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Survival of Xanthoria parietina in simulated space conditions: vitality

assessment and spectroscopic analysis

Article in International Journal of Astrobiology · March 2022

DOI: 10.1017/S1473550422000076

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 International Journal of Astrobiology (2022), 1–17
doi:10.1017/S1473550422000076

R E S E A R C H A RT I C L E

Survival of Xanthoria parietina in simulated space

conditions: vitality assessment and spectroscopic analysis
Christian Lorenz1 , Elisabetta Bianchi2, Renato Benesperi1, Stefano Loppi2, Alessio Papini1,
Giovanni Poggiali3 and John Robert Brucato3
1
Department of Biology, University of Florence, Via La Pira 4, I-50121 Florence, Italy
2
Department of Environmental Sciences, University of Siena, Via P.A. Mattioli 4, I-53100 Siena, Italy
3
INAF-Astrophysical Observatory of Arcetri, Largo E. Fermi, 5, I-50125 Florence, Italy
Author for correspondence: John Robert Brucato, E-mail: [email protected]
Received: 1 November 2021; Revised: 17 December 2021; Accepted: 13 February 2022
Keywords: Astrobiology, chlorophyll fluorescence, FTIR, lichens, NDVI, vitality, Xanthoria parietina

Abstract
Xanthoria parietina is a widespread foliose lichen growing on barks and rocks showing a broad spectrum of
tolerance to air pollutants such as NOX and heavy metals, and resistance to UV-radiation because of the screening
properties provided by the secondary metabolite parietin. The aim of this study was to evaluate the ability of this
lichen species to survive in the following simulated space conditions, UV-radiation in N2 atmosphere and UV-radi-
ation in vacuum. The efficiency of the photosynthetic apparatus was used as an indicator of vitality, and was
expressed in terms of chlorophyll a fluorescence (FV/FM) and Normalized Difference Vegetation Index (NDVI),
which were measured within 72 h from the exposure. Additionally, during the irradiation, the IR reflectance spec-
trum of the lichen was monitored in situ to assess changes in spectral bands. The results showed significant differ-
ences in physiological recovery trends between the treatments, highlighting that UV-radiation in vacuum causes
stronger effects on FV/FM values. The IR analysis revealed several spectral band changes in the fingerprint region.
The most visible variation was the 5200 cm−1 water band that disappeared in the overtone region. Nevertheless, X.
parietina was able to survive UV-radiation in N2 atmosphere and in vacuum, and for this reason it may be
considered a candidate for further evaluations on its survival capacity in extreme conditions.

Contents
Introduction 2
Materials and methods 3
Lichen material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Experimental design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Photosynthetic efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Spectroscopic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Results 5
Physiological response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
IR analysis: band assignments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
IR analysis: spectral changes in UV N2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
IR analysis: spectral changes in UV VAC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Discussion 11
Conclusions 14

© The Author(s), 2022. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative
Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, pro-
vided the original article is properly cited.
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 2 Christian Lorenz et al.

Introduction
One of the main topics of astrobiology research is the study of life limits in stressful environments. The
aim of this research field is to explore the physiological and biochemical effects of extreme conditions –
such as space – on unprotected biological samples (de la Torre Noetzel et al., 2020). At the ecological
and physiological level, the study of organisms in extreme environments might give an indication about
their potential adaptive plasticity, in the view of a climate change perspective, the terrestrial geological
past (Wassmann et al., 2010) and future scenarios, as well as extra-terrestrial habitats such as Mars
surface. Several experiments have been run in Low Earth Orbit (LEO) with satellites (LICHENS
experiment performed on the BIOPAN-5 facility on the FOTON-M2 satellite, see e.g. Sancho et al.,
2007; Raggio et al., 2011) and on the International Space Station (ISS), specifically on the EXPOSE
facilities (Onofri et al., 2012), to test organism survival capacity in space conditions, characterized by
high vacuum, solar radiation and galactic ionizing radiation, solar UV-radiation and extreme temperature
range (de la Torre Noetzel et al., 2018; de Vera et al., 2019). Studies were conducted on different eukaryotic
and prokaryotic organisms. Bacteria such as Bacillus subtilis and Deinococcus radiodurans can survive
space radiation and vacuum (Horneck et al., 2001; de la Vega et al., 2007). Rotschild and Mancinelli
(2001) reported that 25–40% of halophilic Archea tested samples were able to survive 2 weeks on the
FOTON-9 spacecraft (BIOPAN-1 mission), exposed to the unfiltered Sun spectrum. It has been discovered
that akinetes of Cyanobacteria were tolerant to extra-terrestrial conditions and survived in dried state for
long periods (Billi, 2009; Billi et al., 2019). Furthermore, it has been shown that tardigrades (Metazoa)
in a desiccated state can survive space condition, while organisms belonging to other phyla of
Metazoa, such as nematodes, die when exposed directly to solar radiation (Bertolani et al., 2001;
Jönsson et al., 2008).
Several studies focused on lichens, which are pioneer species – organisms able to colonize extreme
environments first – proved to be resistant in space conditions too. Lichens are able to colonize almost
all terrestrial habitats, including the most extreme ecological niches (Green et al., 2012; Zedda and
Rambold, 2015). The symbiotic association of two bionts, represented by a mycobiont and a photo-
biont (this last an alga and/or a cyanobacterium), allows them to survive in a wide range of conditions,
tolerating dry environments, cold habitats, salt spray, water immersion, from high latitudes to the tro-
pics (Kappen, 1973; Nash, 1996; de Vera et al., 2004). This ability to survive and thrive under very
stressful conditions is determined by their poikilohydry and anhydrobiosis, allowing long periods in
desiccated state, and to the high variety of synthesis pathways due to the symbiotic association. The
products of lichen secondary metabolism mostly determine the tolerance of these organisms to stressful
conditions such as high doses of UV-radiation and rapid variations in the environmental context (de
Vera et al., 2004). The BIOPAN experiments showed that 70–100% of Rusavskia elegans (Link)
S.Y. Kondr. & Kärnefelt subsp. elegans (synonym of Xanthoria elegans (Link) Th. Fr. var. elegans),
Rhizocarpon geographicum (L.) DC. s. l. and Aspicilia fruticulosa (Eversm.) Flagey samples survived
for 10–14 days in space conditions, being metabolically active and able to grow after the exposure (de
la Torre Noetzel et al., 2007; Sancho et al., 2007, 2008; Raggio et al., 2011; de Vera, 2012). The LiFE
experiment onboard the EXPOSE-E facility was performed exposing different biological samples for
1.5 years to space conditions. The tested lichen species were R. elegans and R. geographicum and were
exposed to space vacuum with a UV-shield (0.1% transmission neutral density filter) and without it,
receiving 100% space insolation (Onofri et al., 2012; Brandt et al., 2016). Rusavskia elegans showed
a high recovery in post-flight fluorescence measurements of photosystem II (PSII) activity. In particular,
R. elegans showed the highest post-flight viability compared to the other tested species. UV-shielded
samples showed fluorescence value of 99% of pre-flight control, while 100% transmission insolated
samples showed 45% of the pre-flight control (Onofri et al., 2012).
The main difference between space-exposing experiments and ground-based studies is that the latter
allow to simulate extreme conditions for the biological sample and to immediately test viability parameters
after the exposure (de Vera et al., 2003, 2004, 2010). In the light of many different studies in literature
about lichen astrobiological experiments, in this paper we decided to select Xanthoria parietina (L.) Th.
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 International Journal of Astrobiology 3

