Chemical Constituents Isolated from the Lichen Biome of Selected Species Native to North America.

A lichen is a symbiotic association composed of a primary mycobionts and one or more photobionts living mutualistically together, forming a distinct morphological entity beneficial to their partnership and to other associated fungi, photobionts, and bacteria that collectively make up the lichen biome. The taxonomic identification of a lichen species often requires determination of the primary mycobiont's secondary metabolites, the key morphological characteristics of the thallus, and how it relates to other lichen species as seen in DNA phylogeny. This chapter covers lichens and their bionts, taxonomic identification, and their chemical constituents as exemplified by what is found in lichen biomes, especially those endemic to North America. Extraction and isolation, as well as updates on dereplication methods using mass spectrometric GNPS and NMR spectroscopic spin network fingerprint procedures, and marker-based techniques to identify lichens are discussed. The isolation and structure elucidation of secondary metabolites of an endolichenic Penicillium species that produces bioactive compounds will be described in detail.

The symbiotic alga Trebouxia fuels a coherent soil ecosystem on the landscape scale in the Atacama Desert.

Biocrusts represent associations of lichens, green algae, cyanobacteria, fungi and other microorganisms, colonizing soils in varying proportions of principally arid biomes. The so-called grit crust represents a recently discovered type of biocrust situated in the Coastal Range of the Atacama Desert (Chile) made of microorganisms growing on and in granitoid pebbles, resulting in a checkerboard pattern visible to the naked eye on the landscape scale. This specific microbiome fulfills a broad range of ecosystem services, all probably driven by fog and dew-induced photosynthetic activity of mainly micro-lichens. To understand its biodiversity and impact, we applied a polyphasic approach on the phototrophic microbiome of this biocrust, combining isolation and characterization of the lichen photobionts, multi-gene phylogeny of the photobionts and mycobionts based on a direct sequencing and microphotography approach, metabarcoding and determination of chlorophylla+b contents. Metabarcoding showed that yet undescribed lichens within the Caliciaceae dominated the biocrust together with Trebouxia as the most abundant eukaryote in all plots. Together with high mean chlorophylla+b contents exceeding 410 mg m-2, this distinguished the symbiotic algae Trebouxia as the main driver of the grit crust ecosystem. The trebouxioid photobionts could be assigned to the I (T. impressa/gelatinosa) and A (T. arboricola) clades and represented several lineages containing five potential species candidates, which were identified based on the unique phylogenetic position, morphological features, and developmental cycles of the corresponding isolates. These results designate the grit crust as the only known coherent soil layer with significant landscape covering impact of at least 440 km2, predominantly ruled by a single symbiotic algal genus.

Isolation and Identification of Lichen Photobionts Collected from Different Environments in North of Portugal and Evaluation of Bioactivities of Their Extracts.

Lichens are organisms constituted by a symbiotic relationship between a fungus (mycobiont) and a photoautotrophic partner (photobiont). Lichens produce several bioactive compounds; however, the biotechnological exploitation of this organism is hampered by its slow growth. To start studying the possibility of exploiting lichens as alternative sources of bioactive compounds, eighteen lichens were collected in the north of Portugal in order to isolate and study the bioactivity of their photobionts. It was possible to isolate and cultivate only eight photobionts. Three of them, LFR1, LFA2 and LCF3, belong to the Coelastrella genus, the other two (LFA1 and LCF1) belong to the Chlorella genus and for the remaining three photobionts, LFS1, LCA1 and LCR1, it was impossible to isolate their microalgae. These only grow in consortium with bacteria and/or cyanobacteria. All extracts showed antioxidant activity, mainly at a concentration of 10 mg.mL-1. LFS1, a consortium extract, showed the highest antioxidant power, as well as the highest concentration of phenolic compounds (5.16 ± 0.53 mg of gallic acid equivalents (GAE).g-1). The extracts under study did not show significant antibacterial activity against Escherichia coli, Listeria or Salmonella. The Coelastrella sp. and LFA1 extracts showed the highest hyaluronidase inhibition. The LFR1 extract at a concentration of 5 mg.mL-1 showed the highest anti-inflammatory activity (79.77 ± 7.66%). The extracts of Coelastrella sp. and LFA1 also showed greater antidiabetic activity, demonstrating the high inhibitory power of α-amylase and α-glucosidase. LFR1 at a concentration of 5 mg.mL-1, due to its selective cytotoxicity inhibiting the growth of cancer cells (Caco-2 cells), is a promising anticancer agent.

