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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

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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

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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

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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

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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. 57 58 59 60 5

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1 2 3 4 5 6 Organic compounds give a multi-band Raman spectrum, from which three or four 7 8 bands, usually the strongest, are used as characteristic Raman signatures; however, the 9 10 relative intensity of the observed peaks can change with regard to the wavelength of the laser 11 12 33,39 13 used for exciting the Raman effect. This means that sometimes, what seems to be a major 14 15 feature important could exhibit a significantly reduced intensity using a different laser 16 17 wavelength other than that reported in the literature database; the actual interpretation of the 18 19 spectral data and consequent identification of the biomolecules present becomes difficult 20 21 especially in a complex biological system such as that of a lichen colony and its attendant Manuscript 22 23 24 mineral encrustation. 25 26 27 28 29 30 In this work, we make a summary of the main chemicals produced by lichens over the

31 Accepted 32 world detected by Raman spectroscopy with the goal of connecting lichen species, climate 33 34 and lichen protective biomolecules. From our compendium, the relationship between oxalates 35 36 and lichens in dry and wet environments will be examined and the production of protective 37 38 39 pigment admixtures in different climates discussed.

40 Methods 41 42 43 44 45 There are many articles in which studies of lichen pigments have been carried out 46 47 using different analytical techniques but the focus of this paper is where Raman spectroscopy 48 49 has been used for the molecular characterization. The focus on Raman spectroscopy is owing 50 Analytical 51 to the increasing interest in using a nondestructive technique. Despite the increased adoption 52 53 54 of this technique for lichenological systems analysis the resulting spectrum is often complex 55 56 to interpret for nonspecialists. This will be the first time that a data compendium of lichen 57 58 pigments and calcium oxalates related with climate parameters has been published and hence 59 60 6

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1 2 3 will provide a good Raman spectral database for the interpretation of lichen behaviour, 4 5 especially for lichenologists using Raman spectroscopy for the first time for chemical 6 7 identification in lichen systems. 8 9 10 11 12 13 MATERIAL AND METHODS 14 15 16 17 18 19 Because of the change in relative intensity of the spectral signatures observed under 20 21 Manuscript 22 different operational experimental conditions, we have selected for our database (Table 1) 23 24 only the most significant spectral features observed using either 1064 or 785 nm laser 25 26 excitation as fluorescence or resonance Raman effects generally become more significant in 27 28 the visible region of the electromagnetic spectrum, such as that found using 532 or 514 nm 29 30 laser radiation. Although the wavenumber region between 1700-1000 cm -1 is, normally, the

31 Accepted 32 33 most representative area in a Raman spectrum for biomolecular identification, in our 34 35 experience, we have seen that, for some compounds, characteristic bands are detected in the 36 37 700-200 cm -1 region and these too can be quite definitive for molecular or molecular ion 38 39 identification.

40 Methods 41 42

43 44 45 46 Lichens have been grouped under six different climates (Table 1 and Table 2a, 2b, 2c, 47 48 2d; see references in table 1): Polar (cold and dry), Oceanic (temperate and wet), 49 50 Mediterranean (temperate but with wet and dry seasons), Sub-desert (hot and dry), High 51 Analytical 52 mountain+desert (high diurnal temperature variation but very dry in the case of the analyzed 53 54 specimens) and, finally, Tropical (warm and wet). 55 56 57 58 59 60 7

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1 2 3 From all literature references analysed for this study, we have included in our table 4 5 eighty-eight lichen specimens, comprising sixty-six different species belonging to thirty-seven 6 7 different genera. Each species cited in the revised papers has been given a different number; 8 9 10 additionally, for clarity when one particular species appears in different papers with clearly 11 12 identified different pigment mixtures, we have assigned to it the same number but we have 13 14 added a suffix letter (one species, several specimens). For a genus sp, we have assigned 15 16 different identifying numbers each time it appears, in the absence of any further literature 17 18 taxonomic clarification. 19 20 21 Manuscript 22 23 24 25 In some of the studies analyzed for this work, authors have characterized only the 26 27 most common compounds, such as carotene, chlorophyll, cellulose or oxalates; this could be 28 29 explained because a) occasionally, no other molecule gives Raman bands in a lichen analysis 30

31 owing to particular lichen environmental protective strategies; b) because of the analytical Accepted 32 33 parameters chosen for the study or c) because of the analytical spectrometer limitations. 34 35 36 Sometimes, weak Raman spectral signatures remain unassigned, as can be seen in some 37 38 figures in the original articles. When wavenumbers do not appear in the text or tables and no 39

40 spectral assignments have been forthcoming it is impossible to assess what compounds have Methods 41 42 then been detected by Raman spectroscopy - in this case, sometimes they are shown as an 43 44 45 “unknown compound”; here we have not attempted to reassign the work of others in our 46 47 analysis and "unknown compounds" remain as such, although they are discussed further later 48 49 in an appropriate section in this paper. 50 51 Analytical 52 For this work, we do not distinguish spatially where a compound was found, i.e in the 53 54 lichen thallus, apothecia, encrustation etc. All pigments were detected using Raman 55 56 spectroscopy, although from other studies, using different and destructive analytical 57 58 59 60 8

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1 2 3 techniques, more pigments may have been reported, the goal of this review is to compare 4 5 those bio-molecules identified non-destructively using Raman spectroscopy. 6 7 8 9 10 11 RESULTS AND DISCUSSION 12 13 14 15 16 17 Results of the lichen specimens, climates and biomolecules found by Raman 18 19 spectroscopy are shown in tables 2a, 2b, 2c and 2d. 20 21 Manuscript 22

23 24 25 26 CAROTENOIDS AND CHLOROPHYLL 27 28 29 Although chlorophyll is ubiquitously found in lichens, it has been only detected using 30 Raman spectroscopy, in forty two specimens (47,7%) of those compiled for this review 31 Accepted 32 33 (Tables 2a, 2b, 2c, 2d; Table 3). It has been appreciated that chlorophyll is difficult to detect 34 35 in natural samples when a green or blue laser was used for Raman analyses. 33 36 37 38 Contrary to the case of chlorophyll, carotenes were generally found (92%) and yet not 39

40 described in only seven specimens (8%); this is perhaps surprising since the carotenoids are a Methods 41 42 43 group of pigments which give a strong Raman spectroscopic response, particularly when a 44 45 green or blue laser was used because of the resonance Raman enhancement. An electronic 46 47 absorption band for carotenoids occurs near 500 nm and when green laser excitation is used 48 49 the characteristic Raman bands (centred near 1510, 1150 and 1000 cm -1) are resonantly 50 Analytical 51 33,41 52 enhanced. Carotenes fulfil a multiple role: they act as UV-radiation screen, are 53 54 antioxidants and DNA repairers. Despite the presence of this multifunctional pigment, lichens 55 56 also produce other protective biomolecules (Tables 3 and 4). 57 58 59 60 9