Fr. as test organism owing to its close phylogenetical relation to R. elegans. The choice of this species
was also related to its ecological features: X. parietina is a cosmopolitan foliose lichen growing on
barks and rocks, which can colonize almost all habitats from the seashore up to the treeline (Nimis,
2016). This species is also frequent in anthropized environments, being tolerant to air pollutants
such as NOX and heavy metals (Silberstein et al., 1996; Bačkor et al., 2003) and by virtue of these
features, it is often used as a bio-accumulator of persistent airborne pollutants (Loppi et al., 2006).
Xanthoria parietina is tolerant to UV-radiation because of the orange pigment parietin, a product of
its secondary metabolism (Gauslaa and Ustvedt, 2003). This anthraquinone protects the photosystem
from high light influx (Solhaug and Gauslaa, 1996) and its production is stimulated by UV-B radiation
(Solhaug et al., 2003; Solhaug and Gauslaa, 2004). In the astrobiological context, X. parietina has been
investigated by de Vera et al. (2004) in a ground-based experiment, along with R. elegans and
Gyalolechia bracteata (Hoffm.) A. Massal. (synonym of Fulgensia bracteate (Hoffm.) Räsänen). In
particular, the lichens ascospores germination capacity was evaluated after simulated space conditions
in different culture soil media. Generally, 50% of the recovered ascospores were viable and R. elegans
ascospores showed the highest survival (de Vera et al., 2004). Also X. parietina showed a remarkable
germination rate of irradiated ascospores.
The aim of this study was to examine X. parietina recovery through eco-physiological analysis after
exposure to simulated space conditions. The tested treatments were UV-radiation in N2 atmosphere and
UV-radiation in vacuum. These two conditions – simulated in laboratory – can be associated to differ-
ent space environments. Vacuum condition at room temperature is analogous to the environmental situ-
ation found in the proximity of Earth orbit and other planetary bodies with a comparable distance from
the Sun. Saturated nitrogen atmosphere, as an extreme environment, may be related to several planetary
conditions (i.e. early Earth, Mars, possible altered terrestrial scenarios, exoplanets). Chlorophyll a
fluorescence and reflectance were evaluated within 72 h from treatments to assess the recovery capacity
of the photosynthetic apparatus. To contribute to a deeper understanding of the lichen response to the
simulated space conditions, the Fourier-transform infrared reflectance (FTIR) spectra of lichen samples
were monitored in situ during treatments. In this way, it was possible to appreciate in real time changes
in spectral bands, providing information about chemical bonds involved in photodegradation too.
Consequently, the main purposes of the current study were: (i) to evaluate and verify X. parietina
recovery capacity after exposure to simulated space conditions, (ii) to obtain the lichen FTIR reflect-
ance spectrum in not-irradiated conditions and (iii) to monitor the lichen FTIR reflectance spectrum
during treatment to highlight eventual changes in the spectrum itself.

