Lichen acclimation to changing environments: Photobiont switching vs. climate‐specific uniqueness in Psora decipiens

Abstract Unraveling the complex relationship between lichen fungal and algal partners has been crucial in understanding lichen dispersal capacity, evolutionary processes, and responses in the face of environmental change. However, lichen symbiosis remains enigmatic, including the ability of a single fungal partner to associate with various algal partners. Psora decipiens is a characteristic lichen of biological soil crusts (BSCs), across semi‐arid, temperate, and alpine biomes, which are particularly susceptible to habitat loss and climate change. The high levels of morphological variation found across the range of Psora decipiens may contribute to its ability to withstand environmental change. To investigate Psora decipiens acclimation potential, individuals were transplanted between four climatically distinct sites across a European latitudinal gradient for 2 years. The effect of treatment was investigated through a morphological examination using light and SEM microscopy; 26S rDNA and rbcL gene analysis assessed site‐specific relationships and lichen acclimation through photobiont switching. Initial analysis revealed that many samples had lost their algal layers. Although new growth was often determined, the algae were frequently found to have died without evidence of a new photobiont being incorporated into the thallus. Mycobiont analysis investigated diversity and determined that new growth was a part of the transplant, thus, revealing that four distinct fungal clades, closely linked to site, exist. Additionally, P. decipiens was found to associate with the green algal genus Myrmecia, with only two genetically distinct clades between the four sites. Our investigation has suggested that P. decipiens cannot acclimate to the substantial climatic variability across its environmental range. Additionally, the different geographical areas are home to genetically distinct and unique populations. The variation found within the genotypic and morpho‐physiological traits of P. decipiens appears to have a climatic determinant, but this is not always reflected by the algal partner. Although photobiont switching occurs on an evolutionary scale, there is little evidence to suggest an active environmentally induced response. These results suggest that this species, and therefore, other lichen species, and BSC ecosystems themselves may be significantly vulnerable to climate change and habitat loss.


| INTRODUCTION
Biological Soil Crusts (BSCs) are biologically modified soil surfaces composed of an amalgamation of organisms which include lichens, bryophytes, microalgae, (cyano) bacteria, and microfungi. They are often the dominant vegetation type in areas limited by either water availability or temperature, and provide vital ecosystem services such as soil stabilization and nutrient acquisition (Belnap, 2003).
is one such contributing lichen, being a generalist species found to dominate in climatically distinct BSC regions around the world (Büdel, 2003;Galun & Garty, 2003;Rosentreter & Belnap, 2003;Timdal, 1986). Regardless of its worldwide distribution, research on this important lichen species has been minimal.
Lichens are a symbiotic relationship between fungal and algal (photobiont) partners, allowing colonization of habitats where the individual organism could not survive. Numerous studies have investigated the relationships between the fungi and its photobiont (e.g., Dal Grande et al., 2014;Fernández-Mendoza et al., 2011;Kroken & Taylor, 2000;O'Brien, Miadlikowska, & Lutzoni, 2005;Piercey-Normore, 2004) and many lichen families, genera, and species have been shown to associate with an array of algal partners (e.g., Beck, Kasalicky, & Rambold, 2002;Muggia, Baloch, Stabenteiner, Grube, & Wedin, 2011;Muggia et al., 2013;Nyati, Scherrer, Werth, & Honegger, 2014;O'Brien, Miadlikowska, & Lutzoni, 2013;Romeike, Friedl, Helms, & Ott, 2002;Thüs et al., 2011). Although never conclusively shown, this can be assumed to allow the lichen to adapt to different environments (Blaha, Baloch, & Grube, 2006;Yahr, Vilgalys, & Depriest, 2006) and may allow a widening of their ecological niche. Photobiont switching is the mechanism which allows a specific lichen fungus to associate with a new algal partner and has been shown to occur throughout lichen evolution (Henskens, Green, & Wilkins, 2012;Magain & Sérusiaux, 2014;Muggia, Grube, & Tretiach, 2008;Nelsen & Gargas, 2008; Piercey-Normore & Depriest, 2001). However, many questions around photobiont switching are unanswered, how a lichen selects an algal partner is unknown, whether a lichen can actively choose a photobiont from a local pool remains unclear, and nothing is known about time scales over which photobiont switching can occur. Being able to switch photobionts actively would allow lichens to acclimate to changing environmental conditions, presumably by selecting an algal partner that is specifically adapted to those conditions. Acclimation refers to the ability of an organism to modify its gene expression, and hence, physio-morphological features, in response to the environment. This is in contrast to adaption, which refers to actual changes in an organism's genome (Giordano, 2013). To some extent, the ability of lichens to acclimate to their environment has been of interest to lichenologists for many years. Larson and Kershaw (1975) found evidence for acclimation in arctic lichens, discovering rapid acclimation to temperature, light and thallus moisture content. In more recent years, lichens have been found to acclimate their respiration in response to seasonal temperatures (Lange & Green, 2005), and transplants were found to acclimate to high light by increasing thallus thickness and chlorophyll a/b-ratio (Gauslaa, Lie, Solhaug, & Ohlson, 2006). A closely related topic discusses phenotypic plasticity in lichens: the ability of a genotype to develop various phenotypes in response to different environmental conditions (Vallardes, Gianoli, & Gómez, 2007). Many lichen species have been shown to have differing ecophysiological and morphological traits dependent on the ecological niche inhabited (e.g., Muggia, Pérez-Ortega, Fryday, Spribille, & Grube, 2014;Pérez-Ortega et al., 2012;Pintado, Valladares, & Sancho, 1997;Printzen, Domaschke, Fernández-Mendoza, & Pérez-Ortega, 2013;Tretiach & Brown, 1995). Phenotypic plasticity and the ability to actively acclimate to environmental conditions would allow species to withstand pressure from climate change, human disturbance, and habitat loss.
Recently, two climatically distinct populations of P. decipiens were studied in order to assess whether ecophysiological and/or morphological traits could explain the ability to thrive in diverse habitats (C. Colesie , L. Williams, & B. Büdel, submitted). The results suggested that the regulation of thallus water content allowed individuals to be specifically adapted to conditions in a semi-arid region compared to those of a wet, alpine region. This contribution intends to explore whether genetically fixed adaption or acclimation is responsible for this variability. By installing a transplant experiment between climatically variable sites in western Europe, the ability of P. decipiens to acclimate across its range can be investigated. It is expected that the transplanted lichens acclimate to the new environment by associating with a locally adapted photobiont and by modifying their morpho-physiological traits.

