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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.
Introduction
Symbiotic associations of fungi and algae are more common in terrestrial biomes than generally assumed, and curiously, some of the most common are not included in the current review on algal-fungal symbioses1. The largest omitted group are the so-called “green biofilms”2. These are communities of aerophytic algae3 which are widespread across most terrestrial biomes. Although these communities are generally dominated by algal biomass, fungal hyphae closely surrounding algal cells are almost always present (Fig. 4 in2; Fig. 1, S1, here). Green biofilms without fungi occur, according to our observations, only in habitats not subject to heavy periodic drying, e.g. at the base of tree trunks near the soil.
Fig. 1 [Images not available. See PDF.]
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.
Another large group of algal-fungal symbioses, generally overlooked by science, are the semilichens. Although these symbiotic systems were introduced to the scientific community more than a century ago (as Halbflechten; [25]), it is unlikely that a single study has been devoted to semilichens since then. Semilichens represent a kind of intermediate stage between green biofilms and lichens. Their basis is a single dominant fungal species (mycobiont), which produces specific fruiting bodies and thus the individual species of semilichens can be recognized by classical taxonomy. Semilichens, or more precisely their mycobionts, have long been recognized and studied by mycologists and lichenologists, usually as non-lichenised or facultatively lichenised fungi occurring in habitats together with lichens. They differ from true lichens in the absence of a conspicuous and stratified thallus with a recognizable algal layer. Therefore, the prevailing current opinion is that they are saprophytic/endophytic, non-lichenised fungi. The close coexistence of mycobiont with algae is however evident in semilichens, but it takes place in spatially separated colonies of algae.
It is possible that the close co-occurrence of algae and fungi is a necessary condition for the existence of these organisms in semilichens and biofilms on the surface of plants (epiphytes) or on the surface of rocks and stones (epilithes), which are exposed to extreme fluctuations in life conditions, especially frequent desiccation. The anhydrobiotic model4 represents an evolutionary hypothesis for the origin of algal-fungal symbiosis (specifically lichens) under terrestrial conditions based on the assumption that fungi, in order to expand their ecological niche by appearing “on the surface”, were forced to enter into a long-term symbiotic relationship with algae, which provide the fungi with substances necessary to survive the constant alternation of moisture and complete desiccation. A large group of such substances are polyols, which are widespread5,6. In contrast to sugars, polyols are more stable molecules employed in long-term carbon storage and osmoprotection7. Aerophytic algae produce several polyols: glycerol (C3), erythritol (C4), arabitol (C5), mannitol (C6) and rarely volemitol (C7) in Trentepohliales, Ulvophyceae8, ribitol (C5) in most Trebouxiophyceae9 and sorbitol (C6) in Prasiolales and the Botryococcus-clade of Trebouxiophyceae6. Fungi associated with algae in terrestrial habitats utilize algal sugars and polyols (typically ribitol) and convert them into arabitol and mannitol10. Arabitol, with fast turnover, may be important for respiration/growth and mannitol, with slow turnover, may be used as osmoprotectant to survive fungal anhydrobiosis4. According to our observations, the anhydrobiotic model (elaborated further at the end of the Discussion) is applicable to most aerophytic algal-fungal associations, including semilichens and green biofilms, and is relevant to algae as well as fungi. Algae, originally aquatic organisms, expanded their range to terrestrial habitats, where wetness and dryness alternate, apparently thanks to coexistence with fungi.
In this study, we provide a definition of semilichens in the context of other aerophytic algal-fungal associations, and we describe several aspects of their biology: (1) phylogenetic identities of the algae and fungi involved, (2) morphology, especially the close coexistence of the two symbionts, (3) ecological aspect, (4) viability and photosynthetic activity of associated algae and (5) carbon transfer from symbiotic algae to the mycobiont.
