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Lichens are remarkable and classic examples of symbiotic organisms that have fascinated scientists for centuries. Yet, it has only been for a couple of decades that significant advances have focused on the diversity of their green algal and/or cyanobacterial photobionts. Cyanolichens, which contain cyanobacteria as their photosynthetic partner, include up to 10% of all known lichens and, as such, studies on their cyanobionts are much rarer compared to their green algal counterparts. For the unicellular cyanobionts, i.e. cyanobacteria that do not form filaments, these studies are even scarcer. Nonetheless, these currently include at least 10 different genera in the cosmopolitan lichen order Lichinales. An international consortium (International Network of CyanoBionts; INCb) will tackle this lack of knowledge. In this article, we discuss the status of current unicellular cyanobiont research, compare the taxonomic resolution of photobionts from cyanolichens with those of green algal lichens (chlorolichens), and give a roadmap of research on how to recondition the underestimated fraction of symbiotic unicellular cyanobacteria in lichens.
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The Lobaria pulmonaria holobiont comprises algal, fungal, cyanobacterial and bacterial components. We investigated L. pulmonaria's bacterial microbiome in the adaptation of this ecologically sensitive lichen species to diverse climatic conditions. Our central hypothesis posited that microbiome composition and functionality aligns with subcontinental-scale (a stretch of ~1100 km) climatic parameters related to temperature and precipitation. We also tested the impact of short-term weather dynamics, sampling season and algal/fungal genotypes on microbiome variation. Metaproteomics provided insights into compositional and functional changes within the microbiome. Climatic variables explained 41.64% of microbiome variation, surpassing the combined influence of local weather and sampling season at 31.63%. Notably, annual mean temperature and temperature seasonality emerged as significant climatic drivers. Microbiome composition correlated with algal, not fungal genotype, suggesting similar environmental recruitment for the algal partner and microbiome. Differential abundance analyses revealed distinct protein compositions in Sub-Atlantic Lowland and Alpine regions, indicating differential microbiome responses to contrasting environmental/climatic conditions. Proteins involved in oxidative and cellular stress were notably different. Our findings highlight microbiome plasticity in adapting to stable climates, with limited responsiveness to short-term fluctuations, offering new insights into climate adaptation in lichen symbiosis.
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Clima , Líquenes , Microbiota , Líquenes/microbiología , Líquenes/fisiología , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Simbiosis , Hongos/clasificación , Hongos/genética , Hongos/aislamiento & purificación , Hongos/fisiología , Estaciones del Año , GenotipoRESUMEN
Molecular sequence data have transformed research on cryptogams (e.g., lichens, microalgae, fungi, and symbionts thereof) but methods are still strongly hampered by the small size and intermingled growth of the target organisms, poor cultivability and detrimental effects of their secondary metabolites. Here, we aim to showcase examples on which a modified direct PCR approach for diverse aspects of molecular work on environmental samples concerning biocrusts, biofilms, and cryptogams gives new options for the research community. Unlike traditional approaches, this methodology only requires biomass equivalent to colonies and fragments of 0.2 mm in diameter, which can be picked directly from the environmental sample, and includes a quick DNA lysis followed by a standardized PCR cycle that allows co-cycling of various organisms/target regions in the same run. We demonstrate that this modified method can (i) amplify the most widely used taxonomic gene regions and those used for applied and environmental sciences from single colonies and filaments of free-living cyanobacteria, bryophytes, fungi, and lichens, including their mycobionts, chlorobionts, and cyanobionts from both isolates and in situ material during co-cycling; (ii) act as a tool to confirm that the dominant lichen photobiont was isolated from the original sample; and (iii) optionally remove inhibitory secondary lichen substances. Our results represent examples which highlight the method's potential for future applications covering mycology, phycology, biocrusts, and lichenology, in particular.IMPORTANCECyanobacteria, green algae, lichens, and other cryptogams play crucial roles in complex microbial systems such as biological soil crusts of arid biomes or biofilms in caves. Molecular investigations on environmental samples or isolates of these microorganisms are often hampered by their dense aggregation, small size, or metabolism products which complicate DNA extraction and subsequent PCRs. Our work presents various examples of how a direct DNA extraction and PCR method relying on low biomass inserts can overcome these common problems and discusses additional applications of the workflow including adaptations.