Materials and methods

Lichen material
For this study, thalli of X. parietina were randomly collected in a remote area of Florence province
(Tuscany, Italy 43°59′ 25.09′′ N 11°13′ 24.65′′ E, at 300 m a.s.l.) in June and July 2020. Thalli were
dehydrated for 24 h at room temperature (25°C) and stored at −18°C until treatment, as this procedure
ensures that thalli remain healthy for later physiological measurements (Paoli et al., 2013). Three days
before the treatment, thalli were allowed to slowly recover their normal metabolic conditions in a
growth chamber at 25°C and 70 μmol m−2 s−1 PAR photons; overnight, thalli were covered with a
black cotton cloth and kept moistened by spraying with distilled water (Paoli et al., 2019; Bianchi
et al., 2019a).

Experimental design
Six samples (replicates) of about 1 cm2 (the size was determined by the sample holder in the
UV-radiation apparatus, Fig. S1) were used for each experimental condition: (i) UV-radiation in nitro-
gen atmosphere at room temperature and atmospheric pressure (UV N2); (ii) UV-radiation in vacuum
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 4 Christian Lorenz et al.

(100–10−2 Pa) at room temperature (UV VAC). Six unexposed samples were used as control, kept in a
growth chamber at 25°C, under 70 μmol m−2 s−1 PAR photons. The UV irradiation time periods were 1 s,
2 s, 5 s, 10 s, 15 s, 30 s, 60 s, 90 s, 120 s, 5 min, 10 min and 15 min. Between one irradiation time period
and another one, it was possible to perform in situ spectroscopic analyses (see Spectroscopic analysis
section). The time recovery for one spectrum was of 60 s. The total UV irradiation period was
∼36 min. The total UV irradiance was 1.34 MJ m−2 for each exposed sample in the lamp wavelength
range 185–2000 nm. We simulated UV space radiation using a Newport 300 W Ozone Free
Xe-enhanced UV lamp with a sun-like emission spectrum (wavelength range 185–2000 nm, Fig. S2).
Because of its sun-like emission spectrum, the Xenon lamp is used for simulating solar radiation outside
the atmosphere. The above described atmospheric conditions (N2 and VAC) were chosen to set up an
extreme and dehydrating environment for the lichen. The conditions of irradiation in situ did not allow
to reach lower pressure values.
Air-dried samples were weighed before and immediately after the treatments. To evaluate sample via-
bility, photosynthetic efficiency measurements (NDVI and FV/FM, see further) were carried out at different
times: before treatment (pre_exp), immediately after (post_exp) and 24, 48 and 72 h after the exposure.
After UV irradiation samples were kept in a growth chamber at 25°C and 70 μmol m−2 s−1 PAR photons,
covering overnight the thalli with a black cotton cloth and keeping them moist by spraying with distilled
water (Bianchi et al., 2019a; Paoli et al., 2019).

Photosynthetic efficiency
The efficiency of the photosynthetic system of the lichen X. parietina was assessed in terms of spectral
reflectance and chlorophyll a fluorescence emission. The spectral reflectance of chlorophyll pigments
was expressed in terms of Normalized Difference Vegetation Index (NDVI). The NDVI was evaluated
with a portable reflectance-based device (Plant Pen NDVI 310 – Photon System Instruments, Czech
Republic, 2016). NDVI is directly related to PSII integrity, being associated with the difference in
plant reflectance in the visible and near-infrared wavelengths. These differences were used to calculate
the NDVI as follows: NDVI = ((NIR740–VIS660)/(NIR740 + VIS660)). VIS and NIR are the spectral
reflectance measurements acquired respectively in the visible and near-infrared regions (Miloš et al.,
2018).
The analysis of the chlorophyll a fluorescence was carried out with a portable fluorimeter (Plant
Efficiency Analyzer – Handy PEA, Hansatech Ltd, Norfolk, UK). In all experiments, samples were
hydrated and dark-adapted for 10 min before fluorescence measurements (Bianchi et al., 2019b).
Samples were exposed to a flashing light for 1 s with an excitation pulse (3000 μmol s−1 m−2) of
red light (650 nm) and the maximum quantum yield of PSII was determined with the parameter FV/
FM = ((FM–F0)/FM) where FV is the variable fluorescence yield, FM is the maximal fluorescence
yield and F0 is the minimal fluorescence yield (van Kooten and Snel, 1990). The maximum quantum
yield and variations in F0, related to the chlorophyll a content of the light harvesting complex (Baruffo
and Tretiach, 2007), were evaluated to quantify lichen viability and PSII efficiency.

Spectroscopic analysis
Spectroscopic analyses were performed in situ without moving the samples. IR spectra have been
recorded after each period of UV irradiation to monitor changes in the IR spectrum (see
Experimental design section). The time required to scan one IR spectrum was 60 s. The irradiation
setup consisted of the Harrick High Temperature Reaction Chamber (vacuum chamber, Fig. S1)
inserted in the Harrick Praying Mantis™ Diffuse Reflectance Accessory, interfaced to the Brucker
VERTEX 70v FTIR (Fornaro et al., 2020), where lichen samples were irradiated and monitored in
situ with infrared spectroscopy. The technique used to obtain the infrared spectra was the FTIR –
Fourier-transform infrared spectroscopy. Spectra were acquired in the wavenumber range from 8000
to 400 cm−1 with the resolution of 4 cm−1. Spectroscopic analysis was performed with the OPUS
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 International Journal of Astrobiology 5

software. The UV source was a Newport Xe-enhanced UV Ozone Free 300 W lamp with purified Xe at
5–20 bar (wavelength range 185–2000 nm, Fig. S2). Xe arc lamps have a relatively smooth emission
curve in the UV-VIS spectrum, with sun-like emission spectrum and 5800 K colour temperature. The
Xe lamp is hosted in the Newport Research Arc Lamp Housing 50–500 W (Corazzi et al., 2020). The
UV-radiation emitted by the lamp is collimated through an optical system consisting of a first mirror
that reflects the radiation coming from the lamp to a grade fused silica collimating condenser lens
(type Fused Silica Asphere, f-number f/2.2), which directs UV-radiation towards an optical fibre with
an aperture of 800 μm (Poggiali et al., 2020).

