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Introduction

Stromatolites are layered organo-sedimentary structures formed by microbial communities (Burne and Moore, 1987

Burne, R.V., Moore, L.S. (1987) Microbialites; organosedimentary deposits of benthic microbial communities. Palaios 2, 241–254. https://doi.org/10.2307/3514674

). They contain diverse organic molecules as remnants of the microorganisms that formed them, including lipids, proteins, carbohydrates, especially those composing extracellular polymeric substances (EPS), a variety of pigments and nucleic acids (Uveges et al., 2018

Uveges, B.T., Teece, M.A., Fulton, J.M., Junium, C.K. (2018) Environmental controls on pigment distributions in the freshwater microbialites of Fayetteville Green Lake. Organic Geochemistry 125, 165–176. https://doi.org/10.1016/j.orggeochem.2018.08.012

). These biomolecules play crucial roles, influencing mineral precipitation, sediment binding and the overall structure of the microbial mats (Dupraz et al., 2009

Dupraz, C., Reid, R.P., Braissant, O., Decho, A.W., Norman, R.S., Visscher, P.T. (2009) Processes of carbonate precipitation in modern microbial mats. Earth-Science Reviews 96, 141–162. https://doi.org/10.1016/j.earscirev.2008.10.005

). Thus, the preservation of these organic molecules is crucial to understand, since they may provide valuable information about paleoenvironments and the evolution of microbial communities. For this purpose, EPS (e.g., Benzerara et al., 2006

Benzerara, K., Menguy, N., López-García, P., Yoon, T.-H., Kazmierczak, J., Tyliszczak, T., Guyot, F., Brown Jr, G.E. (2006) Nanoscale detection of organic signatures in carbonate microbialites. Proceedings of the National Academy of Sciences 103, 9440–9445. https://doi.org/10.1073/pnas.0603255103

) and lipid biomarkers (e.g., Johnson et al., 2018

Johnson, D.B., Beddows, P.A., Flynn, T.M., Osburn, M.R. (2018) Microbial diversity and biomarker analysis of modern freshwater microbialites from Laguna Bacalar, Mexico. Geobiology 16, 319–337. https://doi.org/10.1111/gbi.12283

) have been prime targets searched for in the lithified portion of microbialites.

Luminescence spectroscopy offers a powerful, non-destructive method to probe biological molecules in mineral–microbe assemblages (Lepot et al., 2008

Lepot, K., Benzerara, K., Brown Jr, G.E., Philippot, P. (2008) Microbially influenced formation of 2,724-million-year-old stromatolites. Nature Geoscience 1, 118–121. https://doi.org/10.1111/gbi.12096

). Luminescence is a general term encompassing the emission of light by a substance as a result of some form of energy input. Fluorescence is a specific type of luminescence occurring when a substance absorbs light at one wavelength and re-emits at a longer wavelength. Here, the terms 'fluorescence’ and 'luminescence’ will be used interchangeably. Many biological molecules autofluoresce, making luminescence an ideal tool for studying organic components in stromatolites without the need for staining. While several studies have used confocal laser scanning microscopy (CLSM) to study luminescence in modern microbial mats and biofilms (e.g., Kawaguchi and Decho, 2002

Kawaguchi, T., Decho, A.W. (2002) In Situ Microspatial Imaging Using Two-Photon and Confocal Laser Scanning Microscopy of Bacteria and Extracellular Polymeric Secretions (EPS) Within Marine Stromatolites. Marine Biotechnology 4, 127–131. https://doi.org/10.1007/s10126-001-0073-7

; Zippel and Neu, 2011

Zippel, B., Neu, T.R. (2011) Characterization of Glycoconjugates of Extracellular Polymeric Substances in Tufa-Associated Biofilms by Using Fluorescence Lectin-Binding Analysis. Applied and Environmental Microbiology 77, 505–516. https://doi.org/10.1128/AEM.01660-10