Materials and methods
Sampling and morphological observations
Green biofilms were collected from bark and stones in the urban landscape of České Budějovice (Czech Republic) in 2023. Observations of fungi and algae were made by light microscopy and fluorescence microscopy. Semilichens have been studied for a long time by the first author and the studied specimens come from different places in Europe from the years 2015–2025 and are deposited in the herbarium PRA (Supplementary table 1). Observations of semilichens were performed by light microscopy in combination with fluorescence in blue light and UV excitation. Samples were fixed in lactoglycerol cotton blue prior to microscopy to enhance negative staining (fluorescence absorption) of otherwise often colourless hyphae. Furthermore, red chlorophyll autofluorescence in UV and blue light was used for visualization (Olympus BX 61). Semilichens were observed mainly on the surface of the substrates, but in some cases the substrate (i.e. usually young smooth bark of trees) was subjected to horizontal sections in order to observe the occurrence of hyphae and algae also inside the substrate.
DNA metabarcoding and bioinformatics
DNA of the semilichen specimens (c. 1 mm3 of dry biomass scraped from the bark in each sample) was isolated using a cetyltrimethylammonium bromide (CTAB)-based protocol11. ITS2 amplicons (Internal transcribed spacer 2 of the ribosomal DNA) were produced by PCR with the barcoded primers ITS3 (5’-GCA TCG ATG AAG AAC GCA GC-3’) and ITS4 (5’-TCC TCC GCT TAT TGA TAT GC-3’). The PCRs were performed using the Q5 High-Fidelity DNA polymerase (BioLabs Inc.), they were run in 35 cycles and the conditions were: initial denaturation at 98°C for 30 s, 98°C denaturation for 10 s, 52°C amplification for 45 s and 72°C elongation for 1 min, with a final 72°C extension for 2 min. Each sample was run in two replicates, and both negative controls (distilled water as a template) and multiplexing controls (unused combinations of left and right barcodes) were used. The PCR products were purified with SPRI AMPure XP paramagnetic beads (Beckman Coulter), pooled equimolarly and sent for library preparation and sequencing to Fasteris (Plan-les-Ouates, Switzerland). Sequencing was performed on the Illumina MiSeq platform with paired end mode (2 × 300 bp). Quality control of the Illumina MiSeq paired-end reads was carried out using FastQC v. 0.11.812. Raw reads were processed according to13, including quality filtering, paired-end assembly, removing primer artifacts, extracting reads by barcodes, reorienting reads to 5′-3′, demultiplexing, dereplicating, OTU clustering (this step carried out using Swarm v. 214, with denoising set to d = 3) and chimera filtering. Only OTUs that were found in at least 10 reads in both replicates, and that were absent from the negative controls, were considered. The data are available at: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA1184125. The OTUs were identified by BLAST searches in SEED215, and only Viridiplantae and Fungi sequences were further processed. The obtained algal sequences were aligned with the closest BLAST matches and a maximum likelihood (ML) tree was constructed in IQ-TREE v. 1.6.116 using GTR + I + G substitution model and ultrafast bootstrapping with 2,000 replications. Monophyletic clades were assigned to individual species based on historically defined species boundaries for each genus3. Morphological identifications of mycobionts were confirmed by the Blast search against NCBI.
Chrorophyll a fluorescence imaging
Aside fluorescence microscopy, FluorCam FC 800-C (Photon System Instrument, Drásov, Czech Republic) was used for 2D fluorescence imaging of viability and photosynthetic process. Pulse amplitude modulation (PAM) fluorescence was used. Measuring light averaged intensity was below 1 µmol m−2 s−1, pulse duration 20 µs, actinic light intensity 150 µmol m−2 s−1 and saturating pulse intensity 1000 µmol m−2 s−1. Chlorophyll fluorescence was used to estimate: (1) chlorophyll abundance and distribution within sample, (2) Maximum quantum yield (Fv/Fm), which is a measure of algal viability/activity and (3) kinetics of photochemical quenching, which is a measure of relative photosynthesis rate. Plant twigs bearing semilichens, however, contain substantial amount of chlorophyll in their tissues (particularly in pheloderm layer). Via gentle drying for several days and subsequent rewetting, plant tissues were killed (Fv/Fm close to zero) and surface algae resurrected (Fv/Fm of healthy phototrophs range about 0.6–0.8). Thus, algal colonies were easily visible on the plant surfaces and their physiology could be further examined. In next step, we removed symbiotic algae from a part of the plant surface using a scalpel and compared it with native sample.
