Freezing and desiccation tolerance in the Antarctic bangiophyte Pyropia endiviifolia (Rhodophyta): a chicken and egg problem?

ABSTRACT Antarctic macroalgal communities of the upper intertidal zone are particularly poor compared with other coastal regions. Exposure to desiccation and freezing combined with the abrasive effect of ice threatens the life of sessile organisms. One of the few species able to colonize this environment is the rhodophyte Pyropia endiviifolia. It belongs to the Bangiales, one of the oldest extant clades of photosynthetic eukaryotes, which has occurred for more than one billion years with basically the same morphological structure. Considering that the extent of Antarctic glaciation is a geologically recent event, we hypothesized that pre-adaptations to desiccation in bangiophytes may have contributed to the success of P. endiviifolia in Antarctica. To test this, we compared its photosynthetic performance and tolerance to desiccation and freezing with those from a related intertidal species, the temperate Atlantic species Porphyra linearis. As evidenced by gas exchange and chlorophyll fluorescence, P. endiviifolia is more adapted to high irradiances than P. linearis. The former species was also more desiccation-tolerant, and showed a higher glass transition temperature. Both species did not differ in chlorophyll content per dry weight, and tolerance to −20°C, even though the ice-nucleation temperature was much higher in P. endiviifolia. Membrane integrity may depend on fatty acid composition in P. endiviifolia, while on enhanced tocopherol in P. linearis. Overall, both species show different strategies to deal with freezing temperatures: supercooling in P. linearis vs. freezing-tolerance in P. endiviifolia. This matches with the probability of being subjected to sub-zero temperatures in their natural environments (lower in the case of P. linearis). Surprisingly, the higher risk of dehydration in the natural habitat of P. linearis is not matched by a higher desiccation tolerance. This observation does not support the initial hypothesis of the study but suggests the opposite: the acclimation to a cold environment results in higher desiccation tolerance. HIGHLIGHTS ● Porphyra linearis and Pyropia endiviifolia are remarkably tolerant to desiccation and freezing.● Antarctic P. endiviifolia is remarkably tolerant to desiccation and freezing.● Mechanisms of freezing tolerance could induce a higher tolerance to desiccation.