K E Y W O R D S
biological soil crusts, environmental change, Europe, genetic diversity, green algae, latitudinal gradient, morphological variability, Myrmecia, plant-climate interactions, plasticity Climatic factors have been shown to be significant determinants of Asterochloris lineages associated with lichens of the genera Lepraria and Stereocaulon (Peksa & Skaloud, 2011). Therefore, we suggest that if a P. decipiens transplant can acclimate, a new photobiont will be required.

| Study sites
In order to cover a broad range of different macro-climatic conditions, four study sites were selected across Europe. The four sites in this study have previously been described in full Williams et al., 2016), and therefore, the following provides only a brief introduction. All climate data were obtained from weather stations installed at each site and covers a period of 2 years (2012-2014).
The site is situated ca. 20 m a.s.l and has a maritime climate, with roughly 500 mm annual precipitation and an average temperature of 8°C.
Annual precipitation is 600 mm.
Situated at ca. 2,600 m a.s.l, the site is located near the Großglockner High Alpine Road. Annual precipitation is between 1,750 mm and 2,000 mm of which 70% falls as snow and the mean annual temperature is −1°C.
Spain and Austria are sites where BSC occurs naturally due to the environmental conditions; in comparison, the Swedish and German sites are, at least to some extent, maintained through human intervention, such as cattle grazing. Due to the extremities in temperature and precipitation at the natural sites, they are here referred to as the extreme sites compared to the milder, temperate sites.

| Transplantation
Psora decipiens samples were collected from every site in 2012. Each sample was at least a 9 cm 2 section of intact BSC dominated by multiple P. decipiens thalli (Figure 1). Samples were air-dried at room temperature and stored at −20°C before use. Five replicates from each site were transplanted between all four sites, including a control which entailed transplantation within the site, to test for any effect of the process itself (number of transplant combinations was 15, see Table 1). All samples were installed in the new site within 6 months of collection and remained in the field for 1.5-2 years. All samples were collected between May and August 2014, once again air-dried, and stored at −20°C before further investigation.

