Photosynthesis measurements on the upper and lower side of the thallus of the foliose lichen Nephroma arcticum (L.) Torss.

The measurements of chlorophyll fluorescence play an important role in studies of lichen physiology. Usually, for foliose lichens fluorescence kinetics is recorded from the upper thalline side often exhibiting green color reflecting the presence of photosynthetic pigments. The lower side of such lichens is grey, dark-brown or black. At the first time, we evaluated photosynthetic activity distribution by chlorophyll fluorescence analysis on both lower and upper thallus sides for the foliose lichen Nephroma arcticum. We have demonstrated that photosynthesis proceeds not only on the green-colored upper side, but also on the gray lower side of the curled growing edges of the thallus lobes. These sides were differed in terms of PSII photochemical quantum yield, activity of non-regulatory dissipation and non-photochemical quenching of excited chlorophyll states (NPQ). Upper side was characterized by higher maximal PSII efficiency, whereas the lower one of the curled edges was characterized by higher actual photochemical quantum yield during actinic light acclimation. NPQ was higher on the upper surface, whereas, on the lower side (of the curled edges) non-regulatory dissipation was predominant. In terms of photosynthetic activity measurements, these results show, that actinic and measuring light reached the layer of phycobiont despite its shielding by mycobiont hyphae. On the melanized lower side in the basal thalline zone attached to the substratum photosynthesis was not detected. Lower side demonstrated higher level of light scattering in the reflectance spectra. We believe that different photoprotective mechanisms against high light are crucial on the upper and lower sides: NPQ on the upper surface, and light scattering and shielding by mycobiont on the lower side. Possible biological role of photosynthesis on the lower side is discussed.

Global distribution of Trebouxiophyceae diversity explored by high-throughput sequencing and phylogenetic approaches.

Trebouxiophyceae are a ubiquitous class of Chlorophyta encountered in aquatic and terrestrial environments. Most taxa are photosynthetic, and many acts as photobionts in symbiotic relationships, while others are free-living. Trebouxiophyceae have also been widely investigated for their use for biotechnological applications. In this work, we aimed at obtaining a comprehensive image of their diversity by compiling the information of 435 freshwater, soil and marine environmental DNA samples surveyed with Illumina sequencing technology in order to search for the most relevant environments for bioprospecting. Freshwater and soil were most diverse and shared more than half of all operational taxonomic units (OTUs), however, their communities were significantly distinct. Oceans hosted the highest genetic novelty, and did not share any OTUs with the other environments; also, marine samples host more diversity in warm waters. Symbiotic genera usually found in lichens such as Trebouxia, Myrmecia and Symbiochloris were also abundantly detected in the ocean, suggesting either free-living lifestyles or unknown symbiotic relationships with marine planktonic organisms. Altogether, our study opens the way to new prospection for trebouxiophycean strains, especially in understudied environments like the ocean.

Clarifying the Relationships between Microsporidia and Cryptomycota.

Some protists with microsporidian-like cell biological characters, including Mitosporidium, Paramicrosporidium, and Nucleophaga, have SSU rRNA gene sequences that are much less divergent than canonical Microsporidia. We analysed the phylogenetic placement and environmental diversity of microsporidian-like lineages that group near the base of the fungal radiation and show that they group in a clade with metchnikovellids and canonical microsporidians, to the exclusion of the clade including Rozella, in line with what is currently known of their morphology and cell biology. These results show that the phylogenetic scope of Microsporidia has been greatly underestimated. We propose that much of the lineage diversity previously thought to be cryptomycotan/rozellid is actually microsporidian, offering new insights into the evolution of the highly specialized parasitism of canonical Microsporidia. This insight has important implications for our understanding of opisthokont evolution and ecology, and is important for accurate interpretation of environmental diversity. Our analyses also demonstrate that many opisthosporidian (aphelid+rozellid+microsporidian) SSU V4 OTUs from Neotropical forest soils group with the short-branching Microsporidia, consistent with the abundance of their protist and arthropod hosts in soils. This novel diversity of Microsporidia provides a unique opportunity to investigate the evolutionary origins of a highly specialized clade of major animal parasites.