Introduction
Red algae (Rhodophyta) occupy a basal evolutionary position among the photosynthetic eukaryotic organisms.Thus, rhodophytes gave rise by secondary endosymbiosis to the highest diversity of algal clades, including among others brown algae, diatoms or dinoflagellates (Archibald & Keeling, 2002).The fossil record supports that a red alga (Bangiomorpha pubescens), dated 1200 Mya, represents the oldest multicellular organism (Butterfield, 2000).Furthermore, recent studies even antedate the oldest rhodophytes by 400 million years (Bengtson et al., 2017).Bangiomorpha pubescens has been assigned to the extant group of the Bangiales.This group includes several genera, Pyropia and Porphyra being among the most represented (Sutherland et al., 2011;Yang et al., 2018).Among Bangiales, the gametophytes of some of them typically live in the upper intertidal and splash zone, including the well-known edible seaweed 'nori' (Neoporphyra haitanensis and Neopyropia yezoensis).The colonization of such intertidal habitats requires adaptation to extremely variable environmental conditions.Specifically, the algae species occurring in the upper tidal zone are exposed cyclically to variable salinity, strong solar radiation and severe desiccation.As a result, many of them have developed a desiccation-tolerant strategy (Contreras-Porcia et al., 2017) together with a complete arsenal of protection mechanisms against high light, thermal stress and reactive oxygen species (Brawley et al., 2017).
Desiccation tolerance has been largely characterized in a wide variety of taxa, including bacteria, tardigrades, plants and algae (Gaff & Oliver, 2013).Essentially, dramatic loss of water from photosynthetic tissues (e.g. to water potentials of ≤ −100 MPa) can easily compromise cell integrity mechanically and biochemically, affecting its functioning irreversibly and ultimately leading to death (Oliver et al., 2020).Thus, desiccation tolerant (DT) organisms are characterized by a set of constitutive and inducible mechanisms that efficiently prevent and/or repair that damage (Fernández-Marín et al., 2016;Oliver et al., 2020), and by the ability to enter into (and withstand) the so-called glassy state: a highly viscous intracellular condition upon severe dehydration, in which metabolic activity is virtually impeded (Fernández-Marín et al., 2013;Ballesteros et al., 2020).Species representing four genera have been described as DT within the Rhodophyta so far (Gaff & Oliver, 2013), but glass transition temperatures (e.g.temperature at which tissue enters the glassy state for a given water content) have never been studied before, to the best of our knowledge.In response to desiccation species of the Bangiales display a metabolic arsenal, which includes the expression of genes encoding polypeptides with chaperone function (Na et al., 2018), heat shock proteins (Celis-Plá et al., 2020) and antioxidant enzymes (Contreras-Porcia et al., 2011;Flores-Molina et al., 2014), together with others associated with basal metabolism (Fierro et al., 2017) and the synthesis of compatible solutes such as floridoside (Qian et al., 2015).When desiccation stress is combined with exposure to high irradiance, the mechanisms of energy dissipation protect the integrity of the photosynthetic apparatus (Bose et al., 1988;Herbert, 1990).
At mid and high latitudes the exposure to freezing temperatures during emersion adds a new component to this cocktail of environmental stresses that characterize the intertidal life at those latitudes (Zacher et al., 2009).Freezing generates cell dehydration, as ice crystals first nucleate in the apoplast (Pearson & Davison, 1994).As a consequence, water is pulled out of the living symplast, sometimes dehydrating the cells to less than 10% water content if cooling is slow enough (Wolfe & Bryant, 1999).Physiological responses to freezing have been less studied than desiccation tolerance in algae, but a series of reports confirms that some seaweeds can tolerate tissue freezing (Kanwisher, 1957;Pearson & Davison, 1994), including the examples within the Bangiales (Lin et al., 2010;Green & Neefus, 2014), that can survive after exposure to temperatures as low as −196°C (Wang et al., 2016).Another unexplored aspect is which strategy or strategies might these freezing-tolerant Bangiales follow to withstand such low temperatures.In vascular plants, two main (and non-exclusive) processes are generally distinguished: freezing tolerance, characterized by a relatively high ice nucleation temperature (i.e.around −3 to −5°C) and supercooling, characterized by ice-free tissues associated with a dramatic drop in the ice nucleation temperature (down to < −10°C or even much lower) (Wisniewski et al., 2014).
In polar waters, the abrasive effect of ice blocks adds a new adverse factor to the upper intertidal and splash zones, creating a particularly harsh environment.As a consequence, very few seaweed species inhabit the intertidal zone in polar regions (Becker et al., 2009).This is particularly the case in maritime Antarctica where the lowest algal coverage and diversity is found in the upper intertidal zone (Marcías et al., 2017).This habitat is dominated by the foliose bangiophyte Pyropia endiviifolia (A.Gepp & E.Gepp) H.G.Choi & M.S.Hwang (Marcías et al., 2017).
Bangiophytes have occurred with basically the same morphology during more than one billion years (Butterfield, 2000).During this period several glaciations have shaped the physiological evolution of photosynthetic organisms (Becker, 2013).Current Antarctic glaciation is a relatively recent event that started during the Cenozoic (Eocene-Oligocene transition, 35 Mya) (Barker et al., 2007).Given the isolation of Antarctica, ice sheet expansion led to the massive extinction of organisms present on the continent.Among the present flora, there is a mix of colonizers that arrived after the retreat of the maximum Pleistocene glaciations and others that were able to survive in isolated ice-free refuges in the extremely harsh Antarctic environment (Peat et al., 2007).In contrast with the terrestrial biota, oceanic communities, including intertidal species, remained more connected and probably were less affected by progressive climate cooling.Therefore, we speculate that Pyropia endiviifolia has been able to succeed in the harsh upper intertidal zone in maritime Antarctica thanks to the existence of pre-adaptations developed in response to multiple stressors in its natural habitat, particularly the constitutive desiccation tolerance of the genus.To give support to this hypothesis, in this study, we have investigated the responses of P. endiviifolia gametophytes to freezing and desiccation and compared them with another species from the related genus Porphyra (Porphyra linearis Greville).This species, found on temperate coasts of Europe and North America, was investigated in the southern part of its distribution range, where it very unlikely would be naturally exposed to freezing temperatures.