Data analysis
The data of lichen physiological parameters (NDVI and FV/FM) were analysed fitting linear
mixed-effects models (LMMs) in a repeated measurement ANOVA design, using thallus identity as
a random effect factor to account for the temporal correlation of observations. Time was used as an
ordinal variable because the relationship between NDVI or FV/FM values and time was not a simple
linear regression. The physiological effects of the treatment were investigated using NDVI and
FV/FM values as response variables and the two treatment conditions explored (UV N2 and UV VAC)
and time of recovery as explanatory variables in a full factorial design. ANOVA type III table, with
Satterthwaite’s method was used to verify the significance of the fixed effects and of associated inter-
action factors. The analyses were run with the free software R. LMM computations were performed
using the lmer function of the lme4 package version 1.1–12 for fitting the models. Significant changes
in lichen mass at the end of the experiment by different treatment conditions were also examined by
means of ANOVA with Tukey’s post-hoc test (with at least p < 0.05 as the significance level). For
each analysis, data normality was checked with the Shapiro–Wilk test.

Results
Physiological response
The results of the physiological response of X. parietina samples under the different experimental con-
ditions are shown in Fig. 1 (NDVI) and Fig. 2 (FV/FM). Once samples were removed from the Harrick
High Temperature Reaction Chamber, they showed significantly lower NDVI values (mean ± SD:
0.2898 ± 0.07 UV N2 and 0.2647 ± 0.01 UV VAC) compared with the respective pre-exposure values
(0.3278 ± 0.06 UV N2, 0.3251 ± 0.08 UV VAC) for both treatments ( p < 0.001) (Tables S1 and S2).
After 72 h in the recovery conditions, samples that reached significantly higher values (0.4041 ±
0.07) were those treated in UV N2, compared to UV VAC samples (0.3682 ± 0.06) and control ones
(0.3277 ± 0.02) ( p < 0.001) (Tables S1 and S2). The maximum quantum yield of primary photochem-
istry (FV/FM) changed significantly after the treatment in both the experimental conditions ( p < 0.001)
(Table S3). FV/FM values of the samples treated in UV N2 showed a significant decrease compared to
pre-exposure ones (0.607 ± 0.02, pre_exp and 0.375 ± 0.1, post_exp) ( p < 0.001) (Fig. 2 and Table S3).
The same trend was observed for the UV VAC samples, which reached lower values (0.042 ± 0.02) than
the initial ones (0.611 ± 0.07, with p < 0.001) (Tables S3 and S4), crossing the threshold of 0.200,
which is considered consequent to photoinhibition (Long et al., 1994). Once the recovery period
began, FV/FM values increased significantly depending on the treatments ( p < 0.001) (Table S3).
Samples exposed to UV N2 recovered already after 24 h, reaching values comparable to the starting
ones after 72 h (Fig. 2). On the other hand, the efficiency of the samples exposed to UV VAC showed
a gradually increasing trend, reaching 0.163 ± 0.1 after 24 h, 0.251 ± 0.1 after 48 h and 0.273 ± 0.1 after
72 h, without reaching initial values (Fig. 2 and Table S3). Control samples were daily hydrated and
kept in dark-adapted conditions. FV/FM of control values ranged during the experiment between
0.579 ± 0.05 and 0.638 ± 0.03 (Fig. 2 and Table S4). F0 values changed significantly after the exposure
in both the experimental conditions ( p < 0.001) (Tables S5, S6 and Fig. S3). A relative increase in F0
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 6 Christian Lorenz et al.

Fig. 1. Normalized Difference Vegetation Index (NDVI) of Xanthoria parietina before (pre_exp),
immediately after (post_exp) and 24 h, 48 h and 72 h after the exposure. Cyan line = control; magenta
line = UV N2; green line = UV VAC. Error bars stand for confidence intervals. See Table S1 for ANOVA
results (Supplemental Information).

Fig. 2. Variation in Xanthoria parietina of the photosystem II (FV/FM) efficiency before (pre_exp),
immediately after (post_exp) and 24 h, 48 h and 72 h after the treatment. Cyan line = control; magenta
line = UV N2; green line = UV VAC. Error bars stand for confidence intervals. See Table S3 for ANOVA
results (Supplemental Information).

followed the exposure of UV N2 until the initial values were reached after 72 h of recovery. F0 values
strongly decreased in samples exposed to UV VAC compared with those treated with UV N2 treatment
and control ones. UV VAC F0 values declined even in the recovery period reaching significantly lower
values (225 ± 32) (Table S8 and Fig. S3). The samples were also weighed before and after the treatments
(Table S7) and it turned out that there is no statistical difference in mass variation between treatments
(Table S8).

