Differential Gene Expression of Mycobionts and Photobionts in Peltigera Britannica Photosymbiodemes

Differential gene expression of mycobionts and photobionts in Peltigera britannica photosymbiodemes

Masterarbeit

zur Erlangung des akademischen Grades eines Master of Science an der Naturwissenschaftlichen Fakultät der Karl-Franzens-Universität Graz

vorgelegt von Jasmin ALMER, BSc

am Institut für Pflanzenwissenschaften Begutachterin: Prof. Dr. Silke Werth

Graz, 2019

Acknowledgements First and foremost, I want to thank my supervisor Prof. Dr. Silke Werth for giving me the opportunity to conduct this research and for offering her guidance, support and not least her patience throughout. I would also like to thank Prof. Dr. Ólafur S. Andrésson, Dr. Denis Warshan and Hörður Guðmundsson from the University of Iceland for helping me with the practical work and for providing facilities for my research work. A special thanks goes out to Dr. Philipp Resl for his extensive help in data analysis, which was much-needed, and to Dr. Fernando Fernández-Mendoza for always offering his advice and assistance. I also want to thank the Icelandic Research Fund for funding this project (grant number 174307-051), the Biomedical Sequencing Facility in Vienna for conducting the sequencing with the HiSeq system, the Icelandic Institute of Natural History for the permission to collect and export lichens, and all the people from the Institute of Plant Sciences at the University of Graz for their support. Last but not least, I would like to thank my family and friends for their continuous encouragement, motivation, and guidance they have given me throughout my studies. I could not have done it without them!

Index Abstract ...... 3 Kurzzusammenfassung ...... 4 Abbreviations ...... 5 List of figures ...... 6 List of tables ...... 6 Introduction ...... 7 Materials and Methods ...... 12 Terminology ...... 12 Biological material ...... 12 Sampling ...... 13 Molecular data ...... 13 Preparation steps ...... 13 Sequencing ...... 15 Bioinformatical analyses ...... 15 Results ...... 16 Quality assessment of MiSeq and HiSeq data ...... 16 De novo transcriptome assembly with Trinity ...... 19 DESeq2 analysis ...... 19 Functional annotation of differentially expressed genes ...... 25 Upregulated ascomycete genes...... 25 Photomorph-mediated differentially expressed ascomycete genes ...... 28 Differentially expressed ascomycete genes in dependency on temperature ...... 30 Differentially expressed cyanobacterial genes in dependency on temperature ...... 33 Differentially expressed chlorophyte genes in dependency on temperature ...... 36 Discussion ...... 39 Photomorph-mediated differentially expressed ascomycete genes ...... 39 Differentially expressed ascomycete genes in dependency on temperature ...... 43 Differentially expressed cyanobacterial genes in dependency on temperature ...... 46 Differentially expressed chlorophyte genes in dependency on temperature ...... 49 Conclusion ...... 53 Literature cited ...... 55

Abstract Photosymbiodemes represent a special case of a lichen symbiosis as the mycobiont enters a symbiosis with two different photosynthetic partners, a cyanobacterium and an alga, developing two distinctly looking photomorphs, a cyanomorph and a chloromorph. These morphs can either grow as two separate individuals or form a single individual. A lichen that is known to grow as a photosymbiodeme is Peltigera britannica. Its photosymbiodemes forming a single individual are found in shadowy and moist habitats whereas the chloromorph forming an independent individual can grow in sunny and dry habitats as well; at least this was observed on P. britannica photosymbiodemes in Iceland. This distribution pattern led to the hypothesis that photosymbiodemes show signs of stress when exposed to higher temperatures; with the chloromorph being more tolerant to heat stress than the cyanomorph. This hypothesis was tested by exposing photosymbiodeme-forming P. britannica specimens to temperatures of 4°C, 15°C and 25°C. Even though the mycobiont is of the same species in both photomorphs, the fungus’ gene expression might be influenced by its interaction with its photosynthetic partners, possibly resulting in differential fungal gene expression. Both the mycobiont’s gene expression and the effects of thermal stress on the organisms of a lichen symbiosis were examined conducting a differential gene expression analysis. For that, RNA libraries were constructed which were sequenced and used for a de novo transcriptome assembly. Differential gene expression analysis was conducted using the program DESeq2. The most significantly differentially expressed genes were functionally annotated. In dependency on the temperature a number of fungal, green algal and cyanobacterial genes were differentially expressed. High temperatures led to an upregulation of heat shock and heat- shock like proteins as well as genes responsible for DNA repair mechanisms in all organisms involved. The fungus and the cyanobacteria appear to experience thermal stress already at 15°C, the green algae only at 25°C. This indicates that the green algal photobiont is more tolerant to heat stress. Furthermore, photosynthetic activity was increased in both photobionts at high temperatures. Various ascomycete genes were differentially expressed in the different photomorphs. In the cyanomorph the upregulated fungal genes encoded an isopenicillin synthetase as well as SUN domain proteins; the latter are responsible for cell wall synthesis. The upregulated fungal genes in the chloromorph encoded a glutathione-S-transferase and proteins responsible for the provision of carbon. These results indicate that the fungal, cyanobacterial and green algal partners of a lichen symbiosis react differently to and cope differently with stress. Additionally, the fungus seems to be affected by its photosynthetic partners regarding its gene expression, as the fungal gene expression in the chloromorph and in the cyanomorph is differentially.

Kurzzusammenfassung Photosymbiodeme stellen einen speziellen Fall einer Flechtensymbiose dar, in welcher der Mykobiont eine Symbiose mit zwei unterschiedlichen Photobionten eingeht, nämlich mit einem Cyanobakterium und einer Alge. Dadurch entstehen zwei unterschiedlich aussehende Photomorphe, Cyanomorphe und Chloromorphe genannt. Diese Morphe wachsen entweder als zwei getrennte Individuen oder bilden ein einziges Individuum. Peltigera britannica ist eine der Flechten, die Photosymbiodeme hervorbringen können. Die Photosymbiodeme dieser Flechte, die als ein einzelnes Individuum wachsen, finden sich in schattigen und feuchten Habitaten. Die Chloromorphe, die als separate Individuen wachsen, kommen jedoch auch in sonnigen und trockenen Habitaten vor; zumindest wurde dies an P. britannica-Exemplaren in Island beobachtet. Aufgrund dieses Verbreitungsmusters wurde die Hypothese aufgestellt, dass Photosymbiodeme auf erhöhte Temperaturen gestresst reagieren, die Chloromorphen Hitze aber besser vertragen als die Cyanomorphen. Diese Annahme wurde untersucht, indem P. britannica- Exemplare, die Photosymbiodeme bilden, Temperaturen von 4°C, 15°C und 25°C ausgesetzt wurden. Obwohl der Pilzpartner in beiden Morphen derselbe ist, könnte es sein, dass die Genexpression des Pilzes aufgrund der Interaktion mit dessen Photosynthese-betreibenden Partnern beeinflusst wird, was eine differentielle Expression der Pilzgene zur Folge hätte. Die Genexpression des Mykobionten sowie die Auswirkungen von Hitzestress auf die Organismen einer Flechtensymbiose wurden mittels einer differentiellen Genexpressionsanalyse untersucht. Dafür wurden RNA- Bibliotheken erzeugt, die sequenziert und für eine de novo Transkriptom-Assemblierung herangezogen wurden. Die differentielle Genexpressionsanalyse wurde mit dem Programm DESeq2 durchgeführt. Die signifikantesten differentiell exprimierten Gene wurden funktionell annotiert. In Abhängigkeit von der Temperatur fanden sich eine Vielzahl von Pilz-, Cyanobakterien- und Grünalgengenen, die differentiell exprimiert wurden. Erhöhte Temperaturen führten zu einer Hochregulierung von Hitzeschockproteinen, Hitzeschock-ähnlichen Proteinen und Genen, die für die DNA- Reparatur von Bedeutung sind – in allen Organismen der Flechtensymbiose. Der Pilz und die Cyanobakterien scheinen bereits bei 15°C hitzegestresst zu sein, die Grünalgen erst bei 25°C. Dies deutet darauf hin, dass die Morphe mit den Grünalgen toleranter gegenüber Hitzestress ist als die Cyanomorphe. Des Weiteren zeigten beide Photobionten bei hohen Temperaturen eine erhöhte Photosyntheseaktivität. Mehrere Pilzgene waren in Abhängigkeit vom Photomorph differentiell exprimiert. In der Cyanomorphe codierten die hochregulierten Pilzgene für eine Isopenicillin-Synthetase sowie für Proteine der SUN Domäne; letztere ist für die Zellwandsynthese von Bedeutung. Die hochregulierten Pilzgene der Chloromorphe codierten für eine Glutathion-S-Transferase sowie für Proteine, die für die Kohlenstoffversorgung im Organismus verantwortlich sind. Diese Resultate implizieren, dass die Pilz-, Cyanobakterien- und Grünalgenpartner einer Flechtensymbiose unterschiedlich auf Stress reagieren und unterschiedlich mit diesem umgehen. Des Weiteren scheint die Genexpression des Pilzes durch dessen Photosynthese-betreibende Partner beeinflusst zu werden, da die Genexpression des Pilzes der Chloromorphe sich von jener der Cyanomorphe unterscheidet.

Abbreviations Amt ammonium transporter ANOVA analysis of variance ARPC5 actin-related protein 2/3 complex subunit 5 C carbon CO2 carbon dioxide DE genes differentially expressed genes dds DESeqDataSet DGE differential gene expression DNA deoxyribonucleic acid GO gene ontology GSH glutathione GSSG glutathione disulphide GST glutathione-S-transferase Hsf heat shock factor Hsp heat shock protein LHC light-harvesting complex log2FC log2Fold Change mRNA messenger RNA N nitrogen NH4+ ammonium NO3- nitrate OEC oxygen-evolving complex PSI photosystem I PSII photosystem II RNA ribonucleic acid ROS reactive oxygen species RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase rpoE RNA polymerase, extracytoplasmic E SUMO small ubiquitin-like modifier TE transposable element tRNA transfer RNA

List of figures Figure 1: Photosymbiodemes of the lichen Peltigera britannica...... 10 Figure 2: A lava field at Heiðmörk reserve in Iceland...... 12 Figure 3: The four specimens of P. britannica (1.2, 2.2, 3.2 and 4.2 – from top to bottom)...... 14 Figure 4: MA plots of differentially expressed genes according to DESeq2 analysis...... 20 Figure 5: Enhanced volcano plots of photomorph-mediated DE genes...... 22 Figure 6: Enhanced volcano plots of DE genes in dependency on temperature...... 23 Figure 7: Enhanced volcano plots of DE genes in depdendency on individuals...... 24 Figure 8: Molecular functions of upregulated ascomycete genes in the chloromorph...... 26 Figure 9: Molecular functions of upregulated ascomycete genes in the cyanomorph...... 27 Figure 10: Specific hydrolase and transferase activity of the upregulated ascomycete genes in the cyanomorph...... 27 Figure 11: Photomorph-mediated DE ascomycete gene TRINITY_DN48557_c0_g1...... 28 Figure 12: Photomorph-mediated DE ascomycete gene TRINITY_DN47170_c11_g1...... 28 Figure 13: Photomorph-mediated DE ascomycete gene TRINITY_DN48101_c1_g1...... 29 Figure 14: Photomorph-mediated DE asocmycete gene TRINITY_DN24613_c0_g1...... 30 Figure 15: DE ascomycete gene TRINITY_DN44171_c0_g1 in dependency on temperature...... 30 Figure 16: DE ascomycete gene TRINITY_DN35635_c0_g1 in dependency on temperature...... 31 Figure 17: DE ascomycete gene TRINITY_DN35336_c0_g1 in dependency on temperature...... 31 Figure 18: DE ascomycete gene TRINITY_DN25204_c0_g1 in dependency on temperature...... 32 Figure 19: DE ascomycete gene TRINITY_DN46772_c7_g4 in dependency on temperature...... 32 Figure 20: DE cyanobacterial gene TRINITY_DN46027_c3_g10 in dependency on temperature...... 33 Figure 21: DE cyanobacterial gene TRINITY_DN46638_c0_g1 in dependency on temperature...... 33 Figure 22: DE cyanobacterial gene TRINITY_DN46972_c1_g1 in dependency on temperature...... 34 Figure 23: DE cyanobacterial gene TRINITY_DN48753_c1_g2 in dependency on temperature...... 35 Figure 24: DE cyanobacterial gene TRINITY_DN44076_c0_g1 in dependency on temperature...... 35 Figure 25: DE chlorophyte gene TRINITY_DN47627_c2_g1 in dependency on temperature...... 36 Figure 26: DE chlorophyte gene TRINITY_DN46800_c0_g1 in dependency on temperature...... 36 Figure 27: DE chlorophyte gene TRINITY_DN43942_c0_g1 in dependency on temperature...... 37 Figure 28: DE chlorophyte gene TRINITY_DN48593_c1_g2 in dependency on temperature...... 38 Figure 29: DE chlorophyte gene TRINITY_DN43001_c0_g4 in dependency on temperature...... 38

List of tables Table 1: MiSeq data...... 17 Table 2: MultiQC report for the HiSeq data...... 18 Table 3: Statistics of the Trinity assembly...... 19 Table 4: Number of DE genes of ascomycetes, chlorophytes and cyanobacteria...... 22

Introduction A lichen is a symbiotic organism, the main partner in the symbiosis being a lichen-forming fungus, also called mycobiont. The mycobiont enters a symbiotic relationship with a photosynthetic partner (a photobiont) which can either be an alga and/or a cyanobacterium (Lumbsch, 2016). The most abundant cyanobacterial partner in a lichen symbiosis is the genus Nostoc whereas the most common algal partner is the green algal genus Trebouxia (Rikkinen, 2002). While lichens are generally considered a mutualistic symbiosis, the lichen-forming fungus could also act as a parasite, as it obtains photosynthetic products and other compounds from its partner whereas it is not entirely established to what extent the photosynthetic partner benefits from this relationship. The mycobiont might be responsible for the optimal positioning of the thallus for proper illumination and gas exchange and thereby supplying the photobiont with adequate conditions (Honegger, 1993). Besides, the fungal partner is in charge of water transport and storage as well, supplying the photobiont with the water it requires for photosynthesis (Rikkinen, 2002). Hyvärinen et al. (2002) however describe the mycobiont as an “optimal harvester” as it seems to maximise the photosynthetic activity and/or nitrogen fixation activity of the photobiont by controlling its growth and reproduction as well as the frequency of heterocysts (cells for nitrogen fixation) in the cyanobacterial partner. Furthermore, it has been described, that it is the mycobiont who is choosing the photosynthetic partner rather than the other way around. This has been referred to as “photobiont recruitment” and underlines the more dominant role of the mycobiont in lichen symbiosis (Hill, 2009). Even though the lichen-forming fungi can belong to the division of the Basidiomycota the vast majority of the fungi – about 98% – belong to the Ascomycota (Werth, 2011). The algae mostly belong to the green algae, but other algae can also be found as partners in lichen symbiosis. About 20% of all known fungi form lichens. Considering this high number, it is not surprising that lichens can be found in every ecosystem and numerous habitat types (Lumbsch, 2016). Their wide range of distribution can also be attributed to their poikilohydric nature, which enables lichens to even survive in harsh environments like the polar regions and withstand extreme temperatures (Werth, 2011). Furthermore, the presence of the photobiont is of relevance as well, as host specialisation – depending on the species of lichen-forming fungus – can be high or low, and as a consequence, the range of a lichen species can either be narrow or wide (O'Brien, et al., 2005). The morphology of lichens is diverse. In general, three main growth forms can be distinguished: crustose, foliose and fruticose. In a crustose lichen, the thalli grow in a crust-like form, usually tightly attached to the substrate. A foliose lichen has a leaf-like, lobate shape, horizontal growth and is more or less loosely attached to its substrate. Fruticose lichens form more complex shrub-like structures, as their thalli grow three- dimensionally and frequently are branched (Wirth & Kirschbaum, 2014). The thallus of a lichen mainly consists of the fungal hyphae which are responsible for building various morphological structures – possibly due to stimulus by the photosynthetic partner – and which accommodate the photobiont. The cells of the photobiont are either randomly distributed in the thallus (homomerous thallus) or occur in an algal layer (heteromerous thallus); the latter is the most abundant pattern (Ahmadjian, 1987).

