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In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/methods Page 1 of 35 Analytical Methods 1 2 3 4 5 6 7 8 9 LICHENS AROUND THE WORLD: A COMPREHENSIVE STUDY OF LICHEN 10 11 SURVIVAL BIOSTRATEGIES DETECTED BY RAMAN SPECTROSCOPY 12 13 14 15 16 I. Miralles 1,2 ; H.G.M. Edwards 3; F. Domingo 1; S.E. Jorge-Villar 4,5* 17 18 19 1 Estación Experimental de Zonas Áridas, Consejo Superior de Investigaciones Científicas, 20 21 Manuscript 22 2 Georges Lemaître Centre for Earth and Climate Research, Université Catholique de 23 24 25 Louvain-La-Neuve, Belgium. 26 27 3 28 Department of Chemical and Forensic Sciences, School of Life Sciences, University of 29 30 Bradford, UK 31 Accepted 32 33 4Area de Geodinámica Interna, Facultad de Humanidades y Educacion, Universidad de 34 35 Burgos, C/ Villadiego s/n, 09001-Burgos (Spain). E-mails: [email protected]; 36 37 [email protected] ; phone +34 947 258 772; Fax: +34 947 258 723 38 39 40 Methods 5National Research Center on Human Evolution (CENIEH), Burgos (Spain). 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 1 Analytical Methods Page 2 of 35 1 2 3 ABSTRACT 4 5 6 A list of the lichen biomolecules detected by Raman spectroscopy has been compiled and 7 8 their appearance related with the environmental conditions operating in the lichen habitat over 9 10 the world. The adaptative climatic strategies of lichens have been analysed as a whole and 11 12 13 some interesting and contradictory conclusions arise with regard to other research conclusions 14 15 reported in the literature, such as the presence of hydrated calcium oxalates and their 16 17 relationship with desiccated environments or the correlation between climatic conditions and 18 19 protective pigments or pigment mixtures. The results of this exercise will be useful for our 20 21 understanding of the biochemical synthetic strategies being employed for the survival of the Manuscript 22 23 24 lichen colonies in hostile terrestrial environments and the prediction of Raman spectral data 25 26 for extremophiles in a range of novel hot and cold desert conditions. Additionally, a database 27 28 of all key lichen biomolecules identified by Raman spectroscopy and their characteristic 29 30 Raman wavenumbers are given for further unambiguos identification. 31 Accepted 32 33 34 35 36 Keywords : Adaptative strategies, climate, biomolecule, extremophile, calcium oxalate 37 38 39 40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 2 Page 3 of 35 Analytical Methods 1 2 3 INTRODUCTION 4 5 6 7 8 9 Lichens are formed by a symbiotic relationship between micro-algae and fungi; the 10 11 first of these provides chlorophyll and permits a photosynthetic function whereas the fungus 12 13 provides shelter and humidity. Lichens have colonized most terrestrial environments, even the 14 15 16 most hostile cold and hot deserts or high altitude habitats and appear on most substrata, such 17 1-8 18 as rocks, soil, wood, brick, leaves, roofing, paint, walls, etc. They are, after 19 20 microorganisms, pioneer colonizers of sterile areas through physical and chemical mineral 21 Manuscript 22 substratum degradation and by the addition of organic matter to new soil production. 23 24 25 26 27 28 The capability of lichens to survive under harsh climatic factors and on different 29 30 substrata has been ascribed to the development of different types of adaptive strategies: 31 Accepted 32 9 33 Charles Darwin emphasised this in his identification that adaptation of species to a changing 34 35 environment was the key to its successful survival. Although some endolithic colonisations 36 37 have been described as lichens 10 , most lichens are epilithic and live on the substratum surface 38 39 and then their survival strategies are necessarily related to the availability of food and water 40 Methods 41 42 and protection from hazardous external features, such as high or low temperature, desiccation, 43 44 chemical toxicity or low wavelength, high energy UV-radiation by producing different 45 46 chemical compounds.1,3,4,11-15 Dormant and active stages have been described as phases of the 47 48 life of lichen species, related to the environmental conditions, and can be evaluated by the 49 50 16-17 51 measurement of their photosynthetic activity. Analytical 52 53 54 55 56 57 There is a wide range of biomolecules, either organic pigments and oxalates, produced 58 59 60 3 Analytical Methods Page 4 of 35 1 2 4,8,18-19 3 by lichens from different