1 | Introduction
A lichen is a symbiotic organism composed of a fungal partner, the mycobiont, and a photosynthetic partner, the photobiont, which can be an alga and/or a cyanobacterium (Schwendener, 1868; Armaleo & Clerc, 1991). Lichens can be found in virtually every terrestrial ecosystem and due to their poikilohydric nature, they manage to survive even in harsh environments such as polar regions and coastal deserts and can withstand extreme temperatures as well as other abiotic stress factors (Werth, 2011). By association with locally adapted photobionts a lichenized fungus may be able to persist under diverse environmental conditions and to occupy large geographic ranges (Werth & Sork, 2014). For some lichen genera (e.g. Peltigera ), an association with a broad range of photobiont strains has been reported (Jüriado et al., 2019; Lu et al., 2018; O’Brien et al., 2005). Lichen-forming fungi are also able to switch photosynthetic partners, to form a stable symbiosis with a more compatible partner; incompatible symbiosis impedes thallus development and lichen growth (Beck et al., 2002; Insarova & Blagoveshchenskaya, 2016). The photobiont type can also determine the lichenized fungus’s fitness by impacting its tolerance of ecological conditions (Ertz et al., 2018; Hyvärinen et al., 2002; Casano et al., 2011). A lichenized fungus’s flexibility in choosing a suitable partner might therefore promote wide geographic distributions and it might also affect the establishment of the symbiosis within its environment (Ertz et al., 2018; Magain & Sérusiaux, 2014; Casano et al., 2011).
For several genera of lichenized fungi belonging to the Peltigerineae, including Peltigera , two distinct morphs have been described – chloromorphs containing green algae and cyanomorphs containing cyanobacteria as main photosynthetic partner; a form of lichen symbiosis referred to as a ‘photosymbiodeme’ (Green et al., 2002). These two morphs can grow as separate individuals but they may also grow as one single compound thallus with green algal and cyanobacterial sectors. Chloromorphs and cyanomorphs of lichens often show pronounced morphological and ecological differences (Hyvärinen et al., 2002; Green et al., 1993; Holtan-Hartwig, 1993), although they contain the same fungal species (Armaleo & Clerc, 1991). The type of photobiont can profoundly affect the ecology of the symbiotic association. For example, depending on its photobiont type, a lichen is able to tolerate stress to a greater or lesser extent; this has been shown for light stress (Demmig-Adams et al., 1990; del Hoyo et al., 2011) and oxidative stress (del Hoyo et al., 2011). Moreover, photobiont types can influence the photosynthetic performance (Green et al., 1993; Henskens et al., 2012), and enable the colonization of nutrient-poor habitats in the case of a cyanobacterial partner thanks to its nitrogen fixation (Goffinet & Hastings, 1994; Almendras et al., 2018; Hitch & Millbank, 1975). Thus, photobiont types can determine stress responses and ecology of lichens.
There are large differences between green algae and cyanobacteria with respect to physiology and cell morphology, which impact the way in which these photobionts can interact with their lichenized fungi. First of all, green algal photobionts are most often photosynthetically active at high ambient relative humidity (96.5%), while cyanobacterial photobionts require the lichen thallus to be hydrated by liquid water (Lange et al., 1986). Secondly, green algal and cyanobacterial photobionts also differ markedly in the photosynthates which are transferred to the lichenized fungi, sugar alcohols like ribitol versus glucose (Richardson & Smith, 1968; Hill, 1972). Thirdly, they additionally differ in the structure and chemistry of their cell envelopes. The sturdy green algal cell walls contain cellulose (Domozych et al., 2012) and in the case of Coccomyxa the exceptionally resilient biopolymer sporopollenin (Honegger, 1984; Honegger & Brunner, 1981). In contrast, cyanobacterial cell envelopes are made of peptidoglycan encapsulated in a polysaccharide sheath (Hoiczyk & Hansel, 2000; Woitzik et al., 1988). Fourthly, although the formation of various chemotypes also depends on environmental factors (Cornejo et al., 2017; Culberson, 1986; Hale, 1957; Skult, 1997), photobiont type can affect the composition and content of carbon-based secondary compounds of lichens and chloromorphs have been reported to contain different secondary metabolites than cyanomorphs of the same fungal species (Kukwa et al., 2020; Tønsberg & Holtan-Hartwig, 1983). The different partners involved in the symbiosis can individually produce different secondary metabolites, and certain fungal metabolites are only produced in symbiosis with a specific photosynthetic partner. For instance, cyanobacteria – free-living and symbiotic ones – are able to produce toxins, e.g. when stressed (Kaasalainen et al., 2012; Gagunashvili & Andrésson, 2018). The effects of these toxins on the fungal and – in the case of tripartite lichens – the green algal partner as well as on other components of the lichen are still poorly known (Kaasalainen et al., 2009; Ivanov et al., 2021; Vančurová et al., 2018; Kaasalainen et al., 2012). Taken together, these marked physiological and structural differences imply that there must be different ways of interaction among partners, which should be reflected at the molecular level e.g., with respect to the fungal gene regulation depending on the interaction with specific symbiotic partners.
Stress responses are vital for survival and persistence of species in different environments, yet it is still not well understood under which conditions the different partners involved in lichen symbioses experience stress. In studies reporting gene expression of lichens exposed to temperature treatments, cyanobacterial photobionts expressed heat shock genes at lower temperatures than lichenized fungi (Steinhäuser et al., 2016), but green algal photobionts expressed heat shock at the same temperature as the lichenized fungus (Chavarria-Pizarro et al., 2021). Yet, to our knowledge, no study has so far addressed stress responses of cyanobacterial and algal photobionts simultaneously within the same compound lichen thallus. For symbiodemes, it is an important open question if the two photobiont types exhibit distinct stress responses at different temperatures.
Because they contain the same fungus and grow under the same environmental conditions, compound thalli with green algal and cyanobacterial sectors represent an ideal study system to explore photobiont-mediated differences in gene expression. The compound thalli can be exposed to identical experimental conditions as a closed system, which enables the examination of photobiont-mediated fungal gene expression and of photobiont-mediated gene expression of the symbiosis as a whole. Compound thalli are also ideally suited to address the question if the symbiotic partners share ecological optima. Previous observational field studies of lichen photosymbiodemes have shown morph-dependent habitat preferences (Elvebakk et al., 2008; Green et al., 1993; Holtan-Hartwig, 1993; Tønsberg & Holtan-Hartwig, 1983), which suggests that the symbiosis partners may differ in their ecological optima.
Here, we investigated differential gene expression in a photosymbiodeme-forming lichen that we exposed to different temperatures, including putatively stressful conditions, to test the hypothesis that the lichenized fungi and the green algal and cyanobacterial photobionts of compound thalli differ in ecological optima, causing them to experience thermal stress at different temperatures. Because of pronounced physiological and structural differences between green algal and cyanobacterial photobionts, we moreover hypothesized that the lichenized fungus exhibits differential gene expression mediated by the type of its photosynthetic partner. The results of this study are key to better understand how different partners influence the ecology of these enigmatic symbiotic organisms.