure 7c) are associated with sheathed filamentous microstructures (cf. Faria et al. 1997 and 2007). In addition to the iron oxides, there is also a peak at 1560-1580 cm\u003csup\u003e-1\u003c/sup\u003e that could potentially be assigned to the G-band of refractory carbon associated with the microstructures. Any D-band of refractory carbon (~1350 cm-1) is obscured by the strong peak produced by hematite at 1320 cm\u003csup\u003e-1\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eBecause a biological origin of a fossil-like microstructure is not always evident, it is necessary to have criteria for what can be interpreted as either a biological or an abiotic trace. Ivarsson et al. (2021) presented a flowchart where the most compelling evidence for biology, including syngenetic biomarkers (known only to occur during biological processes) and complex morphologies that are unlikely to result from abiotic processes, should be compared to abiotic signatures. Should a microstructure show compelling but inconclusive evidence for both biological and abiotic origin, it may be described as a dubiofossil (Ivarsson et al. 2021). Morphology is overall a less convincing character but may in some situations be the only available data (Ivarsson et al. 2021). Morphology alone is still able to provide unique data, i.e. the physical characters of a putative fossil. Problems may arise due to abiotic processes by chance producing microstructures that appear to be biological in origin (Ivarsson et al. 2021). In such a scenario it is helpful to know the taphonomic processes involved in fossilisation or diagenetic processes that might form a host rock. Morphology is most useful when combined with chemical analyses and should not be the sole argument for a microstructure being biotic in origin (Ivarsson et al. 2021).\u003c/p\u003e \u003cp\u003eMcMahon (2019) proposed an abiotic explanation of hyphae and hyphal networks pointing to irregularities in filaments and shows that in laboratory experiments, branches will grow out of a central origin and show a similar appearance to hyphal growth. This shows that abiotic growth of filaments can occur and that such processes may also explain the filaments present in the Arnstein and Kahlleite samples. However, the structures described in McMahon (2019) differ from those described herein, with the possible exception of the apparent hyphal tip in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee. The vesicular pillow basalts from Germany yielded single filaments eventually growing into bulbous shapes on the ends rather than the bulbs being the origin points with several points of branching. Furthermore, the central strands of the Devonian filaments do not consist of iron oxyhydroxide minerals but clay minerals consistent with clay remineralisation on an organic matrix (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; cf. Konhauser \u0026amp; Urrutia 1999).\u003c/p\u003e \u003cp\u003ePeckmann et al. (2008) and Eickmann et al. (2009) did not distinguish morphological or chemical characters among the fossilised structures diagnostic for biological affiliation, so resorted to labelling them as microfossils. They pointed towards several morphological traits in support of a biological origin, including the size range being similar to that of microorganisms, a distinct lack of variation, their positioning, and growth being independent of cleavage planes as well as morphological structures pointing to cell division or differentiation. Filament size and growth direction were also arguments for their biological affiliation. Furthermore, they argued for the observed septa as evidence of biology and that they may agree with a fungal affiliation. The filaments described herein are identical to fossilised hyphae described from similar environments in previous studies (Ivarsson et al. 2020). Both the internal structure with sheaths and the mycelial like appearance are in accordance with fossil fungi rather than prokaryotic remains (Ivarsson et al. 2020). Iron-oxidising bacteria commonly mineralize as homogenous iron oxides, sometimes with secondary, irregular growth. Conversely, fossilised hyphae from fungi usually appear as more complex ultrastructures. Thus, our interpretation is that the filaments represent fossilised fungal hyphae.\u003c/p\u003e \u003cp\u003eIn the context of fungi, the hyphae with bulbous ends are identical to fungal sporophores (Webster \u0026amp; Weber 2007). The seamless transition from filamentous hyphae to a terminal bulb with a slightly irregular surface are all characteristic for fungal sporophores. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, it is possible that the contents visible are remnants of unreleased spores. While the size ranges partly coincide, there are clear size differences between the filaments with an outer layer of a ferric iron mineral and the filaments without (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), but EDS analysis revealed a similar composition of clay minerals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Filaments with the outer layer of ferric iron minerals were more commonly found as a single type of filament within a vesicle and would commonly take up a lot of space within the vesicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e); large clusters with abundant microstructures without iron minerals occur but are less common than filaments with iron mineral coatings. Further studies might reveal new information about these apparently distinctive morphologies.