Studied species and field sites
Two species belonging to the Bangiales were studied: P. endiviifolia and P. linearis.P. endiviifolia inhabits rocky shores in maritime Antarctica and sub-Antarctic islands, occupying the cracks and crevices of the upper intertidal zone in the study site.P. linearis is mostly found on the Atlantic coast of Europe (from Spain to Norway), Iceland and North America (Guiry & Guiry, 2022).Pyropia endiviifolia was collected from the surroundings of the Spanish Antarctic Research Station 'Base Antártica Española Juan Carlos I (BAE JCI)' on Livingston Island, South Shetland Islands (62°40ʹS, 60°23ʹW) in February-March 2018.P. linearis was sampled and studied at Azkorri Beach in the Bay of Biscay (43°23ʹN, 2°58ʹW, Bizkaia, Spain) in January-February 2020.During the study months mean air temperature was +0.8°C at the site on Livingston Island and +11.9°C at Azkorri Beach.Absolute minimum temperatures were in the range of −3.7°C to −5.8°C over the last 6 years on Livingston Island, while at Azkorri Beach they only occasionally dropped below zero (one year during the last 6 years) (see Supplementary table S1).Maximal tidal amplitude during the study period was around four metres at Azkorri and two metres on Livingston.Mean vapour pressure deficit (VPD) was four times higher at Azkorri (see Supplementary fig.S1) than on Livingston during the study period.Thus, while VPD was usually lower than 0.25 kPa on Livingston, at Azkorri VPD was frequently higher than 1 kPa.

Experimental design
Comparison between P. endiviifolia and P. linearis was conducted at three main levels.First, we characterized both species photosynthetically by assessing, on the one hand, photosynthetic pigment composition and, on the other hand, electron transport and carbon assimilation rates against increasing irradiance.Second, we evaluated tolerance of both species to desiccation and determined their glass transition temperatures (e.g.temperature for which molecular mobility is tremendously reduced and thus metabolic activity virtually blocked in a dry sample).Third, we assessed their tolerance to sub-zero temperature and determined the ice nucleation temperature of fully hydrated thalli.Tolerances to desiccation and freezing were tested through analyses of chlorophyll fluorescence (e.g.changes in maximal photochemical efficiency, F v /F m ) and of photosynthetic pigments and tocopherols.Each biological replicate was obtained from a different thallus.All weights were recorded using a Mettler Toledo 0.1 mg precision balance.Blade areas were measured with ImageJ 1.5 software (Wayne Rasband National Institutes of Health, USA).

Pigments and antioxidants
Photosynthetic pigments and tocopherols were analysed by HPLC following the method described by García-Plazaola & Becerril (1999, 2001) with minor modifications.Briefly, approximately 75 mg fresh weight (FW) of thalli were immediately frozen in liquid nitrogen and stored at −80°C until extraction.Samples were extracted in 1 ml of pure acetone buffered with CaCO 3 (0.5 g l -1 ).Supernatant was then filtered through a 0.2 µm polytetrafluoroethylene filter (Teknokroma, Barcelona, Spain) before being analysed by HPLC.Tocopherol and pigments detection and quantification were conducted with a scanning fluorescence detector Waters 474 operating in series with a photodiode array detector Waters 996 (Waters, Milford, Massachusetts, USA) (García-Plazaola & Becerril, 1999, 2001).