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1 2 3 4 5 6 Although it is tempting to suggest that the assignment of specific carotenoids in 7 8 lichens and other organisms is possible from their Raman spectrum, Oliveira et al. (2010) 41 9 10 have shown that the precise characterization of those compounds in natural samples is 11 12 13 ambiguous because of the interaction of these molecules with other compounds in the cell, 14 15 producing significant wavenumber shifts of the characteristic Raman bands, so the 16 17 interpretation of the Raman spectrum through comparison with pure carotenoid standards 18 19 must be undertaken with caution. Hence in this study, we have replaced specified 20 21 “carotenoids” with the generic term in our table since in some cases authors have apparently Manuscript 22 23 24 not considered the consequence of such potential wavenumber shifts in their assignments. 25 26 27 28 29 30 CALCIUM OXALATES

31 Accepted 32 33 34 35 36 Calcium oxalates are compounds which provide a strong Raman spectrum whichever 37 38 laser wavelength is used for exciting the sample. However, for 35,2% of specimens no oxalate 39

40 was detected (Figure 1). Methods 41 42 43 44 45 46 In Figure 1, the percentage of oxalates related to the environmental conditions are 47 48 49 shown. The Polar climate is characterized by cold temperatures and low relative humidity in 50 51 air; what we have called High-mountain/desert shows some similarities with this Polar Analytical 52 53 climate since those lichens were collected in the Atacama desert 4,33,39 with some of the lowest 54 55 terrestrial humidities. It is interesting to note that, despite the extreme desiccation experienced 56 57 by lichens in both areas, there are large numbers of specimens without oxalates detected. 58 59 60 10

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1 2 3 Subdesert climates cannot be used for establishing conclusions here because only four 4 5 specimens were analysed from these regions. 6 7 8 9 10 11 Tropical regions show similar proportions of specimens with and without oxalates to 12 13 the Polar climate, despite the high rainfall. The temperate Oceanic climate shows a high ratio 14 15 16 of specimens with oxalates in comparison with those showing an absence of the calcium 17 18 oxalates. In Mediterranean areas, with a wet and cold season and another one hot and drier, all 19 20 specimens showed that oxalate was present (Figure 1). 21 Manuscript 22 23 24 25 26 The role played by calcium oxalates in the lichen survival strategy is controversial: 27 28 although they are clearly generated as secondary products by lichen metabolic processes, 29 30 some authors have attributed to them a water storage function.1,4,5,12,20,42-46 Furthermore,

31 Accepted 32 33 weddellite, calcium oxalate dihydrate, is metastable at temperatures in excess of 5 degrees C 34 35 and thermodynamically reverts in time to the more stable whewellite, calcium oxalate 36 37 monohydrate. It has been proposed by some authors that the excess of water could be used by 38 39 the lichen in drought periods. However, this suggestion does not appear to be supported by

40 Methods 41 42 the observation that lichens with weddellite detected in the Oceanic and Mediterranean 43 44 climates involve 47,8% and 80% respectively (Figure 1), whereas in dry climates such as 45 46 High mountains/desert (14,4%) and in Polar regions (27,8%) this is lower (Figure1). In 47 48 Subdesert climates 50% of lichens show the presence of weddellite but, as we have previously 49 50 51 pointed out, the low number of lichen specimens studied makes this conclusion non Analytical 52 53 representative. 54 55 56 57 58 59 60 11

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1 2 3 In summary, taking into account the total number of specimens; 71,7% of lichens 4 5 (38/53 spcms) from Oceanic, Mediterranean and Tropical climates show the presence of 6 7 either calcium oxalate monohydrate and dihydrate or both whereas only 45,7% of specimens 8 9 10 from the more extreme desert climates (subdesert, high mountains/desert and polar, 16/35 11 12 specimens) produce the same compounds; i.e. almost three quarters of lichens living under 13 14 wet climates produce oxalates, whereas less than half of the lichens living under dry climates 15 16 produce those molecules; therefore, the hypothesis that oxalates could work as a water storage 17 18 must be revised. 19 20 21 Manuscript 22 23 24 25 OTHER BIOMOLECULES 26 27 28 29 30 From the biomolecules identified by Raman spectroscopy (Table 4), the

31 Accepted 32 33 characterization of parietin as a lichen pigment is outstanding since it appears in no fewer 34 35 than twenty-one specimens (18,42%). The production of this pigment is apparently unrelated 36 37 with any specific climate. The next most common lichen pigments identified by Raman 38 39 spectroscopy are rhizocarpic acid, which appears in 9,65% of specimens and lecanoric acid

40 Methods 41 42 (7,89%) followed by calycin, gyrophoric acid and usnic acid (7,02% each). 43 44 45 46 47 48 Most commonly, it seems that only one pigment is identifiable if at all in any climate 49 50 (Figure 2), but it is significant that the production of four or more pigments is commonly 51 Analytical 52 found in the oceanic climate. Again, the hypothesis that the most inhospitable environmental 53 54 conditions are associated with the most complex pigment mixture synthesised by lichen 55 56 47 57 systems is questionable. 58 59 60 12

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1 2 3 4 5 6 CONCLUSIONS 7 8 9 10 11 12 Several conclusions may be made as a result of the current study. 13 14 15 Biomolecules produced by lichens under different climatic conditions and in a range of 16 17 environments detected using Raman spectroscopy and reported in the literature have been 18 19 reviewed. As a result, from this assimilation of spectral data and a comparative analysis, it is 20 21 Manuscript 22 clear that some hypotheses made on the basis of more localised studies are not supportable. 23 24 25 26 Raman spectroscopy is a suitable technique for lichen biomolecule identification without 27 28 sample manipulation or pretreatment. In particular, the ability to detect carotenoids alone and 29 30 in admixture without extraction is valuable. The detection of chlorophyll is rather more

31 Accepted 32 33 problematic and this can be ascribed to the laser excitation wavelengths used. 34 35 36 37 Calcium oxalate, either as a mono- or dihydrate, appears more often in specimens from wet 38 39 climates, such as oceanic, tropical or Mediterranean than in specimens from drier areas, such

40 Methods 41 42 as polar, desert and subdesert regions. From this work, we conclude that the hypothesis made 43 44 in the literature that whewellite and weddellite act as water reservoirs for lichens under 45 46 drought conditions is not sustainable and that more studies should be carried out to understand 47 48 properly their function. it is also very interesting that only calcium oxalates are detected in the 49 50 51 spectral data and there is no evidence for the presence any other metal oxalate or oxalic acid, Analytical 52 53 even under conditions of calcium deficiency, such as on granite rock substrates: it is believed 54 55 this reflects the insolubility of the calcium oxalate compared with its magnesium or alkali 56 57 58 59 60 13