13C-transfer from algal to fungal polyols
This study uses isotope labelling, which has been used successfully in alcobioses17 and much earlier in classical papers on lichen physiology10. Briefly, the stable carbon 13C, which behaves physiologically the same as widespread 12C, is scarce in nature. Thus, it is a sensitive label of new assimilate production and its fate. We added 13CO2 (99 At%, Cambridge isotope laboratories) into our labelling device used previously17,18 to reach about twice atmospheric CO2 concentration (≈ 850 µmol CO2 mol−1) and let algae/symbioses assimilate it under mild light (200 µmol of photons m−2 s−1) for one or two hours. After that, part of the biological system (about 100 mg of fresh weight representing 0.5 to 2 cm2 patches of more or less planar symbiotic systems) was removed from the substrate, using a scalpel, and quickly killed in 2 mL of boiling methanol and dichloromethane (1:1) mixture. The rest of the system was left in normal conditions to translocate/convert assimilates from algae to fungi or to different metabolites (so called “chase period”), and harvested thereafter (see results).
Then, the supernatant was filtered, evaporated under nitrogen stream and OH groups of sugars and polyols silylated using 25 µL of BSTFA in 100 µL of pyridine and heated at 85°C for one hour. Finally, n-hexane was added to reach a total sample volume of 1 mL. Compound specific isotope analysis was performed using gas chromatography (Trace 1310, Thermo Scientific, Bremen, Germany) and Isotope ratio mass spectrometry (Delta V Advantage, Thermo Scientific, Bremen, Germany). Using chromatographic column ZEBRON ZB-1 (30 m × 0.25 mm ID and 0.25 µm film thickness) we were able to separate all principal polyols (erythrytol, ribitol, arabitol, manitol, sorbitol) and main sugars (glucose and saccharose).
We calculated the 13C excess as a difference between the 13C content of the sample/compound and its natural abundance (1.070 ± 0.005 At%). The relative amount of new carbon in a particular compound may thus be estimated as the 13C excess in that particular compound multiplied by the abundance of this compound in the sample and standardised to 100% for sum of all compounds.
However, this relative partitioning of new carbon tells us nothing about the absolute rate of CO2 assimilation (a measure of whole system activity). Therefore, we calculated the average 13C excess in the pool of all compounds. In short, this is the amount of 13C (above its natural abundance) in the “average metabolite”. Since nearly pure 13CO2 is used, the 1% excess of 13C implies that 1% of all mobile carbon has been replaced due to our labelling. The drop in this value one and four days after labelling represents both respiratory loss and incorporation of new carbon into structural polymers no longer accessible to gas chromatography. Owing to the abovementioned low variability of natural 13C content and high precision of isotope ratio mass spectrometry, the appearance of new carbon in particular compounds is well confirmed. On the other hand, physiological/ecological significance should be considered critically (see Discussion).
Results
Phylogenetic perspectives
Semilichen mycobionts are present within the Ascomycota in Arthoniomycetes, Dothideomycetes, Eurotiomycetes, Lecanoromycetes (in Ostropomycetidae) and Lichinomycetes (sensu Díaz-Escandón et al.)27. According to our long-term observations in Europe, semilichen mycobionts are found in the following taxa:
Arthoniomycetes:Arthonia, Arthothelium and Naevia. Dothideomycetes:Alloarthopyrenia, Arthopyrenia, Cyrtidula, Julella, Karschia, Lichenothelia, Melaspilea, Melaspileella, Monascostroma, Mycoporum, Naetrocymbe and Tomasellia. Eurotiomycetes:Atrodiscus, Blastodesmia, Chaenothecopsis, Leptorhaphis, Mycocalicium, Phaeocalicium and Stenocybe. Lecanoromycetes, Ostropomycetidae:Absconditella, Absconditonia, Cryptodiscus, Eopyrenula, Epigloea, Exarmidium inclusum, Karstenia, Microcalicium, Sphaeronema truncatum and Xerotrema. Lichinomycetes (s.lat.):Steinia, Symbiotaphrina, Trizodia and Thelocarpon.