IR analysis: band assignments

During the experiment, the FTIR spectrum was evaluated for each lichen sample. Slight differences in
bands’ peak intensities were noticed between samples spectra but all the mentioned bands were noticed
in all the lichens spectra. Band assignments of X. parietina spectrum were based on theoretical data
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 International Journal of Astrobiology 7

Table 1. Band assignments for Xanthoria parietina FTIR reflectance spectrum

Peak position
[cm−1] Band assignments Reference
5200 v(H2O) + δ(H2O) (H2Om) Bishop et al. (2013); Gerber et al. (2011);
Granlund et al. (2018); McIntosh et al. (2017)
3500 v(H2O) + v(OH) Bishop et al. (2013); Coates (2000);
McIntosh et al. (2017); Stuart (2004)
2930 v(−CH) asym./sym. + v(=CH2) Coates (2000); Stuart (2004)
asym./sym.
1730 v(C=O) Stuart (2004)
1675 v(C=O) conjugated, free Edwards et al. (2003); Stuart (2004)
1630 δ(H2O) (H2Om) Bishop et al. (2013); McIntosh et al. (2017);
Stuart (2004)
1595–1570–1560 v(C=C) aromatic Coates (2000); Edwards et al. (2003);
Stuart (2004)
1483 Ring stretch, in plane Edwards et al. (2003)
1395–1373 v(C–O) phenyl
1333 Ring stretch, in plane
1172 δ(OH) phenyl, free, in plane
1107 v(C–CH) in plane
1045 v(C–O) aromatic ether Coates (2000); Edwards et al. (2003)
983 Ring breathing, δ(C–H) out of Coates (2000); Edwards et al. (2003);
plane Stuart (2004)
928 δ(C–H) out of plane
765 v(=C–H) aromatic, out of plane Edwards et al. (2003)
Vibrational modes legend: v, stretching; δ, bending. H2Om stands for molecular water.

from the literature (Stuart, 2004; Coates, 2000) and experimental data from Edwards et al. (2003) IR
spectroscopy characterization of the lichen substance parietin, extracted from R. elegans and X. parietina
samples. Gerber et al. (2011) and Granlund et al. (2018) were used as reference to assign 5200 cm−1
band to H2O. Band assignments are reported in Table 1. The lichen FTIR spectrum can be seen in
Fig. S4 and ranged between 8000 and 400 cm−1 wavenumbers. The reported vibrational modes ‘v’ and
‘δ’ stand for stretching and bending. Bands at 5200 and 1630 cm−1 were assigned to H2Om (molecular
water). In particular, 5200 cm−1 was assigned to the stretch and the bend of H2O modes (Gerber et al.,
2011; Bishop et al., 2013; McIntosh et al., 2017; Granlund et al., 2018). The band at 1630 cm−1 was
referred to the H2O bend mode (Stuart, 2004; McIntosh et al., 2017) and the 3500 cm−1 band is char-
acteristic for bend H2O and OH mode. The band at 2930 cm−1 was assigned to the v(−CH) and v
(=CH2) antisymmetric and symmetric modes. Furthermore, it was noticed that there were vibrational
modes distributed in a particular region from 1750 and 400 cm−1 (Fig. S5). From Edwards et al.
(2003), it comes out that the aforementioned region is particularly relevant for parietin vibrational
modes. The band at 1675 cm−1 was assigned to the v(C=O) conjugated and free vibrational modes
(Stuart, 2004; Edwards et al., 2003) and the 1595, 1570 and 1560 cm−1 bands were referred to the aro-
matic (C=C) stretching mode (Stuart, 2004; Edwards et al., 2003; Coates, 2000). The two bands at
1483 and 1333 cm−1 were both assigned to the ring stretch, the first one coupled with OH and the
other one with in-plane bending mode (Edwards et al., 2003). Bands at 1395 and 1373 cm−1 are
due to the phenyl v(C–O) (Edwards et al., 2003). A strong band at 1172 cm−1 was referred to the phe-
nyl, free and in plane OH bending (Edwards et al., 2003). 1107 cm−1 band was specified for (C–CH)
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 8 Christian Lorenz et al.

Fig. 3. (a) FTIR reflectance spectrum of the lichen Xanthoria parietina in the range 8000–400 cm−1
and (b) in the range 1750–400 cm−1 before and after UV exposure in N2. Band changes were evaluated
normalizing the pre-exposure spectrum and the after-exposure spectrum at 7000 cm−1.

stretching in plane (Edwards et al., 2003) and 1045 cm−1 band was assigned to aromatic ether (C–O)
stretching (Edwards et al., 2003; Coates, 2000). The band at 983 cm−1 was referred to the ring breathing
and the out of plane (C–H) bending mode (Stuart, 2004; Edwards et al., 2003; Coates, 2000), while 928
and 765 cm−1 were assigned respectively to the out of plane δ(C–H) (Stuart, 2004; Edwards et al., 2003;
Coates, 2000) and the out of plane and aromatic v(=C–H) vibrational modes (Edwards et al., 2003).