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Ecosistema , Líquenes , Biomasa , Hongos/genética , Líquenes/genética , Reacción en Cadena de la Polimerasa , ADNRESUMEN
Over the decades our understanding of lichens has shifted to the fact that they are multiorganismic, symbiotic microecosystems, with their complex interactions coming to the fore due to recent advances in microbiomics. Here, we present a mutualistic-parasitic continuum dynamics scenario between an orange lichen and a lichenicolous fungus from the Atacama Desert leading to the decay of the lichen's photobiont and leaving behind a black lichen thallus. Based on isolation, sequencing, and ecophysiological approaches including metabolic screenings of the symbionts, we depict consequences upon infection with the lichenicolous fungus. This spans from a loss of the lichen's photosynthetic activity and an increased roughness of its surface to an inhibition of the parietin synthesis as a shared pathway between the photobiont and the mycobiont, including a shift of secondary metabolism products. This degree of relations has rarely been documented before, although lichenicolous fungi have been studied for over 200 years, adding an additional level to the view of interactions within lichens.
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Chlorophyta , Líquenes , Líquenes/microbiología , Filogenia , Hongos , SimbiosisRESUMEN
Exposing their vegetative bodies to the light, lichens are outstanding amongst other fungal symbioses. Not requiring a pre-established host, 'lichenized fungi' build an entirely new structure together with microbial photosynthetic partners that neither can form alone. The signals involved in the transition of a fungus and a compatible photosynthetic partner from a free-living to a symbiotic state culminating in thallus formation, termed 'lichenization', and in the maintenance of the symbiosis, are poorly understood. Here, we synthesise the puzzle pieces of the scarce knowledge available into an updated concept of signalling involved in lichenization, comprising five main stages: (1) the 'pre-contact stage', (2) the 'contact stage', (3) 'envelopment' of algal cells by the fungus, (4) their 'incorporation' into a pre-thallus and (5) 'differentiation' into a complex thallus. Considering the involvement of extracellularly released metabolites in each phase, we propose that compounds such as fungal lectins and algal cyclic peptides elicit early contact between the symbionts-to-be, whereas phytohormone signalling, antioxidant protection and carbon exchange through sugars and sugar alcohols are of continued importance throughout all stages. In the fully formed lichen thallus, secondary lichen metabolites and mineral nutrition are suggested to stabilize the functionalities of the thallus, including the associated microbiota.
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Líquenes , Líquenes/microbiología , Simbiosis , FotosíntesisRESUMEN
Lichen genus Endocarpon in biological soil crust form was chosen as a model to investigate the bacterial communities for the first time across four vertically distinct strata. Key bacterial microbiota in lichen thallus were discovered, which were gradually filtered and mainly derived from the crust soil, with clear host selection tendency. The study provided key information to better understand the homeostasis maintenance mechanism of the lichen symbiont and community assembly of desert lichen crust.
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Lichens are well-known examples of complex symbiotic associations between organisms from different Kingdoms. Microfungi in particular, establish diverse associations with the hosting lichen thallus, as species-specific parasites or transient co-inhabitants. The whole community of lichen-associated fungi constitute the 'lichen mycobiome' comprising both ascomycetes and basidiomycetes, including filamentous and yeast taxa. Metabarcoding results and microscopy analyses show that in some thalli, basidiomycetes are frequent lichen-associated fungi but still only a few species could be axenically isolated and morphologically characterized. Within a broad project aiming at characterizing the mycobiome diversity by culture-dependent and independent approaches in two lichen species selected as reference models - Rhizoplaca melanophthalma and Tephromela atra, we succeed in isolating and culturing 76 new strains of basidiomycetous yeasts. The lichen thalli were collected in different mountain regions worldwide and at relatively high elevation. The yeast strains were isolated on different growth media and were studied for their morphological and genetic diversity. Nuclear internal transcribed spacer (ITS) and ribosomal large subunit (LSU) sequence analyses identified them to belong to ten families within the orders Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes, Tremellomycetes and Ustilaginomycetes. The yeasts here detected showed patterns of host-preference in a few cases and they are potentially related to the ecological conditions.