Freeze Substitution Accelerated via Agitation: New Prospects for Ultrastructural Studies of Lichen Symbionts and Their Extracellular Matrix.

(1) Background: Lichens, as an important part of the terrestrial ecosystem, attract the attention of various research disciplines. To elucidate their ultrastructure, transmission electron microscopy of resin-embedded samples is indispensable. Since most observations of lichen samples are generated via chemical fixation and processing at room temperature, they lack the rapid immobilization of live processes and are prone to preparation artefacts. To improve their preservation, cryoprocessing was tested in the past, but never widely implemented, not least because of an extremely lengthy protocol. (2) Methods: Here, we introduce an accelerated automated freeze substitution protocol with continuous agitation. Using the example of three lichen species, we demonstrate the preservation of the native state of algal photobionts and mycobionts in association with their extracellular matrix. (3) Results: We bring to attention the extent and the structural variability of the hyphae, the extracellular matrix and numerous crystallized metabolites. Our findings will encourage studies on transformation processes related to the compartmentation of lichen thalli. They include cryopreserved aspects of algal photobionts and observations of putative physiological relevance, such as the arrangement of numerous mitochondria within chloroplast pockets. (4) Conclusions: In summary, we present accelerated freeze substitution as a very useful tool for systematic studies of lichen ultrastructures.

Recent advances in research for potential utilization of unexplored lichen metabolites.

Several research studies have shown that lichens are productive organisms for the synthesis of a broad range of secondary metabolites. Lichens are a self-sustainable stable microbial ecosystem comprising an exhabitant fungal partner (mycobiont) and at least one or more photosynthetic partners (photobiont). The successful symbiosis is responsible for their persistence throughout time and allows all the partners (holobionts) to thrive in many extreme habitats, where without the synergistic relationship they would be rare or non-existent. The ability to survive in harsh conditions can be directly correlated with the production of some unique metabolites. Despite the potential applications, these unique metabolites have been underutilised by pharmaceutical and agrochemical industries due to their slow growth, low biomass availability and technical challenges involved in their artificial cultivation. However, recent development of biotechnological tools such as molecular phylogenetics, modern tissue culture techniques, metabolomics and molecular engineering are opening up a new opportunity to exploit these compounds within the lichen holobiome for industrial applications. This review also highlights the recent advances in culturing the symbionts and the computational and molecular genetics approaches of lichen gene regulation recognized for the enhanced production of target metabolites. The recent development of multi-omics novel biodiscovery strategies aided by synthetic biology in order to study the heterologous expressed lichen-derived biosynthetic gene clusters in a cultivatable host offers a promising means for a sustainable supply of specialized metabolites.

Airborne ascospore discharge with co-dispersal of attached epihymenial algae in some foliicolous lichens.

PREMISE: Lichen-forming fungi that colonize leaf surfaces must find a compatible algal symbiont, establish lichen symbiosis, and reproduce within the limited life span of their substratum. Many produce specialized asexual propagules that appear to be dispersed by rain and runoff currents, but less is known about dispersal of their meiotic ascospores. In some taxa, a layer of algal symbionts covers the hymenial surface of the apothecia, where asci discharge their ascospores. We examined the untested hypothesis that their ascospores are ejected into air currents and carry with them algal symbionts from the epihymenial layer for subsequent lichenization. METHODS: Leaves bearing the lichens Calopadia puiggarii, Sporopodium marginatum (Pilocarpaceae), and Gyalectidium viride (Gomphillaceae) were collected in southern Florida. The latter two species have epihymenial algal layers. Leaf fragments with apotheciate thalli were affixed in petri dishes, with glass cover slips attached inside the lid over the thalli. Subsequent discharge of ascospores and any co-dispersed algae was evaluated with light microscopy. RESULTS: All three species discharged ascospores aerially. Discharged ascospores were frequently surrounded by a halo-like sheath of transparent material. In the two species with an epihymenial algal layer, most dispersing ascospores (>90%) co-transported algal cells attached to the spore sheath or wall. CONCLUSIONS: While water may be the usual vector for their asexual propagules, foliicolous lichen-forming fungi make use of air currents to disperse their ascospores. The epihymenial algal layer represents an adaptation for efficient co-dispersal of the algal symbiont with the next genetic generation of the fungus.