\u003c/p\u003e \u003cp\u003eOur re-evaluation of the studied samples suggests that some of the filamentous microstructures represent sheathed hyphae in mycelial networks, similar to the fossils presented in Ivarsson et al. (2020), some surrounded by a layer of a ferric mineral. We did not, however, find anastomoses in these samples. The Raman image scans of the Thuringian hyphae (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec) reveal potential refractory carbon together with hematite. Iron oxide minerals have a high affinity to carbon (Ivarsson et al. 2015), yet microfossils consisting of iron minerals from oceanic basalt are not necessarily associated with carbon (Qu et al. 2023). Some samples were difficult to analyse with ESEM due to being carbon coated, and the G bands from the Raman analysis are consistent with refractory carbon (Zhao et al. 2011). However, because this carbon is present throughout the scanned filaments and not found outside of them, it is likely remaining organic carbon from the organisms rather than remnants of a carbon coating. EDS analysis suggested a clay-like phase, but this could not be confirmed by Raman spectroscopy. Thus, it is likely a clay-phase that is poorly crystalline.\u003c/p\u003e \u003cp\u003eBiogenicity is also supported by the presence of a sporophore-like structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), with the confocal view confirming that the observed detail represents one cohesive structure and not an accumulation of unrelated fossilised structures. The widefield image in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows spheres within these microstructures that are separate from the body of the terminal bulbs, consistent with the presence of spores. The possible identification of spores within the sporophore-like structures supports the assignment of the filamentous Arnstein and Kahlleite microfossils to fungi. The presence of these spheres in association with sporophore-like structures agrees with the interpretation of the microstructures as fossilised fungi. The leftmost sporophore in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea appears ruptured and likely contained spores that were released at some point, and at least one spherule is present outside of it. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef shows a morphology in a Kahlleite sample resembling a sporophore, similar to the ones in the Arnstein samples. Regrettably, only few filaments exist that show all of each of these characteristics. The chemically studied filaments in Kahlleite have not yet shown up alongside sporophores. The presence of sporophore-like microstructures in both the Arnstein and Kahlleite samples from two different mountain ranges indicates that vesicular basalt provided a common subseafloor habitat for fungi in the Devonian period. The structure shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed resembles a dolipore septum, such as the one presented in Lima-Zaloumis et al. (2022). In their study, this type of microstructure has been suggested to represent evidence of basidiomycetes.\u003c/p\u003e \u003cp\u003eThe findings presented in this paper suggest that a suboceanic, basaltic bedrock could have hosted fungi as early as the Devonian period. Further research into the biological affinity of life within the basaltic oceanic crust should include investigations of traces of fossil life within ophiolites, for instance either with confocal microscopy or X-ray tomography as well as isotope analysis.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eFilamentous microstructures within vesicles of Devonian pillow basalt from the Rheinisches Schiefergebirge and the Th\u0026uuml;ringer Wald, previously reported by Peckmann et al. (2008) and Eickmann et al. (2009), were revisited herein. Our re-evaluation of the microstructures as well as identification of new microstructures suggests that many of them plausibly represent fungi, in one case basidiomycetes specifically. While morphology as a criterion sometimes comes with ambiguities, several of the identified microstructures have not yet been shown to result from abiotic processes. Some experiments with abiotic growth result in suspiciously biomorphic structures, however none of which resembles the microstructures observed in the Devonian pillow basalts. These findings support further investigations of submarine basalt as a hub for well-preserved microfossils. Furthermore, our findings reinforce the idea to target basaltic bedrock in extraterrestrial missions such as the search for life on Mars.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eCompeting interests:\u003c/strong\u003e \u003cp\u003eThe authors did not receive support from any organization for the submitted work.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Dan Str\u0026ouml;mb\u0026auml;ck, Sandra Siljestr\u0026ouml;m, and Magnus Ivarsson. The first draft of the manuscript was written by Dan Str\u0026ouml;mb\u0026auml;ck and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003e- M.I. acknowledges funding from Swedish Research Council (No. 2022\u0026ndash;03030). A.K. acknowledges funding from Knut and Alice Wallenberg Foundation (Sweden) (KAW 2020.0145 grant to Vivi Vajda).