; Rouillard et al., 2020

Rouillard, J., García-Ruiz, J.M., Kah, L., Gérard, E., Barrier, L., Nabhan, S., Gong, J., van Zuilen, M.A. (2020) Identifying microbial life in rocks: Insights from population morphometry. Geobiology 18, 282–305. https://doi.org/10.1111/gbi.12377

), its application to the more lithified portions of stromatolites remains limited. Furthermore, although CLSM provides high resolution hyperspectral data (Sinclair et al., 2006

Sinclair, M.B., Haaland, D.M., Timlin, J.A., Jones, H.D.T. (2006) Hyperspectral confocal microscope. Applied Optics 45, 6283–6291. https://doi.org/10.1364/AO.45.006283

), it is primarily used for qualitative imaging, with limited analysis of the spectral information it offers (Gérard et al., 2013

Gérard, E., Ménez, B., Couradeau, E., Moreira, D., Benzerara, K., Tavera, R., López-García, P. (2013) Specific carbonate–microbe interactions in the modern microbialites of Lake Alchichica (Mexico). The ISME Journal 7, 1997–2009. https://doi.org/10.1038/ismej.2013.81

, 2018

Gérard, E., De Goeyse, S., Hugoni, M., Agogué, H., Richard, L., Milesi, V., et al. (2018) Key Role of Alphaproteobacteria and Cyanobacteria in the Formation of Stromatolites of Lake Dziani Dzaha (Mayotte, Western Indian Ocean). Frontiers in Microbiology 9, 796. https://doi.org/10.3389/fmicb.2018.00796

; Stigliano et al., 2023

Stigliano, L., Caumartin, J., Benzerara, K. (2023) Micro-and nanoscale techniques for studying biofilm-mineral interactions. In: Gurtler, V., Patrauchan, M. (Eds.) Biofilms. Academic Press, London, 143–192. https://doi.org/10.1016/bs.mim.2023.04.001

). Here, we investigated the autofluorescence of modern lagoon stromatolites from Mari Ermi, a coastal pond in Western Sardinia, using CLSM. This site experiences seasonal droughts and salinity variations, reflected in the stromatolites’ complex mineralogical composition at the submicrometre scale (Debrie et al., 2022

Debrie, J., Prêt, D., Menguy, N., Estève, I., Sans-Jofre, P., Saint Martin, J.-P., Benzerara, K. (2022) Mapping mineralogical heterogeneities at the nm-scale by scanning electron microscopy in modern Sardinian stromatolites: Deciphering the origin of their laminations. Chemical Geology 609, 121059. https://doi.org/10.1016/j.chemgeo.2022.121059

). Our study aimed to 1) characterise the spectral variability of the fluorescence signal, potentially indicating different types of organic molecules and 2) map the distribution of these spectral variations across entire depth sections of stromatolites at a submicrometre resolution. By thoroughly analysing the hyperspectral CLSM data, we highlight the pervasive nature of fluorescence in these stromatolites and discuss the origins and implications of this signal. This approach allows us to gain new insights into the distribution and preservation of organic molecules within stromatolites, furthering our understanding of these important geological structures and the microbial communities that create them.

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Results

Pervasive luminescence and variations of luminescence intensity across laminations in Mari Ermi stromatolites. Luminescence is ubiquitous in Mari Ermi stromatolites, as evidenced by correlated transmitted light microscopy and epifluorescence mosaics (Figs. 1, 2). This pervasive luminescence sharply contrasts with the absence of detectable signal in both the underlying carbonate pebble substrate and the resin-filled porous regions within the stromatolites (Fig. 1). As further explained in the discussion, most of this luminescence is likely due to the preservation of organics in the lithified portion of the stromatolites. Spectral analysis of the luminescence reveals significant spatial heterogeneity at the submillimetre scale (Fig. 1), with certain variations correlating with the stromatolite’s laminar structure (Fig. 2a, b). Notably, laminae appearing bright under transmitted light microscopy predominantly exhibit green luminescence, whereas darker laminae display lower overall intensities (Fig. 1) and occasionally manifest a redder luminescence signature (Fig. 2b). At higher magnification, the spatial distribution of luminescence intensity becomes more nuanced. Regions of elevated intensity are observed, with some fluorescence signals associated with microbial filaments proximal to the stromatolite surfaces (Fig. 2c, d). These changes are likely related to the molecule content, but possibly also to variations in the characteristics of the fluorescing molecules.