In fourteen semilichens, the identities of the fungi and the algae were verified by the ITS2 barcoding (Fig. 2). Each symbiotic system was generally dominated by a single fungus (mycobiont), but frequently more OTUs (up to six) of abundant algae were present. Associated algae were mostly from Trebouxiophyceae and many of them are known from lichen symbioses (e.g. Apatococcus lobatus, Elliptochloris antarctica or Trebouxia gelatinosa). More rarely, the systems were dominated by algae of the genus Trentepohlia (Ulvophycae). A total of 25 algal OTUs were found (only species abundant in at least one sample are counted) from genera Apatococcus (4 OTUs), Coccomyxa (2), Elliptochloris (1), Symbiochloris (5), Trebouxia (9), Trentepohlia (3), and Tritostichococcus (1).
Fig. 2 [Images not available. See PDF.]
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.
Morphological observations of algal-fungal co-existence
Detailed morphological observations were made on the semilichens Arthopyrenia analepta (Fig. 3), A. cerasii (Fig. 4), A. salicis (Fig. S2), Cryptodiscus tabularum (Fig. S3A,B), Cyrtidula quercus (Fig. S4), Karstenia idaei (Fig. S3C,D), Naetrocymbe punctiformis (Fig. 5) and Naevia punctiformis (Fig. S5). In most cases, a significant part of the fungal hyphae is exposed to the external environment on the substrate surface. These hyphae are associated with algae that occur on the surface and usually are clustered in scattered colonies (Fig. 3, 5).
Fig. 3 [Images not available. See PDF.]
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 [Images not available. See PDF.]
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 [Images not available. See PDF.]
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.
In some cases, the hyphae network is spread beneath the surface, under a layer of smooth scaly bark several tens of micrometres thick, and is thus invisible by simple surface observation (Fig. 4A,B). However, if we observe in an appropriate layer below the surface we find similar structures as in semilichens with hyphae on the surface (Fig. 4C-E). It is similar to the hypophloedal thallus of some lichens, but here the algae do not form a conspicuous continuous layer.
Fungal hyphae are either colourless or melanized to varying degrees. In both cases, they are difficult to observe with classical light optics (Fig. 3–5), which is probably why the symbiosis of semilichens has escaped attention for so long. However, mycobiont hyphae are readily observable by fluorescence microscopy, especially after cotton-blue staining, as they absorb blue light and UV (false dark coloration), contrasting with the distinctly paler surrounding substrate.
Ecology—semilichens occupy open niches
Most known semilichens are epiphytes, only a small proportion occur on rocks or stones (e.g. genus Lichenothelia) and some are part of microbial crusts covering various substrata (Steinia, Thelocarpon). In this study we are mainly concerned with epiphytic semilichens which are widespread in Europe, both in natural habitats and in habitats heavily disturbed by humans. Their ecology is to some extent similar to lichens or corticolous green biofilms, but their niches often differ, and semilichens fill the space not occupied by other symbiotic systems.
Some semilichens find a specific niche on young substrates (sprouts, twigs), where more complex lichens are less competitive because of their slower development. The two most common semilichens in Central Europe are Naetrocymbe punctiformis and Naevia punctiformis, which primarily overgrow thin twigs with smooth bark, but in heavily acidified areas with impoverished epiphytic lichen communities (i.e. in “lichen deserts”19), they often overgrow entire tree trunks, replacing lichen communities.
Other semilichens are able to grow in conditions that are too shady to allow lichens to grow, or only to a limited extent. For example, Cyrtidula quercus and Leptorhaphis maggiana have been observed on rods of Corylus avellana in very dark forest conditions. Semilichens are also often able to grow in micro-sites that are too dry and sunny, where lichens cannot grow. An extreme case is Microcalicium loraasii, which grows on sunny and parched conifer bark. Life in extreme conditions is probably made possible for semilichens by the minimalist lifestyle and the low requirements of the low-biomass partners.