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1 2 3 metal congeners by which means the oxalic acid waste product is removed more effectively 4 5 from the growth area. 6 7 8 9 10 From our results, there is no apparent relationship between pigments or pigment mixtures 11 12 produced and climate; More specific studies should be carried out to clarify what mechanism 13 14 dictates the adoption of a particular survival strategy related to stressed or temperate 15 16 environmental conditions. 17 18 19 20 21 It has also not been observed previously that the presence of a single pigment is the most Manuscript 22 23 common strategy regardless of mild climate. Also, whereas it could have reasonably been 24 25 thought that lichens would have produced four or more pigments in the most hostile and 26 27 extreme environmental conditions, in reality this mixture is produced only in rather more 28 29 30 gentle oceanic climates; of the literature surveyed, only one subdesert specimen produced four

31 Accepted 32 pigments in admixture. 33 34 35 36 The most common lichen pigment is parietin, followed by rhizocarpic acid and then lecanoric 37 38 acid, calycin, gyrophoric acid and usnic acid. It doesn’t seem to have any relationship 39

40 Methods 41 between any climate and a pigment type. The most common pigments appear in different 42 43 climatic conditions. 44 45 46 47 48 ACKNOWLEDGES 49 50 Analytical 51 The authors are grateful for support from the Fellowships Juan de la Cierva 2008-39669 and 52 53 the Marie Curie Intra-European Fellowship (FP7-PEOPLE-2013-IEF, Proposal nº 54 55 56 623393). The authors are also grateful for support from CARBORAD proyect (CGL2011- 57 58 27493) funded by the Spanish Ministerio de Ciencia e Innovación and the proyect 59 60 14

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1 2 3 "Variaciones de pigmentos y otros metabolitos causadas por el microclima en especies clave 4 5 de costras biológicas del suelo" funded by the Asociación de Ecología Terrestre Española 6 7 (AEET). 8 9 10 REFERENCES 11 12 13 1 I. Miralles, S.E. Jorge-Villar, Y. Canón, F. Domingo, Astrobiology 2012, 12, 743–753. 14 15 16 2 C.S. Cockell, J. Knowland, Biol. Rev . 1999, 74 , 311–345. 17 18 19 3 D.L. Dickensheets, D.D. Wynn-Williams, H.G.M. Edwards, C. Crowder, E.M. Newton , J. 20 21 Manuscript 22 Raman Spec. 31:633–635. 23 24 25 4 S.E. Jorge-Villar, H.G.M. Edwards, M.R.D. Seaward, Analyst, 2005, 130 ,730–737. 26 27 28 5 S.E. Jorge-Villar, H.G.M. Edwards, J. Raman Spectrosc. 2010, 41, 63–67. 29 30 6 D.D. Wynn-Williams, J.M. Holder, H.G.M. Edwards, in New Aspects in Cryptogamic 31 Accepted 32 33 Research , eds. B. Schroeter, M. Schlensog, T.G.A. Green, der Gebruder Borntraeger 34 35 Verlagsbuchhandlung, Berlin-Stuttgart, 2000. 36 37 38 7 H.G.M. Edwards, L.F.C. de Oliveira, M.R.D. Seaward, Lichenol., 2005, 37 , 181–189. 39

40 Methods 41 8 P. Ropret, S. Tavzes, K. Retko, L. Legan, T. Spec, N. Ocepek, Preservation– 42 43 44 EUROMED2012, pp. 325–329. 45 46 47 9 C. Darwin, The Origin of Species by Means of Natural Selection: The Preservation of 48 49 Favoured Races in the Struggle for Life, 1859. 50 51 Analytical 52 10 H.G.M. Edwards, N.C. Russell, D.D. Wynn-Williams, J. Raman Spectrosc., 1997, 28 , 53 54 685–690. 55 56 57 11 H.G.M. Edwards, Spectrochim. Acta, Part A, 2007, 68 ,1126–1132. 58 59 60 15

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1 2 3 12 S.E. Jorge-Villar, H.G.M. Edwards, M.R.D. Seaward, Spectrochim. Acta Part A , 2004, 60 , 4 5 1229–1237. 6 7 8 13 H.G.M. Edwards, C.S. Cockell, E.M. Newton, D.D. Wynn-Williams, J. Raman Spectrosc., 9 10 2004, 35 , 463–469. 11 12 13 14 H.G.M. Edwards, E.M. Newton, D.D. Wynn-Williams, R.I. Lewis-Smith, Spectrochim. 14 15 16 Acta Part A , 2003, 59:2301–2309. 17 18 19 15 H.G.M. Edwards, J.M. Holder, M.R.D. Seaward, D.A. Robinson, J Raman Spectrosc., 20 21 2002, 33 , 449–454. Manuscript 22 23 24 16 S. Pannewitz, M. Schlensog, T.G. Allan-Green, L.G. Sancho, B. Schroeter, Oecologia , 25 26 2003, 135 , 30–38. 27 28 29 17 T.G.A. Green, B. Schroeter, L.G. Sancho, Plant life in Antarctica . In: Functional Plant 30

31 Ecology, eds. F. Pugnaire, F. Valladares, CRC Press, Boca Raton, Florida, 2007. Accepted 32 33 34 18 J.A. Elix, Biochemistry and Secondary Metabolites . In: Lichen Biology, ed. T. Nash, 35 36 37 Cabridge University Press, Cambridge, 1996. 38 39 19 S. Huneck, I. Yoshimura, Identification of Lichen Substances . Springer-Verlag, Berlin,

40 Methods 41 42 1996. 43 44 45 20 J.M. Holder, D.D. Wynn-Williams, F. Rull-Perez, H.G.M. Edwards, New Phytol., 2000, 46 47 145 , 271–280. 48 49 50

21 H.G.M. Edwards, E.M. Newton, D.D. Wynn-Williams, S.R. Coombes, J. Mol. Struct., Analytical 51 52 2003, 648 , 49–59. 53 54 55 22 K.A. Solhaug, Y. Gauslaa, L. Nybakken, W. Bilger, New Phytol., 2003, 158 , 91–100. 56 57 58 59 60 16

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1 2 3 23 L.P. Choo-Smith, H.G.M. Edwards, H.P. Endtz, J.M. Kros, F. Heule, H. Barr, J.R. 4 5 Robinson, H.A. Bruining, G.J. Puppels, Biopolymers , 2002, 67 , 1–9. 6 7 8 24 A. Caudron, C. Tfayli, M. Monnier, P. Manfait, D. Prognon, J. Pradeau, J. Pharmaceut. 9 10 Biomed. Anal., 2011, 54 , 866–868. 11 12 13 25 W.P. Findlay, D.E. Bugay, J. Pharmaceut. Biomed. Anal., 1998, 16 , 921-930. 14 15 16 26 Y. Roggo, K. Degardin, P. Margot, Talanta , 2010, 81 , 988–995. 17 18 19 27 E.S.B. Ferreira, A.N. Hulme, H. Mcnab, A. Quye, Chem. Soc. Rev.,2004, 33 , 329–336. 20 21 Manuscript 22 28 I. Miralles, F. Domingo, Y. Cantón, C. Trasar-Cepeda, M.C. Leirós, F. Gil-Sotres, Soil 23 24 25 Biol. Biochem., 2012, 53 , 124–132. 26 27 28 29 I. Miralles, F. Domingo, E. García-Campos, C. Trasar-Cepeda, M.C. Leirós, F. Gil-Sotres, 29 30 Soil Biol. Biochem., 2012, 55 , 113–121.