Gas exchange and chlorophyll fluorescence measurements
Simultaneous gas exchange and chlorophyll fluorescence were measured in freshly collected samples of Pyropia endiviifolia using a GFS-3000 system coupled with an IMAGING-PAM fluorometer (Heinz Walz, Effeltrich, Germany) (Perera-Castro et al., 2020a).Measurements in Porphyra linearis were done using a LI-6800 system (LI-COR Inc., Lincoln, Nebraska, USA) and chlorophyll fluorescence was measured with a Mini-PAM fluorometer (Heinz Walz, Effeltrich, Germany).Samples that were collected and fully hydrated (n = 5), were maintained within a wet tissue inside Petri dishes in lab conditions for 2-7 h after their measurement.Samples were introduced into a custom-made cuvette consisting of a gasket affixed to a piece of thin polyester stocking fabric (Perera-Castro et al., 2020b).The size of these gaskets was equal to the chamber size to ensure its proper closure.CO 2 concentration was standardized at 400 µmol CO 2 mol -1 air, sample temperature was set to +20°C and relative humidity at 60-75% (Pyropia endiviifolia) or 80% (Porphyra linearis).The flow rate within the chamber was 750 μmol s -1 for Pyropia endiviifolia or 600 μmol s -1 for Porphyra linearis.After 10 min inside the chamber in dark conditions, dark respiration (R D ) was measured.Then, the sample was exposed to different intensities of irradiance (I = 20, 40, 75, 165, 370, 580, 922, 1465 and 1855 µmol photons m -2 s -1 for Pyropia endiviifolia and I = 50, 100, 250 and 500 µmol photons m -2 s -1 for Porphyra linearis) in intervals of 5 min.After each interval, net CO 2 assimilation rate (A N ) was recorded and a saturating pulse was applied to determine steady-state and maximal fluorescence at lightadapted conditions (F s and F m ', respectively).Actual quantum yield (ф PSII ) was calculated according to Genty et al. (1989): ф PSII = (F m '−F s )/F m '.Relative electron transport rate (rETR) was calculated according to Krall & Edwards (1992): rETR = ф PSII •I.The maximum light saturated A N (A sat ) and rETR (rETR max ) was obtained by fitting each light curve to Michaelis-Menten (Ritchie & Prvan, 1996a, 1996b), rational (Smith, 1936), exponential (Goudriaan, 1982) or hyperbolic tangent (Jassby et al., 1976) models by using the Microsoft Excel Solver tool (adapted to ETR light curves from Lobo et al. (2013)).The lowest square sum errors were obtained with the rational model of Smith (1936) (eq. 1 and 2): Where AQE A and AQE ETR are the Apparent Quantum Efficiency for A N and rETR-light curves, also fitted by the model.AQE A and AQE ETR are indicators of the potentially fixed μmol CO 2 m −2 s −1 or potentially transported μmol electrons m −2 s −1 per each μmol photons m −2 s −1 , respectively.Calculations of the light compensation point at which A N equals zero (LCP) and the irradiance at which 90% of A sat and rETR max are achieved (I A90 and I ETR90 , respectively) were done for the rational model (eq.3, 4 and 5, respectively):

Desiccation treatments
To evaluate tolerance to desiccation of P. endiviifolia and P. linearis, thalli pieces of approximately 80 mg were incubated during 48 h in closed 50 ml Falcon tubes at three different relative humidities (80%, 50% and 5%).These were obtained by equilibrium of the tube atmosphere with saturated solutions of NaCl (80%) and MgCl 2 (50%) and with silica gel (5%) (for a detailed description of the protocol see López-Pozo et al., 2019).After incubation, blades were gently remoistened with seawater and damage was estimated by the depression of F v /F m compared with control values measured before the start of the experiment.The whole experiment was performed in darkness.Three biological replicates were used per species and treatment

Dynamic mechanical thermal analysis (DMTA)
Mechanical analyses were conducted on desiccated thalli of P. endiviifolia and P. linearis after equilibration with silica gel.This measurement enabled comparison between both species about the extent of molecular mobility when in the dry state.Measurements were conducted with a DMA/ SDTA861e mechanical thermal analyser (Mettler Toledo, Greifensee, Switzerland) in the shear mode.The method used is similar to that previously described in Fernández-Marín et al. (2013, 2019).Briefly, measurements from −50 to +150°C at a heating rate of 2°C min −1 were carried out in the dynamic mode.Each sample was scanned at three different frequencies, 1 Hz, 3 Hz and 10 Hz, and 50 µm strain.Shear storage modulus (G´), shear loss modulus (G") and the loss tangent (tanδ = G"/ G´) were calculated using the Mettler Toledo START e software during the scans.For each biological replicate, the temperature value at the maximum tanδ coincident with the α-relaxation measured at 1 Hz, was considered for estimating of the glass transition temperature (T g ).Three biological replicates were measured per species.

