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.