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Luminescence spectral variations in Mari Ermi stromatolites. A first analysis of CLSM hyperspectral images demonstrates that most of the emitted luminescence corresponds to green light (∼530–560 range; Fig. 1b). However, some spatial variations of the luminescence spectral properties (“colours”) are observed. First, a red luminescence (∼670 nm in wavelength) is detected at the surface of the stromatolites. A red with some green luminescence is also observed deeper in some dark laminations (Figs. 1, 2b). The spectral variability was further analysed based on hyperspectral data acquired over a 4 × 4 mm² area close to the stromatolite surface, encompassing an alternation of bright and dark laminae (Fig. 3). Three wavelength intervals were defined after inspection of the dataset spectral variability and analysis of end member spectra generated by non-negative multivariate statistical analyses (Figs. S-1, S-2): a green interval, C1 (430 to 537 nm), a yellow one, C2 (556 to 624 nm) and a red one, C3 (644 to 722 nm). The choice of three instead of two components was also supported by multivariate statistical analyses (Fig. S-2). The relative contributions of these intervals were calculated for the spectrum of each pixel of the image. All pixels were finally plotted in a ternary scatterplot (Fig. S-1a). Most of the pixels spread between C1 and C2, indicating that most luminescence spectra mainly show a green emission contribution mixed with a, more or less, intense yellow luminescence. The remaining pixels are located closer to the red (C3) component. Overall, all pixels spread between three different spectral components, which explains all the observed spectral variability (Figs. 3b, c and S-1b). The measured spectra of the pixels the most representative of these three spectral components (purest pixels) are shown in Figure 3c (similar to end member spectra obtained by multivariate analyses, Fig. S-2). None of these spectra can be explained by a linear combination of the other two. Moreover, pixels representative of these three spectral components were back projected to the hyperspectral map to assess their spatial distribution (Fig. 3b). The green spectral component (C1) (Fig. S-1c) shows a broad peak with a maximum intensity at 530 nm. The spatial distribution of this component is the most widespread in the stromatolite, corresponding to the pervasive signal initially observed. The yellow spectral component (C2) (Fig. S-1d) looks spectrally similar but with a broad peak centred at a higher wavelength (∼560 nm) and an additional small emission peak at ∼ 670 nm (red fluorescence). This component appears in some dark laminae of the stromatolite. In some deeper laminae (Figs. 1, 2), the red fluorescence peak can be relatively higher. Last, the red component (C3) (Fig. S-1e) shows a maximum intensity at 670 nm with little to no green. This component was spatially restricted to spheres or filaments measuring ∼10 to 30 μm in diameter, located near the surface of stromatolites, with a high fluorescence intensity. Three similar “virtual” spectral end members were obtained by a non-negative matrix factorisation (NNMF) statistical analysis but with a noisier appearance (Fig. S-2).

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Discussion

The luminescence is not only observed as a strong signal in living biofilms at the surface of Mari Ermi stromatolites (Fig. 1), but it is also pervasive in their deeper lithified portion. This fluorescence shows some spectral variability, and three different spectral components are identified (Fig. 4a), with a tentative interpretation below.