Parenchymatous cell division characterizes the fungal cortex of some common foliose lichens.

PREMISE OF THE STUDY: Lichen-forming fungi produce diverse vegetative tissues, some closely resembling those of plants. Yet it has been repeatedly affirmed that none is a true parenchyma, in which cellular compartments are subdivided from all adjacent neighbors by cross walls adjoining older cross walls. METHODS: Using transmission electron microscopy (TEM), we tested this assumption by examining patterns of septum formation in the parenchyma-like cortex of three lichens of different phylogenetic affinities: Sticta canariensis, Leptogium cyanescens, and Endocarpon pusillum. KEY RESULTS: In the cortex of all three lichens, new septa adjoined perpendicularly or obliquely to previous septa. Septal walls possessed an electron-transparent core (median) layer covered on both sides by layers of intermediate electron density. At septal junctures, the core layer of the newer septum was not continuous with that of the older septum. Amorphous, electron-dense material often became deposited in the core region of older septal walls, and the septum gradually delaminated along its median into what could then be recognized as the distinct walls of neighboring cells. However, cells maintained continuity at pores, where adjacent remnants of the electron-transparent core layer suggested septal partition rather than secondary establishment of a lateral wall connection via anastomosis. CONCLUSIONS: Although fungal tissues first arise by the coalescence of filaments early in lichen ontogeny, the mature cortical tissues of some lichens are comparable to true parenchyma in the unrestricted orientation of their septal cross walls and the resulting ontogenetic relationship among neighboring cell compartments.

Spatial organization of the three-component lichen Peltigera aphthosa in functional terms.

The cephalolichen Peltigera aphthosa (L.) Willd. is characterized by lateral heterogeneity, which manifests itself in the presence of three thallus zones, referred to as the apical, basal and medial zone. These zones differ in terms of interaction between lichen bionts and their physiological activity. The apical thallus zone is more efficient in establishing a contact with cyanobacteria, because of a higher lectin content and a larger overall thallus surface area due to the presence of numerous mycobiont hyphae. Cephalodia are formed in this zone. The interaction between the mycobiont and cyanobiont is more intense in the medial zone. However, the establishment of the contact with cyanobacteria in this zone less probable. The spatial distribution of lectins in the thallus was determined. To reveal the differences in photosynthetic activity in three thallus zones, transient analysis of chlorophyll a fluorescence and the assessment of non-photochemical quenching of excited chlorophyll states were performed. Assimilation of absorbed light energy was more effective in the medial zone. The basal zone was characterized by decreased photosynthetic activity, lichen dissociation and thallus death.

From one to six: unrecognized species diversity in the genus Acantholichen (lichenized Basidiomycota: Hygrophoraceae).

We present a taxonomic revision of the lichenized basidiomycete genus Acantholichen, species of which produce a characteristic blue-gray, microsquamulose thallus with spiny apical hyphal cells known as acanthohyphidia. Since its discovery, the genus was thought to be monospecific, only including the generic type, A. pannarioides. However, a detailed morphological and anatomical study of recently collected specimens from the Galápagos, Costa Rica, Brazil and Colombia, combined with a molecular phylogenetic analysis of the internal transcribed spacer (ITS1-5.8S-ITS2) region and 28S of the nuc rDNA and RPB2 sequences, revealed a much more diverse and widespread species assemblage. Based on the results of these analyses, we describe five new species in the genus: A. albomarginatus, A. campestris, A. galapagoensis, A. sorediatus and A. variabilis. We also provide an identification key to all species, anatomical and morphological descriptions, photographs and a table comparing main characters of each species.

Structure and in situ development of the microlichen Gyalectidium paolae (Gomphillaceae, Ascomycota), an overlooked colonist on palm leaves in southwest Florida.