Peltigera is a cosmopolitan genus of lichen-forming fungi, its terricolous (soil inhabiting) and muscicolous (moss inhabiting) species forming small to large foliose lichen thalli. Species of Peltigera can either contain cyanobacteria (Nostoc sp.) or green algae (Coccomyxa sp.) as photobionts (O'Brien, et al., 2009). Some Peltigera fungi can enter a symbiosis with cyanobacteria and green algae, or only one type at the time (Elvebakk, et al., 2008), then referred to as chloromorphs for the green-algal type or cyanomorphs for the cyanobacterial type (Goffinet & Hastings, 1994). These species of Peltigera are mostly found in boreal ecosystems because their distribution is likely to be limited by the occurrence of Coccomyxa, whereas lineages of the cyanobacterial partner Nostoc are more common throughout various ecozones (Pardo-De la Hoz, et al., 2018). Tripartite thalli contain green algae plus another symbiotic partner, in the form of cyanobacteria that are housed in special structures called cephalodia which grow on the upper surface of the thallus. As these lichens have a cyanobacterial partner, they can fix nitrogen, which is an advantage in colonising nutrient-poor habitats (Goffinet & Hastings, 1994). In contrast, bipartite lichens like Peltigera membranacea have only one photobiont partner (Hyvärinen, et al., 2002). When a mycobiont enters a symbiosis with two different photosynthetic partners, it forms two so-called photomorphs. The chloromorph contains only green algae while the cyanomorph contains only cyanobacteria as a symbiosis partner. These photomorphs might either develop separately and form independent individuals, or they might be attached to one another. Even though they might differ in morphology, anatomy, chemistry and other properties both photomorphs are considered to belong to the same species, as the mycobiont in this symbiosis is the same in both morphs – and the mycobiont is the species-determining partner in a lichen symbiosis (Goffinet & Hastings, 1994). The distinct photomorphs are therefore considered to be “phenotypes of a single genotype” (Goffinet & Bayer, 1997). The thallus of Peltigera species is heteromerous, this means that the structure of the thallus is layered, consisting of the upper cortex, the algal layer, and the medulla (containing hyphae only). The upper surface of Peltigera species is corticated whereas the lower surface is without a cortex, but has veins and rhizines, which can be used for the identification of species. The cortex of the Peltigera species can differ considerably in its texture, ranging from smooth to wrinkled and glabrous to hairy (Galloway, 2000). Some species have a tomentum on their upper surface, a cluster of short and fine hyphae that resemble hair and give the surface a felted appearance (Goffinet & Hastings, 1994). The lobes and their margins also vary, being either plane or up- or downturned. Usually, the lobes are rounded, but they can be of an elongated shape as well. Moreover, they do vary in their thickness, with some species having thin and rather brittle lobes (Peltigera membranacea), while others have extremely thick ones (P. aphthosa) (Goffinet & Hastings, 1994). The colour of the cortex also varies from species to species, but cannot be used in determining species, as it is dependent on environmental factors like sunlight. Usually, specimens exposed to the sun are rather brown, whereas the ones growing in shady habitats are blue-grey or blue-green (in species with cyanobacteria as a photobiont) (Galloway, 2000). The veins on the lower surface can be indistinct, broad or narrow; their relief can be low, raised or overlapping (Goward, et al., 1995). The colour of the veins can range from whitish and pinkish to brown and black. Also, the spaces in between the veins, the so-called interstices, are useful in the identification of species, as they often differ in shape (oval, polygonal, lenticular). The rhizines are hyphal extensions with which the lichen attaches to the substrate. They can differ in colour and shape (simple to brush-like) but are not a good indicator for species delimitation (Galloway, 2000) as rhizines at the margin and in the centre can look differently within one species. Furthermore, the

8 appearance of rhizines seems to be depending on locality and possibly the substrate (Goffinet & Hastings, 1994). Many species of Peltigera do develop apothecia, which generally are discs of various brown colours located on the lobe margins. They can be of various shapes, including flat, saddle-shaped or round. Their heterogeneity can be used for the identification of species, but apothecia are not entirely reliable in species identification (Galloway, 2000). Structures like isidia, phyllidia, regeneration lobules, and soredia serve vegetative reproduction (Goffinet & Hastings, 1994). However, thallus fragmentation seems to be a relevant mode of vegetative dispersion as well (Magain, et al., 2018). Regarding their chemistry, Peltigera species either lack lichen substances or contain compounds produced in the acetate-polymalonate pathway and/or the mevalonic acid pathway (Galloway, 2000). Despite the vast amount of research on the Peltigera genus the identification of its species is notoriously difficult and there are still many disputed species, with their exact number not being conclusive (Goward, et al., 1995); so far at least 66 species of Peltigera have been acknowledged (Martínez, et al., 2003). Morphology and chemistry are often not sufficient for correctly identifying a Peltigera species, and instead, molecular DNA sequence data is primarily used nowadays. However, as several recent studies have shown, these data do not necessarily clarify species delimitation and phylogeny but rather reveal the overall complexity of the genus Peltigera (e.g. Magain et al., 2018; Miadlikowska & Lutzoni, 2000; Miadlikowska et al., 2014; Lumbsch & Leavitt, 2011).

The lichen used in this study is Peltigera britannica (Gyelnik) Holtan-Hartwig & Tønsberg, first described in 1983 (Holtan-Hartwig, 1993). It is a holarctic species but does seem to prefer oceanic climates as it has been primarily described for north-western and western Europe as well as north-western North America (Martínez, et al., 2003). Like other species of the Peltigera group, P. britannica forms two different morphotypes: one containing cyanobacteria (Nostoc sp.), the other green algae (Coccomyxa sp.). The two morphs of P. britannica do look very distinctively. The photomorph with the green algae is of a greyish green colour when dry (Holtan-Hartwig, 1993) and of an apple green colour when wet (Goffinet & Hastings, 1994). This photomorph is tripartite, housing cyanobacteria in so-called cephalodia – this means, it has two photosymbiotic partners, a green alga and a cyanobacterium. Since many other Peltigera species do have tripartite morphs, the cephalodia can be used in the identification of species. In P. britannica, the cephalodia are peltate and can be found on the upper cortex, forming dark spots of about 2mm in diameter. They are shell-shaped and fall off rather easily, leaving white scars on the upper surface. In this, P. britannica can be distinguished from similar species like Peltigera aphthosa or Peltigera leucophlebia which do not have shell-shaped cephalodia and which lack the white scars, as their cephalodia do not tend to fall off. The lower side is usually white on the margins (Holtan-Hartwig, 1993), however, it turns abruptly brown or black towards the centre (Goward, et al., 1995). The rhizines are dark as well, their form varying from simple to bush-shaped. The thallus can reach a diameter of up to 40cm, and individual lobes are about 2 to 3 cm wide. The edges of the lobes have been described as involute (Holtan-Hartwig, 1993). The morphotype containing the cyanobacteria is of a dark grey colour, its upper cortex being blue-grey to brown. The lower side resembles that of the green algal photomorph in being white on the margins and dark in the centre. The rhizines are dark and simple to bush-shaped in the cyanobacterial photomorph. The thallus itself can become rather large, reaching a size of up to 30cm in diameter. The lobes usually have a width of about 1.5 to 2cm, their edges being denticulated (Holtan-Hartwig, 1993). 9

These photomorphs can grow as separate individuals, but they have also been found to grow as one individual. Usually, the morph with the cyanobacteria can be found attached to the green algae morph, seemingly developing from its cephalodia (Goward, et al., 1995). One thallus can, therefore, carry both photomorphs attached to each other, forming a so- called photosymbiodeme. But it is not just the cyanobacterial lobes growing on the green algae ones, as green algal lobes can also emerge on cyanobacterial ones (figure 1). The independent growth of the cyanomorph seems to be facilitated by the loosely attached cephalodia on the tripartite thallus – as cephalodia break off, they can be distributed to a new habitat (Holtan-Hartwig, 1993).

Figure 1: Photosymbiodemes of the lichen Peltigera britannica. In the left picture, green-algal and tripartite lobes emerge from a bipartite thallus that has a cyanobacterium as a photosynthetic partner; in the right picture, small cyanobacterial lobes develop from the cephalodia of a tripartite thallus.

The chemotypes of both morphs are similar, as both contain Tenuiorin-aggregate, phlebic acid A and B, unidentified terpenoids (Holtan-Hartwig, 1993), methyl gyrophorate, Figuregyrophoric 2: Photosymbiodemes acid, dolichorrhizin of the lichen Peltigera and zeorinbritannica (Goward,. In the left picture et al.,, gre en 1995)-algal and. P. tripartite britannica lobes emerge is a fromhygrophyte, a bipartite thallus regardless that has a of cyanobacterium the photomorph as a photosynthetic in question. partner; The in the chloromorph right picture, small grows cyanobacterial over lobes develop from the cephalodia of a tripartite thallus. moss and seems to prefer sheltered and shady coastal habitats. It also occurs further inland, if the area is humid enough (Goward, et al., 1995). The cyanomorph seems to prefer humid and shady habitats to a greater extent than the chloromorph. Moisture also seems to be the driving factor for the development of photosymbiodemes, as cephalodia stay rather small in drier habitats, whereas they get bigger in damp conditions (Holtan- Hartwig, 1993). Miadlikowska & Lutzoni (2000) have organised the genus Peltigera into eight sections. According to their research, P. britannica belongs to the section Peltidea within the genus Peltigera. Three other species are assigned to this section: P. aphthosa, P. malacea and P. frippii.

Lichen-forming fungi that are able to enter a symbiosis with a cyanobacterium, as well as with a green alga, and which form photomorphs are referred to as photosymbiodemes. A photosymbiodeme may consist of both tripartite and bipartite thalli (Hyvärinen, et al., 2002). Even though the thalli of photosymbiodemes usually carry two kinds of distinctively looking lobe types, the lichen species is the same (Armaleo & Clerc, 1991). Photosymbiodemes occur in several lichen genera, including Peltigera, Sticta, Nephroma, Lobaria, and Pseudocyphellaria (Green, et al., 2002).

In general, lichen species that carry either a cyanobacterial partner or an algal partner in two different thalli do not grow under the same environmental conditions. Lichens with cyanobacteria occur in rather wet habitats whereas lichens with green algae are usually more tolerant towards drier, although still humid, habitats. As photosymbiodemes contain both photobionts it, therefore, seems plausible for them to be restricted to certain ecological niches, in which both photobionts are able to benefit to a certain extent from the environmental factors, or, at least, where they are not confronted with major disadvantages (Green, et al., 1993). As described by Green et al. (2002), photosymbiodemes usually grow on forest margins, as these habitats offer liquid water (in the form of rainfall), which is of relevance for the photosynthetic activity of cyanobacteria, as well as humidity, which green algae need for a positive net photosynthesis (and which they do prefer over liquid water). This circumstance could also be observed in our study, as the P. britannica samples collected only grew in damp, relatively hidden spots, for example in small cavities or under branches of shrubs growing on an incline – as long as there were enough light and air circulation to promote the growth of an organism. Out in the open field where the lichens were more exposed to the elements (as there is less shading, greater temperature fluctuation and a higher risk of wind and desiccation) photosymbiodemes of P. britannica could not be found; even though tripartite thalli of P. britannica – so thalli with green algae and cephalodia – were growing there. Thalli with only a cyanobacterial partner, or with cyanobacterial lobes, were missing. Light and humidity gradients, among other factors, seemingly influence the occurrence of lichens as either photosymbiodemes or separate individuals (Elvebakk, et al., 2008).

I performed a thermal stress experiment with photosymbiodemes of Peltigera britannica and transcriptome sequencing to assess if chloromorph and cyanomorph lobes show a differential stress response. The observation that cyanobacterial thalli prefer rather stable environmental factors and wet and cold habitats led to the hypothesis that the cyanomorphs of the photosymbiodemes show increased stress response when exposed to higher temperatures than the chloromorphs. This was tested by exposing photosymbiodeme-forming specimens to three temperatures (4°C, 15°C and 25°C) and conducting differential gene expression analysis. Considering the lichens were sampled in Iceland, a country with comparatively cold climate conditions, an increase in temperature should result in an overexpression of stress genes in both photomorphs, but more so in the cyanomorph. The gene expression of the fungal partner in both photomorphs is also of interest in this study, as it is assumed that the mycobiont is being influenced in one way or another by its photosynthetic partner, which is likely to influence fungal gene expression. According to this hypothesis, the mycobiont of a chloromorph should differ in its gene expression from the mycobiont of a cyanomorph, despite the mycobiont being of the same fungal individual in both morphs. Furthermore, it was examined if there are any major genetic differences between the four investigated individuals of the lichen P. britannica when exposed to higher temperatures. It is expected that there are differences between individuals, even though they were collected from neighbouring sites, because each specimen is likely to react to its environment or stress situations in a different and individual-dependent manner.

Materials and Methods Terminology As the P. britannica samples used in this study are photosymbiodemes it does seem to be necessary to establish some rules regarding nomenclature respectively vocabulary to avoid confusion – especially considering that many options of nomenclature in lichenology have been proposed over the years (James 1975; James & Henssen 1976; Renner & Galloway 1982; Galloway 1988; Purvis et al. 1992; Laundon 1995 & 1996, Jørgensen 1991, 1996 & 1997). The fungal partner is referred to as mycobiont, as it is common in the field of lichenology. The photosynthetic partner, regardless of it being an alga or a cyanobacterium, will be referred to as photobiont. To tell apart the cyanobacterial partner from the algal partner the terms cyanomorph and chloromorph will be used, respectively. In regard to P. britannica, the different photobionts will occasionally be called P. britannica/cyan and P. britannica/chlor, the photosymbiodeme P. britannica/chlor+cyan (Heiðmarsson, et al., 1997). As P. britannica does not have thalli with only a green algal photobiont, the terms chloromorph and P. britannica/chlor in this study refer to the tripartite thalli with both green algae and cephalodia. Biological material The Peltigera britannica specimens were collected at Heiðmörk reserve in southwestern Iceland, about 10km southeast of Reykjavík (specimen 1.2 and 2.2: 64°4'5.04728''N, 21°43'38.87947''W; specimen 3.2: 64°4'5.19143''N, 21°43'39.02599''W; specimen 4.2: 64°4'5.44984''N, 21°43'40.89662''W) on the 13th of August 2017. The day temperatures were about 10°C to 12°C. Each of the four thalli used for this study were photosymbiodemes. The four lichen thalli were growing among mosses in shady and moist sites, usually in cavities of lava rocks (figure 2) or under branches of shrubs that are enrooted in these cavities. The lichens were identified as Peltigera britannica by Silke Werth (Ludwig Maximilian University of Munich). Three of the four specimens collected were rather big (approximately 10-12 cm) and consisted mainly of cyanomorphs with small to moderate sized lobes of the chloromorph attached to them (specimens 1.2, 2.2 and 3.2). The fourth sample was only about 6cm in diameter but was a tripartite morph with a small cyanormoph lobe ascending from it (specimen 4.2) (see Figure 3: A lava field at Heiðmörk reserve in Iceland. In the cavities of such lava rocks, near trees and shrubs, photosymbiodemes of P. britannica could be found. figure 3). As photosymbiodemes were rare in Iceland, they were only taken from those sites, where other photosymbiodemes Figurewere 4 :present A lava field as at well, Heiðmörk to prevent reserve in Iceland.overharvesting. In the cavities of such lava rocks, near trees and shrubs, photosymbiodemes of P. britannica could be found. 12

Sampling After collecting the P. britannica specimens, they were kept in a cold room under a diurnal light-dark cycle (12 hours of light and 12 hours of darkness) at a temperature of 4°C to avoid temperature stress. It was also at this temperature that they were first sampled: pieces of tissue (about 1cm² each) of the cyanomorph as well as the chloromorph were cut off from the four specimens (except specimen 4.2 from which only the chloromorph was used as the cyanomorph was too small). The samples were immediately shock frozen in liquid nitrogen and then stored at -80°C. In figure 3 the process of sampling is displayed; these samples are labelled as Chloro_4°C 1 and Cyano_4°C 1 respectively. The lichens were kept in the cold room at 4°C and were watered regularly with distilled water to prevent them from desiccating. Once a week they were left to dry out however, as this is more natural than a permanent oversaturation with water. After an adaption period of 14 days at 4°C, the lichens were sampled again. As in the first run pieces of lobe were taken from each photomorph of every specimen. This time however, the cyanomorph of the specimen 4.2 was also sampled. The sampling can be seen in figure 3 (labelled as Chloro_4°C 2 and Cyano 4°C 2). The obtained samples were frozen in liquid nitrogen and stored at -80°C as well. Afterwards the lichens were sprayed with distilled water and were transferred to a plant growth chamber with 15°C where they were left for two hours before they were sampled in the same manner as before. In the last run the temperature in the plant growth chamber was raised to 25°C and the lichens were left there for two hours as well before sampling them in the same way as before. At the higher temperatures, the specimens desiccated far quicker so they had to be watered with distilled water occasionally in the course of these two hours. The sampling at 15°C and 25°C is depicted in figure 3 as well. Just as in the first two steps, the samples were frozen in liquid nitrogen and stored at -80°C. As specimen 1.2 had a larger thallus more samples were taken at the temperatures of 4°C (after two weeks in the cold chamber), 15°C and 25°C; these are referred to as A, B and C and were taken from three different sites of the thallus. However, only the samples that offered the best reads after sequencing were used for further analysis steps – and it is only these samples that are denoted in figure 3. Molecular data Preparation steps The green algal genus of Coccomyxa contains sporopollenins in its cell wall, exceptionally resilient biopolymers that are commonly found in the cell walls of pollen. These biopolymers can withstand multiple alkali and acid treatments which is why the disruption of Coccomyxa cell walls for the purpose of RNA isolation is hampered (Brunner & Honegger, 1985). Therefore, the samples first had to be processed to enable the cell wall disruption by means of various procedures in order to render them useful for RNA isolation. To make the cell wall of the algae more brittle water has to be removed – this was done by lyophilising the samples at -58°C while applying a vacuum of 31µbar for 12 hours (VirTis Sentry 2.0 – SP Scientific). After lyophilisation the tubes with the samples were closed immediately as a thawing of the samples had to be averted. The samples were shock frozen in liquid nitrogen once again, a metal bead was added to each tube and the lobe samples were then ground with a tissue lyser bead mill (QIAGEN® TissueLyser II).

