Semilichen, an unjustly neglected symbiotic system between green biofilms and true lichens

Abstract

Symbiotic systems of photosynthetic microorganisms and fungi are widespread in terrestrial biomes and lichens are probably the most advanced and complex. Conversely, the least complex systems are "green biofilms" with a completely unexplored mycobiome. We describe here a new system intermediate between green biofilms and lichens-semilichens. Light and fluorescence microscopy, eDNA sequencing, molecular phylogeny, Chlorophyll a fluorescence and ¹³C labelling/metabolomics were used to study algal and fungal identity, morphology and physiology of the symbiosis. Tight contact between algae and a single predominant fungus (mycobiont) is revealed in semilichens. The algae are from the symbiotic lineages of Trebouxiophyceae and Ulvophyceae, the fungi belong to Arthoniomycetes, Dothideomycetes, Eurotiomycetes, Lecanoromycetes and Lichinomycetes. Algae are alive and perform substantial photosynthetic activity. ¹³C labelled photosynthates are partially converted into specific fungal polyols (arabitol, mannitol) demonstrating the C-flow from algae to fungi. The new symbiotic system was defined and compared with other terrestrial algal-fungal symbioses. It is characterized by minimalist environmental requirements and extremely low production of biomass. As a result, it also inhabits environments unfavourable for lichens. Our research supports the hypothesis that the long-term existence of algae and fungi in terrestrial conditions affected by frequent and repeated drying is likely dependent on their mutual coexistence.

Keywords: 13C isotope labelling; Algal-fungal symbiosis; Anhydrobiosis; Metabolomics; Photosynthesis; Trebouxiophyceae.

Fig. 1

Corticolous green biofilm on bark of Tilia in urbanised landscape. A, observed in visible light where algal coating is conspicuous but the mycobiom is invisible. B, observed with fluorescence in blue light where the associated mycobiome (dark hyphae) is well recognised. Scales, 0.5 mm.

Fig. 2

Fungal and algal systematic units involved in the fourteen examined semilichens. Algal species/OTUs previously known from lichen symbioses are in bold. Specific relationships between mycobiont species and algal species/OTUs are illustrated by the deep purple squares.

Fig. 3

Semilichen Arthopyrenia analepta (Dothydeomycetes, Trypethelliales) with perithecioid fruiting bodies and associated with Trebouxiophyceae. A, C, observed in visible light, hyphae of mycobiont invisible. B, D, observed with fluorescence in blue light where the algal-fungal association is readily visible. Specimen: Vondrák 27331 (PRA). Scales, 0.2 mm.

Fig. 4

Semilichen Arthopyrenia cerasi (Dothydeomycetes, Trypethelliales) with Trentepohlia. Algal cells occur under a layer of smooth scaly bark and are invisible by observation of the surface. A, B, fruiting bodies (perithecia) observed on the surface of bark of Corylus avellana. A, observed in visible light; B, observed with fluorescence in UV. C–E, algal-fungal association observed on a bark layers several tens of micrometres below the surface. Specimen: Vondrák 27680 (PRA). Scales, A, B, 0.2 mm; C, D, 50 µm; E, 10 µm.

Fig. 5

Semilichen Naetrocymbe punctiformis (Dothydeomycetes, Capnodiales) with Trebouxiophyceae. A, B, fruiting bodies (perithecia) on the surface of bark. A, observed in visible light, hyphae of mycobiont invisible; B, observed with fluorescence in blue light, fungal hyphae surrounding algal colonies readily visible. C, D, details of algal-fungal association with fluorescence in blue light. Specimen: Vondrák 28343 (PRA). Scales, A, B, 0.2 mm; C, D, 50 µm.