PREMISE OF THE STUDY: Nondeciduous leaves of warm, humid climates can host highly specialized communities of diminutive lichens. The rarely reported Gyalectidium paolae, locally abundant on palm leaves in southwest Florida, may reproduce when as small as 0.15 mm diameter. We examined structural and developmental features to better understand the lifestyle of this extreme ephemeral. METHODS: Blocks containing resin-embedded thalli were sectioned and examined with TEM and SEM-BSE. Propagule development was studied with light microscopy applied to inoculated and naturally colonized plastic coverslips placed in the field. KEY RESULTS: Thallus areolae showed a heterogeneous covering that varied from cellular cortex to a simpler structure derived from fungal wall materials and sparse fungal cells of reduced diameter. Plates of crystalline deposits seemed to interrupt thallus structure, elevating the surface layer. No organized algal layer was present. Symbiont interactions were limited to appositional wall contacts with no haustorial penetration observed. Symbiotic propagules germinated promptly, but relative growth of fungal vs. algal components varied considerably. Smaller photobiont cells released from sporangia were present at the periphery of the thallus, or escaped to some distance. Fully formed hyphophores with abundant propagules appeared within 5 months, although there was evidence that propagule formation in Gyalectidium might occur much sooner. CONCLUSIONS: Gyalectidium paolae builds relatively simple thalli with limited fungal structure, prioritizing rapid formation of asexual propagules. Codispersal of algal symbionts permitted propagules to develop directly into thalli, but microenvironmental conditions may strongly influence survival and developmental equilibrium between the two symbionts necessary for success as a lichen.

Complete life cycle of the lichen fungus Calopadia puiggarii (Pilocarpaceae, Ascomycetes) documented in situ: propagule dispersal, establishment of symbiosis, thallus development, and formation of sexual and asexual reproductive structures.

PREMISE OF THE STUDY: The life histories of lichen fungi are not well known and cannot be readily studied in laboratory culture. This work documents in situ the complete life cycle of the widespread crustose lichen Calopadia puiggarii, which reproduces sexually and asexually on the surfaces of leaves. METHODS: Plastic cover slips held in a mesh frame were placed over leaves in the field and successively removed for microphotography of colonizing lichens. KEY RESULTS: Macroconidia produced within campylidia encircled photobiont cells and codispersed with them, a feature not reported previously for C. puiggarii. Dispersed macroconidia readily germinated and lichenized the photobionts. Algal cells were often dislodged from the encircling macroconidia, providing a likely source for the free-living populations observed. Aposymbiotically dispersed ascospores germinated and lichenized nearby algal cells soon after dispersal. Thallus areolae merged readily in early development, although adjacent mature thalli were often separated by growth inhibition zones. Pycnidia are reported for the first time in Calopadia; their pyriform microconidia probably function as male gametes (spermatia). Pycnidia, apothecia, and campylidia began development similarly as darkly pigmented primordia on the fungal prothallus. CONCLUSIONS: Abundant dispersal of ascospores, conidia, and photobionts allows C. puiggarii to quickly colonize leaves with the dual advantages of sexual and asexual reproduction, and with the added convenience of having its algal partner on hand. Fusions and prothallic capture of additional algae provide many opportunities for multiple mycobiont and photobiont genotypes to be combined in a single thallus, but the outcomes of such events remain to be explored.

The earliest records of internally stratified cyanobacterial and algal lichens from the Lower Devonian of the Welsh Borderland.

Lichenization is assumed to be a very ancient mode of fungal nutrition, but fossil records are rare. Here we describe two fragments of exceptionally preserved, probably charred, lichen thalli with internal stratification. Cyanolichenomycites devonicus has a cyanobacterial and Chlorolichenomycites salopensis a unicellular, presumably green algal photobiont. Fruiting bodies are missing. Cyanolichenomycites devonicus forms asexual spores in a pycnidium. All specimens were examined with scanning electron microscopy techniques. The fossils were extracted by maceration. Extant lichens and free-living cyanobacteria were either experimentally charcoalified for comparison or conventionally prepared. Based on their septate hyphal structure, both specimens are tentatively interpreted as representatives of the Pezizomycotina (Ascomycota). Their presence in 415 million yr (Myr) old rocks from the Welsh Borderland predates existing Late Cretaceous records of pycnidial conidiomata by some 325 Myr and Triassic records of lichens with broadly similar organization by some 195 Myr. These fossils represent the oldest known record of lichens with symbionts and anatomy as typically found in morphologically advanced taxa today. The latter does not apply to Winfrenatia reticulata, the enigmatic crustose lichen fossil from the Lower Devonian, nor to presumed lichen-like organisms such as the Cambrian Farghera robusta or to the Lower Devonian Spongiophyton minutissimum.