Opening the Gap: Rare Lichens With Rare Cyanobionts - Unexpected Cyanobiont Diversity in Cyanobacterial Lichens of the Order Lichinales.

The last decades of research led to a change in understanding of lichens that are now seen as self-sustaining micro-ecosystems, harboring diverse microbial organisms in tight but yet not fully understood relationships. Among the diverse interdependencies, the relationship between the myco- and photobiont is the most crucial, determining the shape, and ecophysiological properties of the symbiotic consortium. Roughly 10% of lichens associate with cyanobacteria as their primary photobiont, termed cyanolichens. Up to now, the diversity of cyanobionts of bipartite lichens resolved by modern phylogenetic approaches is restricted to the filamentous and heterocytous genera of the order Nostocales. Unicellular photobionts were placed in the orders Chroococcales, Pleurocapsales, and Chroococcidiopsidales. However, especially the phylogeny and taxonomy of the Chroococcidiopsidales genera remained rather unclear. Here we present new data on the identity and phylogeny of photobionts from cyanolichens of the genera Gonohymenia, Lichinella, Peccania, and Peltula from a broad geographical range. A polyphasic approach was used, combining morphological and cultivation-depending characteristics (microscopy, staining techniques, life cycle observation, baeocyte motility, and nitrogen fixation test) with phylogenetic analyses of the 16S rRNA and 16S-23S ITS gene region. We found an unexpectedly high cyanobiont diversity in the cyanobacterial lichens of the order Lichinales, including two new genera and seven new species, all of which were not previously perceived as lichen symbionts. As a result, we describe the novel unicellular Chroococcidiopsidales genera Pseudocyanosarcina gen. nov. with the species Pseudocyanosarcina phycocyania sp. nov. (from Peltula clavata, Australia) and Compactococcus gen. nov. with the species Compactococcus sarcinoides sp. nov. (from Gonohymenia sp., Australia) and the new Chroococcidiopsidales species Aliterella compacta sp. nov. (from Peltula clavata, Australia), Aliterella gigantea sp. nov. (from Peltula capensis; South Africa), Sinocapsa ellipsoidea sp. nov. (from Peccania cerebriformis, Austria), as well as the two new Nostocales species Komarekiella gloeocapsoidea sp. nov. (from Gonohymenia sp., Czechia) and Komarekiella globosa sp. nov. (from Lichinella cribellifera, Canary Islands, Spain). Our study highlights the role of cyanolichens acting as a key in untangling cyanobacterial taxonomy and diversity. With this study, we hope to stimulate further research on photobionts, especially of rare cyanolichens.

Trentepohlialean Algae (Trentepohliales, Ulvophyceae) Show Preference to Selected Mycobiont Lineages in Lichen Symbioses.

The main aims of this work were to assess phylogenetic relationships of the trentepohlialean photobionts in tropical, mainly sterile, lichens collected in Bolivia, to examine their genetic diversity, host specificity, and the impact of habitat factors on the occurrence of Trentepohliales. Based on rbcL marker analysis, we constructed a phylogenetic tree with eight major clades of Trentepohliales, of which seven free-living species are intermingled with lichenized ones. Our analyses show that the studied photobionts are scattered across the phylogenetic tree and algae from temperate and tropical regions do not form monophyletic groups, except within one clade that seems to be restricted to the tropics. There is no significant occurrence pattern of lichenized Trentepohliaceae on a specific substratum, except Cephaleuros spp. and Phycopeltis spp., which are restricted to leaves, while some clades with lichenized algae may be specialized to tree bark and wood. Moreover, we found two patterns of associations: first, closely related algae can associate with distantly related mycobionts; second, some other trentepohlioid algae associate with selected lineages of fungi (e.g., Arthoniaceae or Graphidaceae). We also found that some lineages of photobionts are even more selective and associate exclusively with one species (e.g., Dichosporidium nigrocinctum, Diorygma antillarum) or closely related lichen-forming fungi (Herpothallon spp.). Concluding, we found that occurrence of some trentepohlialean photobionts may correlate with the particular type of the mycobiont.