metabolic pathways. Some of these carry out a protective role 4 5 and, at the same time, act as pigments, giving a characteristic colour to the lichen.7,8,14,20-22 6 7 Oxalates are described as wasted products as a result of methabolic activity; they can play a 8 9 1 10 dual role. By one hand, as water storage under low humidity levels; by the other hand, the 11 12 calcium oxalate formation process can also help lichens to rock disintegration and to grow 13 14 into the rock. Although there is a wide range of pigments which often occur in admixture, it 15 16 has already been reported that there is not a direct relationship between pigments and species 17 18 in the way that different species use the same protective pigments 4; also, there is no 19 20 21 correlation between the lichen genus and the pigment mixture since different genera can Manuscript 22 23 produce the same pigment composition and different species from the same genus could 24 25 produce different pigment mixtures. 4-5 This means that naturally there will be inherent 26 27 difficulties in using spectral pigment data for taxonomic purposes. In a similar way, protective 28 29 30 biomolecules are not specific for climatic parameters, that is: lichens living under analogous 31 Accepted 1,5 32 environmental conditions can adopt a different pigment patronage. This means that pigment 33 34 mixtures per se cannot be used for lichen species identification or for environmental 35 36 parameter characterization. 37 38 39 40 Methods 41 42 Lichen pigments have been used from ancient times for the manufacture of inks, 43 44 23-27 45 medicines and perfumes, and for dyeing textiles and garments. Lichens play also an 46 47 essential role in ecosystems by improving biogeochemical soil cycles and increasing the 48 49 carbon stocks in the soils.28-31 Nowadays, lichens have a renewed research interest as 50 Analytical 51 extremophilic organisms in life and space sciences 32-37 which has itself directed the adoption 52 53 of new analytical techniques in lichenology. One of these novel analytical techniques is 54 55 56 Raman spectroscopy, which has been applied to lichenological studies along with the older 57 58 infrared and mass spectroscopic techniques. Raman spectroscopy is a non-destructive 59 60 4 Page 5 of 35 Analytical Methods 1 2 3 technique based on the use of laser radiation in the visible or near infrared region of the 4 5 electromagnetic spectrum for analysing the vibrational spectra in molecules; the basis of this 6 7 technique relies upon the probing of the chemical bonds in organic molecules and inorganic 8 9 10 molecular ions whose Raman spectral signatures are characteristic of each compound or 11 12 molecular ion. Hence each chemical compound, either organic or inorganic, displays a 13 14 specific Raman spectral pattern related with its composition and molecular structure and in a 15 16 particularly advantageous way micro- and macro- analysis can be carried out directly on the 17 18 sample without the necessity for any physical or chemical pretreatment or manipulation to be 19 20 33-34 21 carried out on the specimen. Because the Raman spectra are obtained using a microscope Manuscript 22 23 or lens illuminator then the characteristic molecular information is also derived with a surface 24 25 spatial resolution of the order of several microns, which gives Raman spectroscopic analysis 26 27 an added advantage for the examination of heterogeneous lichen encrustations on mineral 28 29 30 substrates without chemical extraction or separation being effected from the substrate. 31 Accepted 32 33 34 35 36 Although obtaining a Raman spectrum is intrinsically not difficult, the characterization 37 38 of each compound in a mixture from its characteristic Raman bands is complex because of 39 40 several problems, such as the weakness of the Raman signal, fluorescence background Methods 41 42 emission, band overlapping, wavenumber shifts, etc.33,38 Furthermore, several technical 43 44 45 parameters can affect the observed spectrum, such as laser wavelength, which can influence 46 47 the onset of fluorescence or resonance effects; spectral resolution, related to changes in band 48 49 width caused by molecular or ionic environmental effects and so result in band overlapping or 50 Analytical 51 asymmetry; laser power, since high laser irradiance at the specimen in Watts per square cm 52 53 can burn the sample or result in chemical degradation or the induction of molecular or 54 55 38-40 56 structural changes.