International Journal of Astrobiology cambridge.org/ija Research Article *Authors have equally contributed to coordinate the research. Cite this article: Pacelli C et al (2021). Fungal biomarkers are detectable in Martian rock- analogues after space exposure: implications for the search of life on Mars. International Journal of Astrobiology 20, 345–358. https:// doi.org/10.1017/S1473550421000240 Received: 4 June 2021 Revised: 25 August 2021 Accepted: 26 August 2021 Key words: Biomarkers; exploration missions; life detection; Mars; pigments; spectroscopy Author for correspondence: Alessia Cassaro, E-mail: cassaro@unitus.it © The Author(s), 2021. Published by Cambridge University Press Fungal biomarkers are detectable in Martian rock-analogues after space exposure: implications for the search of life on Mars Claudia Pacelli 1,2 , Alessia Cassaro 1 , Mickael Baqué 3 , Laura Selbmann 1,4 , Laura Zucconi 1 , Alessandro Maturilli 3 , Lorenzo Botta 1 , Raffaele Saladino 1 , Ute Böttger 5 , René Demets 6 , Elke Rabbow 7 , Jean-Pierre P. de Vera 8, * and Silvano Onofri 1, * 1 Department of Ecological and Biological Sciences, University of Tuscia, Largo dell’Università snc, 01100 Viterbo, Italy; 2 Italian Space Agency, Scientific Research Unit, Via del Politecnico snc, 00133 Rome, Italy; 3 Planetary Laboratories Department, German Aerospace Center (DLR), Institute of Planetary Research, Ruthefordstraße 2, Berlin, Germany; 4 Italian Antarctic National Museum (MNA), Mycological Section, 16128 Genoa, Italy; 5 Department Terahertz and Laser Spectroscopy, German Aerospace Center (DLR) Berlin, Institute of Optical Sensor Systems, Rutherfordstr. 2, 12489 Berlin, Germany; 6 European Space Agency, ESTEC, Keplerlaan 1, 2201AZ Noordwijk, The Netherlands; 7 German Aerospace Center (DLR) Cologne, Institute of Aerospace Medicine, Radiation Biology, Linder Höhe, 51147 Cologne, Germany and 8 MUSC, German Aerospace Center (DLR), Space Operations and Astronaut Training, Linder Höhe, Cologne-Porz, Germany Abstract Mars is a primary target of astrobiological interest: its past environmental conditions may have been favourable to the emergence of a prebiotic chemistry and, potentially, biological activity. In situ exploration is currently underway at the Mars surface, and the subsurface (2 m depth) will be explored in the future ESA ExoMars mission. In this context, BIOlogy and Mars EXperiment was performed to evaluate the stability and detectability of organic bio- markers under space and Mars-like conditions. Our data suggested that some target mole- cules, namely melanin, azelaic acid and nucleic acids, can be detected even after 16 months exposure to Low Earth Orbit conditions by multidisciplinary approaches. We used the same techniques as onboard the ExoMars rover, as Raman and infrared spectroscopies and gas chromatograph-mass spectrometer, and polymerase chain reaction even if this is not planned for the imminent mission to Mars. These results should be taken into account for future Mars exploration. Introduction The question of whether Mars ever hosted life remains unanswered. Theoretically, various kinds of metabolisms could have been supported there (Stamenković et al., 2018), especially in the Noachian when conditions were supposedly more favourable to life. If life ever evolved on Mars, nowadays it could still develop underground, given the harsh irradiation conditions on the surface (Cockell, 2014). The upcoming missions to Mars aim to detect traces of extant or recently extinct life, namely biomarkers. A biomarker definition is still under development and debate; but it should be considered as a fingerprint of past or pre- sent biological activity (McMahon, 2021) and thus, be strictly biogenic (not found in the abiotic organics in nature). Indeed, it should be an integral component of any water-based life form, and should possess universal chemical and physical properties required for any form of life as we know it. Diverse organic biomarkers have been classified based on the biological features that belong to all known life forms on Earth, such as a compartmenta- lized shape, energy-producing and storage mechanisms, and the use of biomolecules to store hereditary information (Parnell et al., 2007; Summons et al., 2008). These biomarkers are made of simple monomers that constitute the building blocks of cells, such as amino acids, sugars, pigments and lipids, as well as more complex biopolymers such as nucleic acids. A critical consideration in biomarker detection is the compound’s resiliency. For example, the persistence of possible biomarkers on the surface of Mars could be affected by the current hostile environmental conditions, such as the unshielded flux of UV and ion- izing radiation (Dartnell et al., 2007, 2012a) and strongly oxidizing conditions. In this framework and with the aim of achieving a list of biomarkers to serve as reference in the ExoMars mission, the BIOlogy and Mars EXperiment (BIOMEX) has exposed for 16 months extremophiles and molecules outside the ISS (de Vera et al., 2019) to investigate their resistance and stability. Among them, the cryptoendolithic fungus Cryomyces antarcticus, living in the McMurdo Dry Valleys rocks, Antarctica, is a relevant test organism (Onofri et al., https://doi.org/10.1017/S1473550421000240 Published online by Cambridge University Press