Algae thrive in semilichens
The maximum quantum yield of PSII (Fv/Fm), powerful indicator of photosynthetic apparatus activity/viability, shows that surface dwelling symbiotic algae are viable, contrasting with green plant tissues (pheloderm), particularly after drying and rewetting (Fig. 6). Despite the low abundance of algal chlorophyll in comparison to green plant tissues, we can see Fv/Fm about 0.6 in rewetted algal colonies in contrast to near zero in the dead plant tissues (Fig. 6, right column). These values for semilichens (Fig. 6F, I, L) are comparable to algae in green biofilms (Fig. 6C) and in true lichens (Fig. 6O), although chlorophyll abundance is clearly highest in the true lichen Graphis scripta (the largest area with high Fv/Fm). Fv/Fm, however, indicates the potential for photosynthesis, but not the actual rate of photosynthesis. Thus, we measured also chlorophyll fluorescence kinetics (Kautsky induction) in the semilichen Cyrtidula quercus (Fig. 7), a green biofilm, the semilichen Arthopyrenia salicis and the true lichen Graphis scripta (Fig. S6). The upper plot in each graph demonstrates the visual situation and selected pixels for analysis (separately: symbiosis, green plant tissue and calibration plate), middle plot is absolute value kinetics and bottom plot shows normalised fluorescence kinetics to Fo = 1. We see a comparable decline of fluorescence (= increase of photosynthesis) between 20 and 90 s where actinic light (= light driving the photosynthesis) was applied in all algal-fungal systems but not in killed plant tissue nor calibration plate. Algae in semilichens, green biofilms and true lichens express comparable photosynthesis activity, but are spatially scattered in semilichens (while omnipresent in true lichens and some green biofilms).
Fig. 6 [Images not available. See PDF.]
Maximum quantum yield of photosystem II (Fv/Fm) 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 Fv/Fm) has dimension of 20 × 20 mm.
Fig. 7 [Images not available. See PDF.]
Chlorophyll a fluorescence kinetics of the semilichen Cyrtidula quercus. Visual situation (A), absolute values fluorescence (B) and fluorescence standardised to Fo = 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 (Fv/Fm). 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.
13C-transfer from algal to fungal polyols
Fractions of new carbon (13C) in different sugars and polyols at three times after labelling are shown in Fig. 8 (green biofilm, semilichens) and Fig. 9A-D (true lichens). Owing to the specificity of particular polyols to algae or to fungi, putative carbon transfer between partners may be estimated. A slight but conclusive transfer of carbon from algal ribitol and sucrose to fungal mannitol and arabitol was observed in a green biofilm (Fig. 8A) and in examined semilichens (Fig. 8B-F). A similar pattern is visible for four true lichens (Fig. 9A-D). Typically, the dominant fraction of new carbon is in ribitol and sucrose immediately after labelling (2h), and progressively moving into arabitol and mannitol during four-days chase period (assimilation in normal air). Some systems also produce substantial amount of four-carbon erythritol, which probably does not distinguish between algal and fungal pools (see discussion). The category “REST” represents all other GC-amenable metabolites, which were not identified.
Fig. 8 [Images not available. See PDF.]
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 [Images not available. See PDF.]
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.
Since we hypothesise that algal carbon (polyol) requirements are significantly lower in the small fungi in green biofilms and semilichens than in the biomass-rich true lichens, we calculated average 13C excess. Four exemplified true lichens reached higher 13C enrichment than five semilichens at all times after labelling, with green biofilm being intermediate (Fig. 9E).
All these and some additional systems are shown also in Fig. S7 and Fig. SS88. Apart from the percentage of new carbon in particular compounds (Fig. S7, right column), we present the fraction of these compounds in the total metabolite pool (Fig. S7, left column). By comparing the percentage of each compound and the percentage of new carbon in it, we can estimate the turnover rate of the respective compound (higher 13C percentage and lower percentage of substance in total metabolite pool—faster turnover). Average 13C excess of all systems studied is in Fig. S8.
In the case of symbiotic systems with Trentepohlia, it is difficult to trace the carbon flux from the alga to the mycobiont, as demonstrated in the semilichen Arthopyrenia salicis (Fig. S7). This is because the algae of Trentepohliales produce both mannitol and arabitol8, i.e. polyols that are exclusively fungal in symbiotic systems with other algae.
Discussion
Aerophytic algal-fungal associations
The only symbiotic system of aerophytic algae and fungi that has been generally accepted so far is lichens. We emphasize here that there are other systems that can be distinguished from lichens (see below).
