Figure legends
Figure 1. Photosymbiodemes of the lichen Peltigera britannica. (A) green-algal tripartite lobes (apple green) emerge from a bipartite thallus (bluish-gray) that has a cyanobacterium as a photosynthetic partner; (B) small cyanobacterial lobes develop from the cephalodia of a tripartite thallus.
Figure 2. Experimental setup to study differential gene expression in Peltigera britannica photomorphs. Dark green parts represent the cyanomorph, light green parts the tripartite morph. Lichen thalli were exposed to 4 °C for five days (treatment “4 °C_1”), then to 4 °C for two weeks (treatment “4 °C_2”), then to 15 °C for two hours (treatment “15 °C”), and finally to 25 °C for two hours (treatment “25 °C”). After each step, pieces of thallus (ca. 1 cm2) of the cyano- and the tripartite morph were cut off from all four specimens for RNA extraction.
Figure 3. PCA plot for the overall gene expression variance of the mycobiont. The principal component of variance in the mycobiont was temperature-mediated rather than photomorph-mediated (circle = tripartite morph; triangle = cyanomorph), with clusters for low (4 °C_1; green and 4 °C_2; blue), medium (15 °C; orange) and high temperatures (25 °C; red).
Figure 4. (A) 200 most significantly differentially expressed fungal genes of the cyanomorph and the tripartite morph. Lighter shades show DEGs that could not be functionally annotated. 123 DEGs were assigned to the cyanomorph (teal color) and 77 to the tripartite morph (green color). (B) Temperature-mediated differential gene expression of mycobiont, chlorophytes and cyanobacteria (comparing 25 °C with 4 °C_1). Bars include the 200 most significantly differentially expressed genes of each organism. Red bars show genes upregulated at 25°C, blue bars genes downregulated at 25°C. Lighter shades show genes that could not be functionally annotated. 103 of the 200 most significantly differentially expressed fungal genes were downregulated at 25 °C, 97 were upregulated; in the photobionts, 199 genes were upregulated and one was downregulated at 25 °C, respectively.
Figure 5. Visualization of the main biological processes (BP) attributed to differentially expressed ascomycete genes of the cyanomorph and the tripartite morph of Peltigera britannica. Different BPs are shown with distinct colors; section sizes correspond to the number of genes associated with a GOterm. In the cyanomorph, the main BPs were oxidation-reduction process (62%), protein carbohydrate metabolic process (18%) and protein phosphorylation (18%); a minor BP not shown in the graph is GPI anchor biosynthetic process. In the tripartite morph, the majority of fungal DEGs was annotated to oxidation-reduction process (60%), followed by transmembrane transport (32%), tricarboxylic acid cycle (1.55%) and phospholipid biosynthesis (1.49%). Minor BPs of the tripartite morph not depicted are protein peptidyl-prolyl isomerization, threonyl-tRNA aminoacylation, inositol biosynthetic process and cellular potassium ion homeostasis. Analysis based on GO annotation (Bioconductor package topGO).
Figure 6. Expression of stress proteins in the three lichen symbiosis partners based on the counts of the corresponding genes at each temperature. In the mycobiont (orange), upregulation of stress proteins begins at 15 °C already and it shows high stress at 25 °C; the same is true for the cyanobacteria (teal), although a higher number of stress related genes are expressed at lower temperatures (4 °C_1 and 4 °C_2) than in the mycobiont. Chlorophytes (green) appear to be stressed mainly at 25 °C.