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Origin of red fluorescence in the Sardinian stromatolites. The spectrum of the red component (C3) exhibits a maximum value at 670 nm and is co-localised with filaments measuring approximately 10–15 μm in diameter and 100 to 150 μm in length. These filaments have been interpreted as cyanobacteria entombed by calcite precipitation (Saint Martin and Saint Martin, 2015

Saint Martin, J.-P., Saint Martin, S. (2015) Discovery of calcareous microbialites in coastal ponds of western Sardinia (Italy). Geo-Eco-Marina 21, 35–53. https://journal.geoecomar.ro/geo-eco-marina/article/view/03_2015

; Debrie et al., 2022

Debrie, J., Prêt, D., Menguy, N., Estève, I., Sans-Jofre, P., Saint Martin, J.-P., Benzerara, K. (2022) Mapping mineralogical heterogeneities at the nm-scale by scanning electron microscopy in modern Sardinian stromatolites: Deciphering the origin of their laminations. Chemical Geology 609, 121059. https://doi.org/10.1016/j.chemgeo.2022.121059

). While it is possible that a yet unidentified inorganic fluorochrome may contribute to this fluorescence, we note that phycocyanin and chlorophyll emit at a similar wavelength, approximately 650 and 685 nm, and that the emission spectrum of a cyanobacterium (Dartnell et al., 2011

Dartnell, L.R., Storrie-Lombardi, M.C., Mullineaux, C.W., Ruban, A.V., Wright, G., Griffiths, A.D., Muller, J.-P., Ward, J.M. (2011) Degradation of Cyanobacterial Biosignatures by Ionizing Radiation. Astrobiology 11, 997–1016. https://doi.org/10.1089/ast.2011.0663

) looks very similar to the C3 component in Mari Ermi stromatolites. Moreover, similar spectra were measured in the biofilms covering Mari Ermi microbialites (Fig. 4b), further supporting the assignment of this spectral component to chlorophyll and/or phycocyanin.

This type of spectrum was detected in the filaments of the upper part of the stromatolite section only. Chlorophyll degradation occurs through various processes, such as intense irradiation (Dartnell et al., 2011

Dartnell, L.R., Storrie-Lombardi, M.C., Mullineaux, C.W., Ruban, A.V., Wright, G., Griffiths, A.D., Muller, J.-P., Ward, J.M. (2011) Degradation of Cyanobacterial Biosignatures by Ionizing Radiation. Astrobiology 11, 997–1016. https://doi.org/10.1089/ast.2011.0663

), a likely minor factor under the conditions present at Mari Ermi. Other degradation mechanisms include grazing and partial hydrolysis by various enzymes, including chlorophyllases produced by diatoms (Villanueva et al., 1994

Villanueva, J., Grimalt, J.O., de Wit, R., Keely, B.J., Maxwell, J.R. (1994) Chlorophyll and carotenoid pigments in solar saltern microbial mats. Geochimica et Cosmochimica Acta 58, 4703–4715. https://doi.org/10.1016/0016-7037(94)90202-X

). By contrast, chlorophyll was unlikely degraded during data collection, since the 2 % laser power employed is a standard setting for capturing pristine spectra of chlorophyll and phycocyanin (e.g., Gerard et al., 2018

Gérard, E., De Goeyse, S., Hugoni, M., Agogué, H., Richard, L., Milesi, V., et al. (2018) Key Role of Alphaproteobacteria and Cyanobacteria in the Formation of Stromatolites of Lake Dziani Dzaha (Mayotte, Western Indian Ocean). Frontiers in Microbiology 9, 796. https://doi.org/10.3389/fmicb.2018.00796

). Chlorophyll degradation in Mari Ermi stromatolites produces a variety of byproducts, with varying fluorescence spectra or no fluorescence at all for the most advanced ones (Hörtensteiner, 2006

Hörtensteiner, S. (2006) Chlorophyll Degradation During Senescence. Annual Review of Plant Biology 57, 55–77. https://doi.org/10.1146/annurev.arplant.57.032905.105212

). Overall, the occurrence of this red spectral component down to a limited depth suggests that there is a maximum depth, above which chlorophyll/phycocyanin with intact spectral characteristics is preserved. Sun et al. (1991)