Freezing treatments
Fresh thalli samples of approximately 75 mg FW were introduced in closed Petri dishes (to avoid desiccation) and incubated at −20°C for 48 h in darkness.Subsequently, a first subset of samples was incubated at +20°C in darkness to monitor the recovery, while a second subset of samples was immediately immersed in liquid nitrogen to test for freezinginduced variations in photosynthetic pigments and tocopherols.During freezing (48 h) and recovery (6 h), the F v /F m was monitored at different time intervals.

Determination of ice nucleation temperature
The freezing temperature of P. endiviifolia and P. linearis was analysed in hydrated samples by Differential Scanning Calorimetry (DSC, using a DSC 822e from Mettler Toledo, Greifensee, Switzerland).Before the measurements, the DSC equipment was calibrated with zinc, indium and pure water as standards.Samples (≈ 10 mg per replicate) were first sealed in aluminium pans, then equilibrated at 0°C and afterwards cooled at a rate of 0.1°C min -1 from 0 to −20°C under constant nitrogen flow (20 ml min -1 ).Subsamples of the same set were used to determine absolute water content.DSC measurements were performed in five different thalli per species.

Statistical analysis
Statistical analyses were performed with SPSS v25.0 at a significance level of α = 0.05.A one-way ANOVA, with HSD Tukey test as a post hoc, was applied to test significant differences among treatments in both species, after testing normal distribution and variance of the data.For data without normal distribution or variance (e.g.Supplementary fig.S2), Kruskal-Wallis, followed by Mann-Whitney U test as post hoc, was applied.

Physiological characterization of Porphyra linearis and Pyropia endiviifolia
Pyropia endiviifolia and P. linearis thalli (Figs 1, 2) were morphologically very similar under the bright field microscope, the exception being thicker cell walls and blade cross section in P. endiviifolia (Figs 3,4).This explained the higher ratio of dry mass per area in P. endiviifolia that was almost twice that of P. linearis (Table 1).In spite of it, water content at turgor was similar in both species (Table 1).
Pigment composition was remarkably similar in both species, whether in quantitative (when expressed per mass unit) as well as qualitative (pigment composition) terms (Table 1, Fig. 5).It essentially consisted of chlorophyll a (Chl a) plus two major carotenoids (lutein and β-carotene), and small amounts of zeaxanthin and α-carotene (Table 1, Fig. 5).As blade mass area was 85% higher in P. endiviifolia, when pigments were expressed on an area basis, their concentrations were approximately two-fold higher in this species (Table 1).In contrast to pigments, dry mass-based contents of the antioxidant α-tocopherol were more than two-fold higher in P. linearis (42.3 ± 2.6 and 101.7 ± 17.6 nmol g -1 DW in P. endiviifolia and P. linearis, respectively, data not shown, difference significant at p < 0.05).
Light response curves of net carbon assimilation (A N ) and relative electron transport (rETR) were adjusted to the rational model of Smith (1936) for both species (Fig. 6) and the derived parameters are shown in Table 1.Pyropia endiviifolia showed significantly higher A sat , R D and rETR max : 2-, 2.7-and 1.3-fold higher than P. linearis, respectively.Furthermore, saturating levels of A N and rETRconsidered at 90% of the maximum values -were reached at significantly lower irradiances in P. linearis than in P. endiviifolia.Similarly, lower LCP between photosynthesis and (photo)respiration was obtained in P. linearis than in P. endiviifolia.For the lowest irradiances, significantly higher AQE rETR were observed for P. linearis, although quantum efficiency did not differ for A N .A sat was significantly higher in P. endiviifolia when expressed on an area basis (Fig. 6), but this parameter showed similar values in both species when referred to dry mass (0.184 ± 0.017 and 0.151 ± 0.014 µmol CO 2 g -1 DW s -1 in P. endiviifolia and P. linearis, respectively) or to Chl a content (88 ± 8 and 69 ± 7 mmol CO 2 mol −1 Chl a s -1 in P. endiviifolia and P. linearis, respectively).On the contrary, R D in P. endiviifolia was almost twice that of P. linearis even when expressed per unit of dry matter (0.054 ± 0.004 and 0.033 ± 0.003 µmol CO 2 g -1 DW s -1 in P. endiviifoliaand P. linearis, respectively).