Figure 5: For legend see opposite

Figure 6: For legend see opposite The RNA was isolated with the innuPREP Plant RNA Kit (Analytik Jena AG) according to the manufacturer’s instructions. However, instead of using the RL lysis buffer, the PL lysis buffer was used, as it resulted in a higher concentration of isolated RNA in previous test runs. The RNA libraries were constructed – with the help of Hörður Guðmundsson from the University of Iceland – using TruSeq® Stranded mRNA Library Prep (Illumina®). The libraries were poly-A selected. Sequencing The library samples were processed further at the University of Iceland by Denis Warshan, Hörður Guðmundsson and Ólafur S. Andrésson who sequenced the samples with a MiSeq system (Illumina®) producing paired-end reads (2x150b). The quality of the MiSeq results was checked by Philipp Resl (Ludwig Maximilian University of Munich) who mapped the reads to the fungal, algal and cyanobacterial genomes and assessed how the libraries should be pooled for further analysis on a HiSeq system. The pooling itself was conducted by Denis Warshan, who afterwards sent the libraries to the Biomedical Sequencing Facility in Vienna. The Biomedical Sequencing Facility sequenced the libraries using the HiSeq system HiSeq 3000/4000 SR (Illumina ®) and dual indexing, producing single-end reads. Bioinformatical analyses The quality of the MiSeq data as well as the HiSeq data was assessed using the tool FastQC (version 0.11.5; URL: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The FASTX-toolkit (version 0.0.13; URL: http://hannonlab.cshl.edu/fastx_toolkit/) was used for quality filtering and removal of bases with bad quality (e.g. noise). Adapters were removed by trimming the reads with the tool Trimmomatic (version 0.36; URL: http://www.usadellab.org/cms/?page=trimmomatic). After quality filtering, the overall quality of the reads was assessed once again, using the tool MultiQC (version 1.1; URL: https://multiqc.info/). As a reference genome was not available, the processed MiSeq data was used for a de novo transcriptome assembly, using the software Trinity (version 2.4.0; URL: https://github.com/trinityrnaseq/trinityrnaseq/wiki). The quality of the assembly was assessed with TrinityStats.pl, a Trinity script. With the RNA-seq quantification program kallisto (version 0.45.0; URL: https://pachterlab.github.io/kallisto/) the HiSeq data was pseudoaligned to the novo transcriptome assembly. Coding regions were identified with TransDecoder (http://transdecoder.github.io). Statistical analyses were executed with R (version 3.5.2; R Core Team (2018)). Differential gene expression (DGE) analysis was conducted using the program DESeq2 (version 1.22.2 (Love, et al., 2014)). MA plots were constructed to assess data quality and to check if any differentially expressed genes were present. DESeq2 results were visualized with the package EnhancedVolcano (version 1.0.1 (Blighe, 2019). Using MEGAN6 (version 6.13.1; URL: http://ab.inf.uni-tuebingen.de/software/megan6/) the genes were taxonomically assigned; this step was conducted by Philipp Resl. Only genes belonging either to an ascomycete, a chlorophyte or a cyanobacterium were used for DGE analysis. Figure 7: The four specimens of P. britannica (1.2, 2.2, 3.2 and 4.2 – from top to bottom). The pictures were taken just before the first sampling took place (left column). On the right-hand side the spots from where the samples were taken are pictured. The blue font indicates the sampling from the cyanomorph, the black font from the chloromorph. Cyano_4°C 1 and Chloro_4°C 1 respectively refer to the first sampling at 4°C right after the specimens were collected – in later analysis these first samples will be labeled as control samples. Cyano_4°C 2 and Chloro_4°C 2 respectively refer to the second sampling at 4°C and will simply be labeled as 4°C samples in later analysis steps. For specimen 4.2, the sample Chloro_25°C is in brackets as this sample was not used for the analysis; as only a small amount of RNA was isolated from Chloro_25°C, a library could not be constructed. 15

Differentially expressed genes were analysed with a two-way ANOVA to determine the statistical significances of the differences; these calculations were done by Silke Werth. Only upregulated genes were used for this statistical analysis – downregulated ones were not incorporated. The differentially expressed genes were sorted by their p-values – the 200 most significantly differentially expressed genes of each organism and each parameter in question were used for functional annotation. Photomorph-mediated differential gene expression was evaluated with the ascomycete genes but not with the chlorophyte or cyanobacteria genes. The effect of temperature on gene expression was examined using both ascomycete, chlorophyte and cyanobacteria genes. With DIAMOND (version 0.9.24.125; URL: https://github.com/bbuchfink/diamond) the sequences of the differentially expressed genes were aligned. Blast2GO 5 Basic (version 5.2.4; Götz et al. 2008; URL: https://www.blast2go.com/) was used to functionally annotate the genes, based on the Gene Ontology (GO) vocabulary. To determine the role of the four to five most significantly differentially expressed genes of each organism, the genes were aligned using the BLAST web interface by NCBI (Sayers et al. 2019; URL: https://blast.ncbi.nlm.nih.gov/Blast.cgi). The most significant alignment was used to discuss the probable role of the gene and how it might be affected by heat stress.

Results Quality assessment of MiSeq and HiSeq data After MiSeq the percentage and number of reads mapped to the fungus, the algae and the cyanobacteria, as well as the unmapped reads were assessed for each sample in order to ascertain the quality of the reads and to check which can be used for HiSeq. Furthermore, the gene coverage of the reference genes (tub (β-tubulin; an algal and fungal reference gene), gpd1 (glycerol-3-phosphate dehydrogenase 1; a fungal reference gene), secA (protein translocase subunit secA; a cyanobacterial reference gene) and rnpB (RNase P RNA component; a cyanobacterial reference gene)) was calculated to determine how the libraries need to be normalized and pooled for HiSeq. This part of the analysis was conducted by Philipp Resl. The results are summarised in table 1. The table on top displays the results for the chloromorph samples, the bottom table those for the cyanomorph samples. Grey fields indicate those samples that were not used for HiSeq as the data quality was not sufficient enough. This generally concerns the samples of the individual 1.2. As the thallus of this individual was larger than the thalli of the other three individuals more samples were taken (A, B and C). Yet, not all of these samples were used for further analysis, only one was chosen. In the chloromorph, the 1.2A samples were considered best for the HiSeq run, in the cyanomorph 1.2A was chosen for the control temperature, 1.2C for the temperature of 4°C, 1.2B for 15°C and 1.2C for 25°C. The decision on whether samples were used for HiSeq or not was based on the number of unmapped reads, as too many reads that do not map to either organism of interest are undesirable. A gene coverage of 0 was also a factor to omit samples as it indicates that no reads had mapped to the reference genes. Furthermore, samples that had reads mapping to all organisms (fungus, Nostoc and Coccomyxa) – even if it was just a small percentage – were preferred over samples that had reads mapping to only one organism. Moreover, the chloromorph sample of individual 4.2 at 25°C was not used for HiSeq either, as the data quality for this sample was too poor, mainly due to the fact that the overall number of genes that had reads mapping to them was too small (only 200 genes).

Table 8: MiSeq data: the gene coverage of the reference genes (tub, gpd1, secA and rnpB), the percentage and numbers of reads mapped to the fungus, the alga (Coccomyxa) and the cyanobacterium (Nostoc) as well as the percentage and number of unmapped reads has been determined following MiSeq. The upper table includes the result of the chloromorph samples, the bottom table those of the cyanomorph samples. Rows in grey depict those samples that were not used for HiSeq, all others were used for HiSeq.

The quality of both the MiSeq and the HiSeq data was assessed using the tool FastQC. In general, the reads were of adequate quality. Some reads contained adapters, which were removed using Trimmomatic. The Per Base Sequence Content section issued a warning for all samples which is due to the library (TruSeq Stranded Total RNA) as it produces a bias at the read starts. Therefore, the differences between the different bases A, G, C and T are increased in the first positions of the read. This bias was removed by cutting the first positions using the FASTX-toolkit. Furthermore, the sequence duplication level was high as well, likely a result of over-sequencing of the library. The duplicates might be PCR duplicates or biological duplicates (e.g. differences in gene expression), yet a distinction between these two types is not possible at this stage. In order to avoid underestimation of gene expression levels, the duplicates were not removed.

Table 2: MultiQC report for the HiSeq data: General statistics include duplicate reads (%), average GC content (%) and total sequences (millions). Sample Name % Duplicate reads % GC content Total sequences (millions) Chloro_1.2A_04°C_S42748 57.1% 51% 13.8 Chloro_1.2A_15°C_S42752 73.2% 51% 19.9 Chloro_1.2A_25°C_S42753 90.8% 49% 14.7 Chloro_1.2A_control_S42749 63.4% 49% 10.3 Chloro_2.2A_04°C_S42746 61.3% 54% 8.1 Chloro_2.2A_15°C_S42741 87.9% 56% 18.5 Chloro_2.2A_25°C_S42742 86.3% 51% 14.0 Chloro_2.2A_control_S42743 73.7% 53% 7.9 Chloro_3.2A_04°C_S42766 80.1% 52% 36.0 Chloro_3.2A_15°C_S42767 71.2% 51% 14.0 Chloro_3.2A_25°C_S42764 70.4% 52% 12.7 Chloro_3.2A_control_S42763 70.7% 52% 15.2 Chloro_4.2A_04°C_S42757 81.5% 50% 44.2 Chloro_4.2A_15°C_S42758 68.8% 50% 11.4 Chloro_4.2A_control_S42756 77.5% 49% 17.4 Cyano_1.2A_control_S42750 71.9% 49% 20.0 Cyano_1.2B_15°C_S42751 88.1% 49% 5.8 Cyano_1.2C_04°C_S42745 82.9% 46% 7.1 Cyano_1.2C_25°C_S42744 93.9% 48% 10.7 Cyano_2.2A_04°C_S42769 60.6% 48% 9.9 Cyano_2.2A_15°C_S42747 67.6% 49% 6.7 Cyano_2.2A_25°C_S42740 86.2% 48% 18.0 Cyano_2.2A_control_S42768 54.6% 48% 4.4 Cyano_3.2A_04°C_S42761 67.7% 48% 9.2 Cyano_3.2A_15°C_S42765 63.2% 48% 4.3 Cyano_3.2A_25°C_S42762 93.7% 48% 18.1 Cyano_3.2A_control_S42760 71.5% 47% 8.7 Cyano_4.2A_04°C_S42754 60.6% 48% 5.9 Cyano_4.2A_15°C_S42755 60.3% 49% 5.4 Cyano_4.2A_25°C_S42759 77.6% 47% 11.2

After quality filtering, the overall quality of the reads was assessed once again using MultiQC. The general statistics of the MultiQC report for the HiSeq data are displayed in table 2. Trimming the adapters and cutting the first read positions improved the quality scores of the data. There still are many duplicate reads, but these were tolerated. The average GC content ranged between 46% and 56% and was normally distributed for all samples. This means that the GC distribution fits the reference distribution calculated by the program, indicating that the libraries were not contaminated (e.g. with adapters). The total number of sequences varied greatly between samples with a minimum of 4.3 million reads and a maximum of 44.2 million reads, but altogether there were plenty of sequences available to conduct differential gene expression analysis. De novo transcriptome assembly with Trinity The processed MiSeq data was used for a de novo transcriptome assembly, using the software Trinity. The statistics of the Trinity assembly were assessed with the Trinity script TrinityStats.pl and are displayed in table 3. Trinity assembly produced 274600 transcripts, including isoforms. The N50 is 1983, when all transcript contigs are considered, and 1240 when only the longest isoform per 'gene' is taken into consideration. The processed HiSeq data was pseudoaligned to the de novo assembly, using the RNA-seq quantification program kallisto.

Table 3: Statistics of the Trinity assembly. The quality of the assembly was assessed with TrinityStats.pl, a Trinity script. Statistics of the Trinity assembly Number of 'genes' 161023 Number of transcripts 274600 GC content [%] 51.90 N10 contig (based on all transcript contigs) 4459 N10 contig (based on the longest isoform per 'gene') 3757 N50 contig (based on all transcript contigs) 1983 N50 contig (based on the longest isoform per 'gene') 1240

DESeq2 analysis The data was analysed using DESeq2, a program for differential gene expression (DGE) analysis. The evaluation was conducted according to the DESeq2 manual (Love, et al., 2014). To assess the data quality at the beginning of the analysis and in order to check if any differentially expressed genes (DE genes) are present in the samples, MA plots were constructed. The y-axis of the MA plot shows the log2Fold Change (log2FC) of the samples in the DESeqDataSet (dds), the x-axis the normalised gene count. The red dots indicate significantly differentially expressed genes of the DESeqDataSet – the p-value is set to 0.1 by default -, the grey dots are not differentially expressed genes and the triangles depict genes that do not lie within the chosen log2FC values. The MA plots were constructed with all genes of the dataset; the genes had not been taxonomically assigned yet. These plots, therefore, are just interim results that serve to get an overview of the processed data. The main parameters compared to assess the DESeq2 output were “cyanomorph vs chloromorph”, “04°C vs control (4°C)”, “15°C vs control (4°C)” and “25°C vs control (4°C)” (figure 4). There is a large number of genes that are differentially expressed in the chloromorph than in the cyanomorph. The same is true when comparing a temperature of 25°C with the control temperature of 4°C. Differential gene expression is minor when comparing the control temperature of 4°C with a temperature of 15°C as well as with a

19 temperature of 4°C. The latter result indicates, that after two weeks at 4°C the lichens’ gene expression was almost the same as at the beginning. The lichen specimens did not have to acclimatise to temperatures of 4°C, as the laboratory conditions were similar to the conditions of their natural environment.

Figure 4: MA plots of differentially expressed genes (DE genes) according to DESeq2 analysis. Red dots indicate significantly differentially expressed genes; grey dots not significantly differentially expressed genes. Top left: DE genes between cyanomorph and chloromorph. Top right: DE genes between 4°C and control temperature (4°C). Bottom left: DE genes between 15°C and control temperature (4°C). Bottom right: DE genes between 25°C and control temperature (4°C). 20

It cannot be ascertained yet, to which organisms these differentially expressed genes belong, what proteins they encode or what function they have. In order to identify all the aforementioned characteristics further analysis steps were necessary. Using MEGAN6, the genes were taxonomically assigned (the taxonomical assignment was performed by Philipp Resl). Only genes belonging either to an ascomycete, a chlorophyte or a cyanobacterium were used for differential gene expression analysis. The main points of interest were the DGE in dependence on the photomorphs, the temperature as well as the individuals. Therefore, DESeq2 was applied to determine both the differentially expressed ascomycete genes, the differentially expressed chlorophyte genes and the differentially expressed cyanobacteria genes for all three parameters in question. The results were visualised with volcano plots (EnhancedVolcano (Blighe, 2019)). In the graphs below (figures 5, 6 and 7), one can see the differentially expressed genes (ascomycete, chlorophyte and cyanobacteria) in dependency on the morphs, the temperature and the individuals. Red dots indicate genes that are differentially expressed, passing the thresholds for both the p-value and the log2Fold Change (log2FC). By default, the p-value threshold is chosen to be 10e-6 and the log2FC threshold is > |2|. These are also the cut-offs that are depicted in the graphs. The blue dots represent those genes that are differentially expressed according to the p-value but not the log2FC; with the green dots it is the other way round. Grey dots depict genes that are not differentially expressed. There are several photomorph-mediated differentially expressed ascomycete genes (figure 5), even though the thresholds for statistical significance were rather strict. There are downregulated DE genes (negative log2FC) as well as upregulated ones (positive log2FC). The numbers of up- and downregulated of all samples are summarised in table 4. A vast number of the chlorophyte genes in the two photomorphs were differentially expressed. This is to be expected, as the chlorophyte genes in the chloromorph are compared to the chlorophyte genes in the cyanomorph – which does not contain chlorophytes. In figure 5 one can see two clusters of differentially expressed (and downregulated) chlorophyte genes, with the one on the left side consisting of genes that are highly significantly differentially expressed (log2FC of -20 or less and a p-value < 0.01). This high significance is the result of the absence of chlorophyte genes in the cyanomorph. Therefore, almost all of the chlorophyte genes are differentially expressed in dependency on the photomorphs. The result is similar in the cyanobacterial genes in the photomorphs. A large number of genes is differentially expressed. This is due to the fact that the chloromorph should not contain cyanobacteria. However, as the chloromorph is tripartite and does have cephalodia it also contains cyanobacteria. Yet the number of cyanobacterial genes in the chloromorph is still rather low, as their distribution in the morph is restricted to the cephalodia. Hence, most differentially expressed cyanobacterial genes are likely to be found in the cyanomorph rather than the chloromorph. The majority of the differentially expressed cyanobacterial genes in the photomorphs are upregulated.