IR analysis: spectral changes in UV N2

The full FTIR spectrum of the lichen X. parietina, before and after irradiation for UV N2 treatment, is
reported in Fig. 3(a) and the change in the spectrum in the range 1750–400 cm−1 is shown in Fig. 3(b).
Table 2 reports the peak shifting (Δỹ) and the qualitative assessments of the changes of the assigned
band features due to UV N2 treatment (∼36 min irr.). The changes in the spectrum continuum were
evaluated normalizing the pre-exposure spectrum and the post-exposure spectrum at 7000 cm−1.
Figure 4 shows the details of UV N2 spectral changes. As it is reported in Figs 3(a) and 4(a), there
is a remarkable variation of the peak reflectance in 5200 cm−1 band (v(H2O) + δ(H2O)), which disap-
pears completely after treatment. A general spectrum upshifting is observed. Bands at 3500 cm−1
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 International Journal of Astrobiology 9

Table 2. FTIR reflectance spectrum of Xanthoria parietina after ∼36 min of UV irradiation in N2
Peak position [cm−1] Δỹ Qualitative bands
before UV-irr. [cm−1] changes assessment Band assignments
5200 Complete disappearance v(H2O) + δ(H2O) (H2Om)
3500 −8 Intensity decrease v(H2O) + v(OH)
2930 −2 Slight intensity decrease v(−CH) asym./sym. + v(=CH2)
asym./sym.
1730 – Intensity decrease v(C=O)
1675 −2 Intensity decrease v(C=O) conjugated, free
1630 +2 Intensity decrease δ(H2O) (H2Om)
1595 – Slight intensity decrease v(C=C) aromatic
1570 – Intensity decrease
1560 −3 Intensity decrease
1483 – Slight intensity decrease Ring stretch, in plane
1395 −1 Slight intensity decrease v(C–O) phenyl
1373 – Slight intensity decrease
1333 – Slight intensity increase Ring stretch, in plane
1172 – Slight intensity increase δ(OH) phenyl, free, in plane
1107 – Slight intensity decrease v(C–CH) in plane
1045 – Slight intensity decrease v(C–O) aromatic ether
983 – Slight intensity increase Ring breathing, δ(C–H) out of plane
928 – Intensity decrease δ(C–H) out of plane
765 −2 Highly intensity decrease v(=C–H) aromatic, out of plane
Band positions were evaluated normalizing the pre-exposure spectrum and the after-exposure spectrum at 7000 cm−1. Vibrational modes legend: v,
stretching; δ, bending. H2Om stands for molecular water. Δỹ stands for wavenumber shift with respect to the same peak before UV irradiation. Blank
cells stand for too weak differences to evaluate or null variations

(v(H2O) + v(OH)) and 2930 cm−1 (v(−CH) asym./sym. + v(=CH2) asym./sym.) show respectively −8
and −2 cm−1 variation in the wavenumber peak. In Fig. 4(c), the formation of new bands in the
range 1550–1450 cm−1 is observed. The new observed bands are 1529 and 1512 cm−1 (already recognized
as shoulders in the pre-exposure spectrum), 1501, 1493, 1462, 1441 and 1423 cm−1. Furthermore, the
1400–900 cm−1 spectral region shows a general down peak shifting (Table 2 and Fig. 3(b)).

IR analysis: spectral changes in UV VAC

The full FTIR spectrum of the lichen X. parietina before and after UV VAC treatment is shown in
Fig. 5(a) and the global change in spectrum in the range 1750–400 cm−1 is reported in Fig. 5(b).
Table 3 reports the peak shifting (Δỹ) and qualitative assessment of the identified band changes
after treatment. Changes in the spectrum continuum were evaluated normalizing the pre-exposure spec-
trum and the post-exposure spectrum at 7000 cm−1. Differently from UV N2 treatment, it was decided
that UV irradiation in vacuum should start when pressure reached 0.60 Pa to normalize the starting con-
ditions for each sample. The UV-waiting time period necessary to reach 0.60 Pa could be 20–60 min
since the pump was turned on. The sample reported in Table 3, Figs 5 and 6 had a UV-waiting period
of 24 min. As it is shown in Fig. 6(a), 5200 cm−1 band (v(H2O) + δ(H2O)) disappeared before starting
to UV-irradiate the sample. Bands at 3500 cm−1 (v(H2O) + v(OH)) and 2930 cm−1 (v(−CH) asym./
sym. + v(=CH2) asym./sym.) showed respectively null and −6 cm−1 variation in wavenumber peak
shifting with a visible change in bands area. It is noticeable that the upshifting of the continuum is
higher than the UV N2 one (Fig. 5(a)). Observing the range 1700–1450 cm−1 (Fig. 6(c)), from the
UV-waiting period spectra to the first 10–20 s of UV-radiation spectra, a down peak shifting trend
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 10 Christian Lorenz et al.

Fig. 4. Details of the lichen IR spectrum changes for UV N2 treatment. The legend of the lines’ colours
showed in (a) is also referred to (b) and (c). (a) Band at 5200 cm−1 (v(H2O) + δ(H2O)); (b) band at
3500 cm−1 (v(H2O) + v(OH)) and band at 2930 cm−1 (v(−CH) asym./sym. + v(=CH2) asym./sym.);
(c) general changes in the spectral range 1700–1450 cm−1. Band changes were evaluated normalizing
the pre-exposure spectrum and the after-exposure spectrum at 7000 cm−1.
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 International Journal of Astrobiology 11

Fig. 5. (a) FTIR reflectance spectrum of the lichen Xanthoria parietina in the range 8000–400 cm−1
and (b) in the range 1800–400 cm−1 before and after UV exposure in VAC. Band changes were
evaluated normalizing the pre-exposure spectrum and the after-exposure spectrum at 7000 cm−1.

was noticeable, then the spectrum continued to upshift. Furthermore, in the range 1400–400 cm−1, a
general wavenumber peak shifting is observed, as it is reported in Table 3 and Fig. 5(b). In the
range 1400–950 cm−1, a down peak shifting in reflectance was recorded (Fig. 5(b)).