| Morphological analysis
Thalli that had grown during transplantation were first identified

| Molecular analysis
Lichen thalli were thoroughly washed to remove any epiphytic fungi, algae, and loose soil. Individual thalli were detached from any remaining soil particles under a binocular microscope. Total genomic DNA was extracted using a CTAB method followed by phenol-chlo- from Tunisia, and one from Portugal were also included to act as comparisons. See Table A1 in Appendix for specimen lists and accession numbers.
Each DNA extraction contained both the fungal and algal symbionts, and general green algal primers were used. Therefore, the results confirm that the obtained sequences come from the intended lichens and not from epiphytic green algae because a contamination would have resulted in a mixed sequence. The algal DNA was amplified using the green algae-specific forward primer All500af

| RESULTS
Initially, Psora decipiens samples appeared to not only survive being transplanted to a new environment but to thrive, as can be seen by the substantial new growth in Figure 1 and the numbers of replicates with new growth per transplant combination in Table 1. Therefore, the discovery of thalli transplanted to Austria and Spain without an algal layer once the morphological investigation began was unexpected ( Figure 2). Thalli, from each transplanted sample, were examined, and the algal layer was found to be missing in both the transplanted and new-grown thalli of all replicates that had been transplanted to a site where the climate was more extreme than the original, for example, Sweden (temperate) to Austria (alpine) ( Table 2). In three transplant combinations (Sweden to Austria, Sweden to Spain, and Germany to Spain), small pockets of remaining algae were discovered in protected positions and used in downstream investigation (Table 2). Transplantation between sites where the climatic change was not toward either the semi-arid or alpine sites, for example, Spain with 100% bootstrap and posterior probability values ( Figure 6). In addition, in this experiment, the photobiont did not change when transplanted to a new site, even though in some cases it survived. In contrast, the analysis for Germany, Sweden, and Spain samples did not resolve any clear clades or gain high support. Sequence identity within this group, for both genes, was also found to be 99%. When the sequences from the Germany, Sweden, and Spain group were compared to the Austrian sequences, similarity was only found to be 94%; however, within the Austrian group, sequences are identical. The African *Within very small areas of a replicate living algae was located, these thalli were within folded dense areas of thalli and surrounded by those with dead algal layers. Algae within the new-grown thalli were always found to have died.