31 Accepted 32 33 30 I. Miralles, C. Trasar-Cepeda, M.C. Leirós, F. Gil-Sotres, Soil Biol. Biochem., 2013, 58 , 34 35 1–8. 36 37 38 31 I. Miralles, B. van Wesemael, Y. Cantón, S. Chamizo, R. Ortega, F. Domingo, G. 39

40 Methods Almendros, Geoderma , 1012, 189 –190 : 227–235. 41 42 43 32 H.G.M. Edwards, C.D. Moody, S.E. Jorge-Villar, D.D. Wynn-Williams, Icarus , 2005, 44 45 46 174 , 560–571. 47 48 49 33 S.E. Jorge-Villar, H.G.M. Edwards, Anal. Bioanal. Chem., 2006, 384 , 100–113. 50 51 Analytical 52 34 S.E. Jorge-Villar, I. Miralles, C. Capel, V. Hernández-Jolín, Anal. Methods , 2011, 3, 53 54 2783–2791. 55 56 57 35 D.D. Wynn-Williams, H.G.M. Edwards, Icarus , 2000, 144 , 486–503. 58 59 60 17

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1 2 3 36 D.D. Wynn-Williams, Antarctic as a model for ancient in Mars . In: The Search for Life on 4 5 Mars, ed. J. A. Hiscox, British Interplanetary Society, London, 1999. 6 7 8 37 L.G. Sancho, R.T. Torre, A. Pintado, Fungal Biol. Rev., 2008, 22 , 103–109. 9 10 11 38 S.E. Jorge-Villar, L.G. Benning, H.G.M. Edwards, Amase Team., Geochem. Transac ., 12 13 2007, 8, 1–11. 14 15 16 39 S.E. Jorge-Villar, H.G.M. Edwards, M.R. Worland, Origins Life Evol. Biosph., 2005, 35 , 17 18 19 489–506. 20 21 Manuscript 22 40 S.E. Jorge-Villar , H.G.M. Edwards, Int. J. Astrobiol., 2004, 3, 165–174. 23 24 25 41 V.E. de Oliveira, H.V. Castro, H.G.M. Edwards, L.F.C. de Oliveira, J. Raman Spectrosc., 26 27 2010, 41 , 642–650. 28 29 30 42 H.G.M. Edwards, D.W. Farwell, M.R.D. Seaward, Lichenologist , 1997, 29 , 83–90.

31 Accepted 32 33 43 H.G.M. Edwards, M.R.D. Seaward, S.J. Attwood, S.J. Little, L.F.C. de Oliveira, M. 34 35 Tretiach, Analyst , 2003, 128 , 1218–1221. 36 37 38 44 M.R.D. Seaward, H.G.M. Edwards, J. Raman Spectrosc., 1997, 28 , 691–696. 39

40 Methods 41 45 S.E. Jorge-Villar, H.G.M. Edwards, L.G. Benning, Icarus , 2006, 184 , 158–169. 42 43 44 46 R.L. Frost, Anal. Chim. Acta , 2004, 517 , 207–214. 45 46 47 47 D. Amico, T. Collins, J.C. Marx, G. Feller, C. Gerday, Psychrophilic microorganisms: 48 49 50 challenges for life Salvino, University of Liege, Liege, Belgium, 2006. 51 Analytical 52 53 48 H.G.M. Edwards, E.M. Newton, D.D. Wynn-Williams, D. Dickensheets, C. Schoen, C. 54 55 Crowder, Int. J. Astrobiol., 2003, 1, 333–348. 56 57 58 59 60 18

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1 2 3 49 P. Vítek, E.M.A. Ali, H.G.M. Edwards, J. Jehlicka, R. Cox, K. Page, Spectrochim. Acta 4 5 Part A , 2012, 86 , 320–327. 6 7 8 50 H.G.M. Edwards, D.D. Wynn-Williams, S.J. Little, L.F.C. de Oliveira, C.S. Cockell, J.C. 9 10 Ellis-Evans, Spectrochim. Acta Part A , 2004, 60 , 2029–2033. 11 12 13 51 H.G.M. Edwards, E.M.Newton, D.L. Dickensheets, D.D. Wynn-Williams, Spectrochim. 14 15 16 Acta Part A , 2003, 59 , 2277–2290. 17 18 19 52 H.G.M. Edwards, J.M. Holder, D.D. Wynn-Williams, Soil Biol. Biochem., 1998, 30 , 1947– 20 21 1953. Manuscript 22 23 24 53 H.R.D. Seaward, H.G.M. Edwards, D.W. Farwell, Nova Hedwigia , 1998, 66 , 463–472. 25 26 27 54 H.G.M. Edwards, N.C. Russell, M.R.D. Seaward, D. Slark, Spectrochim. Acta Part A , 28 29 1995, 51 , 2091–2100. 30

31 Accepted 32 55 H.G.M. Edwards, F. Rull-Perez, Biospectrosc ., 1999, 5, 47–52. 33 34 35 56 M.R.D. Seaward, H.G.M. Edwards, D.W. Farwell, FT-Raman microscopic studies of 36 37 Haematomma ochroleucum var. porphyrium. Studies in lichenology with emphasis on 38 39 chemotaxonomy, geography and phytochemistry. Festschrift Ch. Leuckert. (eds: Knoph, J.G.,

40 Methods 41 42 Schrüfer, K., and Sipman, H.J.M.)- Bibliotheca Lichenologica 57:395–407. J. Cramer in der 43 44 Gebrüder Borntraeger Verlagsbuchhandlung, Berlin-Stuttgart, 1995. 45 46 47 57 H.G.M. Edwards, N.C. Russel, M.R.D. Seaward, Spectrochim. Acta Part A , 1997, 53 , 99– 48 49 105. 50 51 Analytical 52 58 B. Prieto, H.G.M. Edwards, M.R.D. Seaward, Geomicrobiol. J., 2000, 17 , 55–60. 53 54 55 59 B. Prieto, M.R.D. Seaward, H.G.M. Edwards, T. Rivas, B. Silva, Biospectrosc ., 1999, 5, 56 57 58 53–59. 59 60 19