Phylotypic Characterization of Mycobionts and Photobionts of Rock Tripe Lichen in East Antarctica.

Saxicolous rock ripe lichens that grow on rocks in the East Antarctic fellfields were sampled for phylotypic characterization of its constituent mycobionts (fungi) and photobionts (algae and cyanobacteria). The rock tripe lichen-forming fungal and algal phylotypes were classified under the common lichen-forming genera of ascomycetes, namely, Umbilicaria, and green algae, namely, Trebouxia and Coccomyxa. However, phylotypes of the green algal chloroplasts and the lichen-associated cyanobacteria showed unexpectedly high diversity. The detected chloroplast phylotypes were not fully affiliated with the green algal genera Trebouxia or Coccomyxa. The predominant chloroplast phylotype demonstrated maximum resemblance to Neglectella solitaria, which is neither a known Antarctic species nor a typical lichen photobiont. Another dominant chloroplast phylotype belonged to the atypical Antarctic green algae family. Cyanobacterial phylotypes were dominated by those affiliated with the Microcoleus species rather than the well-known lichen-associates, Nostoc species. The occurrences of these Microcoleus-affiliated cyanobacterial phylotypes were specifically abundant within the Yukidori Valley site, one of the Antarctic Specially Protected Areas (ASPA). The ASPA site, along with another 50 km-distant site, yielded most of the cryptic diversity in the phylotypes of chloroplasts and cyanobacteria, which may contribute to the phenotypic variability within the rock tripe lichen photobionts.

Survey of the occurrence of desiccation-induced quenching of basal fluorescence in 28 species of green microalgae.

MAIN CONCLUSION: Desiccation-induced chlorophyll fluorescence quenching seems to be an indispensable part of desiccation resistance in the surveyed 28 green microalgal species. Lichens are desiccation tolerant meta-organisms. In the desiccated state photosynthesis is inhibited rendering the photobionts potentially sensitive to photoinhibition. As a photoprotective mechanism, strong non-radiative dissipation of absorbed light leading to quenching of chlorophyll fluorescence has been proposed. Desiccation-induced quenching affects not only variable fluorescence, but also the so-called basal fluorescence, F0. This phenomenon is well-known for intact lichens and some free living aero-terrestrial algae, but it was often absent in isolated lichen algae. Therefore, a thorough screening for the appearance of desiccation-induced quenching was undertaken with 13 different aero-terrestrial microalgal species and lichen photobionts. They were compared with 15 aquatic green microalgal species, among them also three marine species. We asked the following questions: Do isolated lichen algae show desiccation-induced quenching? Are aero-terrestrial algae different in this respect to aquatic algae and is the potential for desiccation-induced quenching coupled to desiccation tolerance? How variable is desiccation-induced quenching among species? Most of the aero-terrestrial algae, including all lichen photobionts, showed desiccation-induced quenching, although highly variable in extent, whereas most of the aquatic algae did not. All algae displaying quenching were also desiccation tolerant, whereas all algae unable to perform desiccation-induced quenching were desiccation intolerant. Desiccation-induced fluorescence quenching seems to be an indispensable part of desiccation resistance in the investigated species.

Fungal Diversity in Lichens: From Extremotolerance to Interactions with Algae.

Lichen symbioses develop long-living thallus structures even in the harshest environments on Earth. These structures are also habitats for many other microscopic organisms, including other fungi, which vary in their specificity and interaction with the whole symbiotic system. This contribution reviews the recent progress regarding the understanding of the lichen-inhabiting fungi that are achieved by multiphasic approaches (culturing, microscopy, and sequencing). The lichen mycobiome comprises a more or less specific pool of species that can develop symptoms on their hosts, a generalist environmental pool, and a pool of transient species. Typically, the fungal classes Dothideomycetes, Eurotiomycetes, Leotiomycetes, Sordariomycetes, and Tremellomycetes predominate the associated fungal communities. While symptomatic lichenicolous fungi belong to lichen-forming lineages, many of the other fungi that are found have close relatives that are known from different ecological niches, including both plant and animal pathogens, and rock colonizers. A significant fraction of yet unnamed melanized ('black') fungi belong to the classes Chaethothyriomycetes and Dothideomycetes. These lineages tolerate the stressful conditions and harsh environments that affect their hosts, and therefore are interpreted as extremotolerant fungi. Some of these taxa can also form lichen-like associations with the algae of the lichen system when they are enforced to symbiosis by co-culturing assays.