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBengtson S, Ivarsson M, Astolfo A, Belivanova V, Broman C, Marone F, Stampanoni M (2014) Deep-biosphere consortium of fungi and prokaryotes in Eocene subseafloor basalts. 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Proceedings of the Royal Society B 286:20192410. https://doi.org/10.1098/rspb.2019.2410\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrcutt BN, LaRowe DE, Biddle JF, Colwell FS, Glazer BT, Reese BK, Kirkpatrick JB, Lapham LL, Mills HJ, Sylvan JB, Wankel SD, Wheat CG (2013) Microbial activity in the marine deep biosphere: progress and prospects. Frontiers in Microbiology 4. https://doi.org/10.3389/fmicb.2013.00189\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeckmann J, Bach W, Behrens K, Reitner J (2008) Putative cryptoendolithic life in Devonian pillow basalt, Rheinisches Schiefergebirge, Germany. 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Chemical Geology 126:137\u0026ndash;146. https://doi.org/10.1016/0009-2541(95)00114-8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWebster J, Weber R. (2012) Introduction to fungi, 3. ed., 5. print. ed. Cambridge Univ. Press, Cambridge.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao GM., Yang YQ, Zhang, W, Luo X, Zhang RJ, Chen Y (2011) Raman scattering characterization of a carbon coating after low-energy argon ion bombardment. Physica B: Condensed Matter 406:3876\u0026ndash;3884. https://doi.org/10.1016/j.physb.2011.07.016\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"facies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"faci","sideBox":"Learn more about [Facies](http://link.springer.com/journal/10347)","snPcode":"10347","submissionUrl":"https://www.editorialmanager.com/faci/default2.aspx","title":"Facies","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ophiolite, endolith, fungus, Devonian, Rheinisches Schiefergebirge, Thüringer Wald","lastPublishedDoi":"10.21203/rs.3.rs-6512033/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6512033/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe presence of fossilised fungi within deep crustal rock formations has been established based on fossil evidence from 400 Ma continental crust and 81 Ma oceanic basaltic crust. Moreover, the Palaeoproterozoic Ongeluk Formation contains putative fungal remains reaching 2.4 Ga. The resulting gap of 2\u0026nbsp;billion years raises questions regarding the history of fungi in marine subsurface environments, in particular the lack of bona fide fossils in ophiolites, sections of layered basalts from mid-ocean ridges. Devonian examples of endolithic microorganisms preserved in marine pillow basalt stem from the Arnstein locality, Rheinisches Schiefergebirge, and the Kahlleite locality, Th\u0026uuml;ringer Wald, Germany, and have previously been found to contain filaments of microorganisms with uncertain biological affinity. The filamentous fossils were investigated using environmental scanning electron microscopy, Raman spectroscopy, confocal microscopy, widefield microscopy, and optical light microscopy. Energy dispersive spectroscopy analyses of several of the inferred microfossils revealed high carbon content and clay minerals, pointing to a mode of mineralization in association with organic matter and agreeing with a biological origin. Raman spectroscopy revealed that particularly iron oxide minerals are typified by carbon contents. Element compositions similar to younger mineralised fungal remains and morphologies resembling sporophores and hyphae agree with the interpretation of the Arnstein and Kahlleite fossils as marine fungi, shedding new light on many of the previously undetermined fossils and plausibly narrowing the fossil gap of oceanic deep subsurface fungi by at least 300\u0026nbsp;million years.\u003c/p\u003e","manuscriptTitle":"Re-evaluation of endolithic microfossils in pillow basalt of two Variscan orogens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 16:25:50","doi":"10.21203/rs.3.rs-6512033/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-12-30T10:10:30+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-05-09T10:03:00+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-08T19:41:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-25T02:21:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Facies","date":"2025-04-23T07:15:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"facies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"faci","sideBox":"Learn more about [Facies](http://link.springer.com/journal/10347)","snPcode":"10347","submissionUrl":"https://www.editorialmanager.com/faci/default2.aspx","title":"Facies","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9b4b2510-56d0-4efa-bb93-207a0783a011","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T06:24:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 16:25:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6512033","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6512033","identity":"rs-6512033","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}