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1 2 3 60 H.G.M. Edwards, K.A.E. Edwards, D.W. Farwell, I.R. Lewis, M.R.D. Seaward, J. Raman 4 5 Spectrosc., 1994, 25 , 99–103. 6 7 8 61 H.G.M. Edwards, D.W. Farwell, M.R.D. Seaward, Int. Biodeterioration , 1991, 27 , 1–9. 9 10 11 62 H.G.M. Edwards, S.E. Jorge-Villar, M.R.D. Seaward, L.L. St. Clair, Raman Spectroscopy 12 13 of Rock Biodeterioration by the Lichen Lecidea Tessellata Flörke in a Desert Environment, 14 15 16 Utah, USA. In: Biodeterioration of Stone Surfaces, eds. L.L. Clair, M.R.D. Seaward, 2004. 17 18 19 63 L.F.C. de Oliveira, H.G.M. Edwards, J.C. Feo-Manga, M.R.D. Seaward, R. Lücking, 20 21 Lichenologist , 2002, 34 , 259–266. Manuscript 22 23 24 64 L.F.C. de Oliveira, P.C.C. Pinto, M.P. Marcelli, H.F. Dos Santos, H.G.M. Edwards, J. 25 26 Mol. Struct., 920 , 128–133. 27 28 29 65 F.J. Chu J, M.R.D. Seaward, H.G.M. Edwards, Spectrochim. Acta Part A , 1998, 54 , 967– 30

31 982. Accepted 32 33 34

35 36 37 TABLES 38 39

40 Table 1. Climate, lichen genus and species, site locations and specimen numbers. Methods 41 42 43 44 45 Table 2a: Number of lichen specimens and biochemical compounds found by using Raman 46 47 spectroscopy on Polar climate. 48 49 50 Table 2b: Number of lichen specimens and biochemical compounds found by using Raman 51 Analytical 52 spectroscopy on Oceanic climate. 53 54 55 Table 2c: Number of lichen specimens and biochemical compounds found by using Raman 56 57 58 spectroscopy on Mediterranean and Subdesert climates. 59 60 20

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1 2 3 Table 2d: Number of lichen specimens and biochemical compounds found by using Raman 4 5 spectroscopy on High Montain+Desert and Tropical climates. 6 7 8 Table 3: Lichen chemicals by specimens and climates. SPCMS: specimens; SPCS: species; 9 10 Ca OX.: calcium oxalate (unspecified hydration); WEDDE. OX. DIHYDRATE: Weddellite 11 12 13 (oxalate dihydrate); WHEWE. OX. MONOHYDRATE: Wewellite (oxalate monohydrate); 14 15 PIGMENTS: other pigments than carotene and chlorophyll.Table 4: Number of times each 16 17 pigment was found under different climates, by using Raman spectroscopy and total 18 19 percentage of each pigment found in the total specimen number. 20 21 Manuscript 22 FIGURES 23 24 25 Figure 1 . Representation by pie-chart of: a) Percentage of lichen specimens with and/or 26 27 28 without oxalates; b) Type of oxalates found in different world climate regions. The world 29 30 climate regions considered in this study are: Polar (18 specimens [spcms]), Oceanic (23

31 Accepted 32 spcms), Mediterranean (10 spcms), Subdesert (4 spcms), High-mount/desert (13 spcms) and 33 34 Tropical (20 spcms). Climate Map zones of the world: Original uploader was Waitak at 35 36 en.wikipedia Later version(s) were uploaded by Splette at en.wikipedia. - Transferred from 37 38 39 en.wikipedia; transfered to Commons by User:Legoktm using CommonsHelper.

40 Methods 41 42 https://en.wikipedia.org/wiki/Climate#/media/File:ClimateMap_World.png 43 44 45 46 47 Figure 2 . Representation by pie-chart of pigments occurrence: number of pigments in each 48 49 50 region (without carotenoids and chlorophyll). The world climate regions considered in this 51 Analytical 52 study are: Polar (18 speciments [spcms]), Oceanic (23 spcms), Mediterranean (10 spcms), 53 54 Subdesert (4 spcms), High-mount/desert (13 spcms) and Tropical (20 spcms). 55 56 57 58 59 60 21

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1 2 3 Climate Map zones of the world: Original uploader was Waitak at en.wikipedia Later 4 5 version(s) were uploaded by Splette at en.wikipedia. - Transferred from en.wikipedia; 6 7 transfered to Commons by User:Legoktm using CommonsHelper. 8 9 10 https://en.wikipedia.org/wiki/Climate#/media/File:ClimateMap_World.png 11 12 13 14 15 16 17 18 19 20 21 Manuscript 22 23 24 25 26 27 28 29 30

31 Accepted 32 33 34 35 36 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 22

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1 2 3 Table 1: Climate, lichen genus and species, site locations and specimen numbers. (SPEC: Specimen) 4 5 6 CLIMATE GENUS SPECIES SITE SPEC 7 1-Football Saddle, 8 northern Polar Acarospora Acarospora chlorophana 14,20,43,48, Victoria Land, Antarctica; 1a 9 2- Harrow Peaks, Victoria 10 Land. 11 Football Saddle, northern Polar Acarospora Acarospora chlorophana 32 1b 12 Victoria Land. 13 Polar Acarospora Acarospora chlorophana 3 Victoria Land (Antarctica) 1c 14 Signy Island in the Polar Acarospora Acarospora sp. 49 15 maritime Antarctic. 2a 16 Mars O asis on Alexander Polar Buellia Buellia sp. 50 3 17 Island. 18 1-Signy I.; 2- Leonie 19 13,14 Island in Marguerite Bay, Polar Caloplaca Caloplaca sublobulata 4 20 off the Antarctic 21 Peninsula. 1-Crater Cirque, Victoria Manuscript 22 Polar Caloplaca Caloplaca saxicola 14,48,51 5a 23 Land. 14 24 Polar Candelaria Candelaria sp. Victoria Land. 6 14 25 Polar Candelariella Candelariella sp. Victoria Land. 7 14 26 Polar Lecidea Lecidea sciatrapha Signy I. 8 27 Polar Lepraria Lepraria sp. 14 Signy I. 9 28 Polar Rhizocarpon Rhizocarpon geographicum 14 Victoria Land 10 29 Polar Umbilicaria Umbilicaria antarctica 14 Signy I. 11 30 Polar Usnea Usnea antarctica 14 Signy I. 12

31 Harrow Peaks, Victoria Accepted Polar Xanthoria Xanthoria elegans 52 13a 32 Land, Antarctica. 33 1-Harrow Peaks, Victoria 34 Land; 2-Leonie Island in Marguerite Bay, off the 35 Polar Xanthoria Xanthoria elegans 13, 14,32 13b 36 Antarctic Peninsula; 3- 37 Mc-Murdo Valley 38 (Victoria Land). Harrow Peaks, Victoria 39 Polar Xanthoria Xanthoria mawsonii 52 Land, Antarctica. 14a