Lichen (Fig. 10A, B). A symbiotic system formed by a single dominant fungal species (mycobiont) and one or more species of algal or cyanobacterial photobiont. It is characterised morphologically by the existence of a three-dimensional “lichenised thallus” where the photobiont is internally organised, often in a layer. The lichen mycobiont depends on the supply of carbon, in the form of polyols and sugars, from its photosynthetic partner10,20. It is the best known and longest known coexistence of algae and fungi21, and there are several recent reviews summarizing current knowledge4,22, 23–24.
Semilichen (Fig. 10C). A symbiotic system formed by a single dominant fungal species (mycobiont), living in facultative or obligate symbiosis with one or more species of algae. It does not form a three-dimensional and stratified thallus with a recognizable algal layer, as real lichens do, but the close coexistence of mycobiont and algae takes place inconspicuously in spatially separated spots, i.e. algal colonies. This is reflected in the minimalism in biomass production; lichens and green biofilms usually produce noticeably more biomass per area. The algae do not form a conspicuous coating and their biomass does not predominate over that of fungi, which makes semilichens markedly different from green biofilms. Hyphae of semilichen mycobionts are mostly exposed to the surface or are slightly immersed, but do not deeply penetrate the substrate. It is a long-term coexistence in which the algae thrive (so it is not an obvious parasitism of the fungus on the algae). Metabolic exchange between the algae and fungus occurs. The ecology partially overlaps with lichens and green biofilms, but semilichens can inhabit extreme microsites where lichens and green biofilms are at a strong disadvantage (see ecological section in Results). As with lichens, the name used informally to refer to a species of semilichen is the name of the mycobiont, although, strictly speaking, that name refers only to the fungus.
The term was first used by Zukal25, as Halbflechten, for the minute fungi occurring on bryophyte leaves, which, according to his observations, always occurred in the company of algae. Unfortunately, the identity of the fungi that Zukal described in his article is now uncertain. Their names have fallen into oblivion and the type material, supposedly stored in a Viennese herbarium, has not been found. In the following more than a hundred years, no one was interested in Zukal’s concept, but taxonomic research on fungi, which we now consider to be mycobionts in semilichens, gradually proceeded. These fungi were thought to be non-lichenised or possibly associated with algae, but their relationship with algae has not been thoroughly investigated. The term “Halbflechten” has only recently been resurrected in its English form26 and characterised as fungi associated with algae, but without the typical three-dimensional lichenised thallus. A more detailed characterization of the semilichens awaited our current study.
Coexistence with cyanobacteria instead of green algae is rare in semilichens. An example is Trizodia acrobia, a fungus from Lichinomycetes in the broad sense27, which forms a mycelium on Sphagnum leaves and is associated with scattered colonies of Nostoc cyanobacteria28,29.
Semilichens differ from the so-called ‘borderline lichens’ occurring in the marine environment. These symbioses of fungi with algae or cyanobacteria have a reduced crustose thallus30,31, however, their thallus is still more or less continuous, similar to that of endolithic lichens (Fig. 10B), and therefore they should be classified as lichens.
In a few cases, semilichens are hardly distinguishable from green biofilms. The algal coatings associated with e.g. the semilichen genera Microcalicium or Stenocybe have a similar morphology to numerous corticolous green biofilms. From the other side, semilichens are not always sharply delimited from true lichens. For example, the symbiotic system of the tropical fungus Eremithallus costaricensis32 represents an intermediate stage between a semilichen and a lichen. Some temperate symbioses of fungi and algae in microbial mucous crusts, e.g., in Cryptodiscus, Epigloea, Steinia, and Thelocarpon, can also be considered transitional stages.
Green biofilm (Fig. 10D). A community of aerophytic algae co-occurring with a wide range of microscopic fungi (mostly hyphomycetes). Algae form most of the biomass and are prominent at first sight, while fungi are inconspicuous due to their smaller biomass and often colourless or slightly melanised hyphae. The algae and fungi live in a close symbiotic relationship (Fig. 1, S1) and a metabolic exchange between them was demonstrated (Fig. 7A). Outwardly, the algal colonies co-occurring with the mycobiome are thriving and the fungi are not in the role of overt algal parasites, but rather in the role of long-term mutualistic symbionts. The relationship between microscopic algae and hyphomycete mycobiome is, along with the lichens, the most widespread algal-fungal relationship in terrestrial environments subject to periodic desiccation.