Cardinal characters on a slippery slope--a re-evaluation of phylogeny, character evolution, and evolutionary rates in the jelly lichens (Collemataceae s. str).

Recent molecular systematic studies have indicated that the traits currently used for generic delimitation in the jelly lichens (Collemataceae s. str.), may not characterize monophyletic groups. Here we reconstruct the phylogeny of Collemataceae using Bayesian and maximum likelihood analyses based on mitochondrial (mtSSU rDNA) and nuclear (nuLSU rDNA, Beta-tubulin and MCM7) markers of 70 Collemataceae species. We studied the evolution of four morphological and ecological characters traditionally used to delimit genera and infra-generic groups. Finally, we tested if differences in branch-lengths between clades are due to differences in rates of molecular evolution. Eleven strongly supported groups were recovered in the resulting well-resolved and well-supported phylogeny. The presence/absence of a eucortex, which is currently used as the cardinal character to define genera in the group, does not characterize monophyletic groups corresponding to the genera as currently circumscribed. Ancestral state reconstruction indicates that the most recent common ancestor of the jelly lichens most likely was saxicolous/terricolous, lacked a tomentum, and had transversally septate ascospores. Although the cortex state could not be reconstructed for the ancestor of the family, our observations indicate that a lack of cortex may have an evolutionary advantage in saxicolous/terricolous species in semi-arid environments, as non-corticate species tends to be larger and occur in higher frequency and abundance in such regions, compared to corticate species. A significant evidence for faster evolutionary rates was found in a lineage mainly including taxa that occur in the wet tropics and humid temperate regions, compared to other lineages. We suggest that this can explain the greater diversity of Collemataceae in tropical and humid areas.

Honeggeriella complexa gen. et sp. nov., a heteromerous lichen from the Lower Cretaceous of Vancouver Island (British Columbia, Canada).

PREMISE OF THE STUDY: Colonists of even the most inhospitable environments, lichens are present in all terrestrial ecosystems. Because of their ecological versatility and ubiquity, they have been considered excellent candidates for early colonizers of terrestrial environments. Despite such predictions, good preservation potential, and the extant diversity of lichenized fungi, the fossil record of lichen associations is sparse. Unequivocal lichen fossils are rare due, in part, to difficulties in ascertaining the presence of both symbionts and in characterizing their interactions. This study describes an exceptionally well-preserved heteromerous lichen from the Lower Cretaceous of Vancouver Island. METHODS: The fossil occurs in a marine carbonate concretion collected from the Apple Bay locality on Vancouver Island, British Columbia, and was prepared for light microscopy and SEM using the cellulose acetate peel technique. KEY RESULTS: The lichen, Honeggeriella complexa gen. et sp. nov., is formed by an ascomycete mycobiont and a chlorophyte photobiont, and exhibits heteromerous thallus organization. This is paired with a mycobiont-photobiont interface characterized by intracellular haustoria, previously not documented in the fossil record. CONCLUSIONS: Honeggeriella adds a lichen component to one of the richest and best characterized Early Cretaceous floras and provides a significant addition to the sparse fossil record of lichens. As a heteromerous chlorolichen, it bridges the >350 million-year gap between previously documented Early Devonian and Eocene occurrences.

A radical shift in the taxonomy of Lepraria s.l.: molecular and morphological studies shed new light on the evolution of asexuality and lichen growth form diversification.