Table 4: Number of differentially expressed genes of ascomycetes, chlorophytes and cyanobacteria in dependency on photomorphs, temperature and individuals (p-value = 0.01; log2FC >|2|). Differentially expressed genes Upregulated Downregulated Ascomycete genes – photomorphs 84 130 Chlorophyte genes – photomorphs 16 54162 Cyanobacteria genes – photomorphs 611 13 Ascomycete genes – temperature 1020 1097 Chlorophyte genes – temperature 1267 2707 Cyanobacteria genes – temperature 185 / Ascomycete genes – individuals 181 211 Chlorophyte genes – individuals 5994 25875 Cyanobacteria genes - individuals 4 768

Figure 5: Enhanced volcano plots of photomorph-mediated differentially expressed ascomycete (top left), chlorophyte (top right) and cyanobacteria (bottom left) genes. Red dots indicate significantly differentially expressed genes (p-value = 0.01; log2FC >|2|); green dots indicate genes that are differentially expressed according to the log2FC but not to the p-value; blue dots represent differentially expressed genes according to the p-value but not the log2FC; and grey dots indicate genes that are not differentially expressed. There are several differentially expressed ascomycete genes that are up- and downregulated. A vast number of the chlorophyte genes are differentially expressed; these genes are predominantly downregulated. Differentially expressed cyanobacteria genes are abundant as well and mostly are upregulated.

In dependency on the temperature (4°C, 15°C, 25°C and a control temperature (4°C)) there are several differentially expressed ascomycete genes, both upregulated and

22 downregulated (figure 6). Due to the great difference between the temperatures (maximal difference of 21°C) a difference in gene expression was expected. The same is true for the chlorophyte genes in dependency on the temperature. Yet the overall number of differentially expressed chlorophyte genes is higher than for the ascomycete genes (table 4). The differentially expressed cyanobacteria genes in dependency on the temperature are upregulated only. It has not been determined whether these are cyanobacterial genes of the cyanomorph or the chloromorph; most of them probably will be genes of the cyanomorph, but as the chloromorph has cephalodia, there might be differentially expressed cyanobacterial genes in the chloromorph as well. The amount of DE genes is not as high as in the other two samples (table 4), which is expected, as during RNA library construction eukaryotic mRNA was procured using poly-A selection. However, due to carry-over effects cyanobacterial genes are still present in the libraries.

Figure 6: Enhanced volcano plots of differentially expressed ascomycete (top left), chlorophyte (top right) and cyanobacteria (bottom left) genes in dependency on the temperature. Red dots indicate significantly differentially expressed genes (p-value = 0.01; log2FC >|2|); green dots indicate genes that are differentially expressed according to the log2FC but not to the p-value; blue dots represent differentially expressed genes according to the p-value but not the log2FC; and grey dots indicate genes that are not differentially expressed. There are various differentially expressed ascomycete genes that are up- and downregulated. A vast number of the chlorophyte genes are differentially expressed; these genes are both up- and downregulated. Differentially expressed cyanobacteria genes are not as abundant and mostly are upregulated.

Regarding the individuals there are differentially expressed genes for the ascomycete, the chlorophyte and the cyanobacteria as well (figure 7). There are several differentially

23 expressed ascomycete genes which are upregulated as well as downregulated. The DGE for the chlorophyte genes in dependency on the individuals is a lot higher and these genes are both upregulated and downregulated too. For the cyanobacterial genes the DGE in dependency on the individuals is not as pronounced, but there are multiple DE genes; most of them are downregulated, only a few are upregulated.

Figure 7: Enhanced volcano plots of differentially expressed ascomycete (top left), chlorophyte (top right) and cyanobacteria (bottom left) genes in dependency on the individuals. Red dots indicate significantly differentially expressed genes (p-value = 0.01; log2FC >|2|); green dots indicate genes that are differentially expressed according to the log2FC but not to the p-value; blue dots represent differentially expressed genes according to the p-value but not the log2FC; and grey dots indicate genes that are not differentially expressed. There are several differentially expressed ascomycete genes that are up- and downregulated. A vast number of the chlorophyte genes are differentially expressed; these genes are both up- and downregulated. Differentially expressed cyanobacteria genes are not as abundant and mostly are downregulated.

In general, the number of differentially expressed chlorophyte genes is higher in all the parameters tested than the number of differentially expressed ascomycete or cyanobacteria genes (figure 5 to 7). This is probably due to the fact that the number of chlorophyte genes – taxonomically assigned with MEGAN6 – is a lot higher to begin with. 66006 genes were assigned to the taxon Chlorophyta, whereas 34164 genes belong to the Ascomycota and 4407 to the cyanobacteria. As the overall number of cyanobacterial genes and the number of reads mapping to cyanobacteria is low to begin with, the chance of genes being differentially expressed in this sample is reduced as well. This would explain why there are relatively few differentially expressed cyanobacterial genes (apart from those in the photomorphs, which has already been explained above) compared to differentially expressed chlorophyte genes.

Functional annotation of differentially expressed genes After DESeq2 analysis the normalised count data for both the ascomycete, the chlorophyte and the cyanobacteria genes was used for statistical analysis (two-way ANOVA) as well as for functional analyses. The latter was conducted with Blast2GO (GO annotations) and BLAST. The 200 most significantly differentially expressed genes were determined with two-way ANOVA – both for the ascomycete, the chlorophyte and the cyanobacteria genes. This statistical analysis was conducted by Silke Werth. The differences between P. britannica/chlor and P. britannica/cyan were assessed on the basis of the ascomycete genes. As both photomorphs contained the same mycobiont, differences between the morphs are expressed by the mycobiont rather than the photobionts, which are not the same in the photomorphs. Chlorophyte genes are present only in the chloromorph; therefore, the differential gene expression of chlorophyte genes with respect to morphs is increased to begin with. Comparing a morph containing chlorophytes with one that does not is therefore ineffectual, which is why chlorophyte genes were not used for this part of the analysis. Neither were the cyanobacteria genes, even though these are present in both morphs. But as cyanobacteria are restricted to cephalodia in the chloromorph, the overall number of cyanobacteria genes in the chloromorph is small compared to the number of cyanobacteria genes in the cyanomorph. To assess differences in gene expression at different temperatures, the genes of all three organisms were used. This was done to compare how ascomycetes, chlorophytes and cyanobacteria react to an increase in temperature. As not all 200 significantly differentially expressed genes of all samples and for all conditions could be discussed accurately in data analysis due to lack of time only four to five top DE genes – that is those with the greatest significance – were processed further. Orthologous sequences of the genes in question were searched for in protein databases using BLAST. The functions of the sequences with the most significant alignment were determined and described for each organism and the effect of temperature on these genes or proteins was investigated.

Upregulated ascomycete genes These graphs were constructed to get an overview of the molecular functions of the differentially expressed and upregulated ascomycete genes in the chloromorph as well as the cyanomorph. To assess molecular functions the 200 most significantly differentially expressed ascomycete genes (according to the two-way ANOVA) were associated with a GO term (GO annotation) using Blast2GO. The two-way ANOVA as well as the GO annotation were performed by Silke Werth. The Blast2GO algorithm assigns a function to each gene and then clusters the genes according to their functions. In the chloromorph, the ascomycete genes have three main functions: transport, binding and catalysis (figure 8). Most of the upregulated ascomycete genes in the chloromorph have a binding function, with ion binding being the primary binding activity. The other binding activities include organic cyclic compound binding, heterocyclic compound binding and nucleic acid binding. Concerning the catalytic activity of the ascomycete genes in the chloromorph, the activity is the highest for oxidoreductases, followed by transferases and hydrolases. The primary transport activity is that of transmembrane transporters. In the cyanomorph, the differentially expressed upregulated ascomycete genes encode two main molecular functions: binding and catalysis (figure 9); unlike in P. britannica/chlor, there is no transport activity in P. britannica/cyan. The main binding activity in the cyanomorph is ion binding as well. The catalytic activity is the highest for

25 the hydrolase activity followed by the transferase activity – both of which are secondary only in the chloromorph. The oxidoreductase activity, which has the highest catalytic activity in the chloromorph, is of low catalytic activity in the cyanomorph. The catalytic activity of the cyanomorph comprises also enzymes, that act on proteins. In the cyanomorph, the hydrolase and the transferase activity can be divided into subsections (figure 10). The hydrolase activity consists of a peptidase activity, a nucleoside-triphosphatase activity, a phosphatase activity, a hydrolase activity that acts on acid anhydrides as well as a hydrolase activity that acts on acid anhydrides in phosphorus-containing anhydrides. The transferase activity comprises a kinase activity, a methyltransferase activity, a transferase activity that transfers one-carbon groups and a transferase activity that transfers phosphorus-containing groups. Figure 8 and 9 only comprise the differentially expressed ascomycete genes, whose molecular function could be determined. As many genes were hypothetical or unspecified, they could not be functionally annotated. For other significantly differentially expressed genes no GO terms were found; these are also not depicted in the graphs. In general, 16% of the 200 most significantly differentially expressed ascomycete genes in the cyanomorph were hypothetical; 34% of the differentially expressed genes could not be annotated to a GO term. In the chloromorph, 14% of the 200 most significantly differentially expressed ascomycete genes were hypothetical and 29% could not be annotated to GO terms.

Figure 8: Molecular functions of upregulated ascomycete genes in the chloromorph. The main functions include binding, transport and catalysis. Ion binding is the primary binding activity; oxidoreductase activity the primary catalysis activity; and transmembrane transport the primary transport activity.

Figure 9: Molecular functions of upregulated ascomycete genes in the cyanomorph. The main functions include binding and catalysis. The primary catalytic activity is hydrolase activity; the primary binding activity is ion binding.

Figure 10: Specific hydrolase (left) and transferase activity (right) of the upregulated ascomycete genes in the cyanomorph. 27

Photomorph-mediated differentially expressed ascomycete genes

Figure 11: Photomorph-mediated differentially expressed ascomycete gene TRINITY_DN48557_c0_g1 (left; p-value = 2e- 07) including the effects of temperature (right; p-value ≈ 0.0079). The highest differentially expressed Ascomycota gene when comparing the two photomorphs is TRINITY_DN48557_c0_g1 (p-value = 2e-07), which is upregulated in the cyanomorph (figure 11). This gene is not just differentially expressed in the photomorphs but also at different temperatures (p-value ≈ 0.0079). It is upregulated at the control temperature (4°C), 4°C and 25°C and is low in expression at 15°C. The NCBI-Blast of its nucleotide sequence resulted in two significantly similar protein sequences, namely the isopenicillin N synthetase and the 2OG-Fe(II) oxygenase superfamily.

Figure 12: Photomorph-mediated differentially expressed ascomycete gene TRINITY_DN47170_c11_g1 (left; p-value = 2.1e-06) including the effects of temperature (right; p-value ≈ 0.318). TRINITY_DN47170_c11_g1 was the second most significant differentially expressed ascomycete gene (p-value = 2.1e-06). It is upregulated in the cyanomorph (figure 12). The 28 protein products identified for this gene were SUN domain proteins as well as a cell wall synthesis protein. The exact SUN proteins could not be determined but in fungi the SUN domain proteins are responsible for cell wall synthesis as well as fungal morphogenesis in a wider sense (Gastebois, et al., 2013). The gene TRINITY_DN47170_c11_g1 therefore seems to be responsible for fungal cell wall biogenesis.

Figure 13: Photomorph-mediated differentially expressed ascomycete gene TRINITY_DN48101_c1_g1 (left; p-value = 4.6e- 06) including the effects of temperature (right; p-value ≈ 0.00476). A differentially expressed gene that was upregulated in P. britannica/chlor was TRINITY_DN48101_c1_g1 (p-value = 4.6e-06). This gene is also significantly differentially expressed in dependency on temperature (p-value ≈ 0.00476) as it is upregulated at every temperature but especially at 15°C (figure 13). It has high similarity to glutathione S- transferases, a superfamily of enzymes known for their role in detoxification processes as they catalyse the conjugation of reduced glutathione to xenobiotics with an electrophilic centre, resulting in non-toxic and more soluble substances. However, the glutathione S- transferase can also have peroxidase activity, reducing peroxidases during oxidative stress (Morel, et al., 2009).

Another differentially expressed Ascomycota gene that is upregulated in the chloromorph is TRINITY_DN24613_c0_g1 (p-value = 5.8e-06) (figure 14). The protein sequences that exhibited the highest similarity with the query sequence in the NCBI-Blast were galactonate dehydratase and enolase C-terminal domain-like protein. Moreover, TRINITY_DN24613_c0_g1 is not just upregulated in the chloromorph but does have significant differential expression in dependency on the temperature as well (p-value = 5.97e-05), as it is upregulated in both the 4°C and the control sample (4°C), but not in the 15°C or 25°C samples. Galactonate dehydratase (EC 4.2.1.6) is an enzyme that catalyses the cleavage reaction of D-galactonate to 2-dehydro-3-deoxy-D-galactonate and H2O. It is involved in the D- galactonate degradation pathway and the degradation of sugar acids which are responsible for the provision of carbon and therefore energy sources (BRENDA. URL: www.brenda-enzymes.org; date accessed: 29.11.2019). D-galactonate itself is a metabolic intermediate of the D-galactose metabolism (Singh, B. et al. 2019). The enolase C-terminal domain-like protein is of relevance in the carbohydrate catabolic processes (UniProt. URL: 29 https://www.uniprot.org/uniprot/A0A2T4BD53; date accessed: 29.11.2019), thereby contributing to the carbon and energy supply in the organism as well. Subject to these two protein structures encoded by TRINITY_DN24613_c0_g1 this gene seems to play a role in the degradation of carbohydrates and thus the provision of energy.

Figure 14: Photomorph-mediated differentially expressed ascomycete gene TRINITY_DN24613_c0_g1 (left; p-value = 5.8e-06) including the effects of temperature (right; p-value = 5.97e-05).

Differentially expressed ascomycete genes in dependency on temperature

Figure 15: Differentially expressed ascomycete gene TRINITY_DN44171_c0_g1 in dependency on temperature in the chloromorph (left; p-value = 0) and the cyanomorph (middle; p-value = 0) including its differential expression regarding the morphs (right; p-value ≈ 0.7752). TRINITY_DN44171_c0_g1 is the most significantly differentially expressed Ascomycota gene regarding difference in temperature (p-value = 0). It shows considerable upregulation in both morphs at 25°C yet an increased upregulation already occurs at 15°C. At 4°C the gene’s activity is minor (figure 15). According to NCBI-Blast this gene is similar to the ubiquitin-like protein SMT3, a protein belonging to the SUMO (small ubiquitin-like modifier) subfamily of the ubiquitin family. It is responsible for the SUMOylation of proteins, a post-translational modification that is based on the conjugation of a SUMO protein with a target protein (Wilkinson & Henley, 2010). The SUMOylation activity of SUMO proteins increases in stress situations (Zhou, et al., 2004). 30

Figure 16: Differentially expressed ascomycete gene TRINITY_DN35635_c0_g1 in dependency on temperature in the chloromorph (left; p-value = 0) and the cyanomorph (middle; p-value = 0) including its differential expression regarding the morphs (right; p-value ≈ 0.0991). TRINITY_DN35635_c0_g1 is a DE gene (p-value = 0) that is moderately upregulated in both morphs when exposed to temperatures of 4°C and 15°C. At 25°C however, the gene is not expressed in any of the two morphs (figure 16). According to the NCBI-Blast this gene is similar to the mediator complex of the RNA polymerase II (RNA polymerase II mediator complex component SRB4; Mediator complex, subunit Med17). Mediator is a multisubunit complex which is essential for various gene expression steps, particularly DNA transcription and is well investigated for its coregulator role in RNA polymerase II transcription. It is highly conserved in eukaryotes (Jeronimo & Robert, 2017).