Sun, M., Aller, R.C., Lee, C. (1991) Early diagenesis of chlorophyll-a in Long Island Sound sediments: A measure of carbon flux and particle reworking. Journal of Marine Research 49, 379–401. https://doi.org/10.1357/002224091784995927

showed that chlorophyll degrades very rapidly in sediments under oxic conditions, with a half-life of a few tens of days, whereas parameters such as anoxia may extend preservation for more than a few decades (Pirtle-Levy et al., 2009

Pirtle-Levy, R., Grebmeier, J.M., Cooper, L.W., Larsen, I.L. (2009) Chlorophyll a in Arctic sediments implies long persistence of algal pigments. Deep Sea Research Part II: Topical Studies in Oceanography 56, 1326–1338. https://doi.org/10.1016/j.dsr2.2008.10.022

). Lepot et al. (2014)

Lepot, K., Compère, P., Gérard, E., Namsaraev, Z., Verleyen, E., Tavernier, I., et al. (2014) Organic and mineral imprints in fossil photosynthetic mats of an East Antarctic lake. Geobiology 12, 424–450. https://doi.org/10.1111/gbi.12096

detected chlorophyll fluorescence in 160-year-old sediments but suggested that they may have underestimated preservation because of some pigment loss upon sample preparation. The detection of chlorophyll residues with mass spectrometry probes signatures of porphyrin and not intact chlorophyll, which can therefore be at a much more advanced degradation stage than mentioned above. Such signatures have been detected in rocks as old as 1.1 billion years (Gueneli et al., 2018

Gueneli, N., McKenna, A.M., Ohkouchi, N., Boreham, C.J., Beghin, J., Javaux, E.J., Brocks, J.J. (2018) 1.1-billion-year-old porphyrins establish a marine ecosystem dominated by bacterial primary producers. Proceedings of the National Academy of Sciences 115, E6978–E6986. https://doi.org/10.1073/pnas.1803866115

).

Origin of green fluorescence in Mari Ermi stromatolites. Interpreting the origin of the two other spectral components, primarily emitting in the green, is less straightforward. One hypothesis is that they are inorganic in origin, possibly linked with the presence of luminescence activators within calcite, the predominant mineral phase composing Mari Ermi stromatolites (Debrie et al., 2022

Debrie, J., Prêt, D., Menguy, N., Estève, I., Sans-Jofre, P., Saint Martin, J.-P., Benzerara, K. (2022) Mapping mineralogical heterogeneities at the nm-scale by scanning electron microscopy in modern Sardinian stromatolites: Deciphering the origin of their laminations. Chemical Geology 609, 121059. https://doi.org/10.1016/j.chemgeo.2022.121059

). The most well known activator in this context is Mn²⁺, present at trace levels in Mari Ermi stromatolites (see Table S-1). However, Mn²⁺ substitution in calcite typically yields a luminescence spectrum centred at approximately 600–610 nm (Fig. S-2), which significantly diverges from the spectrum observed here (see Figs. 3c and 4a). Alternatively, the two components might have an organic origin. A first hypothesis could be that the observed signal is associated with non-pigmented material of currently unknown origin. However, in the remainder of this discussion, we will focus on a pigment-based origin, since, based on the observation of the biofilms at the surface of these modern stromatolites, pigments appear as an obvious source of fluorescence, likely to be preserved upon lithification. The broad emission bands with maxima at 530 and 560 nm observed for the two Mari Ermi components bear a striking resemblance with the emission spectra of carotenoid pigments (Fig. S-3) (Vermaas et al., 2008

Vermaas, W.F.J., Timlin, J.A., Jones, H.D.T., Sinclair, M.B., Nieman, L.T., Hamad, S.W., Melgaard, D.K., Haaland, D.M. (2008) In vivo hyperspectral confocal fluorescence imaging to determine pigment localization and distribution in cyanobacterial cells. Proceedings of the National Academy of Sciences 105, 4050–4055. https://doi.org/10.1073/pnas.0708090105