40 Methods Polar Xanthoria Xanthoria mawsonii 14 Rothera, Graham Land. 41 14b Oceanic Aspicilia Aspicilia calcarea 12 Vizcainos (Spain). 15 42 12 43 Oceanic Caloplaca Caloplaca holocarpa Vizcainos (Spain). 16 12 44 Oceanic Caloplaca Caloplaca decipiens Tañabueyes (Spain). 17 Jaramillo de la Fuente 45 Oceanic Caloplaca Caloplaca teicholyta 12 18 46 (Spain). 12 47 Oceanic Caloplaca Caloplaca saxicola Villamoron (Spain). 5b 12 48 Oceanic Caloplaca Caloplaca saxicola Olmos de Picaza (Spain). 5c 12 49 Oceanic Candelariella Candelariella medians Tanabueyes (Spain). 19 50 Oceanic Chroodiscus Chroodiscus megalophthalmus 53 New Zealand. 20 Frocester, Gloucester Analytical 51 Oceanic Diploicia Diploicia canescens 54 21 52 (UK) 55 53 Oceanic Diploschistes Diploschistes scruposus Sahagún (Spain) 22 54 Dirina massilie nsis forma Oceanic Dirina 44 Lincolnshire (UK) 23a 55 sorediata Dirina massiliensis forma Laulivery, Cornwall and 56 Oceanic Dirina 42 23b 57 sorediata Norfolk (UK) 58 Oceanic Dirina Dirina massiliensis forma Cape Clear Island, Co. 23c 59 60 23

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1 2 42 3 sorediata Cork, S.W. (Ireland). 4 Haematomma ochroleucum var. Goxhill Hall, Lincolnshire Oceanic Haematomma 56 24 5 Porphyrium (UK). Menithwood, 6 Oceanic Lecanora Lecanora muralis 57 25 7 Worcestershire (UK). Bastavales, La Coruña 8 Oceanic Lecidea Lecidea fuscoatra 58 26 (Spain). 9 San Lorenzo church 10 59 Oceanic Ochrolechia Ochrolechia parella (Santiago de Compostela, 27a 11 Spain). 12 Bridge of Merza 13 (Pontevedra, 14 Spain), Monastery of 15 Oceanic Ochrolechia Ochrolechia parella 59 Toxosoutos (La Coruña, 27b 16 Spain), Os Anxeles 17 church (La Coruña, 18 Spain). Bastavales, La Coruña 19 Oceanic Porpidia Porpidia cinereoatra 58 28 20 (Spain). 21 58 Bastavales, La Coruña Oceanic Porpidia Porpidia macrocarpa 29 Manuscript 22 (Spain). Portpatrick, SW Scotland 23 Oceanic Xanthoria Xanthoria parietina 52 (UK). 30a 24 Oceanic Xanthoria Xanthoria parietina 12 Burgos (Spain). 25 30b S.W. Scotland and west 26 Oceanic Xanthoria Xanthoria parietina 60 31 Yorkshire (UK). 27 Mediterra 28 Acarospora Acarospora oxytona 57 Sierra Nevada (Spain). nean 32a 29 Mediterra 30 Acarospora Acarospora oxytona 20 Picon (Spain). 32b nean 31 Mediterra Sicily and Égadi Islands Accepted Aspicilia Aspicilia calcarea 43 33 32 nean (Italy). 33 Mediterra Caloplaca Caloplaca aurantia 57 Southern Spain. 34 34 nean 35 Mediterra Dirina massiliensis forma sorediata Palazzo Farnese, Dirina 61 23d 36 nean Caprarola (Italy). 37 Palazzo Farnese, 38 Mediterra Dirina massiliensis forma sorediata Caprarola (Central Italy); Dirina 42 23e 39 nean SS. Niccolo e Cataldo near Lecce (S.E. Italy); 40 Methods 41 Mediterra Dirina massiliensis forma Sicily and Égadi Islands Dirina 43 23f 42 nean sorediata (Italy). Mediterra Tremiti Islands, San 43 Dirina Dirina massiliensis f. massiliensis 43 23g 44 nean Nicola (Italy). Mediterra Tremiti Islands, San 45 Lecanora Lecanora sulfurea 43 nean Nicola (Italy). 35 46 Mediterra Monte Sicily, Égadi Tephromela Tephromela atra 43 47 nean Islands (Italy). 36 48 Tabernas Desert (SE, 49 Sub-desert Diploschistes Diploschistes diacapsis 1,34 37 Spain). 50 Dirina massiliensis forma sorediata Analytical 51 Desert Dirina 42 E. of Alexandria (Egypt). 23h 52 Tabernas Desert (SE, Sub-desert Lepraria Lepraria crassissima 1,34 53 Spain). 38 54 Tabernas Desert (SE, Sub-desert Squamarina Squamarina lentigera 1,34 39 55 Spain). 56 High 57 mountain Acarospora Acarospora sp. 33 Atacama Desert (Chile) 2b 58 + desert 59 60 24

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1 2 3 High 4 4 mountain Acarospora Acarospora sp. orange-yellow Atacama Desert (Chile) 40 5 + desert High 6 4 7 mountain Acarospora Acarospora sp. Brown Atacama Desert (Chile) 41 + desert 8 High 9 39 mountain Acarospora Acarospora cf. Schleichera Atacama Desert (Chile) 42a 10 + desert 11 High Acarospora Schleichera greenish- 12 mountain Acarospora 4 Atacama Desert (Chile) 42b yellow 13 + desert 14 High Atlas Mountains High 15 near Oukaimeden, about mountain Aspicilia Aspicilia caesiocinerea agg 15 43 16 48 km south of Marrakech + desert 17 (Morrocco). 18 High 4 19 mountain Candelariella Candelariella genus orange-yellow Atacama Desert (Chile). 44 20 + desert 21 High Utah, Colorado Plateau Manuscript mountain Lecidea Lecidea Tessellata Florke 62 45 22 (USA). 23 + desert High 24 4 25 mountain Rhizocarpa Rhizocarpa sp. White Atacama Desert (Chile) 46 26 + desert 27 High mountain Rhizoplaca Rhizoplaca sp. White 4 Atacama Desert (Chile) 47 28 + desert 29 High 30 mountain Xanthopamundia Xanthopamundia sp. White 4 Atacama Desert (Chile) 48

31 + desert Accepted 32 High 33 mountain Xanthomendoza Xanthomendoza mendozae 32 Atacama Desert (Chile) 49a 34 + desert 35 High 36 mountain Xanthomendoza Xanthomendoza mendozae red 4 Atacama Desert (Chile) 49b 37 + desert 38 Tropical Buslicia Buslicia sp. 5 Kilauea, Hawaii (USA). 50 39 Tortuguero National Park Tropical Calenia Calenia triseptata 63 51