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Ascomicetos , Basidiomycota , Líquenes , Ascomicetos/genética , Basidiomycota/genética , Humanos , Líquenes/microbiología , Filogenia , SimbiosisRESUMEN
Fungal-algal relationships-both across evolutionary and ecological scales-are finely modulated by the presence of the symbionts in the environments and by the degree of selectivity and specificity that either symbiont develop reciprocally. In lichens, the green algal genus Trebouxia Puymaly is one of the most frequently recovered chlorobionts. Trebouxia species-level lineages have been recognized on the basis of their morphological and phylogenetic diversity, while their ecological preferences and distribution are still only partially unknown. We selected two cosmopolitan species complexes of lichen-forming fungi as reference models, i.e., Rhizoplaca melanophthalma and Tephromela atra, to investigate the diversity of their associated Trebouxia spp. in montane habitats across their distributional range worldwide. The greatest diversity of Trebouxia species-level lineages was recovered in the altitudinal range 1,000-2,500 m a.s.l. A total of 10 distinct Trebouxia species-level lineages were found to associate with either mycobiont, for which new photobionts are reported. One previously unrecognized Trebouxia species-level lineage was identified and is here provisionally named Trebouxia "A52." Analyses of cell morphology and ultrastructure were performed on axenically isolated strains to fully characterize the new Trebouxia "A52" and three other previously recognized lineages, i.e., Trebouxia "A02," T. vagua "A04," and T. vagua "A10," which were successfully isolated in culture during this study. The species-level diversity of Trebouxia associating with the two lichen-forming fungi in extreme habitats helps elucidate the evolutionary pathways that this lichen photobiont genus traversed to occupy varied climatic and vegetative regimes.
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Lichens represent self-supporting symbioses, which occur in a wide range of terrestrial habitats and which contribute significantly to mineral cycling and energy flow at a global scale. Lichens usually grow much slower than higher plants. Nevertheless, lichens can contribute substantially to biomass production. This review focuses on the lichen symbiosis in general and especially on the model species Lobaria pulmonaria L. Hoffm., which is a large foliose lichen that occurs worldwide on tree trunks in undisturbed forests with long ecological continuity. In comparison to many other lichens, L. pulmonaria is less tolerant to desiccation and highly sensitive to air pollution. The name-giving mycobiont (belonging to the Ascomycota), provides a protective layer covering a layer of the green-algal photobiont (Dictyochloropsis reticulata) and interspersed cyanobacterial cell clusters (Nostoc spec.). Recently performed metaproteome analyses confirm the partition of functions in lichen partnerships. The ample functional diversity of the mycobiont contrasts the predominant function of the photobiont in production (and secretion) of energy-rich carbohydrates, and the cyanobiont's contribution by nitrogen fixation. In addition, high throughput and state-of-the-art metagenomics and community fingerprinting, metatranscriptomics, and MS-based metaproteomics identify the bacterial community present on L. pulmonaria as a surprisingly abundant and structurally integrated element of the lichen symbiosis. Comparative metaproteome analyses of lichens from different sampling sites suggest the presence of a relatively stable core microbiome and a sampling site-specific portion of the microbiome. Moreover, these studies indicate how the microbiota may contribute to the symbiotic system, to improve its health, growth and fitness.