Figure 17: Differentially expressed ascomycete gene TRINITY_DN35336_c0_g1 in dependency on temperature in the chloromorph (left; p-value = 0) and the cyanomorph (middle; p-value = 0) including its differential expression regarding the morphs (right; p-value ≈ 0.8167).

In both P. britannica/cyan and P. britannica/chlor the gene TRINITY_DN35336_c0_g1 is significantly (p-value = 0) upregulated at temperatures of 25°C. At 4°C and 15°C TRINITY_DN35336_c0_g1 is inactive (figure 17). This gene encodes for heat shock proteins (hsp70-like protein), a family of highly conserved proteins that are expressed in cells in stress situations like heat or in the presence of oxygen radicals or chemicals. Stress can irreversibly damage cells, as it disturbs the structural integrity of proteins and thereby impedes metabolic activities. Many of the Hsps expressed in stressed cells are molecular chaperones, which have various functions in a cell, including binding of unfolded proteins to prevent protein aggregation and assistance in correct protein folding (Walter & Buchner, 2002).

Figure 18: Differentially expressed ascomycete gene TRINITY_DN25204_c0_g1 in dependency on temperature in the chloromorph (left; p-value = 5e-07) and the cyanomorph (middle; p-value = 5e-07) including its differential expression regarding the morphs (right; p-value ≈ 0.3152).

TRINITY_DN25204_c0_g1 is significantly upregulated (p-value = 5e-07) in the mycobiont of both photomorphs at 15°C and especially at 25°C (figure 18). The gene in question is ARPC5 (actin-related protein 2/3 complex subunit 5). It belongs to the multiprotein complex Arp2/3 complex and is contributing to actin polymerisation in cells, which is of relevance for cell motility and cytoskeletal function (Welch, et al., 1997). Besides its occurrence in the cytoplasm the Arp2/3 complex is located in the nucleus as well where it promotes nuclear actin polymerisation. In the nucleus actin polymerisation primarily subserves the movement of DNA double-strand breaks, a result of DNA damage, which are to be repaired by homology-directed repair. Therefore, the Arp2/3 complex is a part of DNA repair pathways (Schrank, et al., 2018).

Figure 19: Differentially expressed ascomycete gene TRINITY_DN46772_c7_g4 in dependency on temperature in the chloromorph (left; p-value = 2e-07) and the cyanomorph (middle; p-value = 2e-07) including its differential expression regarding the morphs (right; p-value ≈ 0.3497). The gene TRINITY_DN46772_c7_g4 is significantly upregulated (p-value = 2e-07) at 25°C in both photomorphs. At 4°C, 15°C and the control temperature (4°C) this gene is not expressed (figure 19). TRINITY_DN46772_c7_g4 is orthologous to Retrovirus-related pol polyprotein from transposon TNT 1-94. Transposons, or transposable elements (TEs) are DNA sequences that can change their location in the genome. TEs are widespread in both prokaryotes as well as eukaryotes. Two main types of transposons are differentiated: retrotransposons and DNA transposons. The former TEs are DNA sequences that undergo transcription into RNA as well as reverse transcription back into DNA before being inserted into the new position within the genome. The latter TEs are not transcribed into RNA (Pray, 2008).

Differentially expressed cyanobacterial genes in dependency on temperature

Figure 20: Differentially expressed cyanobacterial gene TRINITY_DN46027_c3_g10 in dependency on temperature (left; p-value = 2.5e-05) including its differential expression regarding the individuals (right; p-value ≈ 0.2072).

TRINITY_DN46027_c3_g10 is a cyanobacterial gene that is the most significantly differentially expressed (p-value = 2.5e-05) in all four lichen specimens exposed to heat stress. Upregulation of the gene starts at 15°C and is further increased at 25°C; at 4°C the gene does not seem to be active (figure 20). According to NCBI-Blast the gene is highly similar to bleomycin resistance protein. Bleomycin resistance proteins – as the name implies – provide resistance against the antibiotic bleomycin and therefore is part of the antibiotic stress response system (UniProt. URL: https://www.ebi.ac.uk/interpro/entry/InterPro/ IPR000335/; date accessed: 01.12.2019).

Figure 21: Differentially expressed cyanobacterial gene TRINITY_DN46638_c0_g1 in dependency on temperature (left; p-value = 0.0001705) including its differential expression regarding the individuals (right; p-value ≈ 0.0637). 33

The cyanobacterial gene TRINITY_DN46638_c0_g1 is significantly differentially expressed (p-value = 0.0001705) in dependency on temperature. Upregulation can already be observed at 15°C, at 25°C the gene is expressed even to a higher degree. At 4°C and in the control sample (4°C) TRINITY_DN46638_c0_g1 is not expressed. A differential gene expression of TRINITY_DN46638_c0_g1 between the four individuals collected could not be identified (figure 21). The gene in question is orthologous to the RNA polymerase sigma factor RpoE. Sigma factors (σ-factors) are transcription initiation factors. They are responsible for the binding of RNA polymerase to DNA initiation sites (UniProt. URL: https://www.uniprot.org/uniprot/P0AGB6; date accessed: 01.12.2019).

Figure 22: Differentially expressed cyanobacterial gene TRINITY_DN46972_c1_g1 in dependency on temperature (left; p-value = 0.0004215) including its differential expression regarding the individuals (right; p-value ≈ 0.7577).

At 15°C as well as 25°C the cyanobacterial gene TRINITY_DN46972_c1_g1 is upregulated in all four individuals (p-value = 0.0004215). Particularly at 25°C the normalised gene count is increased, at 15°C the normalised gene count is lower; the effect of increased temperature on the upregulation is just at its starting point (figure 22). TRINITY_DN46972_c1_g1 has high similarity with lysine--tRNA ligase (also referred to as lysyl-tRNA synthetase). This enzyme catalyses the reaction: ATP + L-lysine + tRNA(Lys) ⇌ AMP + diphosphate + L-lysyl-tRNA(Lys) (QuickGO. URL: https://www.ebi.ac.uk/ QuickGO/GTerm?id=GO:0004824; date accessed: 01.12.2019). The product of interest is the lysyl-tRNA which transfers lysine into ribosomes for protein synthesis (Wu, et al., 2007).

The cyanobacterial gene TRINITY_DN48753_c1_g2 is significantly differentially expressed (p-value = 0.0029799) in dependency on temperature in all four individuals. At 15°C the gene already shows great upregulation but at 25°C it is upregulation is even more pronounced (figure 23). TRINITY_DN48753_c1_g2 is highly similar to the photosystem I core protein PsaB. Photosystem I is, along with photosystem II, an integral part in photosynthesis. It has two core proteins, PsaA and PsaB, which form a heterodimer to bind the electron donor P700 – a chlorophyll dimer – as well as various electron acceptors (InterPro. URL: http://www.ebi.ac.uk/interpro/entry/InterPro/IPR006244/; date accessed: 02.12.2019).

Figure 23: Differentially expressed cyanobacterial gene TRINITY_DN48753_c1_g2 in dependency on temperature (left; p-value = 0.0029799) including its differential expression regarding the individuals (right; p-value ≈ 0.1389).

Figure 24: Differentially expressed cyanobacterial gene TRINITY_DN44076_c0_g1 in dependency on temperature (left; p-value = 0.0064623) including its differential expression regarding the individuals (right; p-value ≈ 0.2952).

TRINITY_DN44076_c0_g1 is a differentially expressed cyanobacterial gene (p-value = 0.0064623) that is highly upregulated at 25°C and moderately upregulated at 15°C as well as 4°C – at the control temperature of 4°C the gene is not expressed. The expression of this gene is the similar in all four individuals (figure 24). TRINITY_DN44076_c0_g1 is orthologous to chaperonin GroEL, a protein that refolds denatured or misfolded polypeptides and proteins, most notably under stress conditions (Goloubinoff, et al., 1997).

Differentially expressed chlorophyte genes in dependency on temperature

Figure 25: Differentially expressed chlorophyte gene TRINITY_DN47627_c2_g1 in dependency on temperature (left; p-value = 5.65e-05) including its differential expression regarding the individuals (right; p-value ≈ 0.044). The chlorophyte gene TRINITY_DN47627_c2_g1 is significantly differentially expressed (p-value = 5.65e-05) in dependency on temperature. Upregulation of the gene starts at 15°C and is the highest at 25°C. There also is an individual difference in the expression of this gene (p-value ≈ 0.044), TRINITY_DN47627_c2_g1 is upregulated in individual 2.2 and individual 3.2, but not in individual 4.2 and only slightly in individual 1.2 (figure 25). This gene is similar to the light-harvesting chlorophyll a/b-binding protein Lhca2. The light- harvesting complex (LHC) is an essential part of photosynthesis. There are multiple LHC proteins which bind photosynthetic pigments in order to capture light – the main energy source for photosynthesis (Ganeteg, et al., 2001).

Figure 26: Differentially expressed chlorophyte gene TRINITY_DN46800_c0_g1 in dependency on temperature (left; p-value = 0.0002766) including its differential expression regarding the individuals (right; p-value ≈ 0.4872). 36

TRINITY_DN46800_c0_g1 is a chlorophyte gene that is upregulated at 25°C only, while showing no expression at 4°C, 15°C and in the 4°C control group (p-value = 0.0002766). There are no differences in individual gene expression (figure 26). The NCBI-Blast of this gene resulted in two possible sequences with significant alignments: an ammonium transporter (E-value = 3e-29) as well as a class I heat shock-like protein (E-value = 7e- 28). As these two proteins differ greatly in their functions both will be considered in the discussion of heat stress response in chlorophyte genes.

Figure 27: Differentially expressed chlorophyte gene TRINITY_DN43942_c0_g1 in dependency on temperature (left; p-value = 0.0016676) including its differential expression regarding the individuals (right; p-value ≈ 0.7408). The chlorophyte gene TRINITY_DN43942_c0_g1 is significantly differentially expressed (p-value = 0.0016676) as it is upregulated at 25°C but does not show any expression at 4°C or 15°C. Furthermore, there are no individual differences regarding the gene expression (figure 27). TRINITY_DN43942_c0_g1 is similar to the oxygen-evolving enhancer protein 3 and therefore is a component of the oxygen-evolving complex (OEC) of photosystem II. The OEC’s role during photosynthesis is the oxidation of water, producing oxygen as a result (Raymond & Blankenship, 2008).

TRINITY_DN48593_c1_g2 is a chlorophyte gene that is upregulated at 25°C but not at 4°C or 15°C and therefore is significantly differentially expressed (p-value = 0.0027463). On an individual level there is no differential expression of this gene (figure 28). It is similar to a small subunit of chloroplast ribulose 1,5-bisphosphate carboxylase/oxygenase, better known as RuBisCO. RuBisCO is the enzyme that catalyses the fixation of atmospheric carbon dioxide (CO2) to ribulose-1,5-bisphosphate within the Calvin- Benson-Bassham cycle of photosynthesis (Erb & Zarzycki, 2018).

Figure 28: Differentially expressed chlorophyte gene TRINITY_DN48593_c1_g2 in dependency on temperature (left; p-value = 0.0027463) including its differential expression regarding the individuals (right; p-value ≈ 0.3573).

Figure 29: Differentially expressed chlorophyte gene TRINITY_DN43001_c0_g4 in dependency on temperature (left; p-value = 0.0066621) including its differential expression regarding the individuals (right; p-value ≈ 0.5963).

The chlorophyte gene TRINITY_DN43001_c0_g4 is another DE gene that is upregulated at 25°C (p-value = 0.0066621). At the control temperature (4°C), 4°C and 15°C the gene is not upregulated. There are no individual differences in the gene expression (figure 29). The BLAST search aligns TRINITY_DN43001_c0_g4 with an antiviral helicase as well as an RNA helicase. Antiviral helicases are responsible for blocking the translation of antiviral mRNA (UniProt. URL: https://www.uniprot.org/uniprot/P35207; date accessed: 02.12.2019) and therefore are part of the antiviral response system. RNA helicases – including several antiviral helicases – modify RNA secondary structure and therefore are an essential for RNA metabolism (Jankowsky, 2011).

Discussion Differential gene expression analyses have illustrated the effect of temperature stress on all partners involved in a lichen symbiosis as well as a distinct photomorph-mediated gene expression pattern of the mycobiont of the photosymbiodeme. Many ascomycete, chlorophyte and cyanobacterial genes were upregulated at higher temperatures; however, a few were upregulated at colder temperatures. Furthermore, various ascomycete genes were upregulated either in the chloromorph of the lichen specimens or in the cyanomorph, but not in both photomorphs. The photomorph-mediated differential gene expression was affected by the temperature as well, indicating that environmental factors are relevant for the mycobiont’s gene expression in different photomorphs. The ascomycete and the cyanobacteria proved to be more sensitive to temperature fluctuations, as genes were upregulated already at 15°C, whereas for the chlorophyte, genes usually were upregulated at 25°C. When exposed to different temperatures, individual effects on the organisms’ gene expression were negligible. To determine the role of the four to five most significantly differentially expressed genes of each organism, a BLAST analysis was conducted (NCBI BLAST). The most significant alignment was used to discuss the probable role of the gene and how it might be affected by heat stress.

Photomorph-mediated differentially expressed ascomycete genes The mycobiont’s gene expression is differential when in association with different photobiont types. One differentially expressed ascomycete gene is TRINITY_DN48557_c0_g1 (figure 11) which is upregulated in the cyanomorph. This gene is orthologous to the enzyme isopenicillin N synthetase, which is part of the penicillin biosynthesis pathway (KEGG. URL: https://www.genome.jp/dbget- bin/www_bget?ec:1.21.3.1; date accessed: 29.11.2019). The upregulation of proteins from the 2OG-Fe(II) oxygenase superfamily is consistent with the upregulation of the isopenicillin N synthetase as the former is of relevance in the penicillin biosynthesis in fungi and bacteria as well (Aravind & Koonin, 2001). Of course, penicillin is known to be produced by fungi (Holtman, 1947) and it has also been reported that lichen secondary metabolites can have antibiotic properties (Shrestha & Clair, 2013) but the precise reason why a lichen-forming fungus would produce antibiotics is open. High expression of penicillin in this instance is only the case in the cyanomorph and not in the chloromorph, indicating that it might be a reaction to the Nostoc symbiont. However, if this is the case, similar results can be expected from other Peltigera britannica samples or even other Peltigera species, which enter a symbiosis with a cyanobacterial photobiont. The gene in question shows differential expression when exposed to different temperatures, so heat or stress reactions might be the driving factors for penicillin production. It is possible that the upregulation of the isopenicillin N synthetase is a result of a combination of various factors influencing the specimens, including heat stress, the presence of a Nostoc or even the presence of different bacterial strains in the cyanomorph of the lichens. The reaction of the mycobiont could furthermore be dependent on the metabolism of its photosynthetic partner, as the interaction between the mycobiont and the photobiont is of relevance for the production of lichen substances (Shrestha & Clair, 2013). Therefore, the production of antibiotics by the ascomycete genes could be a result of its interaction with its cyanobacterial partner. Considering Nostoc’s preference of wet and cool habitats, it already is stressed at 15°C and has to react to this stress accordingly. This might affect the mycobiont as well – due to the nature of 39 symbiosis – as it is possible that the mycobiont misinterprets the stress response of the cyanobacterium and perceives it as an alien, maybe even intruding, organism instead. But if the reason for the production of antibiotics is due to stress, this should result in an upregulation of other stress genes in the mycobiont as well. Another possibility for the upregulation of the isopenicillin N synthetase might be the presence of toxins, which have been reported to be produced by cyanobacteria in lichen symbiosis. The production of toxins by Nostoc was observed in various lichen species and especially in humid climate types (Kaasalainen, et al., 2012). Toxins can harm the mycobiont, therefore, the mycobiont has to react accordingly to avoid damage. This, at least, could explain the expression of the isopenicillin N synthetase in the fungus of our P. britannica photosymbiodemes. The reason for the production of penicillin could also be due to the method of sampling lichen tissue, as it was cut off. This is likely to cause stress to the lichen and might result in a stress response that induces the upregulation of antibiotics genes. However, this method was used for both photomorphs, so the fungus in the chloromorph should theoretically also show an upregulation of antibiotic-producing genes – but it doesn’t. It cannot be determined with certainty, why the mycobiont upregulates the isopenicillin synthetase gene in the cyanomorph, and at certain temperatures only. Heat stress alone, which according to our results affects P. britannica/cyan to a greater extent than P. britannica/chlor, cannot explain the expression of the isopenicillin N synthetase, as at 15°C, this gene is not expressed. In order to asses on whether penicillin biosynthesis is common in mycobionts associated with Nostoc future studies are needed. At this point – due to the lack of studies on the subject of penicillin production in lichens – the expression of isopenicillin N synthetase has to be considered an exceptional case, likely induced by various stress factors. The TRINITY_DN47170_c11_g1 gene (figure 12), being likely responsible for cell wall synthesis in the fungus (Gastebois, et al., 2013), is the second most significantly differentially expressed Ascomycota gene and is upregulated in the cyanomorph. The reason why fungal cell wall biogenesis occurs in the cyanomorph at a higher rate, is not entirely clear, but there are various options. First, the process of cell wall synthesis could be linked with the production of antibiotics in the cyanomorph. The expression of genes from the penicillin biosynthesis pathway might be a stress response to the cyanobacterial partner or an alien organism. But as could be the expression of cell wall biosynthesis genes, as the fungus might attempt to prepare for a possible infection by an alien organism by restructuring its cell wall, which is a highly dynamical structure in fungi, and therefore helps the fungus to adapt to the new conditions it is confronted with (Hopke, et al., 2018). Secondly, it has been determined that the fungal cell wall plays a relevant part in the infection of host organisms like plants. Due to the diversity of fungi and their complex cell wall compositions, cell wall modifications and synthesis during host infections are still poorly understood; yet, during the process of infection as well as interaction with a host, the fungal cell wall undergoes various alterations – including cell wall synthesis – as it forms so-called penetration hyphae, with which it infects the host organism. Another type of fungal structure are haustoria, special type of hyphae, which serve nutrient acquisition by penetrating or adhering to the photobiont’s cells (Geoghegan, et al., 2017). Haustoria are of relevance not only in the infection of host organisms but in the lichen symbiosis as well, as the fungus interacts with its photosynthetic partner. But haustoria are not present in the symbiosis with the green alga Coccomyxa as this algal genus contains sporopollenin in its cell wall which the fungus cannot degrade or penetrate (Honegger, 1984). Nostoc, despite not having sporopollenin in its cell wall, is not penetrated by the haustoria either. Instead, the hyphae encircle the Nostoc cells tightly and sometimes even invaginate them (Pawlowski & Bergman, 2007). Furthermore, intrawall haustoria have been described in 40