Desiccation tolerance and molecular mobility in the dry state
Blades of both species were desiccated under controlled conditions (by equilibration with three different relative humidities from 5%, 50% and 80%) and desiccation tolerance was thereafter assessed through F v /F m during rehydration.Response differed between both species: while P. endiviifolia was not affected by desiccation, in P. linearis F v /F m decreased concomitantly with the severity of dehydration (Fig. 7).Thus, in P. linearis equilibration of samples with 80% RH induced a slight decrease of the F v /F m (≈ 25%) in comparison to initial values.This decrease was significantly greater after desiccation at 50% RH (F v /F m decreased to half the control values), and dramatic (close to 10% of control values) after desiccation at 5%.
Dynamic mechanical thermal analysis measurements of dried samples equilibrated with silica gel (5% RH), enabled us to evaluate the extent to which severe dehydration may limit molecular mobility within photosynthetic thalli of both species.Thus, higher glass transition temperatures would indicate more stable (e.g. less mobile) molecular mobility at ambient temperature in the dry state.We obtained mean T g ± SE of 49.0 ± 2.9°C for P. endiviifolia and of 39.4 ± 3.3°C for P. linearis (Fig. 8), although the difference was not significant (p = 0.091 in the one-way ANOVA).This indicated slightly higher (but not significant) molecular mobility for P. linearis than for P. endiviifolia at a comparable state of dehydration.

Freezing tolerance
To evaluate the ice nucleation temperature, DSC measurements were conducted in fully hydrated samples.The DSC scans of hydrated blades showed exothermic peaks corresponding to freezing temperatures between −4.3 and −17.8°C (Fig. 9).Overall, Table 1.Summary of selected morphological and physiological traits of the photosynthetic thalli from the two studied species.Data are mean ± SE (n = 5-7).Asterisks denote significant differences between both species at p < 0.05.P. linearis samples froze at significantly lower temperatures and showed greater variability (−13.0 ± 1.7°C) than P. endiviifolia (−6.4 ± 0.8°C) (p < 0.05) (Fig. 9).
Exposure of hydrated blades to temperature well below freezing point (−20°C) caused a progressive decrease of F v /F m in both species.This decrease was nevertheless moderate, and after 48 h, F v /F m values were still around 70% of the initial control values (Fig. 10).This slight decline in F v /F m was fully reversible upon 30 minutes of rewarming at +20°C, remaining stable for, at least, the following 6 h.This pattern indicates that both species were fully tolerant to −20°C under our experimental conditions.

Changes in photosynthetic pigments and tocopherols during desiccation and freezing
Changes in pigment and tocopherol composition were followed in desiccation and freezing treatments (Supplementary fig.S2).While pigment contents did not change significantly with stress treatments in P. endiviifolia, desiccation induced a significant decrease in Chl a, particularly at the intermediate 50% RH treatment in P. linearis.The same trends were observed for the α-tocopherol content, although not significant (p < 0.05).