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BACKGROUND: Antimicrobial resistance (AMR) is a major threat to public health. Microorganisms equipped with AMR genes are suggested to have partially emerged from natural habitats; however, this hypothesis remains inconclusive so far. To understand the consequences of the introduction of exogenic antimicrobials into natural environments, we exposed lichen thalli of Peltigera polydactylon, which represent defined, highly diverse miniature ecosystems, to clinical (colistin, tetracycline), and non-clinical (glyphosate, alkylpyrazine) antimicrobials. We studied microbiome responses by analysing DNA- and RNA-based amplicon libraries and metagenomic datasets. RESULTS: The analyzed samples consisted of the thallus-forming fungus that is associated with cyanobacteria as well as other diverse and abundant bacterial communities (up to 108 16S rRNA gene copies ng-1 DNA) dominated by Alphaproteobacteria and Bacteroidetes. Moreover, the natural resistome of this meta-community encompassed 728 AMR genes spanning 30 antimicrobial classes. Following 10 days of exposure to the selected antimicrobials at four different concentrations (full therapeutic dosage and a gradient of sub-therapeutic dosages), we observed statistically significant, antimicrobial-specific shifts in the structure and function but not in bacterial abundances within the microbiota. We observed a relatively lower response after the exposure to the non-clinical compared to the clinical antimicrobial compounds. Furthermore, we observed specific bacterial responders, e.g., Pseudomonas and Burkholderia to clinical antimicrobials. Interestingly, the main positive responders naturally occur in low proportions in the lichen holobiont. Moreover, metagenomic recovery of the responders' genomes suggested that they are all naturally equipped with specific genetic repertoires that allow them to thrive and bloom when exposed to antimicrobials. Of the responders, Sphingomonas, Pseudomonas, and Methylobacterium showed the highest potential. CONCLUSIONS: Antimicrobial exposure resulted in a microbial dysbiosis due to a bloom of naturally low abundant taxa (positive responders) with specific AMR features. Overall, this study provides mechanistic insights into community-level responses of a native microbiota to antimicrobials and suggests novel strategies for AMR prediction and management. Video Abstract.
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Antiinfecciosos/farmacología , Ascomicetos/efectos de los fármacos , Ascomicetos/genética , Farmacorresistencia Microbiana/efectos de los fármacos , Farmacorresistencia Microbiana/genética , Microbiota/efectos de los fármacos , Microbiota/genética , Colistina/farmacología , Glicina/análogos & derivados , Glicina/farmacología , Pirazinas/farmacología , ARN Ribosómico 16S/genética , Tetraciclina/farmacología , GlifosatoRESUMEN
Lichens host highly diverse microbial communities, with bacteria being one of the most explored groups in terms of their diversity and functioning. These bacteria could partly originate from symbiotic propagules developed by many lichens and, perhaps more commonly and depending on environmental conditions, from different sources of the surroundings. Using the narrowly distributed species Peltigera frigida as an object of study, we propose that bacterial communities in these lichens are different from those in their subjacent substrates, even if some taxa might be shared. Ten terricolous P. frigida lichens and their substrates were sampled from forested sites in the Coyhaique National Reserve, located in an understudied region in Chile. The mycobiont identity was confirmed using partial 28S and ITS sequences. Besides, 16S fragments revealed that mycobionts were associated with the same cyanobacterial haplotype. From both lichens and substrates, Illumina 16S amplicon sequencing was performed using primers that exclude cyanobacteria. In lichens, Proteobacteria was the most abundant phylum (37%), whereas soil substrates were dominated by Acidobacteriota (39%). At lower taxonomic levels, several bacterial groups differed in relative abundance among P. frigida lichens and their substrates, some of them being highly abundant in lichens but almost absent in substrates, like Sphingomonas (8% vs 0.2%), and others enriched in lichens, as an unassigned genus of Chitinophagaceae (10% vs 2%). These results reinforce the idea that lichens would carry some components of their microbiome when propagating, but they also could acquire part of their bacterial community from the substrates.
