Contrasting lipid biomarkers profiles in two prebiotic-astrobiological analogs from Ladakh (Indian Himalayas): a boron-rich geothermal system (Puga) and an alkaline brine lake (Tso Kar).
Authors: Sánchez-García Laura , Carrizo Daniel , Parro Victor 
Published: July 2024

The union territory of Ladakh (northern India) hosts different environment with astrobiological interest due to a number of characteristics derived from their high elevation, such as geothermal systems, desiccated environments, permafrosts, glacial geomorphologies or dust devil formations (Pandey et al., 2019). Located in the Indian Himalayas, this area lying mostly above 3500 (up to 5700) m.a.s.l. shows a cold desert-type climate, with lowered air pressures (~500-580 hPa), low oxygen, and high UV radiation. The rain-shadowed region offers various potential analogues to geological sites and water bodies that existed on the early (Noachian-Hesperian) Mars, such as the Puga geothermal field and Tso Kar.The Puga hot springs are a boron-rich geothermal system located at the mouth of the Puga Valley, in the NW of the Himalayas (33&#186;13&#180;39.38&#180;&#180;N, 78&#186;18&#180;22.98&#180;&#180;E). This geothermal system occupies an area of 4 km x 1 km and exhibits a range of geothermal activity, including geysers, boiling pools, mud pots, and sulfur/boratic mineral deposits. The near-neutral water discharging at about 70&#186;C boils easily due to the high elevation and carries boron, an element thought to have played a key stabilizing role in the synthesis of RNA (Ricardo et al., 2004). Tso Kar is a fluctuating shallow (1&#8211;3 m depth), alkaline brine lake of high turbidity waters (TDS = 190.5 g L&#8722;1) of the Na&#8211;K&#8211;SO4-Cl type (Saxena &amp; D&#180;Amore, 1984). The desiccating lake is located at 4,535 m.a.s.l. (33&#186;18&#180;56.63&#180;&#180;N, 77&#186;57&#180;20.30&#180;&#180;E), in a cold desert environment with permafrost mounds around the palaeo-shoreline, a great daily temperature oscillation, and presence of ostracods (Kramer et al., 2014). The high-altitude lacustrine system contains two types of environments according to their water chemistry, dominated by saline waters near its shoreline or dominated by freshwater in areas receiving streams from glacial melting.Both systems represent astrobiologically relevant settings, with potential as prebiotic (Puga hot springs) or Martian paleolakes (Tso Kar) analogs, whose biogeochemistry is poorly explored. Moreover, the particular physical-chemical properties of both settings provide interesting scenarios hosting extremophilic life, whose fingerprints may be environmentally representative and thus useful for the search for life traces in analogous Martian environments. Notably, the molecular remains of cell membranes upon degradation (i.e. lipid biomarkers) provide a powerful life-diagnostic tool that may be used to detect life for up to billions of years after death. This chemical recalcitrance is indispensable in the field of astrobiology given the harmfulness of the intense radiation outside of a protective atmosphere and the time span that the geological ages of planetary bodies encompass.Thus, we conducted a research project to explore the forensic ability of lipid biomarkers to record representative life fingerprints of relevant Martian analogs. We investigated the lipid biomarker profile of various samples from both analogous scenarios from Ladakh. In Puga, we studied five biofilms of different appearance from two boiling pools, four fresh biofilms from the water immersed, inner wall of the pools (orange, pale green, red-purple, and dark green) and one crusty sample from the emerged shore (whitish). In Tso Kar, five samples were analyzed; two sediment samples from the saline-dominated lake shore (orange and dark grey sediments), one sediment from a freshwater stream flowing to the lake, and two crusty samples (whitish and yellowish) from the surrounding area around the lake.&#160;We aim to obtain a molecular biomarker record characteristic of each of the two analogous scenarios as a benchmark for recognizing traces of possible life in equivalent environments on Mars.In both scenarios, lipid biomarkers were extracted, isolated and characterized at molecular and isotopic level. The prevailing biosources and metabolic pathways involved in their synthesis were identified and, according to their different molecular distribution, two biomarker patterns characteristic of each environment were built. While certain lipidic compounds were ubiquitous in both environments (C16 and C18 fatty acids, n-alkane C17, or chloropyll-derived phytol), some others were characteristically found in one or another system. For instance, bacterial hopanoids, cyclopropyl C21 fatty acid diagnostic of thermophiles (i.e. Aquificales), long chain polyunsaturated alkanes characteristics of non-sulfur bacteria (Chloroflexi), diols and branched alkanols were found only or in relatively higher concentration in Puga. In contrast, a relatively higher proportion of polyunsaturated fatty acids and sterols derived from eukaryotic organisms (e.g. algae, protozoa or fungi) was observed in Tso Kar. In addition, the Puga lipid biomarkers are generally more enriched in 13C (i.e. less negative &#948;13C) than those in Tso Kar, which illustrates also metabolic differences between the two Martian analogs with likely a relatively greater proportion of chemolithotrophs in the geothermal system. Overall, the concentration of lipid biomarkers and bulk biomass (as content of total organic carbon content or TOC) was lower in Tso Kar (0.4-1.1 % vs. 1.9-10 % TOC in Puga), illustrating the relatively lower biological productivity and/or biomarkers preservation in the hypersaline environment.This work is the first to describe molecular and isotopic lipid fingerprints in these unexplored analogs, where the only existing organic data are of ATP or lipopolysaccharides (Pandey et al., 2019). These results are the basis for further biogeochemical research in the analog-rich Himalayan region.&#160;Acknowledgements:This work was funded by MCIN/AEI/10.13039/501100011033 and &#8220;ESF Investing in your future&#8221; through the grant RYC2018-023943-I (L.S.-G.) and projects PID2021-126746NB-I00 (V.P. and L.S.-G.) and&#160;PID2022-140180B-C21 (D.C.). The authors thank PhD. Siddharth Pandey, Pavithra Sekhar, Abul A. Khan, and Bharti Sharma from Amity University; Annalea Beattie from the Mars Society Australia; and Kunal Mehra from Cosmic Adventures for the organization and logistic assistance during the ESEP&#180;22 exploration campaign to Ladakh, as well as Prof. Sudha Rajamani from the Indian Institute of Science Education and Research for her support in the field as an expert in biogeochemistry.&#160;References:Kramer et al. (2014). A late quaternary ostracod record from the Tso Kar basin (North India) with a note on the distribution of recent species. J. Paleolimnol. 51, 549&#8211;565, DOI 10.1007/s10933-014-9773-7.
Ricardo A. et al. (2004). Borate minerals stabilize ribose. Science 303:196.
Saxena V.K. &amp; D&#180;Amore F. (1984). Aquifer chemistry of the Puga and Chumatang high temperature geothermal systems in India. J. Volcanol. Geoth. Res. 21, 333-346.
Pandey S. et al. (2019). Ladakh: diverse, high-altitude extreme environments for off-earth analogue and astrobiology research. Int. J. Astrobiol., 1-21. https://doi.org/10.1017/S1473550419000119.