A combination of molecular phylogenetic analyses of ITS and mtSSU sequences, morphological and chemical analyses were used to investigate the lineages nominally included in the sterile lichen genus Lepraria. A core group (Lepraria s. str.) was resolved as sister to Stereocaulon. Species producing the secondary compounds argopsin, pannarin and usnic acid were found to belong to other lineages of lichen-forming ascomycetes. Study of Leprocaulon revealed that all species, except the type, likely represent members of Lepraria s. str. that have evolved a fruticose growth form. The correct name for the type species of Leprocaulon is shown to be L. quisquiliare, not L. microscopicum, and the genus is redefined to include several species previously placed in Lepraria. Leprocaulon quisquiliare is also shown to comprise two morphologically convergent species. The name is lectotypified and epitypified on material from the type region (Germany) and its application restricted to Old World populations. New World populations of L. quisquiliare are described as L. americanum. Leprocaulon, in its revised sense, is recognized in a new family (Leprocaulaceae) and order (Leprocaulales) sister to the Caliciales and including the genus Halecania. A new genus of Pilocarpaceae, Nelsenium, is introduced to accommodate Lepraria usnica. The status of Lepraria ecorticata is discussed in the context of usnic acid-producing Lecanora species. These nomenclatural novelties are proposed: (i) transfers from Leprocaulon to Lepraria: Lepraria albicans comb. nov., L. arbuscula comb. nov., L. congestum comb. nov., L. gracilescens comb. nov., L. pseudoarbuscula comb. nov., L. subalbicans comb. nov., L. tenellum comb. nov.; (ii) transfers from Lepraria to Leprocaulon: Leprocaulon adhaerens comb. nov., L. coriense, L. santamonicae comb. nov., L. terricola comb. nov. and L. textum comb. nov.; (iii) new taxa: Leprocaulales ord. nov., Leprocaulaceae fam. nov., Nelsenium gen. nov., Leprocaulon americanum sp. nov. and L. knudsenii sp. nov.

Gene expression indicates a zone of heterocyst differentiation within the thallus of the cyanolichen Pseudocyphellaria crocata.

Development of the symbiotic association in the bipartite lichen Pseudocyphellaria crocata was investigated by characterizing two regions of the thallus. Thallus organization was examined using microscopy. A HIP1-based differential display technique was modified for use on Nostoc strains, including lichenized strains. Northern hybridization and quantitative real-time polymerase chain reaction were used to confirm differential display results, and determine expression levels of key cyanobacterial genes. Photosystem II yield across the thallus was measured using pulse-amplitude modulated fluorescence. Microscopy revealed structural differences in the thallus margins compared with the centre and identified putative heterocysts in both regions. Differential display identified altered transcript levels in both Nostoc punctiforme and a lichenized Nostoc strain. Transcript abundance of cox2, atpA, and ribA was increased in the thallus margin compared with the centre. Expression of cox2 is heterocyst specific and expression of other heterocyst-specific genes (hetR and nifK) was elevated in the margin, whereas, expression of psbB and PSII yield were not. Structural organization of the thallus margin differed from the centre. Both regions contained putative heterocysts but gene expression data indicated increased heterocyst differentiation in the margins where photosystem II yield was decreased. This is consistent with a zone of heterocyst differentiation within the thallus margin.

Extreme phenotypic variation in Cetraria aculeata (lichenized Ascomycota): adaptation or incidental modification?

BACKGROUND AND AIMS: Phenotypic variability is a successful strategy in lichens for colonizing different habitats. Vagrancy has been reported as a specific adaptation for lichens living in steppe habitats around the world. Among the facultatively vagrant species, the cosmopolitan Cetraria aculeata apparently forms extremely modified vagrant thalli in steppe habitats of Central Spain. The aim of this study was to investigate whether these changes are phenotypic plasticity (a single genotype producing different phenotypes), by characterizing the anatomical and ultrastructural changes observed in vagrant morphs, and measuring differences in ecophysiological performance. METHODS: Specimens of vagrant and attached populations of C. aculeata were collected on the steppes of Central Spain. The fungal internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GPD) and the large sub-unit of the mitochondrial ribosomal DNA (mtLSUm), and the algal ITS and actin were studied within a population genetics framework. Semi-thin and ultrathin sections were analysed by means of optical, scanning electron and transmission electron microscopy. Gas exchange and chlorophyll fluorescence were used to compare the physiological performance of both morphs. KEY RESULTS AND CONCLUSIONS: Vagrant and attached morphs share multilocus haplotypes which may indicate that they belong to the same species in spite of their completely different anatomy. However, differentiation tests suggested that vagrant specimens do not represent a random sub-set of the surrounding population. The morphological differences were related to anatomical and ultrastructural differences. Large intercalary growth rates of thalli after the loss of the basal-apical thallus polarity may be the cause of the increased growth shown by vagrant specimens. The anatomical and morphological changes lead to greater duration of ecophysiological activity in vagrant specimens. Although the anatomical and physiological changes could be chance effects, the genetic differentiation between vagrant and attached sub-populations and the higher biomass of the former show fitness effects and adaptation to dry environmental conditions in steppe habitats.