Fig. 6

Maximum quantum yield of photosystem II (F_v/F_m) for a green biofilm (A-C), semilichens (D-L) and a true lichen (M–O). Left column shows fluorescence intensity, which is related to chlorophyll content. Photobionts were removed from part of samples using scalpel (areas delimited by white lines). The most intensive fluorescence is visible in patches where rhytidoma, masking green plant tissues, was also removed (especially D,M). Middle column are native samples, whereas right column are samples dried for four days in laboratory conditions to deactivate/kill green plant tissues and rewetted one hour before measurement again. Semilichens are: Arthopyrenia salicis with Trentepohlia (D-F), Cyrtidula quercus with Trebouxiophyceae (G-I) and Naetrocymbe punctiformis with Trebouxiophyceae (J-L). True lichen is: Graphis scripta with Trentepohlia (M–O). Calibration plate (zero F_v/F_m) has dimension of 20 × 20 mm.

Fig. 7

Chlorophyll a fluorescence kinetics of the semilichen Cyrtidula quercus. Visual situation (A), absolute values fluorescence (B) and fluorescence standardised to F_o = 1 (C). Comparison of symbiosis (blue line, area1), plant tissue where photobionts were removed (red line, area2) and calibration plate where no photochemistry occurs (black line, area3) is made. Dark adapted sample was subjected to low intensity measuring light (< 1 µmol m⁻² s⁻¹) for first 4 s, then to saturating flash (≈ 1000 µmol m⁻² s⁻¹ ) for one second allowing us to calculate maximum quantum yield of PS II (F_v/F_m). After short dark relaxation (6 to 19s), in time 20 to 90 s, actinic light (150 µmol m⁻² s⁻¹ ) with five superimposed saturating flashes were applied to find photochemical (photosynthesis) and non-photochemical (photoprotection) quenching. Last part (91-190s) is dark relaxation with three saturating flashes to obtain relaxation rate of photoprotective mechanisms.

Fig. 8

Green biofilm and semilichens—Percentage of new carbon in particular sugars and polyols 2 h, one day and four days after ¹³CO₂ labelling. Gradual incorporation of new carbon into fungal polyols (arabitol and mannitol) is visible. (A), corticolous green biofilm (see Fig. 1); (B, C), two different samples of Leptorhaphis maggiana (Eurotiomycetes, Phaeomoniellales); (D), Naetrocymbe punctiformis (Dothydeomycetes, Capnodiales); (E), Naevia punctiformis (Arthoniomycetes, Arthoniales); (F), Stenocybe pullatula (Ostropomycetidae, Mycocaliciales); (A-F), associated photobionts are Trebouxiophyceae.

Fig. 9

Lichens with Trebouxiophyceae photobionts – Percentage of new carbon in particular sugars and polyols 2 h, one day and four days after ¹³CO₂ labelling. Gradual incorporation of new carbon into fungal polyols (arabitol and mannitol) is visible. (A), Phlyctis argena (Ostropomycetidae, Gyalectales); (B), Ramalina farinacea (Lecanoromycetidae, Lecanorales); (C), Rhizocarpon geographicum (Lecanoromycetidae, Rhizocarpales) and (D), Usnea hirta (Lecanoromycetidae, Lecanorales). E, Average ¹³C enrichment in all metabolites pooled. It is measure of CO₂ assimilation intensity (and reciprocally metabolite turnover) for the whole system. True lichens (in blue), one green biofilm (green) and hemilichens (red) are shown.

Fig. 10

Schematic illustration of the most common aerophytic symbioses in temperate habitats; (A), epilithic crustose lichen; (B), endolithic crustose lichen; (C), semilichen; (D), green biofilm. The typical crustose lichen (A) is composed of a complex multi-layer thallus, consisting of upper cortex (isodiametric fungal cells), algal layer and medulla (loose hyphal tissue adjacent to the substrate). An example of a crustose lichen with a reduced thallus is the endolithic lichen (B), in which the thallus tissues are hidden in the substrate. The semilichen (C) has a simple structure, consisting of a hyphal network growing over the substrate and associated with algal colonies. The green biofilm (D) consists of aggregated algal cells and associated mycobiome.