Discussion
The inference of physiological evolution from extant organisms has to be done with care, but the reconstruction of past climatic conditions can provide clues to what environmental challenges were like that organisms had to face.Thus, given the current habitat preferences of some Bangiophytes for the uppermost intertidal zone, and the morphological similarities of fossil species with the extant ones, it is likely that these algae had to develop adaptations to tolerate a partial desiccation from the very beginning of their evolutionary history (Butterfield, 2000).
In the present study we postulate that the ability to tolerate desiccation of intertidal species of the genus Porphyra spp.(Blouin et al., 2011) could represent a preadaptation that allowed these species to tolerate freezing temperatures (Verhoeven et al., 2018), making their survival possible in the progressively colder Antarctic climate.If this is correct, it could be  expected that temperate or tropical species, rarely or never exposed to negative temperatures, could be constitutively freezing tolerant if they are tolerant to desiccation.To give support to this hypothesis we physiologically characterized two Bangiophyte species: the Antarctic P. endiviifolia and the temperate Atlantic P. linearis and compared them in terms of stress tolerance.
Gas exchange and chlorophyll fluorescence data obtained in this study provide evidence that the studied populations of P. linearis are notably more adapted to low irradiance than P. endiviifolia.This result is counterintuitive if we consider the latitudinal distribution of both populations and could be explained by phenological differences (i.e.P. linearis spreads during wintertime in southern Europe while P. endiviifolia does so in summertime in Antarctica).However, when expressed on a dry mass base, both species were remarkably similar in their assimilation rates, indicating that the observed differences were mostly due to differences in blade morphology rather than to photosynthetic efficiency.Thus, the higher blade mass area in P. endiviifolia (Table 1) could be the result of the enhanced mechanical protection against the abrasive effect of ice blocks.
The gametophytes of both species have a contrasting phenology: Porphyra linearis develops during wintertime while Pyropia endiviifolia develops during summertime only (Oliveira et al., 2009).As a consequence the studied population of the first species is very rarely exposed to sub-zero temperatures (only one record in eight years during the growing season), while freezing events occur routinely at the shore of Livingston Island during the Antarctic summer (Supplementary table S1).Moreover, given the ice nucleation temperatures measured in both species (−6.4 and −13°C in P. endiviifolia and in P. linearis, respectively) and the actual temperatures recorded in nearby meteorological stations, tissue freezing could occur Fig. 8. Dynamic mechanical thermal analysis (DMTA) scans of Pyropia endiviifolia thalli, left, and Porphyra linearis, right, fully desiccated (equilibrated with silica gel).Shaded areas highlight the α-relaxation, which is identified as a peak or step in the Tan δ (upper panels), and as a deep decrease in the storage modulus (G′) (lower panels).The α-relaxation corresponds to the transition from the glassy state (at lower temperatures), where molecular mobility is extremely limited, towards the gel state (at higher temperatures), where molecular mobility is much higher and enzymatic reactions are progressively more likely to occur.Glass transition temperature (T g ) estimated at the peak of the α-relaxation in the Tan δ (scanned at 1 Hz) was 49.0 ± 2.9°C for P. endiviifolia and 39.4 ± 3.3 for P. linearis (average ± SE, n = 3).One representative curve from three independent biological replicates is shown, per species.occasionally in P. endiviifolia, but never in P. linearis.The lower freezing point in P. linearis could be explained by the observation that it grows in winter, when this ice nucleation temperature decreases in a process of seasonal acclimation, as has been described for many other intertidal species (Lundheim, 1997).For example, the terrestrial green macroalga Prasiola crispa, growing in the vicinity P. endiviifolia, also shows a very similar ice nucleation point of −5.1 ± 0.4°C, and also a similar T g of +45.1 ± 1.0°C (Fernández-Marín et al., 2019).Despite this, both rhodophytes in this study were equally tolerant to sub-zero temperatures.More surprising was the finding that both species kept a remarkably high photochemical efficiency under sub-zero temperatures, where the lower freezing temperature of P. linearis was likely to be related to its slower decrease in F v /F m upon freezing.The high concentration of long chain poly-unsaturated fatty acids eicosapentaenoic acid (20:5 n-3) of P. endiviifolia (Santos et al., 2017), as well as for other Porphyra species (Blouin et al., 2006), probably contributes to the maintenance of thylakoid fluidity at temperatures close to or below freezing point.