Discussion
The lichen X. parietina survived the simulated extreme conditions used in this study. UV-radiation, able
to photo-dissociate chemical bonds, and high vacuum, first cause of extreme dehydration (Sancho
et al., 2007), are two of the most lethal factors that make outer space an inhospitable environment.
The two simulated treatments (UV N2 and UV VAC) affected differently the photosynthetic parameters,
showing remarkable differences in X. parietina recovery trends. UV N2 treatment affected post-
exposure values of the photobiont metabolic activity. Noteworthy, in the 72 h of recovery period,
NDVI values recovered more than the 100%, overcoming the starting ones. Actually, the recovery con-
ditions – offering daily water spraying – increased thallus hydration and consequently increased NDVI
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 12 Christian Lorenz et al.

Table 3. FTIR reflectance spectrum of Xanthoria parietina after ∼36 min of UV irradiation in VAC
Peak position [cm−1] Δỹ Qualitative bands changes
before UV-irr. [cm−1] assessment Approximate band assignments
5200 Complete disappearance v(H2O) + δ(H2O) (H2Om)
3500 – Slight intensity decrease and v(H2O) + v(OH)
high decrease in band area
2930 −6 Intensity decrease and high v(−CH) asym./sym. + v(=CH2)
decrease in band area asym./sym.
1730 −2 Intensity increase v(C=O)
1675 Not evaluable v(C=O) conjugated, free
1630 −8 Intensity decrease δ(H2O) (H2Om)
1595 – Highly intensity decrease v(C=C) aromatic
1570 +6 Highly intensity decrease
1560 +2 Highly intensity decrease
1483 – Highly intensity decrease Ring stretch, in plane
1395 Not evaluable v(C–O) phenyl
1373 −2 Slight intensity increase
1333 −8 Slight intensity increase Ring stretch, in plane
1172 −8 Intensity increase δ(OH) phenyl, free, in plane
1107 Not evaluable v(C–CH) in plane
1045 Not evaluable v(C–O) aromatic ether
983 −12 Intensity increase Ring breathing, δ(C–H) out of
plane
928 Not evaluable δ(C–H) out of plane
765 −3 Intensity decrease v(=C–H) aromatic, out of plane
Band positions were evaluated normalizing the pre-exposure spectrum and the after-exposure spectrum at 7000 cm−1. Vibrational modes legend: v,
stretching; δ, bending. H2Om stands for molecular water. Δỹ stands for wavenumber shift with respect to the same peak before UV irradiation. Blank
cells stand for too weak differences to evaluate or null variations. ‘Not evaluable’ stands for those bands that show too high noise or peak
overlapping.

values, as reported in Barták et al. (2018) for the lichen, Dermatocarpon polyphyllizum (Nyl.) Blomb.
& Forssell. According to Miloš et al. (2018), NDVI variation related to thallus water content is species-
specific, such as the positive correlation noticeable in Physconia muscigena (Ach.) Poelt, while in
R. elegans, the NDVI showed constant values for different thallus water contents. In UV VAC conditions,
the same NDVI recovery pattern was observed. Nevertheless, significant differences between treat-
ments were recorded in chlorophyll a fluorescence value. UV N2 treatment produced a decrease in sam-
ples FV/FM post-exposure values, recovering 86% of the starting ones in 24 h and 93% in 72 h. In this
study, the integrity of the photobiont belonging to the lichen symbiosis was evaluated, but it is well
known that the mycobiont is involved in the protection of the photobiont from UV-radiations (see
e.g. Bianchi et al., 2019b and references therein). In the case of X. parietina, this protection is mainly
achieved by the production of the anthraquinone parietin, a compound secreted by the fungus as a
secondary lichen metabolite (Solhaug and Gauslaa, 2004). Photosynthetic activity recovery may be
explained considering also the thallus structure, playing an efficient role in UV rays screening and
avoiding damages to the photobiont layer (Collins and Farrar, 1978; Solhaug et al., 2003; Solhaug
and Gauslaa, 2004). On the other hand, samples treated in UV VAC conditions were strongly photo-
inhibited reaching only 7% of the starting FV/FM values. After that, lichens recovered 45% of the initial
values after the 72 h recovery period. This treatment proved to be more impacting on lichen photosyn-
thetic performance, likely because of the combined effect of UV-radiation and vacuum application, as
suggested by de Vera et al. (2004). A possible explanation of the difference between the recovery
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 International Journal of Astrobiology 13

Fig. 6. Details of the lichen IR spectrum changes for UV VAC treatment. The legend of the lines’ col-
ours showed in (a) is also referred to (b) and (c). (a) Band at 5200 cm−1 (v(H2O) + δ(H2O)); (b) band
at 3500 cm−1 (v(H2O) + v(OH)) and band at 2930 cm−1 (v(−CH) asym./sym. + v(=CH2) asym./sym.);
(c) general changes in the spectral range 1700–1450 cm−1. Band changes were evaluated normalizing
the pre-exposure spectrum and the after-exposure spectrum at 7000 cm−1.
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 14 Christian Lorenz et al.