Peltigera species with Nostoc, which penetrate the membrane of the cyanobacterial cell wall rather than the entire cell wall (Ahmadjian & Koriem, 1986). This process requires cell wall morphogenesis, as the hyphae do have to extend and grow to encompass and/or penetrate the photobiont cells (Feofilova, 2010). This means, that in the case of P. britannica, the lichen used in this study, haustoria formation is of relevance in the symbiosis with the cyanobacterium only. Therefore, the fungus is likely to experience increased cell wall reorganisation and biosynthesis resulting in an upregulation of genes responsible for cell wall synthesis in the cyanomorph. In the chloromorph, the formation of haustoria does not take place, as the hyphae are rather adjacent to the Coccomyxa cells and therefore do not need to undergo substantial cell wall reorganisation. The upregulation of the ascomycete gene TRINITY_DN48101_c1_g1 in the chloromorph (figure 13) showed indications of stress response, as this gene encodes glutathione S-transferases (GST), enzymes that are responsible for metabolic detoxification. These enzymes are active in the presence of xenobiotic substances and during oxidative stress (Morel, et al., 2009). Oxidative stress is rather common in lichens, as the number of reactive oxygen species (ROS) increases in stress conditions like desiccation/rehydration, water saturation and herbivory (Françoise, et al., 2014). Especially during desiccation, an accumulation of ROS in lichens has been observed. This can lead to damage in the lichen wherefore it has to react accordingly. A major driver in the tolerance towards oxidative stress is glutathione (GSH), an antioxidant which reduces ROS, whereby GSH itself is being oxidised into so-called glutathione disulphide (GSSG) (Kranner, et al., 2008). The enzyme GST is a major driver of this detoxification process, as it is responsible for the conjugation of GSH onto ROS to detoxify them (Kammerscheit, et al., 2019). Lichens that tolerate desiccation to a higher degree usually contain an increased GSH pool in their hydrated state, enabling a rapid oxidation to GSSG and therefore detoxification of ROS during desiccation. This results in an increased GSSG pool in desiccation-tolerant lichens during desiccation. GSSG is reduced back to GSH during rehydration (Kranner, 2002). The upregulation of GST in the lichen of this study could therefore be a result of beginning desiccation and desiccation-induced accumulation of ROS. Even though the thalli were watered regularly, they were subject to several desiccation/rehydration cycles – especially during the treatments with increased temperatures – which might have been enough to induce oxidative or desiccation stress. The reason why the expression of GST is upregulated in P. britannica/chlor but not in P. britannica/cyan might be due to an increased tolerance of the former to desiccation. Lichens with green algal photobionts have been described to be more tolerant to desiccation than those with cyanobacterial photobionts (Kranner, 2002). In this case the chloromorph of P. britannica should have an increased GSH pool in its hydrated state. In the case of desiccation, the chloromorph might therefore be able to react quickly by oxidising GSH to GSSG, resulting in an upregulation of GST. P. britannica/chlor might in fact be more tolerant towards desiccation, as chloromorphs can be found growing in the open field, where they are more exposed to the elements, whereas the photosymbiodemes with the cyanomorphs only grow at moist and shady sites (personal observations). However, the reduction of ROS is only one function of the glutathione S-transferase as it is also responsible for the detoxification of xenobiotics (Morel, et al., 2009), including metals and metalloids, agricultural chemicals and polyaromatic hydrocarbons (Watkinson, et al., 2016). Various studies have shown, that GST activities in lichens are increased in areas with high environmental pollution (Rustichelli et al. 2008; Oztetik & Cicek 2011). As the lichen specimens in this experiment were collected in a nature reserve, off of heavily trafficked roads and other infrastructural elements, pollution with xenobiotics should be low in these lichens. Therefore it does not seem to be likely that the 41 upregulation of GST is due to pollution. Furthermore, as the cyanomorph and the chloromorph were growing as one individual (P. britannica/chlor+cyan), the effects of pollution should be noticeable in the Ascomycota genes in both morphs and not just in the chloromorph. The increase in GST activity in the chloromorph appears to be a consequence of abiotic environmental factors rather than pollutants. Besides the already mentioned effects of desiccation and rehydration, light intensity also influences the expression of GSTs, at least in plants. While darkness and shade reduce the levels of GST, high light leads to an increase in GST activity (Gallé, et al., 2019). Even though light alone is unlikely to be responsible for the upregulation of GST genes in our lichen samples – as the chloromorph was exposed to the same light conditions as the cyanomorph – it might be possible that it is the combination of light and desiccation that led to an increased GST activity in the chloromorph. TRINITY_DN24613_c0_g1 is an ascomycete gene that is upregulated in the chloromorph as well as in temperatures of 4°C (figure 14). This gene seems to be responsible for carbohydrate degradation and thus the provision of carbon. Carbohydrates are substantial for the lichen regarding the provision of energy which is required for a range of metabolic processes like respiration and biosynthesis of various compounds (e.g. secondary metabolites or cell wall components) (Palmqvist, 2000). Therefore, the main function of the two protein sequences that are encoded by TRINITY_DN24613_c0_g1, namely galactonate dehydratase and enolase C-terminal domain-like protein, is carbon supply. Yet it is not known what the fungus utilises this carbon for – it could be relevant for growth or cell wall synthesis or possibly even respiration. However, as the definite role is unidentified, one can only hypothesise why this gene is upregulated in P. britannica/chlor as well as at 4°C. It is possible that the cyanomorph does not exhibit an upregulation in proteins relevant for carbohydrate metabolism as it can draw on nitrogen compounds for its metabolic processes. Of course, carbon is essential for the cyanomorph as well, but there might be a preference for nitrogen compounds – at least in the conditions tested in this study. Lichen species that are capable of fixing nitrogen usually do have a lower C:N ratio than lichens with an algal partner (Crittenden & Kershaw, 1978). This would explain, why the gene in question is upregulated in the chloromorph only. Another possibility for the upregulation of ascomycete genes responsible for carbohydrate degradation in the chloromorph, could be the availability of different carbon compounds in the different photomorphs. The algae, for example, can produce ribitol, which is translocated to the mycobiont (Richardson & Smith, 1968). In order to be able to use this carbon compound, the mycobiont might need to degrade it, whereas the mycobiont might be able to use other carbon compounds, including ones it receives from the cyanomorph (e.g. glucans) (Hill, 1972), degrading them only minimally or not at all. The reason why it is upregulated at cool temperatures (4°C) might be cold tolerance. Lichens must be able to withstand cold temperatures, especially lichens growing in Iceland. Carbon metabolism does play a role in tolerance as well as acclimatisation to cold temperatures. Primary as well as secondary carbon metabolites are essential for organisms to withstand cold and freezing temperatures (Fürtauer et al. 2019; Calzadilla et al. 2019; Tarkowski & Van den Ende 2015), at least in photosynthetic organisms. The fungus probably responds similarly to cool temperatures and therefore needs to metabolise the carbohydrates it obtains from its photosynthetic partner. This would explain the upregulation of these genes, which are responsible for carbon supply, at 4°C but not at 15°C and 25°C.

There is a number of photomorph-mediated differentially expressed ascomycete genes – with some genes being upregulated in the chloromorph and others in the cyanomorph. The top four significantly differentially expressed ascomycete genes were discussed in this study, but three of them seem to be affected by abiotic factors and stress, as they are also differentially expressed under different temperatures. Therefore, the upregulation of these genes might not be representative of a general pattern of the ascomycete’s differential gene expression in Peltigera britannica. Especially the upregulation of isopenicillin N synthetase seems to be induced by some external factor, rather than being a fundamental function of the ascomycete in the cyanomorph. The same is true for the galactonate dehydratase, which is upregulated in the chloromorph at 4°C and the glutathione S-transferase that seems to be expressed in the chloromorph due to the presence of reactive oxygen species. However, despite the effect of stress and possible other external factors the results clearly indicate that differential gene expression in the ascomycete is present and that part of this DEG is likely due to the mycobiont’s interaction with the photobiont. The mycobiont seems to react differently to stress when in association with different photobiont types. This is especially evident in the upregulation of the glutathione S-transferase in the ascomycete genes of P. britannica/chlor, which indicates that the mycobiont is more tolerant to desiccation forming a chloromorph than when forming a cyanomorph. The photosynthetic partner does seem to influence the fungal partner’s gene expression. Each photobiont type provides the mycobiont with, for example, different metabolic products and the mycobiont therefore has to interact with its photosynthetic partners in different ways to obtain, transport and process these products (Shrestha & Clair 2013; Singh, G. et al. 2019). Ascomycete genes that are upregulated in one photomorph might therefore not be upregulated in the other. Despite a vast number of differentially expressed ascomycete genes, their functional annotation proves to be complicated. Many of the DE genes of the ascomycete encode hypothetical proteins and proteins of unknown function, whose precise role in an organism is difficult to determine. 16% of the 200 most significantly differentially expressed ascomycete genes in the cyanomorph were hypothetical; in the chloromorph 14% were hypothetical. The annotation of the genes to GO terms proved to be even more difficult, as 34% of the 200 most significantly differentially expressed ascomycete genes could not be annotated to a GO term in the cyanomorph and 29% in the chloromorph. Hence, the greatest difficulty in the determination of fungal gene functions and their role in the organism is the overall lack of functional assignments of the genes in question. Even though the alignment to unspecified sequences hampered the evaluation and assessment of the results in this study there nevertheless is potential for future studies to focus on exactly those obscure genes and identify their biological roles and molecular functions.

Differentially expressed ascomycete genes in dependency on temperature The ubiquitin-like protein SMT3 encoded by the ascomycete gene TRINITY_DN44171_c0_g1 shows considerable activity at both 15°C and 25°C in both P. britannica/cyan and P. britannica/chlor (figure 15). This protein belongs to the SUMO subfamily which consists of proteins being responsible for the SUMOylation of a range of other proteins. SUMOylation of proteins is of great relevance as it renders the targeted proteins useful for various vital biological processes, among other transcription, translation and DNA replication. Besides, stress is a factor that results in an increased activity of SUMO proteins and thereby protein SUMOylation (Zhou, et al., 2004). Heat shock is one of the stress factors that causes protein SUMOylation and leads to an

43 accumulation of SUMO conjugates shortly after a rise in temperature, indicating that SUMOylation might be an early stress response system. The SUMOylation is the starting point of a cascade of cellular processes in reaction to stress as the binding of SUMO proteins activates the target proteins – for example heat shock factors (Hsf) – which in turn activate specific target proteins – for example heat shock proteins (Hsp) (Kurepa, et al., 2003). Stress response is indispensable for the cell as stress causes more or less serious damage, e.g. to protein structures and thus to protein functions (Liebelt & Vertegaal, 2016). Activation of Hsps as a consequence of a SUMOylation process has been described for various organisms, including Arabidopsis (Kurepa, et al., 2003) and Candida albicans (Leach, et al., 2011). The upregulation of the ubiquitin-like protein SMT3 in the Peltigera britannica photomorphs of our study might indeed be a response to thermal stress, as it is expressed at temperatures of 15°C and 25°C, but not at temperatures of 4°C. It is likely that the function of SMT3 is congruent with the functions of similar SUMO proteins which are responsible for the activation of Hsps to protect proteins and cells against thermal stress, as has been reported for the yeast Saccharomyces cerevisiae (Enserink, 2015). The ascomycete gene TRINITY_DN35635_c0_g1 is moderately upregulated at colder temperatures (4°C and 15°C) whereas it is downregulated at higher temperatures, showing only little activity at 25°C in both morphs (figure 16). Its corresponding protein is a subunit of the mediator complex, a multisubunit complex essential for the regulation of DNA transcription by RNA polymerase II. Its main function – as its name implies – is the mediation between transcription factors and the RNA polymerase II machinery to further the assembly of the pre-initiation complex of the RNA transcription. However, mediator complex is involved in a number of other pathways, including almost all stages of the RNA polymerase II transcription (e.g. elongation, termination and mRNA processing) as well as cell growth and differentiation pathways (Yin & Wang, 2014). Considering the essential and multi-layered function of mediator complex, its upregulation in an organism is not surprising. Its downregulation in increased temperature on the other hand can be regarded as unusual – as transcription and various other pathways do not come to a halt due to thermal stress. On the contrary, multiple studies have assessed that certain mediator subunits are responsible in the cell’s response to heat stress, as they are directly recruited by Hsfs to associate with Hsp gene promoters, resulting in Hsp activity (Kim & Gross 2013; Åkerfelt, et al. 2010). A downregulation of mediator complex in heat therefore does seem to be counterproductive. But the function of the mediator complex and its subunits is diverse, and for Arabidopsis thaliana it has been shown that certain mediator complex subunits (MED16, MED14 and MED2) regulate cold-responsive gene expression (Hemsley, et al., 2014). This would explain why the expression of the mediator complex subunit in our study is downregulated in hot temperatures and upregulated in cooler temperatures. After being exposed to 4°C for two weeks the lichen specimens were adapted to cold temperatures and the expression of this particular mediator complex subunit was part of the regular metabolism of the lichens. An increase in temperature however, disturbed this process resulting in a downregulation of this subunit, as it is not crucial for the response to warmer temperatures in the cell. This result illustrates that an organism exposed to heat stress does not solely react by means of overexpression of stress genes but also through downregulation of other genes that are involved in metabolic pathways under normal conditions. Downregulation of such genes may be preferable for a cell exposed to stress as available energy sources can be used for stress response in order to protect the cell from damage rather than for anabolic activities that are not absolutely essential in stress situations and are, as a consequence, restricted in their activity (Aprile-Garcia, et al., 2019). 44