| DISCUSSION
The purposes of this study were to investigate the acclimation potential of a lichen species that is found in climatically diverse BSC ecosystems. This would suggest that an important and widespread BSC species can successfully navigate environmental change. Although the lichen was found to survive and even produce new thalli when transplanted between sites, the algal layer was frequently found to have disappeared. Transplantation between sites, where an extreme climatic shift occurred, resulted in lichens unable to photosynthesise and therefore would be assumed to soon die. Psora decipiens has been shown to be composed of at least four genetically and geographically distinct groups, with a narrow range of algal symbionts that apparently cannot be switched when introduced into a new environment.
The German and Sweden sites sharing a genotype highlights their similarities, they have been shown to be the most climatically similar, share the most lichen species, and have comparable BSC compositions , including cyanobacterial assemblages . This lends supports to the suggestion that the genotypic and morpho-physiological variations found within P. decipiens have a climatic determinant. Austrian P. decipiens was found to have a distinct, single, algal genotype, clearly separate from the other sites; this led to the conclusion that photobiont switching did not take place. However, the lack of genetic diversity, low support, and deficiency of clades based on site, for the rest of the transplants, makes it impossible to completely disprove photobiont switching in this case. Nevertheless, it seems unlikely. Transplant and photobiont survival only occurred in sites that had similar or milder climatic conditions than the original.
Allocation of resources has previously been shown to allow acclimation in lichens on a seasonal, moderate basis (Schofield, Campbell, Funk, & MacKenzie, 2003). The lichens surviving transplantation between Sweden and Germany is unsurprising considering the similar environmental conditions and a corresponding mycobiont genotype.
Therefore, the transplants did not necessarily need to acclimate any further than what would already occur, on a seasonal basis, in their native habitat. Transplantation survival from an extreme to a mild site can also be explained by climatic conditions; an extreme site is not extreme all year around, and conditions at certain times would be very similar to those in a mild site, during which the lichens would be active, compared to harsh periods (high summer in Spain) where lichens are, for the most part, dormant (Raggio et al., 2014;Schroeter et al. 2010).
However, it should be noted that this is grossly simplifying the effect of environmental conditions on lichens and does not take microclimate into consideration. In this case, transplants survived, died, or lost their algal layer and did not show a tendency to acclimate or to switch photobionts over 2 years. Increasing the time period may allow the effects of a milder climate on an extreme climate-adapted sample to be further comprehended.
F I G U R E 5 Maximum-likelihood tree of Psora decipiens using data from the nuITS. Numbers at nodes represent first the ML bootstrap support (values ≥50%) and second the posterior probabilities from the Bayesian analysis (values ≥70%). Ger = Germany, Swe = Sweden, Spa = Spain, Aus = Austria. Examples: Ger2 = Germany replicate 2 control and Ger1-Aus = Germany replicate 1 transplanted to Austria One surprising facet of this study was the transplantation samples' new growth being found to no longer have an algal layer. An explanation for the transplants being able to produce new thalli before environmental conditions became detrimental is due to the time of year the transplantation experiment was set up. The winter months in the arid Spanish site are when BSC organisms are active, with relatively little activity during the extreme summer months (Raggio et al., 2014). The transplants were installed in October 2012, during the growing season, 6-7 months before a harsh summer returned. A similar situation occurred in Austria, the samples were transplanted in July 2012, the middle of the alpine sites' growing season (Colesie, Green, Raggio, & Büdel, 2016). Consequently, it can be assumed that there was sufficient time for new growth before climatic conditions became detrimental to the transplanted lichens. It has been suggested that mycobionts may survive without a photobiont for up to a year (Etges & Ott, 2001) or 8 months when subjected to starvation stress (Zhang & Wei, 2011). However, it is generally believed that mycobionts must associate with an algal partner, even an incompatible one, within a short time period (Honegger, 1992). Therefore, it is possible that over more time a new photobiont could have been incorporated and the results show a potential pool of free-living algae that could be utilized. It has been broadly suggested that lichens would switch their photobiont through a fungal spore associating with a new algal strain (Etges & Ott, 2001;Hedenås, Blomberg, & Ericson, 2007;Sanders & Lücking, 2002) or through taking algae from soredia (Ott, 1987), which are packets of fungi and algae that are easily dispersible and therefore frequently available. Additionally, lichens have also been known to steal a photobiont from other lichens during early stages of thallus development (Friedl, 1987;Lücking & Grube, 2002;Stenroos, 1990;Wedin et al., 2016). With these mechanisms in mind, it seems unlikely, although not impossible, that a fully formed thallus would be able to integrate a new alga, although a longer term transplantation experiment would be required to fully explore this.
When lichens become stressed, respiration has been shown to increase by a significant margin (Kappen & Lange, 1972). A small-scale physiological experiment on the transplanted lichens with no remaining algal layer showed extremely high levels of respiration, and as was expected, no photosynthetic activity ( Figure A1 in Appendix Although the aim of this study was not to discuss photobiont diversity of P. decpiens, a finding which contradicts previous research has been an interesting outcome. Psora decipiens had previously been thought to associate with Asterochloris (Schaper & Ott, 2003) and/or Trebouxia photobionts, and also to be highly diverse (Ruprecht et al., 2014 (Thüs et al., 2011). These lichen species (Placidium sp. and Heteroplacidium sp.) are also associated with BSC, occur in the same habitats as P. decipiens, and share a similar morphology.
In conclusion, Psora decipiens may be considered a cosmopolitan soil crust lichen species; however, these results demonstrate that the species includes different genotypes that apparently cannot acclimate to changing environmental conditions within the species range. This is a small-scale study considering that P. decipiens has a nearly worldwide distribution, and therefore to understand the genetic diversity and biogeography of this species, much larger scale studies are required.
There is no evidence that photobiont switching takes place, and it currently seems that P. decipiens associates with a narrow range of pho-  F I G U R E A 1 Gas exchange of Psora decipiens transplant, from Spain to Austria, and Austrian Control. Upper graph shows the emitted photosynthetic active radiation (PAR) and the lower the gas exchange response. The control sample shows release of CO 2 (Photosynthesis) compared to the transplant, which had no remaining algal layer, which takes up CO 2 (respires)