trends in the physiological parameters (NDVI and FV/FM) may be that NDVI is usually correlated to
chlorophyll content (Garty et al., 2001). The intra-thalline water content in lichens (Granlund et al.,
2018) is the reason why NDVI can be used to make an indirect evaluation of primary photosynthesis
(Bednaříková et al., 2020). On the contrary, FV/FM is more related to chlorophyll damage (Zhang et al.,
2017). Hence, we can suppose that, while carotenoids are similarly affected by both treatments, UV
VAC may affect more deeply the chlorophyll content inducing a delay in photosynthesis reactivation,
with a consequent longer recovery time, as proposed by Kranner et al. (2008). The longer recovery time
may be related to the F0 decreasing values after exposure in UV VAC samples (Fig. S3). These findings
suggest that some lichen species may be considered extremotolerant, representing excellent
test-organism for space survival studies. Their ability to regulate water content depending on the envir-
onmental water availability (poikilohydric organisms) allow them to tolerate wet–dry cycles including
thallus water fluctuations from 20 to 160% water per thallus dry weight (Kappen, 1988). These features
may have contributed to the survival capability of samples in the extreme dehydration conditions such
as the simulated ones here. On the spectroscopical side, the IR band at 5200 cm−1 (v(H2O) + δ(H2O))
disappeared in the two performed treatments, suggesting a water loss from the lichen. Lichens are able
to resist to dehydration cycles, longer desiccation periods may produce permanent cellular collapses
and consequently affect the metabolic activity (Brandt et al., 2016). The treatments of nitrogen and
vacuum are considered as the main cause of dehydration process in the samples here analysed. For
this reason, it can be assumed that the 5200 cm−1 band decrease, associated to thalli water release,
is more strictly related to the Reaction Chamber atmosphere than to the UV-radiation. In both the treat-
ments, it is also noticeable a general upshifting of the spectral continuum due to the UV-radiation
effects, which is higher in vacuum conditions. A particular band change trend is observed in UV
VAC spectral range 1400–950 cm−1, where the band peak intensities become deeper, apparently sug-
gesting an increase related to those vibrational modes during the exposure. Lichen samples were not
able to biosynthesize any secondary metabolic substances during both exposures because they were
presumably inactivated and dehydrated by the extreme simulated conditions. Despite the increasing
of peak intensities, the spectrum showed a decreasing spectral contrast for the overall bands suggesting
a decreasing of the bands area linked to the number of chemical bonds associated to that particular
frequency. This fact may be interpreted as a direct damage of molecular bonds due to UV irradiation.
Physiological parameters suggested that vacuum application may cause stronger damages due to a
higher dehydration effect. According to Gauslaa and Solhaug (1999) air-dried lichen thalli exposed
to high light exposure show a time-lag in fluorescence recovery trends, as observed in this study. As
a consequence of the more intense UV-radiation effects in vacuum, we observed several spectral
band changes related to damage and extensive photodegradation of the thallus. Moreover, we observed
in both treatments a decrease in peak intensity of those bands related to –CH groups on aromatic rings,
which are probably related to parietin. Further studies may be performed with the aim of confirming the
possibility of thallus morphological and ultrastructural damages and parietin photodegradation under
UV-radiation.

Conclusions
Our work analysed two different environmental conditions: (i) vacuum and room temperature and (ii)
saturated nitrogen atmosphere at room temperature and atmospheric pressure. These two conditions –
simulated in laboratory – can be associated to different space environments, such as those found in the
proximity of Earth orbit for vacuum simulation and saturated nitrogen atmosphere may be comparable
to several planetary conditions (i.e. early Earth, Mars, possible altered terrestrial scenarios, exoplanets).
The examination of X. parietina recovery through eco-physiological analysis revealed the capability of
this lichen species to survive in extreme conditions such as those simulated in this investigation. It has
been highlighted the significant difference between treatments about the PSII efficiency (FV/FM) and
PSII integrity (NDVI) recovery trends, finding that UV-radiation in vacuum produces more intense
effects on FV/FM values than UV N2. After 72 h, UV N2 fluorescence mean values recovered up to
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 International Journal of Astrobiology 15

93% of the starting ones, while UV VAC fluorescence recovered up to 45% of the pre-exposure values.
Moreover, the study introduces the first scanned FTIR reflectance spectrum of the lichen species
X. parietina. Besides, the experiment comprised a spectral monitoring during the treatment phase in
order to evaluate band changes due to the exposure conditions. Spectral analysis suggests that the rela-
tion between mass variation and H2O 5200 cm−1 band disappearance after treatment is strictly linked to
the thalli dehydration due to the atmospheric simulated conditions represented by N2 insufflation and
high vacuum application. As stated in the introduction, one of the main objects of this study was to
increase our knowledge about the potential adaptive capability of organisms in extreme environments.
Our results, for the first time, have investigated the survival of X. parietina with multiple analytical
techniques and in particular we performed IR spectroscopy in situ during treatments. The study high-
lighted that the recovery performances of the lichen – kept in a growth chamber at 25°C with 12 h dark
and 12 h light 70 μmol m−2 s−1 PAR photons, daily moistened – were better after the exposure to solar-
like UV-radiation in conditions close to extreme planetary environments (i.e. saturated nitrogen atmos-
phere in our ground-based experiment) than those in pure space conditions (i.e. vacuum).
Supplementary material. The supplementary material for this article can be found at https://doi.org/10.1017/
S1473550422000076.

Acknowledgements. This work was co-founded by INAF and ASI, grant ‘Life in Space n. 2019-3-U.0’.

Conflict of interest. None.

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