Microalgal communities in green biofilms on bark of trees are known to be diverse2,33 consisting of green algae belonging mainly to Trebouxiophyceae and Chlorophyceae34,35. While information on algal communities is rich, the surprising diversity of fungi and other groups, both prokaryotic and eukaryotic, is only recently beginning to be recognized36. Fungal hyphae closely attached to Apatococcus lobatus cells in green biofilms were reported37,38. The latest progress in mycobiome research was achieved on axenic cultures of the alga Elliptochloris and accompanying hyphomycetes from a green biofilm2. Development of these axenic cultures was observed along with the development of the mixed community of fungus and algae. Algae in the company of the fungus multiplied faster and lost chlorophyll more slowly, suggesting that the association with the fungus is beneficial to the algae. The taxonomic identity of fungi in green biofilms is still completely unknown.
In addition to algae, some biofilms also contain cyanobacteria. Recently, a specific symbiotic system of fungi and cyanobacteria, the phyllosymbium, has even been described39, which is, in our opinion, the cyanobacterial alternative to a green biofilm.
Alcobiosis. A hitherto little known but locally abundant symbiotic system in which algae form colonies or a continuous layer within and beneath the fruiting bodies of corticioid basidiomycetes17. Morphologically, this coexistence bears a striking resemblance to lichens, but the dependence of the mycobiont on carbon from the algal partner has not been demonstrated and the benefits of this coexistence for both partners remain a mystery.
Black fungi associated with algae. The coexistence of black fungi (also ‘yeast-like black fungi’ or ‘black meristematic fungi’) with photosynthetic organisms (algae and cyanobacteria) has been repeatedly observed40,41. These melanin-producing fungi, mostly classified as either Dothideomycetes or Eurotiomycetes, have long fascinated scientists with their unique oligotrophic life strategy. They often colonize strongly sunlit, periodically drying rock substrates. The prevailing opinion today is that these fungi survive on inorganic substrates precisely because of their symbiosis with phototrophs. According to our observations, at least some of these symbiotic relationships can be considered semilichens (e.g., in the genus Lichenothellia, Dothideomycetes).
Fig. 10 [Images not available. See PDF.]
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.
Facultative lichens / facultative semilichens
The dependence of lichen mycobionts on carbon supply from their photosynthetic partners is probably not unequivocally true. At least some macrolichens, e.g. Peltigerineae and Usnea, have been shown to have laccases and the saprophytic enzyme activity42, 43–44 and they may therefore partly feed on organic matter from the substrate. Utilization of organic carbon from the substrate is even more likely in microlichens (not studied), and several species of fungi from the Ostropomycetidae are known that can associate with algae to form lichen, but can also exist without them45,46. In our concept, therefore, such optionally lichenised fungi are not semilichens, but facultative lichens. In Ostropomycetidae, but also in other predominantly lichen lineages, some species congeneric with lichens are completely unlichenised47.
In semilichens, the use of organic nutrients from the substrate is probably even more widespread, and some semilichen mycobionts can also occur almost without algae and can be called facultative semilichens (e.g. Arthonia albopulverea, Naevia pinastri or Microcalicium ahlneri). Some genera, in which semilichens predominate, also include representatives in which an association with algae has not been observed (e.g. Naevia pinastri48).
It is likely that the mycobionts of lichens and semilichens live part of their lives in the aposymbiotic phase. In true lichens, this phase seems to be mostly short and represents the spore germination stage, when the fungus seeks a suitable photosynthetic partner4,23. In semilichens, however, it is possible that the aposymbiotic phase may be long-term, in which the mycobiont behaves quite differently than in the symbiotic phase, e.g. as an endophyte, saprophyte or even parasite. This final suggestion is supported by the fact that relatively closely related fungi, such as those of the Dothideomycetes and Eurotiomycetes, exhibit a wide range of life strategies49.