In contrast to freezing, responses to desiccation differed greatly between both species.Thus, P. endiviifolia was fully desiccation tolerant, exhibiting a level of tolerance comparable to other wellcharacterized species of bryophytes, lichens or algae (López-Pozo et al., 2019).However, P. linearis could be considered as intermediate in terms of tolerance to desiccation, suffering severe damage at the higher levels of dehydration.
As observed with pigment analysis, dry mass expressed Chl a content did not differ between both species and carotenoid composition was also similar in quantitative and qualitative terms containing the four major carotenoids found in the order Bangiales: lutein, β-carotene and small amounts of zeaxanthin and α-carotene (Koizumi et al., 2018; Supplementary fig.S2).In contrast to the rapid light-induced changes observed in the xanthophyll cycles of brown or green algae, red algae respond to changes in light environment mostly by synthesis or degradation of photoprotective carotenoids.This is reflected by a long-term adjustment in xanthophyll composition, as has been shown in Gracilaria birdiae or Corallina elongata (Ursi et al., 2003;Esteban et al., 2009).The low plasticity of the photosynthetic apparatus was also evidenced by the lack of readjustment to freezing of pigment composition in both species.In principle, high light favours the synthesis in timeframes of days of the β-carotenoids, zeaxanthin and β-carotene (Xie et al., 2020).In fact, only recently, a functional zeaxanthin epoxidase has been reported in red algae (Dautermann & Lohr, 2017).On the contrary, a decrease in Chl a and carotenoids occurred in response to moderate desiccation (80% and 50% RH).This effect was not observed under severe dehydration probably because, as indicated with the T g of +39°C, molecular interactions, including oxidations, were severely limited at this low water content.The same pattern was observed for the case of the α-tocopherol in P. linearis.The presence of this antioxidant and of its biosynthetic genes have been described previously in red algae (Ferraces-Casais et al., 2012;Brawley et al., 2017), and its consumption implies a strong antioxidant demand even when treatments were conducted in darkness.
Overall, the studied southern population of P. linearis shows a very low freezing point and a surprising tolerance to low temperatures that are not likely in its natural habitat.The cellular and molecular mechanisms that enable this remarkable behaviour under low temperature must differ from those required upon severe dehydration since this species failed to withstand severe dehydration.Based on the T g obtained, more fluid lipid composition (which is useful for low temperatures but puts at risk membrane stability upon desiccation) could be a possible reason behind it, although further studies would be needed.Although the actual risk of ice formation within the tissues was higher in the Antarctic species, the finding that both algae were equally tolerant to freezing (at −20°C, well below ice-nucleation temperature) is in principle consistent with our initial hypothesis that the gain of freezing tolerance was a side-effect of the ancestral desiccation tolerance of these species.However, present results also show that the Antarctic P. endiviifolia was much more tolerant to desiccation than P. linearis.Given that VPD values are on average four times lower on Livingston Island than on Azkorri Beach, the risk of desiccation is much lower in the first site (Supplementary fig.S1).This finding is somehow counterintuitive and clearly does not support our initial hypothesis that freezing tolerance in P. endiviifolia was an exaptation.Furthermore, it could suggest just the opposite: that the adaptation to the low temperatures of the hostile Antarctic environment somehow results in a complete tolerance to desiccation, even when severe desiccation events are unlikely in the Antarctic intertidal habitats.This reciprocal cross-tolerance to freezing and desiccation could be the result of the shared mechanisms involved in the resistance to both stresses (Verhoeven et al., 2018).The association with fungal communities could also be involved in the higher stress resistance of P. endiviifolia (Furbino et al., 2014) and, in general, with the high metabolic resilience of Antarctic intertidal seaweeds (Celis-Plá et al., 2020).

Fig. 5 .
Fig. 5. Pie chart showing pigment composition in Porphyra linearis and Pyropia endiviifolia.Total pigment content is 2.7 µmol g -1 DW in both species.Three major carotenoids are shown: lutein, zeaxanthin and β-carotene.α-Car (not shown) was 0.9% of total pigment content, and 1.8% in Porphyra linearis and Pyropia endiviifolia, respectively.Data are mean for n = 3 for P. endiviifolia and n = 11 for P. linearis.

Fig. 10 .
Fig.10.Effect on F v /F m (expressed as percentage of control values) of 48 h incubation at −20°C for 48 h followed by 6 h of recovery at +20°C.Data are mean ± SE (n = 4-12).Asterisks denote significant differences between both species at p < 0.05.