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.

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.

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.

Variability in carbon uptake and (re)cycling in Antarctic cryptoendolithic microbial ecosystems demonstrated through radiocarbon analysis of organic biomarkers.

Cryptoendolithic lichens and cyanobacteria living in porous sandstone in the high-elevation McMurdo Dry Valleys are purported to be among the slowest growing organisms on Earth with cycles of death and regrowth on the order of 103 -104  years. Here, organic biomarker and radiocarbon analysis were used to better constrain ages and carbon sources of cryptoendoliths in University Valley (UV; 1,800 m.a.s.l) and neighboring Farnell Valley (FV; 1,700 m.a.s.l). Δ14 C was measured for membrane component phospholipid fatty acids (PLFA) and glycolipid fatty acids, as well as for total organic carbon (TOC). PLFA concentrations indicated viable cells comprised a minor (<0.5%) component of TOC. TOC Δ14 C values ranged from -272‰ to -185‰ equivalent to calibrated ages of 1,100-2,550 years old. These ages may be the result of fractional preservation of biogenic carbon and/or sudden large-scale community death and extended period(s) of inactivity prior to slow recolonization and incorporation of 14 C-depleted fossil material. PLFA Δ14 C values were generally more modern than the corresponding TOC and varied widely between sites; the FV PLFA Δ14 C value (+40‰) was consistent with modern atmospheric CO2 , while UV values ranged from -199‰ to -79‰ (calibrated ages of 1,665-610 years). The observed variability in PLFA Δ14 C depletions is hypothesized to reflect variations in the extent of fixation of modern atmospheric CO2 and the preservation and recycling of older organic carbon by the community in various stages of sandstone recolonization. PLFA profiles and microbial community compositions as determined by molecular genetic characterizations and microscopy differed between the two valleys (e.g., predominance of biomarker 18:2 [>50%] in FV compared to UV), representing microbial communities that may reflect distinct stages of sandstone recolonization and/or environmental conditions. It is thus proposed that Dry Valley cryptoendolithic microbial communities are faster growing than previously estimated.

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.

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.

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.

Resistance of the Lichen Buellia frigida to Simulated Space Conditions during the Preflight Tests for BIOMEX--Viability Assay and Morphological Stability.

Samples of the extremotolerant Antarctic endemite lichen Buellia frigida are currently exposed to low-Earth orbit-space and simulated Mars conditions at the Biology and Mars Experiment (BIOMEX), which is part of the ESA mission EXPOSE-R2 on the International Space Station and was launched on 23 July 2014. In preparation for the mission, several preflight tests (Experimental and Scientific Verification Tests, EVT and SVT) assessed the sample preparation and hardware integration procedures as well as the resistance of the candidate organism toward the abiotic stressors experienced under space and Mars conditions. Therefore, we quantified the post-exposure viability with a live/dead staining technique utilizing FUN-1 and confocal laser scanning microscopy (CLSM). In addition, we used scanning electron microscopy (SEM) to investigate putative patterns of morphological-anatomical damage that lichens may suffer under the extreme exposure conditions. The present results demonstrate that Buellia frigida is capable of surviving the conditions tested in EVT and SVT. The mycobiont showed lower average impairment of its viability than the photobiont (viability rates of >83% and >69%, respectively), and the lichen thallus suffered no significant damage in terms of thalline integrity and symbiotic contact. These results will become essential to substantiate and validate the results prospectively obtained from the returning space mission. Moreover, they will help assess the limits and limitations of terrestrial organisms under space and Mars conditions as well as characterize the adaptive traits that confer lichen extremotolerance.

Alectorioid Morphologies in Paleogene Lichens: New Evidence and Re-Evaluation of the Fossil Alectoria succini Mägdefrau.

One of the most important issues in molecular dating studies concerns the incorporation of reliable fossil taxa into the phylogenies reconstructed from DNA sequence variation in extant taxa. Lichens are symbiotic associations between fungi and algae and/or cyanobacteria. Several lichen fossils have been used as minimum age constraints in recent studies concerning the diversification of the Ascomycota. Recent evolutionary studies of Lecanoromycetes, an almost exclusively lichen-forming class in the Ascomycota, have utilized the Eocene amber inclusion Alectoria succinic as a minimum age constraint. However, a re-investigation of the type material revealed that this inclusion in fact represents poorly preserved plant remains, most probably of a root. Consequently, this fossil cannot be used as evidence of the presence of the genus Alectoria (Parmeliaceae, Lecanorales) or any other lichens in the Paleogene. However, newly discovered inclusions from Paleogene Baltic and Bitterfeld amber verify that alectorioid morphologies in lichens were in existence by the Paleogene. The new fossils represent either a lineage within the alectorioid group or belong to the genus Oropogon.

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.

Fluorescence quenching in the lichen Peltigera aphthosa due to desiccation.

Photoprotective mechanisms were studied on the tripartite lichen Peltigera aphthosa that exhibits external cephalodia. Using the methods of steady-state and time-resolved fluorescence microscopy, we studied the dynamics of the rehydration process in different parts of the lichen thalli. It was found that apical, medial and basal parts of the thallus are not only morphologically different, but also show completely different chlorophyll induction curves and other spectral characteristics. In dry state, significant contribution to the fluorescence spectrum of lichen gives a green fluorescence of hyphae forming the upper crust, which is rapidly and almost completely quenched during the rehydration process. Probably this is one of the protective mechanisms that reduce the amount of light reaching the PS II reaction centers in the dry state. In the process of rehydration, we observed an increase in the intensity of the chlorophyll fluorescence of the photobiont at 680 nm, with significant changes of the fluorescence lifetimes and the amplitude ratios of fast and slow components of fluorescence decay kinetics. While in dry state, chlorophyll fluorescence is strongly quenched (opposite to the fluorescence of the hyphae), and the fluorescence time constants recover to the typical decay times of active photosynthetic organisms during rehydration. The quantitative behavior of these changes differs largely between the apical, medial and basal parts of the thallus, probably due to the complex interactions of the fungus, algae and cyanobacteria.

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.

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.

The symbiotic playground of lichen thalli--a highly flexible photobiont association in rock-inhabiting lichens.

The development of characteristic thallus structures in lichen-forming fungi requires the association with suitable photoautotrophic partners. Previous work suggests that fungi have a specific range of compatible photobionts and that selected algal strains are also correlated with the habitat conditions. We selected the rock-inhabiting crust lichen Protoparmeliopsis muralis, which exhibits high flexibility in algal associations. We present a geographically extended and detailed analysis of algal association patterns including thalli which host superficial algal colonies. We sampled 17 localities in Europe, and investigated the photobiont genotypic diversity within and between thalli and compared the diversity of intrathalline photobionts and externally associate algal communities between washed and unwashed thalli by single-strand conformation polymorphism analyses and ITS sequence data. The results show that (1) photobiont population within the lichen thalli is homogeneous; (2) multiple photobiont genotypes occur within single areoles and lobes of individual lichens; and (3) algal communities which superficially colonize the lichen thalli host taxa known as photobionts in unrelated lichens. Photobiont association patterns are extremely flexible in this ecologically versatile crust-forming lichen. We suggest that lichen surfaces represent a potential temporary niche for free-living stages of lichen photobionts, which could facilitate the establishment of further lichens in the proximal area.

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.

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.

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.