). If these components are indeed carotenoids, their spatial distribution suggests that they significantly outweigh undegraded chlorophyll/phycocyanin within Mari Ermi stromatolites. This observation could be explained in two possible ways: either carotenoids are more abundant than chlorophyll/phycocyanin in the microbial mats, which is not observed (Fig. 4b), or there is preferential degradation of phycocyanin/chlorophyll compared to carotenoids. Consistently, carotenoids are known to be resistant to certain processes of degradation due to their antioxidant properties (Nelson and Cox, 2005

Nelson, D.L., Cox, M.M. (2005) Lehninger Principles of Biochemistry. Fourth Edition, W.H. Freeman and Company, New York.

). Future analyses of biomarkers would help to further test these ideas.

Alternatively, a seemingly more parsimonious interpretation for the two green luminescent components is that they correspond to two different degradation stages of chlorophyll and/or phycocyanin. Several studies have shown that chlorophyll-type pigments are frequently preserved with some structural modifications (Tahoun et al., 2021

Tahoun, M., Gee, C.T., McCoy, V.E., Sander, P.M., Müller, C.E. (2021) Chemistry of porphyrins in fossil plants and animals. RSC Advances 11, 7552–7563. https://doi.org/10.1039/D0RA10688G

). Moreover, the breakdown of photosynthetic pigments has been shown to result in the appearance of what has been called a non-specific green autofluorescence (GAF). This process intensifies after cellular death (Schulze et al., 2011

Schulze, K., López, D.A., Tillich, U.M., Frohme, M. (2011) A simple viability analysis for unicellular cyanobacteria using a new autofluorescence assay, automated microscopy, and ImageJ. BMC Biotechnology 11, 118. https://doi.org/10.1186/1472-6750-11-118

) and has been detected for a diversity of microorganisms such as algae, dinoflagellates, diatoms (Tang and Dobbs, 2007

Tang, Y.Z., Dobbs, F.C. (2007) Green Autofluorescence in Dinoflagellates, Diatoms, and Other Microalgae and Its Implications for Vital Staining and Morphological Studies. Applied and Environmental Microbiology 73, 2306–2313. https://doi.org/10.1128/AEM.01741-06

) and cyanobacteria (Roldán et al., 2014

Roldán, M., Ascaso, C., Wierzchos, J. (2014) Fluorescent Fingerprints of Endolithic Phototrophic Cyanobacteria Living within Halite Rocks in the Atacama Desert. Applied and Environmental Microbiology 80, 2998–3006. https://doi.org/10.1128/AEM.03428-13

). GAF emission spectra measured at different degradation stages show a major peak at a wavelength varying between 530 up and 550 nm together with a peak at 670 nm which becomes smaller in intensity at more advanced degradation (Tang and Dobbs, 2007

Tang, Y.Z., Dobbs, F.C. (2007) Green Autofluorescence in Dinoflagellates, Diatoms, and Other Microalgae and Its Implications for Vital Staining and Morphological Studies. Applied and Environmental Microbiology 73, 2306–2313. https://doi.org/10.1128/AEM.01741-06

). The spectra of the two green-emitting components found in Mari Ermi stromatolites are very similar to the two different degradation stages. In this scenario, the spectral component showing a maximum emission at 560 nm, accompanied by a small peak at 670 nm, represent an intermediate stage in the chlorophyll/phycocyanin degradation pathway. Conversely, the component that exhibits a primary peak at 530 nm, with no discernible emission at 670 nm, corresponds to a later stage of degradation. In this context, the relatively higher red signal observed in some of the deeper laminae, as shown in Figures 1 and 2, would be best explained as an additional stage where pigments are best preserved, suggesting a quicker lithification of the laminae containing them. Additionally, spectra with a small peak at 670 nm also show a red-shifted emission maximum (560 nm) compared to spectra with no 670 nm peak (emission maximum at 530 nm). Based on existing spectral references, it remains unclear whether this spectral shift is also correlated with the degradation advance of chlorophyll/phycocyanin or some variability of the initial pigment assemblage.