Heveochlorella (Trebouxiophyceae): a little-known genus of unicellular green algae outside the Trebouxiales emerges unexpectedly as a major clade of lichen photobionts in foliicolous communities.

Foliicolous lichens are formed by diverse, highly specialized fungi that establish themselves and complete their life cycle within the brief duration of their leaf substratum. Over half of these lichen-forming fungi are members of either the Gomphillaceae or Pilocarpaceae, and associate with Trebouxia-like green algae whose identities have never been positively determined. We investigated the phylogenetic affinities of these photobionts to better understand their role in lichen establishment on an ephemeral surface. Thallus samples of Gomphillaceae and Pilocarpaceae were collected from foliicolous communities in southwest Florida and processed for sequencing of photobiont marker genes, algal cultivation and/or TEM. Additional specimens from these families and also from Aspidothelium (Thelenellaceae) were collected from a variety of substrates globally. Sequences from rbcL and nuSSU regions were obtained and subjected to Maximum Likelihood and Bayesian analyses. Analysis of 37 rbcL and 7 nuSSU algal sequences placed all photobionts studied within the provisional trebouxiophycean assemblage known as the Watanabea clade. All but three of the sequences showed affinities within Heveochlorella, a genus recently described from tree trunks in East Asia. The photobiont chloroplast showed multiple thylakoid stacks penetrating the pyrenoid centripetally as tubules lined with pyrenoglobuli, similar to the two described species of Heveochlorella. We conclude that Heveochlorella includes algae of potentially major importance as lichen photobionts, particularly within (but not limited to) foliicolous communities in tropical and subtropical regions worldwide. The ease with which they may be cultivated on minimal media suggests their potential to thrive free-living as well as in lichen symbiosis.

New insights into diversity and selectivity of trentepohlialean lichen photobionts from the extratropics.

Aerial green algae of Trentepohliaceae can form conspicuous free-living colonies, be parasites of plants or photobionts of lichen-forming ascomycetes. So far, their diversity in temperate regions is still poorly known as it has been mostly studied by phenotypic approaches only. We present new insights in the phylogenetic relationships of lichenized representatives from temperate and Mediterranean parts of Europe by analysis of 18S rRNA and rbcL gene fragments, and nuclear ITS sequence data. For this purpose we isolated the trentepohlialean photobionts from lichens representing different genera. Algal cultures from lichenized and free-living Trentepohliaceae were used to design new primers for amplification of the marker loci. We constructed a phylogenetic hypothesis to reveal the phylogenetic placements of lichenized lineages with 18S rRNA and rbcL sequences. ITS variation among the clades was substantial and did not allow including them in the general phylogenetic assessment, yet ITS appears to be a promising marker for DNA-barcoding approaches. Specific algae were found in particular lichen but the overall diversity of photobionts was limited. The multilocus tree does not support the current morphological classification of genera in Trentepohliaceae, suggesting that morphology is more variable than previously thought in this group of algae.

Unequal allocation of excitation energy between photosystem II and I reduces cyanolichen photosynthesis in blue light.

Photosynthesis was compared in two cyanobacterial lichens (Lobaria hallii and Peltigera praetextata) and two green algal lichens (Lobaria pulmonaria and Peltigera leucophlebia) exposed to red, green or blue light. Cyanolichens had substantially lower photosynthetic CO(2) uptake and O(2) evolution than the green algal lichens in blue light, but slightly higher photosynthesis in red and green light. The effective quantum yield of photosystem (PS) II (Φ(PSII)) decreased with increasing red and green light for all species, but in blue light this response occurred in green algal lichens only. Cyanolichen Φ(PSII) increased with increasing blue light at low irradiances, but decreased at stronger exposures. However, after adding red light the efficiency of blue light for photosynthetic O(2) evolution increased by 2.4 times. Because phycobilisomes associated with PSII have a low blue light absorption, our results are consistent with blue light absorption mainly by Chl in PSI. Thereby, unequal allocation of excitation energy between PSII and PSI results in low cyanolichen photosynthesis under blue light. This is new knowledge in the science of lichenology with important implications for e.g. the reliability of using Chl fluorometers with blue light for cyanolichens.