40 (Costa Rica). Methods 41 Tropical Candelariela Candelariela concolor 5 Kilauea, Hawaii (USA). 52 42 Tropical Caloplaca Caloplaca crosbyae 5 Kilauea, Hawaii (USA). 53 43 Tropical Cladonia Cladonia coniocraea 5 Kilauea, Hawaii (USA). 54a 44 Tropical Cladonia Cladonia coniocraea 5 Kilauea, Hawaii (USA). 54b 45 Cantareira, Sao Paulo 46 (Brazil); Pinheiros, Tropical Cryptothecia Cryptothecia rubrocincta 7 55 47 Santuario de Caraja, 48 Minas Gerais (Brazil). 49 Tropical Dirinaria Dirinaria aegialita 5 Kilauea, Hawaii (USA). 56a 50 Tropical Dirinaria Dirinaria aegialita 5 Kilauea, Hawaii (USA). 56b Analytical 51 Tropical Dirinaria Dirinaria applanata 5 Kilauea, Hawaii (USA). 57a 52 Tropical Dirinaria Dirinaria applanata 5 Kilauea, Hawaii (USA). 57b 53 Tropical Dirinaria Dirinaria sp 5 Kilauea, Hawaii (USA). 58 54 Monteverde Biological Tropical Echinoplaca Echinoplaca strigulacea 63 59 55 Reserve, (Costa Rica). 56 Tropical Hyperphysia Hyperphysia adglutinata 5 Kilauea, Hawaii (USA). 60 57 Tropical Parmotrema Parmotrema sp. 5 Kilauea, Hawaii (USA). 61 58 59 60 25

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1 2 3 Campus of the Federal University of Juiz de 4 Parmotrema tinctorum Del. Ex Tropical Parmotrema 64 Fora, in Juiz de Fora city, 62 5 Nyl. 6 Minas Gerais State (Brazil). 7 Tropical Ramalina Ramalina umbilicata 5 Kilauea, Hawaii. 8 63 Near La Selva Biological 9 Tropical Tricharia Tricharia carnea 63 64 Station (Costa Rica). 10 Tropical Xanthoparmelia Xanthoparmelia scabrosa 65 Hong Kong (China). 65 11 5 12 Tropical Xanthoparmendia Xanthoparmendia sp . Kilauea, Hawaii (USA). 66 13 14 15 16 17 18 19 20 21 Manuscript 22 23 24 25 26 27 28 29 30

31 Accepted 32 33 34 35 36 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 26

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1 2 3 4 5 1 Table 2a Number of lichen specimens and biochemical compounds found by using Raman spectroscopy on Polar climate. 6 7 8 POLAR 9 10 MIXTURE 1a 1b 1c 2a 3 4 5a 6 7 8 9 10 11 12 13a 13b 14a 14b 11 Weddellite X X X X X 12 Whewellite X X X X X X X 13 Calcium oxalate X X 14 Carotene X X X X X X X X X X X X X X X X X X 15

16 Chlorophyll Manuscript 17 Cellulose X X 18 Atranorin X X 19 Calycin X X X 20 Fumarprotocetraric acid X X 21 22 Gyrophoric acid X X Parietin X X X X X X X

23 Accepted 24 Pulvinic acid X 25 Pulvinic dilactone X X 26 Rhizocarpic acid X X X X X 27 Usnic acid X 28 C2 (Glycosidic linkage) X X 29 C5 (Metabolic product) X 30 Methods 31 2 32 33 34 35 36 37 38 Analytical 39 40 41 42 27 43

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical Methods Page 28 of 35

1 2 3 4 5 1 Table 2b Number of lichen specimens and biochemical compounds found by using Raman spectroscopy on Oceanic climate. 6 7 8 OCEANIC 9 MIXTURE 15 16 17 18 5b 5c 19 20 21 22 23a 23b 23c 24 25 26 27a 27b 28 29 30a 30b 31 10 11 Weddellite X X X X X X X X X X X 12 Whewellite X X X X X X X X X X 13 Calcium oxalate X 14 Carotene X X X X X X X X X X X X X X X X X X X X X X 15 Chlorophyll X X X X X X X X X

16 Cellulose X X X X X X X X X Manuscript 17 Anthraquinone X 18 Aspicilin X 19 Atranorin X X 20 Calycin X 21 Diploschistesic acid X 22 Emodin X X X X

23 Accepted 24 Erythrin X X X 25 Fragilin X 26 Gyrophoric acid X X 27 Lecanoric acid X X X 28 Parietin X X X X X X X X 29 Parietinic acid X X X X

30 Pulvinic acid anhydride X Methods 31 Rhizocarpic acid X 32 Stictaurin X 33 Stictic acid X 34 Teloschistin X X X 35 C1 (Polyphenolic acid) X 36 C2 (Glycosidic linkage) X 37 C3 (Unknown compound) X 38 Analytical C5 (Metabolic product) X X X 39 40 2 41 42 28 43

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 29 of 35 Analytical Methods

1 21 Table 2c: Number of lichen specimens and biochemical compounds found by using Raman spectroscopy on Mediterranean and Subdesert climates. 3 4 5 MEDITERRANEAN SUB-DESERT 6 7 MIXTURE 32a 32b 33 34 23d 23e 23f 23g 35 36 37 23h 38 39 8 Weddellite X X X X X X X X X X 9 Whewellite X X X X X X X X X X X X 10 Calcium oxalate 11 12 Carotene X X X X X X X X X 13 Chlorophyll X X X 14 Cellulose X X X 15 Anthraquinone X 16 Calycin X Manuscript 17 18 Emodin X 19 Erythrin X X X 20 Fumarprotocetraric acid X 21 Lecanoric acid X X X X X 22 Parietin X X

23 Rhizocarpic acid X X X Accepted 24 Usnic a cid X X 25 26 C3 (Unknown compound) X 27 C5 (Metabolic product) X X X X X X 282 29

30 Methods 313 32 33 34 35 36 37 38 Analytical 39 40 41 42 29 43

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical Methods Page 30 of 35

1 21 Table 2d: Number of lichen specimens and biochemical compounds found by using Raman spectroscopy on High Montain+Desert and Tropical climates. 3 4 5 HIGH MOUNTAIN + DESERT TROPICAL 6 MIXTURE 2b 40 41 42a 42b 43 44 45 46 47 48 49a 49b 50 51 52 53 54a 54b 55 56a 56b 57a 57b 58 59 60 61 62 63 64 65 66 7 Weddellite X X X X X 8 Whewellite X X X X X X X X X X X 9 Calcium oxalate Carotene X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X 10 Chlorophyll X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X 11 Cellulose X 12 Atranorin X X X 13 Calycin X X X 14 Chiodectonic acid X Confluentic acid X 15 Gyrophoric acid X X X X