High temperatures (25°C) result in an upregulation of heat shock proteins in the ascomycete (TRINITY_DN35336_c0_g1; figure 17). The production of Hsps in stress situations is crucial for the cell, as Hsps prevent cell damage and death and renders cellular recovery possible. Many Hsps have chaperone functions, they prevent misfolding of proteins and protein aggregation, and support the correct folding, refolding or renaturation of proteins (Beere, 2004). Heat is one of the stress factors that results in denaturation and misfolding of proteins, which is why the production and activation of Hsps in heat-stressed cells is of great importance (Walter & Buchner, 2002). In order to be able to induce stress tolerance in the cell, Hsps must interact with proteins and/or polypeptides. Many heat shock proteins (e.g. Hsp70 and Hsp 90) have special domains, like a N-terminal ATPase domain and a C-terminal domain, with which they can interact and bind with proteins resulting in the protection of the bound protein (Beere, 2004). Considering the pivotal role of heat shock proteins in contributing to stress tolerance and counteracting potentially damaging processes, the upregulation of heat shock proteins at high temperatures in our sample is plausible. 25°C is a temperature that is already too high for the mycobiont in P. britannica so it produces Hsps. As the lichen in question was collected in Iceland, a country with cold climate conditions that in general does not experience temperatures of 25°C for extended time periods, the lichen is confronted with a severe stress situation when exposed to these high temperatures. The temperature of 15°C did not cause increased expression of heat shock proteins, but temperatures in the range of 15°C can occur in the course of the Icelandic summer and therefore 15°C might not be regarded as stressful for the fungus. Furthermore, lichens have been determined to show flexibility in their ability to tolerate heat stress. Cladonia rangiferina for example tolerates higher temperatures in summer whereas it experiences more stress when exposed to the same temperatures in winter (Tegler & Kershaw, 1981). As the specimens of P. britannica used in our study were collected in summer it is possible that their ability to tolerate higher temperatures was increased; and remained increased even after being exposed to temperatures of 4°C for two weeks. However, the response of lichen to heat stress varies from species to species, as well as from population to population (e.g. in Peltigera canina) (MacFarlane & Kershaw, 1980) so the observed tolerance of P. britannica towards temperatures up to 15°C in our study does not imply that an increase in temperature is perceived likewise in all individuals and populations of P. britannica. Another Ascomycota gene that is upregulated at high temperatures – especially at 25°C – is ARPC5 (TRINITY_DN25204_c0_g1; figure 18), a member of the multiprotein complex Arp2/3 which is responsible for actin polymerisation in the cytoplasm as well as in the nucleus. In the nucleus the Arp2/3 complex contributes to the repair of damaged DNA as it promotes the migration of DNA double-strand breaks which are to be repaired (Schrank, et al., 2018). Heat is a stress factor that can cause DNA damage, particularly due to the fact that hyperthermia can sensitise cells to agents that cause DNA damage, increasing the probability of damage. DNA damage can also be caused directly by heat as it denaturises proteins and affects DNA replication (Oei, et al., 2015). As DNA is likely to be damaged by heat stress an overexpression of genes and proteins that participate in DNA repair mechanisms at high temperatures is apparent. The Arp2/3 complex is one component of these DNA repair mechanisms (besides various heat shock proteins (Sottile & Nadin, 2018)). Its upregulation in the photomorphs at 15°C and especially at 25°C might therefore be a reaction to commencing DNA damage induced by heat. However, hyperthermia can inhibit DNA repair as well (Oei, et al., 2015) but this was not observed in the present experiment. Perhaps, the inhibition of DNA repair is only prevalent at

45 extremely high temperatures and as the heat stress in our study was only moderate, an inhibition might not have taken place yet. TRINITY_DN46772_c7_g4 is an Ascomycota gene that is differentially expressed at different temperatures in both photomorphs. At 25°C the gene is significantly upregulated, whereas it is not expressed at 4°C and 15°C (figure 19). TRINITY_DN46772_c7_g4 has high similarity to Retrovirus-related pol polyprotein from transposon TNT 1-94, this means it is likely to be a transposable element (TE). TEs are DNA sequences that can change their position in the genome. If transposons land inside a gene as a result of their translocation they might induce mutations. However, most TEs are assumed to be silent. This means that their movements within the genome do not result in phenotypic effects. Additionally, not all transposons can move around actively in the genome, as some are inactivated by mutations whereas others are inactivated by epigenetic regulation (e.g. DNA methylation) (Pray, 2008). Activation of TE transcription can occur in stress situations, including heat (e.g. in Arabidopsis thaliana) and UV light (e.g. in Cucumis melo) (Huang, et al., 2018). The activation of transposons elevates their movement rates within the genome which furthermore increases the probability of mutations. These mutations can either be deleterious or advantageous. Advantageous mutations for instance, might be of relevance in an organism’s adaptation to stress (Negi, et al., 2016). A positive effect of transposon translocation has been identified for Arabidopsis thaliana, in which transposon movement, activated by heat stress, resulted in an abscisic acid-insensitive phenotype of Arabidopsis, presumably rendering the plant more tolerant towards stress (Ito, et al., 2016). The upregulation of transposons in our P. britannica specimens at 25°C is induced by thermal stress and is part of the lichen’s stress response. Whether the activation of this transposon is of an advantage for the lichen – as it could enable stress adaptation – or whether it is likely to cause deleterious mutations could be an interesting subject for future studies.

Changes in temperature lead to differential expression of ascomycete genes in this experiment. Of the top five significantly differentially expressed ascomycete genes most are involved in the organism’s heat stress response, like Hsp, which prevent stress induced cell damage, and SMT3, which activates Hsps by SUMOylation. The Arp2/3 complex is part of a DNA repair mechanism and the expression of this gene therefore suggests a damaging effect of heat on DNA. Heat stress also induces the activation of transposable elements. But not all differentially expressed ascomycete genes are the result of heat stress. The mediator complex is upregulated at 4°C and 15°C and likely regulates cold-responsive gene expression. As it is activated in cooler temperatures heat leads to a downregulation of this gene. Heat stress in the ascomycete is induced to a great extent at 15°C. At 25°C, the fungus seems to be experiencing serious heat stress, as all the major genes responsible for an organism’s heat stress response are upregulated. As the specimens of P. britannica were collected in the Icelandic summer, with temperatures of about 10-12°C, a higher tolerance of the mycobiont to 15°C might be the result of the lichen’s seasonal adaptation to increased temperatures.

Differentially expressed cyanobacterial genes in dependency on temperature The most significantly differentially expressed cyanobacterial gene in dependency on temperature is TRINITY_DN46027_c3_g10, which is slightly upregulated at 15°C and highly upregulated at 25°C (figure 20). The gene’s sequence is orthologous to bleomycin resistance protein, which promotes resistance against bleomycin, an antibiotic produced

46 primarily by actinomycetes. Bleomycin breaks DNA compounds, rendering this antibiotic lethal for cells. Actinomycetes protect themselves from the effects of their own compounds with bleomycin resistance proteins (Dumas, et al., 1994). Albeit, genes conferring resistance to bleomycin and other antibiotics have been described for cyanobacteria as well (Urtubia, et al., 2016). In the bacterium Escherichia coli the presence of several antibiotic resistance genes seems likely to have been induced by adaptation to stress situations like thermal stress; a supposition that has been inferred as the tolerance towards antibiotics underlies the same mechanism as the tolerance towards heat (Cruz- Loya, et al., 2019). Therefore, the expression of a bleomycin resistance protein in the cyanobacterial genes of our P. britannica specimens at high temperatures is probably recruited by thermal stress response pathways. As in E. coli, the mechanisms for heat stress adaptation in the Nostoc of P. britannica might be similar to the mechanisms of antibiotic resistance, which would explain why genes coding for bleomycin resistance are upregulated even though there is no evidence of bleomycin actually being present in the samples. The upregulation of antibiotic resistance proteins in this case might simply be a side-effect of thermal stress. The cyanobacterial gene TRINITY_DN46638_c0_g1 is upregulated at 15°C and especially at 25°C in all four P. britannica specimens (figure 21). This gene is highly similar to the RNA polymerase sigma factor rpoE, an initiation factor for transcription. The gene rpoE encodes the sigma factor E. In Escherichia coli, rpoE is responsible for the induction of stress response, which has been tested by exposing E. coli to thermal stress. Mutants without the rpoE gene were not able to deal with high temperatures (Raina, et al., 1995). Under normal conditions rpoE is inactive, heat stress leads to an activation of this gene. Thereupon, it induces the transcription of heat shock genes, which protect the cell from damages that thermal stress can cause (Roncarati & Scarlato, 2017). An increased upregulation of this sigma factor with increasing temperature in our samples underlines its function in an organism’s stress response induced by heat. TRINITY_DN46972_c1_g1 is a cyanobacterial gene upregulated at 15°C as well as at 25°C in all four individuals (figure 22). The orthologous gene is lysine--tRNA ligase (also referred to as lysyl-tRNA synthetase), an enzyme that is responsible for the formation of lysyl-tRNA. Lysyl-tRNA is of relevance in protein synthesis, transferring lysine into ribosomes (Wu, et al., 2007). Besides, the enzyme has another function – which has been assessed for Escherichia coli – as it synthesises various adenyl dinucleotides, particularly AppppA (also referred to as Ap4A). This function of lysyl-tRNA ligase is active only under stress conditions leading to an accumulation of Ap4A as well as the other dinucleotides. Ap4A serves as a modulator of heat shock response, binding to proteins expressed during heat shock. However, its exact role in heat shock response has not yet been determined, a signalling function of these dinucleotides has also been proposed (Onesti, et al., 1995). The upregulation of lysyl-tRNA synthetase at high temperatures in our study is consistent with the above-mentioned findings. Even though it is not sure if the lysyl-tRNA synthetase in our samples synthesises Ap4A as well, a role of this enzyme in cellular stress response cannot be ruled out. Hence, the presence of this enzyme in organisms experiencing thermal stress is not unanticipated. The cyanobacterial gene TRINITY_DN48753_c1_g2 is upregulated at 15°C and 25°C in all four individuals (figure 23). This gene is orthologous to the photosystem I core protein PsaB. Photosystem I (PSI) is along with photosystem II (PSII) a protein complex that is part of the photosynthetic pathway. It has two core proteins, PsaA and PsaB (InterPro. URL: http://www.ebi.ac.uk/interpro/entry/InterPro/IPR006244/; date accessed: 02.12.2019). PSII can sustain serious damage during heat stress. In plants the inactivation of photosynthesis via PSII due to high-temperature stress has been observed. 47

PSI, on the other hand, is said to be more stable. There even are indications of enhancement of function in PSI under stress conditions, compared to PSII (Ivanov, et al., 2017). Switching from PSII to PSI during heat stress and thereby increasing the photosynthetic activity of PSI is a mechanism to avoid damage to PSII (Yamauchi, et al., 2011). Photosystem I, on the other hand, can be damaged under low-temperature stress conditions and therefore undergoes inhibition when exposed to cold temperatures (Tjus et al. 1998; Zhang & Scheller 2004). Consequently, the upregulation of the PSI core protein PsaB in Nostoc genes of the P. britannica specimens in this study might be caused by the higher temperature, possibly along with a (partial) inactivation of PSII, which leads to an increased photosynthetic activity of PSI. At 4°C, the PSI core protein PsaB is not upregulated – this might be the result of inactivation or at least downregulation of PSI at colder temperatures. The cyanobacterial gene TRINITY_DN44076_c0_g1 is strongly upregulated at 25°C and moderately upregulated at 15°C as well as 4°C – at the control temperature of 4°C the gene is not expressed (figure 24). This gene is orthologous to chaperonin GroEL, a heat shock protein that refolds denatured and misfolded proteins (Goloubinoff, et al., 1997). Under stress conditions, including high-temperature stress, proteins are likely to be misfolded which usually interferes with their metabolic activities. A protein’s restriction or even loss of function represent an irreversible damage to cells, which is why stress- induced deleterious effects should be avoided. Heat shock genes like chaperonins are essential to stress tolerance as they prevent protein denaturation and assist in protein refolding (Walter & Buchner, 2002). The heat shock protein GroEL is synthesised as a response to stress, this means it is part of the stress response mechanism and crucial for the cell’s coping with unfavourable conditions (Llorca, et al., 1998). By binding to proteins affected by stress, chaperonins – as well as other heat shock proteins – are able to stabilise these proteins and hamper their denaturation or aggregation with other proteins (Goloubinoff, et al., 1997). In the P. britannica specimens of this study GroEL is already expressed at a temperature of 4°C. This could be caused by other stress factors, e.g. oxidative stress or light stress (Susin, et al., 2006). High light can cause stress reactions, especially in wet thalli. In a desiccated state, lichens show increased stress tolerance (Gasulla, et al., 2012). Thus, light stress might be the reason that the activity of chaperonins is upregulated at 4°C. At the control temperature, which was 4°C as well, the gene is not expressed, but this might be due to the fact that the control group was sampled right after collection and therefore was not exposed to a diurnal light-dark cycle; therefore, light stress and cellular stress response might be reduced in this group. Besides, GroEL is not just expressed in stressed cells but under normal growth conditions as well, involved in the folding of newly synthesised proteins (Susin, et al., 2006) which could also explain its upregulation at 4°C.

A range of cyanobacterial genes show temperature-related differential gene expression in Peltigera britannica. The top five significantly DE genes are responsible for either (heat) stress response or photosynthesis. The upregulation of chaperonins and sigma factors seems to be a direct response to heat stress. The bleomycin resistance protein might be a response to heat stress, but could also have been induced by other stress factors or a combination of various stress factors. The exact function of the lysyl-tRNA synthetase in the cyanobacterial genes of P. britannica is not known but this enzyme seems to be part of the organism’s stress response as well. Lysyl-tRNA synthetase is also responsible for the production of lysyl-tRNA, however, this function does not explain why the enzyme is expressed at 15°C and 25°C only. In general, heat stress is induced in cyanobacterial genes already at a temperature of 15°C. The cyanobacterial partner of the lichen symbiosis 48 therefore seems to be less tolerant to heat stress than the fungal partner. This however, is not surprising, as the cyanomorphs of P. britannica only grow in shady, moist and sheltered habitats in which environmental fluctuations are rare (personal observations). The preference to such sites might simply reflect the cyanobiont’s limited tolerance to increased temperatures. This environmental effect has also been found in other photosymbiodemes (Green et al. 1993; Lange et al. 1988; Purvis 2000). Furthermore, an activation of PSI and therefore photosynthesis at increased temperatures has been determined for one DE cyanobacterial gene. The activation of this PSI gene might be a result of heat stress-induced inactivation of the PSII. However, it has not been proven that PSII is actually inactive. As not all significantly differentially expressed genes have been functionally annotated, there might be differentially expressed cyanobacterial genes encoding proteins of the PSII. In this case, the upregulation of the photosystem I core protein PsaB would not be due to an inactivation of PSII but the result of an overall increase in the cyanobacterium’s photosynthesis at higher temperatures. Whether the temperatures tested induce inactivation of PSII or a general increase in photosynthesis is difficult to determine, as the optimal photosynthetic temperature is dependent on various other environmental factors like light and water availability (Alam, et al., 2015). The photosynthetic activity and therefore the optimal temperature are also subject to seasonal changes. As Kershaw (1977a) has assessed on two Peltigera species, the optimum temperature for net photosynthesis is low in winter and high in summer, corresponding to the ambient temperature. Lichens are therefore capable of photosynthetic acclimation (Kershaw, 1977a). As the Peltigera britannica specimens used in this study were sampled in summer, at a temperature of about 12°C, this temperature might be the optimal temperature for net photosynthesis. This would explain an upregulation of photosynthetic genes at 15°C and 25°C, as well as the upregulation of cold-responsive ascomycete genes (in the form of the mediator complex). Furthermore, Kershaw (1977b) determined that in spring and autumn lichens can acclimate rapidly to changing temperatures. However, in mid-summer, lichens are not able to acclimate to cold temperatures and vice versa (Kershaw, 1977b). The fact that some lichens are not capable of photosynthetic acclimation to cold temperatures in mid- summer could explain why photosynthetic genes in this study are downregulated at 4°C. The temperature of 15°C might be the optimal temperature for net photosynthesis in the photosymbiodemes of P. britannica when acclimated to summer conditions. Whether 25°C is inhibiting overall photosynthesis of the cyanobionts by inactivating PSII or whether this temperature is within the optimal photosynthetic temperature of the organisms and thereby increases overall photosynthesis cannot be determined with certainty. But as 25°C leads to an expression of genes that are relevant for heat shock response, this temperature might also be high enough to partly inactivate the photosynthetic apparatus.