The anhydrobiotic model is applicable to fungi in semilichens
The “nutritional model” of lichen symbiosis, prevailing until recently, assumed that the mycobiont uses the carbon obtained from its photosynthetic partner for respiration and growth50. This model is supported by numerous data showing correlations between the photosynthetic activity of the phototroph and thallus growth51. However, most of these data were obtained from “large” macrolichens, which are only the tip of the iceberg of the entire diversity of lichens and algal-fungal associations overall. Most lichens, however form small and slowly growing crusts, potentially more physiologically similar to semilichens.
(Spribille et al.)4 introduces an alternative “anhydrobiotic model” suggesting that the primary role of fungus-acquired carbon, particularly in the form of polyols, is the ability to survive anabiosis under repeated desiccation. This is particularly important for fungi that exposed the hyphae to aerophytic conditions on the substrate surface. The anhydrobiotic model is based on three key findings: (1) the amount of carbon fixed by a phototroph is 10–20 × greater than is needed to supply the growth and respiratory requirements of the slowly growing fungus52; (2) mass loss of algal and fungal polyols during repeated re-wetting53; (3) little incorporation of fixed carbon in proteins54. This model was outlined much earlier by Smith55 who concluded that the symbiotic polyol transfer represents primarily an adaptation to cyclical desiccation and rewetting.
The average 13C enrichment of the symbiotic system is lower in semilichens and green biofilms than in lichens (Fig. 8E) as is the flux of carbon from algal polyols and sugars to fungal ones (Fig. 7 vs. Figure 8A-D). Thus, a nutritional model is unlikely for semilichen mycobiont, which tends to reduce its biomass to a minimum, whereas an anhydrobiotic model, where the mycobiont uses a common polyol pool specifically to survive repeated desiccation, seems more likely.
Aerophytic algae are frequently associated with fungi
According to our observations, aerophytic algae occurring for a long time under conditions of frequent alternation of moisture and desiccation are associated with fungi. Sometimes algae are parasitized by fungi, e.g. by Athelia56, but more often it is a longer lasting relationship in which the algae thrive. In general, aerophytic algae occur in some of these symbiotic systems: green biofilms, lichens or semilichens. Algal colonies with no apparent fungal presence were observed only under balanced microclimate conditions with permanently raised humidity. The above applies to Trebouxiophyceae, but aerophytic Trentepohlia (Ulvophyceae) often occurs without an apparent mycobiome (our observations). This may be explained by the wide range of polyols (including the generally fungal arabitol and mannitol) produced directly by the algae8.
In a periodically drying environment, Trebouxiophyceae either need a fungal partner, or, at least, find coexistence with the fungus advantageous2. During cyclic desiccation and re-hydration, aerophytic algae are subjected to photo-oxidative stress57, which they cope with by various mechanisms58. Free living aerophytic algae have been shown to be able to survive short-term stress (on the order of tens of days) caused by desiccation59, but the longer-term survival of anhydrobiosis or repeated alternations of desiccation and wetting has not been properly investigated.
Attention is currently turning to the protective role of the drought-induced non-photochemical quenching (d-NPQ60,61). In this context, the transport of fungal arabitol into algae in a lichen was observed62. Arabitol acquired by the alga is then essential for the expression of d-NPQ to dissipate excess captured light energy into heat, protecting the photobiont from photoinhibition. Interestingly, no other polyol studied (ribitol, mannitol) was able to enhance d-NPQ. Furthermore, algae can utilise fungal respiratory CO2 to increase their photosynthesis and reduce photorespiration63. In addition, recent research has revealed that algae from the Trebouxiophyceae, typically included in symbiotic systems with fungi, share genes for the production of a particular enzyme from the glycoside hydrolases group64. This enzyme may be active in breaking down the lichenan, a key component of the mycobiont’s cell wall. These new findings support the view that aerophytic algae are under selection pressure to symbiosis with their fungal partners.
Acknowledgements
Linda in Arcadia kindly revised the manuscript.
Author contributions
JV planned and designed the research. SS and VK performed lab work, PŠ analysed NGS data. JK performed physiological experiments. PŘ created Fig. 2. JV, PŠ and JK wrote the manuscript.
Data availability
Sequences available: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA1184125
Declarations
Competing interests
The authors declare no competing interests.
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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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