Convergent evolution of a symbiotic duet: the case of the lichen genus Polychidium (Peltigerales, Ascomycota).

PREMISE OF THE STUDY: Thallus architecture has long been a powerful guide for classifying lichens and has often trumped photobiont association and ascomatal type, but the reliability of these characters to predict phylogenetic affinity has seldom been tested. The cyanolichen genus Polychidium unites species that have strikingly similar gross morphology but consort with different photobiont genera. If Polychidium were found to be monophyletic, photobiont switching among closely related species would be suggested. If, however, species were found to arise in different lineages, a convergent body plan and ascomatal type evolution would be inferred. METHODS: We tested the monophyly of Polychidium with a multilocus phylogeny based on nuclear and mitochondrial sequence data from all known Peltigeralean families and reconstructed ancestral states for specific thallus architecture and ascomatal ontogeny types relative to Polychidium and other clades. KEY RESULTS: We found that Polychidium consists of two species groups that arose independently in different suborders within the Peltigerales, associated with Nostoc and Scytonema photobionts, respectively. We infer from ancestral character state reconstruction that dendroid thallus architecture evolved independently in these two lineages. CONCLUSIONS: The independent development of similar dendroid thallus architecture in different fungal suborders with different photobionts represents a clear and previously overlooked example of convergent evolution in lichens. Our results also suggest a pattern of character state conservation, loss, and reversion in ascomatal ontogeny types, hitherto considered conserved traits useful for higher level ascomycete systematics.

Expansion of the Stictidaceae by the addition of the saxicolous lichen-forming genus Ingvariella.

The monotypic, lichen-forming genus Ingvariella originally was segregated from Diploschistes and placed within the Thelotremataceae (Ostropales) based on aspects of exciple morphology. However, the I+ hymenium and amyloid ascus wall suggest affinities to families other than the Thelotremataceae. To assess the identity of Ingvariella and to investigate its placement within the Ostropales, we inferred phylogenetic relationships of I. bispora by comparison of mtSSU rDNA and nuLSU rDNA sequences for 59 species encompassing a broad array of ostropalean fungi by means of Bayesian, maximum likelihood and weighted maximum parsimony methods. Here we report that Ingvariella is a member of the Stictidaceae, sister to the mainly saprotrophic genus Cryptodiscus. The inclusion of the first saxicolous lichen-forming fungus within this family expands the broad ecological diversity of the Stictidaceae, where saprotrophic fungi, corticicolous lichen-forming fungi and lichenized and non-lichenized conspecific taxa have been described previously. We also present new insights into the relationships among other families within the Ostropales.

Effects of nitrogen enrichment on phosphatase activity and nitrogen : phosphorus relationships in Cladonia portentosa.

*Relationships between nitrogen deposition in the UK and phosphomonoesterase (PME) activity and nitrogen (N) and phosphorus (P) concentrations in Cladonia portentosa were quantified to understand factors limiting lichen growth and to further develop biomarkers for N pollution. *Lichen was collected from sites differing either in rates of wet N (NH(4)(+) + NO(3)(-)) deposition or in annual mean N concentration in rainfall based on both measured and modelled data sets. The PME activity, and total N and P concentrations were measured in specific horizontal strata in lichen mats and PME activity in the thallus was located using an enzyme-labelled fluorescent phosphatase substrate. *With an increase in modelled N deposition from 4.1 to 32.8 kg N ha(-1) yr(-1), PME activity, thallus N and N : P ratio increased by factors of 2.3, 1.4 and 1.8, respectively. Correlations with modelled data were generally stronger than with measured data and those with N deposition were stronger than those with N concentration in rainfall. The PME activity was located solely in the lichen fungus in outer regions of the thallus. *Nitrogen enrichment changes lichen N : P ratios from values typical of N limitation (for example, 10) to those indicative of P limitation (for example, 26) driving upregulation of PME activity.