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Ascomicetos , Cianobacterias , Líquenes , MicrobiotaRESUMEN
Assessing the ecological impacts of environmental change on biological communities requires knowledge of the factors driving the spatial patterns of the three diversity facets along extensive environmental gradients. We quantified the taxonomic (TD), functional (FD), and phylogenetic diversity (PD) of lichen epiphytic communities in 23 beech forests along Europe to examine their response to environmental variation (climate, habitat quality, spatial predictors) at a continental geographic scale. We selected six traits related to the climatic conditions in forest ecosystems, the water-use strategy and the nutrient uptake, and we built a phylogenetic tree based on four molecular markers. FD and climate determined TD and PD, with spatial variables also affecting PD. The three diversity facets were primarily shaped by distinct critical predictors, with the temperature diurnal range affecting FD and PD, and precipitation of the wettest month determining TD. Our results emphasize the value of FD for explaining part of TD and PD variation in lichen communities at a broad geographic scale, while highlighting that these diversity facets provide complementary information about the communities' response under changing environmental conditions. Furthermore, traits such as growth form, photobiont type, and reproductive strategy mediated the response of lichen communities to abiotic factors emerging as useful indicators of macroclimatic variations.
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The polyphyletic group of black fungi within the Ascomycota (Arthoniomycetes, Dothideomycetes, and Eurotiomycetes) is ubiquitous in natural and anthropogenic habitats. Partly because of their dark, melanin-based pigmentation, black fungi are resistant to stresses including UV- and ionizing-radiation, heat and desiccation, toxic metals, and organic pollutants. Consequently, they are amongst the most stunning extremophiles and poly-extreme-tolerant organisms on Earth. Even though ca. 60 black fungal genomes have been sequenced to date, [mostly in the family Herpotrichiellaceae (Eurotiomycetes)], the class Dothideomycetes that hosts the largest majority of extremophiles has only been sparsely sampled. By sequencing up to 92 species that will become reference genomes, the "Shed light in The daRk lineagES of the fungal tree of life" (STRES) project will cover a broad collection of black fungal diversity spread throughout the Fungal Tree of Life. Interestingly, the STRES project will focus on mostly unsampled genera that display different ecologies and life-styles (e.g., ant- and lichen-associated fungi, rock-inhabiting fungi, etc.). With a resequencing strategy of 10- to 15-fold depth coverage of up to ~550 strains, numerous new reference genomes will be established. To identify metabolites and functional processes, these new genomic resources will be enriched with metabolomics analyses coupled with transcriptomics experiments on selected species under various stress conditions (salinity, dryness, UV radiation, oligotrophy). The data acquired will serve as a reference and foundation for establishing an encyclopedic database for fungal metagenomics as well as the biology, evolution, and ecology of the fungi in extreme environments.
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Bacteria have recently emerged as important constituents of lichen holobionts. Here, 29 bacterial metagenome-assembled genome (MAG) sequences were reconstructed from lichen metagenomes and taxonomically classified in four phyla. These results provide a pivotal resource for further exploration of the ecological roles played by bacterial symbionts in lichen holobionts.
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We have studied the highly oceanic genus Coniocarpon in Norway. Our aim has been to delimit species of Coniocarpon in Norway based on an integrative taxonomic approach. The material studied comprises 120 specimens of Coniocarpon, obtained through recent collecting efforts (2017 and 2018) or received from major fungaria in Denmark, Finland, Norway and Sweden, as well as from private collectors. We have assessed (1) species delimitations and relationships based on Bayesian and maximum likelihood phylogenetic analyses of three genetic markers (mtSSU, nucITS and RPB2), (2) morphology and anatomy using standard light microscopy, and (3) secondary lichen chemistry using high-performance thin-layer chromatography. The results show three genetically distinct lineages of Coniocarpon, representing C. cinnabarinum, C. fallax and C. cuspidans comb. nov. The latter was originally described as Arthonia cinnabarina f. cuspidans and is herein raised to species level. All three species are supported by morphological, anatomical and chemical data.