Variations of the green fluorescence intensity and spectral characteristics between laminae. The green fluorescence intensity differed between laminae; bright laminae had a higher fluorescence intensity than dark laminae (Fig. 1). Debrie et al. (2022)

Debrie, J., Prêt, D., Menguy, N., Estève, I., Sans-Jofre, P., Saint Martin, J.-P., Benzerara, K. (2022) Mapping mineralogical heterogeneities at the nm-scale by scanning electron microscopy in modern Sardinian stromatolites: Deciphering the origin of their laminations. Chemical Geology 609, 121059. https://doi.org/10.1016/j.chemgeo.2022.121059

proposed that bright laminae formed in the presence of denser microbial biofilms compared with dark laminae. This would be consistent with the interpretation of this fluorescence as GAF, resulting from chlorophyll/phycocyanin degradation, with the higher fluorescence intensity in bright laminae resulting from an initially higher microbial cell density.

In addition to the intensity, the fluorescence spectral properties vary between laminae (Figs. 2b, 3b). While an emission peak at 670 nm was observed in dark laminae, sometimes quite deep under the stromatolite surface (Fig. 1), it was negligible in bright laminae. According to the aforementioned hypothesis, this could be explained by a stronger degradation of chlorophyll/phycocyanin in bright laminae than in dark laminae. Similarly, the porosity has been shown to be higher in bright laminae than in dark laminae (Debrie et al. 2022

Debrie, J., Prêt, D., Menguy, N., Estève, I., Sans-Jofre, P., Saint Martin, J.-P., Benzerara, K. (2022) Mapping mineralogical heterogeneities at the nm-scale by scanning electron microscopy in modern Sardinian stromatolites: Deciphering the origin of their laminations. Chemical Geology 609, 121059. https://doi.org/10.1016/j.chemgeo.2022.121059

). Overall, it could be speculated that higher porosity may allow longer contact time between porewater and pigments, resulting in more advanced degradation. Alternatively, if porosity is secondary, this may be indicative of locally more important transformations which may have also impacted pigment degradation.

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Conclusions

Three distinct photoluminescence signals are observed in modern stromatolites from Mari Ermi, Sardinia. While other, yet to be identified, origins may exist, these signals are best interpreted as the results of relatively intact to variably degraded pigments produced by the microorganisms that thrived during the formation of the stromatolites. Notably, well preserved chlorophyll/phycocyanin pigments are mainly associated with morphologically preserved microfossils, predominantly near the stromatolite’s surface, and occasionally in some deeper laminae. Variably degraded chlorophyll/phycocyanin molecules were pervasive within the carbonate matrix, generating a non-specific green autofluorescence (GAF). GAF varied in intensity and spectrally across distinct laminae, possibly due to varying degradation advances. The varying degrees of pigment degradation across laminae may provide clues about the diagenetic history of stromatolites and the environmental conditions they experienced over time. Future research should focus on determining the maximum duration of fluorescent pigment preservation, the effects of environmental factors on organic signature persistence, and the comparison of luminescence characteristics between modern and ancient stromatolites. These investigations will improve our understanding of organic matter preservation in stromatolites, a knowledge useful for better interpreting the fossil record of past life on Earth.

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Acknowledgements

Our work has benefitted from the financial support from the Paris Ile-de-France Region – DIM “Matériaux anciens et patrimoniaux” (MAP) and from the French national INSU programme Interrvie. We thank Imène Esteve, Stéphanie Delbrel and Béatrice Doisneau for support at the SEM facility and Massimo Marras and Roberto Brundu from the marine protected area “Peninsula of Sinis - Island of Mal di Ventre”. We thank two anonymous reviewers and Satish Myneni for their comments that significantly improved the quality of the manuscript.

Editor: Satish Myneni

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References

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Supplementary Information

The Supplementary Information includes:

• Materials and Methods
• Figures S-1 to S-3
• Table S-1
• Supplementary Information References

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