In situ imaging of usnic acid in selected Cladonia spp. by vibrational spectroscopy.

In this study, we demonstrate the first in situ detection of usnic acid (UA) in selected species of the lichen Cladonia, using FPA-FTIR imaging and Raman microscopy. Fruticose lichens present a variety of defensive mechanisms, one of which is the production of UA. This polyketide secondary metabolite, produced by certain lichenized fungi, has a protective function for the lichen that includes a strong absorption in the ultraviolet range. Upon confirming the distinct spectral signature of UA in lichen tissue, we mapped its distribution in Cladonia arbuscula, Cladonia uncialis and Cladonia sulphurina tissues. Spectroscopic images were obtained from cryosectioned lichen fragments embedded in media and from hand-sectioned fragments that were media-free. UA was present in the pycnidia, and younger walls of C. arbuscula and C. uncialis, the spore-producing region of a C. uncialis apothecium, and in both the younger and older soredia of C. sulphurina. The localization of UA in lichens is an important precursor to future work that includes the identification of the gene cluster responsible for its biosynthesis. Our results show that FTIR and Raman imaging can be an effective way to study the distribution of natural products in lichens with micron-scale precision.

Lichen Vitality After a Space Flight on Board the EXPOSE-R2 Facility Outside the International Space Station: Results of the Biology and Mars Experiment.

As part of the Biology and Mars Experiment (BIOMEX; ILSRA 2009-0834), samples of the lichen Circinaria gyrosa were placed on the exposure platform EXPOSE-R2, on the International Space Station (ISS) and exposed to space and to a Mars-simulated environment for 18 months (2014-2016) to study: (1) resistance to space and Mars-like conditions and (2) biomarkers for use in future space missions (Exo-Mars). When the experiment returned (June 2016), initial analysis showed rapid recovery of photosystem II activity in the samples exposed exclusively to space vacuum and a Mars-like atmosphere. Significantly reduced recovery levels were observed in Sun-exposed samples, and electron and fluorescence microscopy (transmission electron microscope and field emission scanning electron microscope) data indicated that this was attributable to the combined effects of space radiation and space vacuum, as unirradiated samples exhibited less marked morphological changes compared with Sun-exposed samples. Polymerase chain reaction analyses confirmed that there was DNA damage in lichen exposed to harsh space and Mars-like environmental conditions, with ultraviolet radiation combined with space vacuum causing the most damage. These findings contribute to the characterization of space- and Mars-resistant organisms that are relevant to Mars habitability.

Dehydration rate and time of desiccation affect recovery of the lichen alga [corrected] Trebouxia erici: alternative and classical protective mechanisms.

The mechanisms involved in desiccation tolerance of lichens and their photobionts are still poorly understood. To better understand these mechanisms we have studied dehydration rate and desiccation time in Trebouxia, the most abundant chlorophytic photobiont in lichen. Our findings indicate that the drying rate has a profound effect on the recovery of photosynthetic activity of algae after rehydration, greater than the effects of desiccation duration. The basal fluorescence (F'(o)) values in desiccated algae were significantly higher after rapid dehydration, than after slow dehydration, suggesting higher levels of light energy dissipation in slow-dried algae. Higher values of PSII electron transport were recovered after rehydration of slow-dried Trebouxia erici compared to rapid-dried algae. The main component of non-photochemical quenching after slow dehydration was energy dependent (q (E)), whereas after fast dehydration it was photoinhibition (q (I)). Although q (E) seems to play a role during desiccation recovery, no significant variations were detected in the xanthophyll cycle components. Desiccation did not affect PSI functionality. Classical antioxidant activities like superoxide dismutase or peroxidase decreased during desiccation and early recovery. Dehydrins were detected in the lichen-forming algae T. erici and were constitutively expressed. There is probably a minimal period required to develop strategies which will facilitate transition to the desiccated state in this algae. In this process, the xanthophyll cycle and classical antioxidant mechanisms play a very limited role, if any. However, our results indicate that there is an alternative mechanism of light energy dissipation during desiccation, where activation is dependent on a sufficiently slow dehydration rate.