Identification and expression of functionally conserved circadian clock genes in lichen-forming fungi

Abstract

Lichen-forming fungi establish stable symbioses with green algae or cyanobacteria. Many species have broad distributions, both in geographic and ecological space, making them ideal subjects to study organism-environment interactions. However, little is known about the specific mechanisms that contribute to environmental adaptation in lichen-forming fungi. The circadian clock provides a well-described mechanism that contributes to regional adaptation across a variety of species, including fungi. Here, we identify the putative circadian clock components in phylogenetically divergent lichen-forming fungi. The core circadian genes (frq, wc-1, wc-2, frh) are present across the Fungi, including 31 lichen-forming species, and their evolutionary trajectories mirror overall fungal evolution. Comparative analyses of the clock genes indicate conserved domain architecture among lichen- and non-lichen-forming taxa. We used RT-qPCR to examine the core circadian loop of two unrelated lichen-forming fungi, Umbilicaria pustulata (Lecanoromycetes) and Dermatocarpon miniatum (Eurotiomycetes), to determine that the putative frq gene is activated in a light-dependent manner similar to the model fungus Neurospora crassa. Together, these results demonstrate that lichen-forming fungi retain functional light-responsive mechanisms, including a functioning circadian clock. Our findings provide a stepping stone into investigating the circadian clock in the lichen symbiosis, e.g. its role in adaptation, and in synchronizing the symbiotic interaction.

Figure 1

Presence of core circadian clock genes in fungi and domain architecture of White Collar-1 (wc-1). (a) Phylogenetic tree based on a WC-1 sequence alignment of 31 lichen-forming fungi (green) and a selection of non-lichen-forming taxa. This is a maximum likelihood tree. Bootstrap support greater than 75% is indicated at the branches. Taxa used in the experimental part of this study are marked by asterisks (***). Presence/absence of putative circadian homologs from Neurospora crassa is shown adjacent to each species. Functional domains annotated according to Pfam (b–d) and PROSITE (e–g) databases in lichen-forming (d, g) and non-lichen-forming lineages in the Ascomycota (c, f) as well as the Basidiomycota and Mucoromycota (b, e).

Figure 2

Homologs of frq in two lichen-forming fungi are responsive to light. (a) Thalli of the lichen-forming fungi Dermatocarpon miniatum and Umbilicaria pustulata, harvested in the Taunus mountain range of central Germany. (b) Lichen thalli were acclimatized to standard laboratory conditions via a 72 h LD 12:12 cycle at 16° C before 24 h in constant darkness, followed by a 20-min light induction at CT 0. (c) RT-qPCR analysis demonstrates broad conservation of light responsiveness in the circadian clock components of lichen-forming fungi. n = 6 for Dmin, n = 5 for Upust per treatment; mean ± SEM are shown. Asterisks represent significance extracted using Šidàk-adjusted contrasts: *P < 0.05; **P < 0.01; ***P < 0.001. Dmin: Dermatocarpon miniatum; Upust: Umbilicaria pustulata; frq: frequency; wc-1: white collar-1.