16 Lecanoric acid X Manuscript 17 Parietin X X X X Pulvinic dilactone X X X X 18 Rhizocarpic acid X X 19 Usnic acid X X X X X 20 C1 (Polyphenolic acid) X X X 21 C3(Unknown compound) X C4 (Unknown compound) X 22 2

23 Accepted 24 253 26 27 28 29

30 Methods 31 32 33 34 35 36 37 38 Analytical 39 40 41 42 30 43

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 31 of 35 Analytical Methods

1 TABLE 3: Lichen chemicals by specimens and climates. SPCMS: specimens; SPCS: species;

1 2 Ca OX.: calcium oxalate (unspecified hydration); WEDDE. OX. DIHYDRATE: Weddellite 2 3 (oxalate dihydrate); WHEWE. OX. MONOHYDRATE: Wewellite (oxalate monohydrate); 3 4 4 PIGMENTS: other pigments than carotene and chlorophyll . 5 Wedde. Whewe. Carotene Speci 6 Climate Cellulose Ox. Ox. Ca Ox. and/or Pigments men 7 dihydrate monohydrate chlorophyll 8 9 Polar 1a X X 1 1 10 18 spcms 1b 1 1 11 14 spcs 1c X 1 2 12 2a X X 1 1 13 14 3 X X 1 0 15 4 1 1 16 5a 1 1 17 18 6 X 1 3 19 7 1 2 20 8 X 1 3 21 9 X 1 2 Manuscript 22 23 10 X X 1 1 24 11 1 1 25 12 1 2 26 27 13a X X 1 1 28 13b 1 1 29 14a X X 1 1 30 14b 1 1

31 Accepted 32 Oceanic 15 X 1 3 33 23 spcms 16 1 2 34 18 spcs 17 X 1 4 35 36 18 X 2 3 37 5b X 2 4 38 5c X 2 4 39 19 X 1 4

40 Methods 41 20 X X 0 1 42 21 X X 1 0 43 22 X 2 4 44 45 23a X X 1 0 46 23b X X 1 2 47 23c X 1 2 48 49 24 X X 1 0 50 25 X 1 0 Analytical 51 26 2 1 52 53 27a X X 2 0 54 27b X X 2 0 55 28 X X X 2 0 56 29 2 0 57 58 30a X X 1 1 59 60 31

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30b X 1 1 1 31 X X 1 2 2 Mediterranean 32a X X 1 1 3 10 spcms 32b X X 1 1 4 6 spcs 5 33 X 1 0 6 34 X X 0 2 7 23d X X X 1 2 8 9 23e X X 1 2 10 23f X X 0 0 11 23g X X 0 0 12 13 35 X 0 0 14 36 X X 0 0 15 Subdesert 37 X 2 4 16 4 spcms 23h X X 1 2 17 4 spcs 18 38 X X 2 2 19 39 X 2 3 20 High 2b 2 2 21 mount+desert Manuscript 22 40 2 2 23 13 spcms 41 X 2 0 11 spcs 24 42a X 1 2 25 26 42b 2 1 27 43 X 2 0 28 44 2 2 29 45 X X 2 0 30 46 2 0 31 Accepted 32 47 2 1 33 48 2 1 34 35 49a 1 1 36 49b 2 1 37 Tropical 50 X 2 0 38 20 spcms 51 X 2 0 39 17 spcs 52 2 1

40 Methods 41 53 2 1 42 54a 2 1 43 54b 2 1 44 55 X X 2 2 45 56a 2 1 46 56b X 2 1 47 48 57a 2 2 49 57b X 2 1 50 58 X 2 1 51 59 X 2 0 Analytical 52 60 2 0 53 61 2 1 54 62 1 1 55 56 63 X 2 0 57 64 X 2 0 58 65 2 0 59 66 X 2 1 60 32

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1 2 3 4 TABLE 4: : Number of times each pigment was found under different climates, by using Raman spectroscopy and total percentage of each pigment 5 found in the total specimen number. 6

7 High mount 8 Compound Polar Oceanic Mediter. Subdesert Tropical Total % total pigments 9 +desert 10 Anthraquinone 1 1 2 1.75 11 Aspicilin 1 1 0.88 12 Atranorin 2 2 3 7 6.14 13 14 Calycin 3 1 1 3 8 7.02 15 Chiodectonic acid 1 1 0.88

16 Confluentic acid 1 1 0.88 Manuscript 17 Diploschistesic acid 1 1 0.88 18 Emodin 5 1 6 5.26 19 Erythrin 3 2 1 6 5.26 20 21 Fragilin 2 2 1.75 22 Fumarprotocetraric acid 2 1 1 4 3.51 Gyrophoric acid 2 2 4 8 7.02

23 Accepted 24 Lecanoric acid 3 2 3 1 9 7.89 25 Parietin 7 8 1 1 3 1 21 18.42 26 Parietinic acid 4 4 3.51 27 Pulvinic acid 1 1 0.88 28 29 Pulvinic acid anhydride 1 1 0.88

30 Pulvinic dilactone 2 3 1 6 5.26 Methods 31 Rhizocarpic acid 5 1 2 1 2 11 9.65 32 Stictaurin 1 1 0.88 33 Stictic acid 1 1 0.88 34 Teloschistin 4 4 3.51 35 Usnic acid 1 2 2 3 7.02 36 8 37 TOTAL PIGMENTS 25 42 8 11 13 15 114 100.01 38 Analytical 39 40 41 42 33 43

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical Methods Page 34 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Manuscript 22 23 24 25 26 27 28 29 30

31 Accepted 32 Representation by pie-chart of: a) Percentage of lichen specimens with and/or without oxalates; b) Type of 33 oxalates found in different world climate regions. The world climate regions considered in this study are: Polar (18 specimens [spcms]), Oceanic (23 spcms), Mediterranean (10 spcms), Subdesert (4 spcms), High- 34 mount/desert (13 spcms) and Tropical (20 spcms). Climate Map zones of the world: Original uploader was 35 Waitak at en.wikipedia Later version(s) were uploaded by Splette at en.wikipedia. - Transferred from 36 en.wikipedia; transfered to Commons by User:Legoktm using CommonsHelper. 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 Page 35 of 35 Analytical Methods

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Manuscript 22 23 24 25 26 27 28 29 30 Representation by pie-chart of pigments occurrence: number of pigments in each region (without

31 carotenoids and chlorophyll). The world climate regions considered in this study are: Polar (18 speciments Accepted 32 [spcms]), Oceanic (23 spcms), Mediterranean (10 spcms), Subdesert (4 spcms), High-mount/desert (13 33 spcms) and Tropical (20 spcms). 34 Climate Map zones of the world: Original uploader was Waitak at en.wikipedia Later version(s) were 35 uploaded by Splette at en.wikipedia. - Transferred from en.wikipedia; transfered to Commons by 36 User:Legoktm using CommonsHelper. 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