Differentially expressed chlorophyte genes in dependency on temperature The chlorophyte gene TRINITY_DN47627_c2_g1 is significantly differentially expressed in P. britannica in dependency on temperature. It is upregulated at 15°C and 25°C as well as in individual 2.2 and 3.2 (and to a smaller extent in individual 1.2) (figure 25). TRINITY_DN47627_c2_g1 has high similarity with the light-harvesting chlorophyll a/b- binding protein Lhca2. Lhca2 is a light-harvesting complex (LHC) protein that, together with other proteins and photosynthetic pigments, forms LHCs which are responsible for capturing light for photosynthesis. LHCs are part of both photosystem I and photosystem II, yet these complexes differ in their protein structure. The LHC protein Lhca2 belongs to

PSI and builds up the LHC I complex with Lhca1, Lhca3 and Lhca4 (Ganeteg, et al., 2001). As already described for the effects of high-temperature stress on the cyanobacterial genes of P. britannica, the function of PSI is enhanced under stress conditions. This is due to the high vulnerability of PSII to heat stress –PSII gets inactivated in these adverse situations, PSI on the other hand gets activated. As a result, potential damage to PSII is being avoided or at least reduced (Ivanov et al. 2017; Yamauchi et al. 2011). The upregulation of Lhca2 in the chlorophyte genes of the lichen specimens in our sample might therefore be the result of PSI activation under high-temperature stress. However, this gene expression is dependent on the individual as well which indicates that not all four individuals react equally to increased temperatures – at least concerning their photosynthetic activity. The chlorophyte gene TRINITY_DN46800_c0_g1 is only expressed at a temperature of 25°C (figure 26). According to NCBI-Blast two different proteins are highly similar to this gene: an ammonium transporter as well as a class I heat shock-like protein. The upregulation of the latter at high temperatures is anticipated, as heat shock proteins are part of organisms’ stress response system. Their purposes in stressed cells have already been discussed in this paper and do not require further clarification (Beere 2004; Walter & Buchner 2002; Llorca et al. 1998; Goloubinoff et al. 1997). The ammonium transporter (Amt) is – as its name implies – responsible for uptake as well as transport of ammonium (Santos, et al., 2017). Like cyanolichens, which contain nitrogen-fixing cyanobacterial photobionts, tripartite lichens are able to fix atmospheric nitrogen as well – as they contain cyanobacteria in special structures called cephalodia. The rate of nitrogen fixation increases with temperature (Millbank & Kershaw, 1969). Chlorolichens, on the other hand, which contain only algae as a photosynthetic partner, are not able to fix nitrogen (N) and rely on the uptake of ammonium (NH4+) or nitrate (NO3-) as nitrogen sources (Dahlman, et al., 2004). Nitrogen is a vital nutrient in plants as well as lichens as it positively affects growth and photosynthetic performance. Its role in photosynthesis is due to the fact that nitrogen is a component of photosynthetic pigments and enzymes. Nitrogen availability also controls the amount and size of chloroplasts (Bassi, et al., 2018). As the chloromorph of P. britannica in our study is tripartite, the green algal photobionts could obtain nitrogen from the cyanobacteria. However, it has been shown that most of the fixed nitrogen is utilised by the mycobiont. Therefore, the algae might obtain their nitrogen sources either from the fungus or use a portion of the fixed nitrogen that the mycobiont did not absorb (Kershaw & Millbank, 1970). Furthermore, the chloromorph’s nitrogen source could be either nitrate or ammonium (Dahlman, et al., 2004), and the latter is obtained and transported via ammonium transporters (Santos, et al., 2017). An upregulation of Amts in the chlorophyte genes of our lichen samples at 25°C could be due to increased nitrogen fixation activity at higher temperatures. If N itself is not available to the algal cells, they might use nitrogen compounds like ammonium for nitrogen metabolism, which could also explain the expression of Amts. Furthermore, various studies have assessed that ammonium has a mitigating effect on cellular damage caused by stress and is likely to enhance defence mechanisms (Cao et al. 2019; Li et al. 2017). Ammonium therefore leads to an increased tolerance towards stress. The upregulation of Amt in P. britannica might hence be part of the organism’s stress response. It cannot be determined with certainty whether increased nitrogen fixation activity at 25°C or reaction to high-temperature stress leads to the expression of the ammonium transporter. The chlorophyte gene TRINITY_DN43942_c0_g1 is upregulated at 25°C but does not show any expression at 4°C or 15°C (figure 27). This gene has high similarity with the oxygen-evolving enhancer protein 3 and is a component of the oxygen-evolving complex 50

(OEC) of photosystem II, being a part of the photosynthetic process. The OEC catalyses the oxidation of water to oxygen, a light-dependent reaction and key element of oxygenic photosynthesis (Raymond & Blankenship, 2008). As PSII is negatively affected by high temperature stress, the oxidising activity of the oxygen-evolving complex is inhibited (Zhao, et al., 2008). This observation contradicts our results, in which the expression of OEC proteins is increased at high temperatures. Hence, the upregulation of OEC proteins cannot be a consequence of the organism’s stress response. On the contrary, it seems much more likely that the photosynthetic apparatus in the chlorophytes benefits from a rise in temperature and therefore enhances its activity. 25°C isn’t an unusually high temperature which is why the photosynthetic complex might not be affected negatively by it. Photosynthetic optimum temperatures are normally distributed; too cold as well as too hot temperatures have negative effects on photosynthesis whereas a temperature range in between promotes an organism’s photosynthetic activity. Moreover, the optimal temperature is species-dependent with some species preferring cold and others warm temperatures (Wagner, et al., 2014). Concerning chloro- and cyanolichens, differences in their photosynthetic activity in dependency on various abiotic factors have been reported. The hydration status of a lichen is of great relevance for its photosynthetic activity. High thallus water saturation increases the net photosynthesis rate in cyanolichens, but depresses it in chlorolichens. Yet, photosynthetic activity in chlorolichens can be induced by high air humidity without the presence of liquid water (Green, et al., 2002). Then again, hydrated cyanolichens are more susceptible to high light and therefore to photoinhibition than hydrated chlorolichens. This being the case, hydration might lead to photosynthetic inactivation in cyanolichens which especially affects PSII. Chlorolichens on the other hand, can withstand high-light photoinhibition when hydrated. However, desiccation together with high-light stress can cause photoinhibition in chlorolichens (Alam, et al., 2015). The lichens in our experimental setup were hydrated and exposed to light 12 hours daily. This, in combination with increased temperatures, could explain why PSII genes are upregulated in the chlorophyte but not in the cyanobacteria. Light, heat and water saturation might have caused a downregulation of PSII in P. britannica/cyan, whereas the green algae appear to have benefited from this experimental setup regarding their photosynthetic activity. TRINITY_DN48593_c1_g2 is a chlorophyte gene that is upregulated at 25°C (figure 28). It has high similarity with a small subunit of chloroplast ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). RuBisCO is the enzyme that catalyses the fixation of atmospheric carbon dioxide (CO2) to ribulose-1,5-bisphosphate which produces two molecules of 3-phosphoglycerate. This reaction is part of the Calvin-Benson-Bassham cycle of photosynthesis. RuBisCO catalyses its reactions only slowly; however, a large number of RuBisCO in the cell compensates for its low turnover frequency (Erb & Zarzycki, 2018). RuBisCO is yet another chlorophyte gene encoding proteins involved in photosynthesis. The upregulation of a set of these genes indicates that 25°C does not inhibit photosynthesis but rather promotes it. Therefore, a temperature of 25°C seems to be within the optimal temperature range of the chlorophyte’s photosynthetic activity. As various components of the photosynthetic apparatus are upregulated and not just those of the PSI, the expression of these genes is unlikely to be a response to heat stress. It is difficult to determine the photosynthetic optimum temperature of an organism as the rate of photosynthesis is dependent on other environmental factors apart from temperature, including light and hydration (Alam, et al., 2015). The combinations of different environmental factors (e.g. due to change of seasons) can lead to an alteration of photosynthetic optimum temperature within an organism as a result of the organism’s adaptation to its environment (Wagner, et al., 2014). The photosynthetic chlorophyte 51 genes of P. britannica/chlor are mostly upregulated at 25°C, some already at 15°C, indicating that the optimal temperature for photosynthesis in the chloromorph includes the temperatures of 15°C to 25°C. While the increase in temperature seems to be advantageous for the lichen’s photosynthetic activity, the expression of heat shock proteins signalises that higher temperatures are not preferable for other cellular processes but rather are considered stress-inducing. Another differentially expressed chlorophyte gene is TRINITY_DN43001_c0_g4, which is upregulated at 25°C (figure 29). The gene is highly similar to RNA helicases and antiviral helicases (which can be RNA or DNA helicases). Therefore, the gene could be encoding an RNA helicase. RNA helicases modify RNA secondary structure and are integral for the RNA metabolism. Abiotic stress, including temperature stress, can influence the expression of RNA helicases, whose pathways are described to be similar to that of heat shock response mechanisms (Rosana, et al., 2012). RNA helicases might be responsible for the initiation of translation, the export of mRNA, the processing of rRNA and transcription of stressed cells. Furthermore, RNA helicases could be relevant for RNA proofreading. Their exact role in cellular stress response has yet to be determined, however (Owttrim, 2006). These results suggesting that RNA helicases are a part of a cell’s stress response, including response to heat stress, are representative of our own findings of increased RNA helicase expression at high temperatures. Despite their precise role in stressed cells being uncertain, an upregulation of these enzymes at 25°C in our samples was not unexpected.

In general, there is a range of differentially expressed chlorophyte genes in dependency on temperature. Most of these genes encode for either proteins responsible for (heat) stress response or photosynthetic proteins. The upregulation of RNA helicases and various Hsps seem to be a direct consequence of increased temperature. The expression of these genes begins at a temperature of 25°C only, suggesting that chlorophyte might tolerate heat to a greater extent than the cyanobacterial and fungal partners of the lichen symbiosis. In the mycobiont and the cyanobacterial photobiont, upregulation of heat shock proteins was induced at 15°C already. This is consistent with the findings of Green et al. (2002) as well as personal observations that P. britannica/chlor can grow in open habitats, that is habitats with are exposed to fluctuations in temperature, light and water availability, whereas P. britannica/chlor+cyan only grow in shady and moist habitats where environmental factors remain relatively constant. This distinct distribution pattern of the two photomorphs implies that P. britannica/chlor is more stress tolerant, including heat stress, than the cyanomorph. This would explain why heat stress proteins are upregulated at 25°C only. Furthermore, photosynthetic activity of the chlorophyte is upregulated at 25°C. Unlike in the cyanomorph in which we could only observe the expression of PSI proteins, in the chloromorph proteins of both the PSI and the PSII were upregulated. 25°C seem to be an optimal photosynthetic temperature in chlorophytes, however, as already mentioned before, the assessment of optimal photosynthetic temperatures is difficult as photosynthesis is also influenced by other environmental factors like light and water availability (Green et al. 2002; Alam et al. 2015). The effect of temperature alone on photosynthetic activity is difficult to determine. Furthermore, seasonal changes also influence the lichen’s optimal photosynthetic temperature. In summer the optimal temperature usually is higher than in winter, lichens are therefore capable of photosynthetic acclimatisation and can react to changing environmental factors (Kershaw 1977a; Kershaw 1977b; Wagner et al. 2014). The chlorophyte’s photosynthesis genes of

52 the Peltigera britannica specimens in our study are mostly upregulated at 25°C, some already at 15°C, indicating that the optimal temperature for photosynthesis in the chloromorph includes a temperature range of 15°C to 25°C. An upregulation of genes responsible for respiration has not been observed, even though respiration is said to increase with temperature. However, it has been shown that lichens are able to acclimate their respiration rates to seasonal changes, resulting in higher respiration rates in winter than in summer (Lange & Green, 2005). At large, an increase in temperature seems to be advantageous for the lichen’s photosynthetic activity, however, the expression of heat shock proteins indicates that a temperature of 25°C is not preferable for other cellular processes.

Conclusion The differential gene expression analyses in this study have illustrated the effect of temperature stress on all partners involved in a lichen symbiosis as well as a distinct photomorph-mediated gene expression pattern of the mycobiont of the photosymbiodeme. The mycobiont, which is the same species in both photomorphs of the photosymbiodeme, does have photomorph-mediated differential gene expression. Ascomycete genes that are upregulated in one photomorph might therefore not be upregulated in the other photomorph. This is likely due to the interaction of the mycobionts with the photobionts (Singh, G. et al., 2019). It has been shown that the mycobiont’s gene expression is different in dependency on different photosynthetic partners, but it could not be definitely determined to what extent these differences effect the fungus or the symbiosis in general. The greatest difficulty in the determination of gene functions and their role in the organism is the overall lack of functional assignments of the genes in question. Many of the ascomycete genes differentially expressed between photomorphs have represented hypothetical and unspecified proteins or functions, hampering the functional characterisation of these DE genes. Even though the evaluation and assessment of differentially expressed ascomycete genes proves to be difficult, there nevertheless is potential for future studies to focus on those unknown genes and identify their biological roles and molecular functions to shed light on differential fungal gene expression in lichen symbiosis. Each organism in a lichen symbiosis, be it the fungus, the alga or the cyanobacterium, reacts to thermal stress – and each organism does so in a different manner. Both the ascomycetes, the cyanobacteria and the chlorophytes have an upregulated expression of heat shock proteins or heat shock-like proteins as a result of increased temperature. The expression of these proteins is part of an organism’s stress response; therefore, this result is expected. But the expression of Hsps reflects the tolerance respectively intolerance of the organism in question to heat stress. Upregulation of Hsps begins at 15°C in the ascomycete and cyanobacteria genes, indicating a higher susceptibility to stress compared to chlorophyte genes, in which the upregulation of Hsps starts at 25°C. The chlorophyte therefore seems to be more tolerant towards heat stress than the other major partners of the Peltigera britannica lichen symbiosis. Yet, the response to thermal stress is not an absolute term as the tolerance and susceptibility to stress is subject to fluctuations. Different populations of the same species might react differently to stress, depending on differential acclimatisation to the environment (MacFarlane & Kershaw, 1980). Furthermore, seasonal changes in stress response have been reported for lichens, as they usually tolerate higher temperatures in summer and lower temperatures in winter as part of seasonal adaption (Tegler & Kershaw, 1981). There is also a variety of other factors that influence an organism’s tolerance to stress, 53 including light conditions and water availability (Green et al. 2002; Gasulla et al. 2012). Increased temperature might be perceived more or less stressful by the organism relative to those other factors. The distribution pattern of different photomorphs of the lichen P. britannica might be indicative of the respective photobiont’s stress tolerance. P. britannica/chlor grow in open areas, where they are more exposed to the elements – as there is less shading, the temperature fluctuations are higher and risk of desiccation is greater. Chloromorphs therefore need to be able to tolerate stress in order to survive in these rather harsh environments. Cyanomorphs, on the other hand, would only grow as photosymbiodemes at relatively sheltered, damp and shady sites – e.g. in small cavities or deeply immersed under shrubs – suggesting that the cyanobacteria are more susceptible to the environmental stresses conveyed by open areas. Furthermore, cyanomorphs need liquid water for photosynthetic activity (Green, et al., 2002); they therefore have to occur at sites with high water availability, where desiccation occurs seldomly. Therefore, P. britannica/chlor+cyan can only grow at sites where both morphs benefit to a certain extent from the environmental conditions and where the environmental factors do not seriously impair the growth or survival of either morph (Green, et al., 2002). In the chlorophytes and cyanobacteria, photosynthetic activity was increased at higher temperatures (15°C and 25°C). In the cyanobacteria, PSI genes were expressed, which might be due to inactivation of PSII under heat conditions, but could also be the result of an overall increase in photosynthetic activity due to a rise in temperature. In the chlorophyte both PSI and PSII genes were upregulated, suggesting that the chlorophyte’s photosynthetic activity increases with higher temperatures. As both photosystems are active in the chlorophyte, the photosynthetic apparatus does not seem to be negatively affected by heat stress. The photosynthetic optimum temperatures of an organism are difficult to determine, as they are species-dependent (Wagner, et al., 2014) as well as dependent on various abiotic factors (Green et al. 2002; Alam et al. 2015). Furthermore, photosynthetic activity and photosynthetic optimal temperature are subject to seasonal changes as well (Kershaw 1977a; Kershaw 1977b). Therefore, it is likely, that the optimal temperature for photosynthesis in our P. britannica specimens is about 15°C, as the lichens were collected in the Icelandic summer at temperatures of about 12°C. A temperature of 25°C could negatively affect photosynthesis and inactivate certain components of the photosynthetic apparatus (like PSII). However, a temperature of 25°C could still be in the range of optimal photosynthetic temperature and enhance photosynthetic activity as a consequence. In general, P. britannica prefers lower temperatures, as the photosynthetic activity of the photobionts is not compensated for by the respiration activity of the mycobiont.

This study offers valuable insights into differential gene expression in photosymbiodemes of the lichen species Peltigera britannica. The analysis of gene expression in all partners involved in the symbiosis – fungi, green algae and cyanobacteria – illustrates that the interaction between the symbiosis partners can lead to differential gene expression; as is the case in the mycobiont, whose gene expression is partly dependent on the type of photosynthetic partner. Yet, despite the close interaction between the symbiosis partners, they still maintain individual characteristics, as they, for example, react differently to stress, regardless of the partner. Especially for the fungal genes, the functional annotation proved to be difficult, as many proteins encoded by these genes are unknown or hypothetical. However, future studies could elaborate on the unknown functions and shed light on the differential fungal gene expression in lichen symbiosis; especially, as these unknown genes might not only be of great relevance for the lichen and its biology but for our understanding of the lichen symbiosis as well. 54

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