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SUMMARY Undisturbed associations of Peltigera membranacea and associated mosses were held under simulated autumn, winter and spring conditions in a specially constructed, controlled-environment chamber providing an atmosphere enriched with labelled nitrogen. Simulated rainfall via a programmable sprinkler system was incorporated, and the liquid run-off was collected during 4 week experimental periods. Dry weight, total nitrogen and 15N labelling of the moss, lichen and run-off liquid were determined, and nitrogenase activity of the lichen thalli was estimated by acetylene reduction at the end of the tests. It was estimated that the mosses derived direct nutritional benefit from nitrogen compounds leached from the lichen thalli, but due to simultaneous nitrogen-fixing activity by epiphytes strictly quantitative estimates of the beneficial effects of the lichen were unobtainable.

SUMMARY The photosynthetic properties of cyanobiont lichens from contrasting habitats were measured to identify whether the increased assimilation rates which characterized Peltigera membranacea (Ach.) Nyl. from an exposed habitat were correlated with increased carbon-concentrating mechanism (CCM) activity. The results were contrasted with data obtained from two populations of Peltigera praetextata (Florke ex Sommerf.) Zopf collected from dry and damp microhabitats within a shaded woodland and Peltigera leucophlebia (Nyl.) Gyelnik, which has been shown to lack a carbon-concentrating mechanism. The differences in assimilation rates between the cyanobiont lichens were not accounted for by differences in chlorophyll content. Peltigera membranacea from the exposed habitat which had the highest assimilation rates had the lowest F and KO.5 values and accumulated the greatest C,-pool indicating that increased C, accumulation contributed towards the higher assimilation rates shown by these species. The convexity of the light response curve for the cyanobiont lichens decreased with increasing assimilation rates. This might have indicated a diversion of electron transport to energize the carbon-concentrating mechanism. The apparent quantum efficiency of CO2 assimilation ((DCO2) was correlated with the genus of lichen photobiont. All cyanobiont lichens had comparable values for (DCO2 which were greater than that of the tripartite Peltigera leucophlebia. Light compensation points reflected the exposure of the habitats with higher compensation points characterizing the cyanobiont population from the exposed crag and the tri-partite population from the open grassland. Carbon isotope discrimination values for organic matter and measured instantaneously were the same for all cyanobiont lichens and were comparable with values recorded for species with a carbon-concentrating mechanism. Carbon isotope measurements for P. leucophlebia were typical of those recorded for species without a carbon-concentrating mechanism. Variation in source isotope signature and refixation of respiratory CO2 were considered to be significant factors in determining organic matter and instantaneous carbon-isotope discrimination. These factors might have masked any subtle variation in carbon-isotope discrimination which resulted from variable CCM activity. The functional significance of increased carbon-concentrating mechanism activity in cyanobiont lichens occupying exposed habitats is discussed.

Burgaz, A. R., Fontecha-Galan, A., Gutierrez-Larruga, B. & Rodriguez-Arribas, C. 2016. The Cladoniaceae and three additional noteworthy lichens from Croatia. — Herzogia 30: 138–151. In the framework of a project on the family Cladoniaceae in the Mediterranean area of Eurasia many new records and the chemical variability of the family in Croatia are discussed. Cladonia conista, C. cyathomorpha, C. dimorpha, C. homosekikaica, C. novochlorophaea, C. peziziformis and C. umbricola are reported as new to Croatia. In addition, Flavoparmelia soredians, Parmelia serrana and Peltigera membranacea were discovered as new to Croatia. A key to all the Cladoniaceae taxa known from Croatia is provided.

Organisms have evolved different cellular mechanisms to deal with environmental stress, primarily through complex molecular mechanisms including protein refolding and DNA repair. As mutualistic symbioses, lichens offer the possibility of analyzing molecular stress responses in a particularly tight interspecific relationship. We study the widespread cyanolichen Peltigera membranacea, a key player in carbon and nitrogen cycling in terrestrial ecosystems at northern latitudes. We ask whether increased temperature is reflected in mRNA levels of selected damage control genes, and do the response patterns show geographical associations? Using real-time PCR quantification of 38 transcripts, differential expression was demonstrated for nine cyanobacterial and nine fungal stress response genes (plus the fungal symbiosis-related lec2 gene) when the temperature was increased from 5 °C to 15 °C and 25 °C. Principle component analysis (PCA) revealed two gene groups with different response patterns. Whereas a set of cyanobacterial DNA repair genes and the fungal lec2 (PC1 group) showed an expression drop at 15 °C vs. 5 °C, most fungal candidates (PC2 group) showed increased expression at 25 °C vs. 5 °C. PC1 responses also correlated with elevation. The correlated downregulation of lec2 and cyanobacterial DNA repair genes suggests a possible interplay between the symbionts warranting further studies.

Although lichens are generally described as mutualistic symbioses of fungi and photosynthetic partners, they also harbour a diverse non-phototrophic microbiota, which is now regarded as a significant part of the symbiosis. However, the role of the non-phototrophic microbiota within the lichen is still poorly known, although possible functions have been suggested, including phosphate solubilization and various lytic activities. In the present study we focus on the bacterial biota associated with the foliose lichen Peltigera membranacea. To address our hypotheses on possible roles of the non-phototrophic microbiota, we used a metagenomic approach. A DNA library of bacterial sequence contigs was constructed from the lichen thallus material and the bacterial microbiota DNA sequence was analysed in terms of phylogenetic diversity and functional gene composition. Analysis of about 30,000 such bacterial contigs from the P. membranacea metagenome revealed significant representation of several genes involved in phosphate solubilization and biopolymer degradation.

We thank Marion Millot, Marylene Chollet, Philippe Uriac, Nina Corlay and Friardi Ismed for providing Stereocaulon alpinum, Peltigera membranacea, S. pileatum, S.vulcani and S. scutelligerum, S. graminosum, S. halei, and S. montagneanum, respectively. We are also grateful to Lydie Sparfel for performing the variance analysis and to Sophie Tomasi for valuable comments to improve the manuscript and Stephen S. Bates for proof reading. The authors thank the Vietnamese government for the grant that supported Vu Thi Huyen.; International audience; The composition of fatty acids (FAs) is known to discriminate families within genera of bacteria, fungi, octocorals and algae. Here, we applied the more sensitive gas chromatographic (GC) analysis to investigate the taxonomic value of using FA composition to discriminate 15 species of lichen, including three chlorolichens (two Cladonia species and Stereocaulon scutelligerum ), nine tripartite lichens ( Stereocaulon species) and three cyanolichens ( Lichina pygmaea , Collema cristatum , Peltigera membranacea ). One macroscopic cyanobacterium ( Nostoc sp.), corresponding to the photobiont partner of Peltigera and Collema cyanolichens, was included for comparison. Five lipid extraction methods were evaluated, using Stereocaulon scutelligerum . Shaking at 80 °C with chloroform/methanol (2:1, v/v) was the most efficient and reproducible method. The total FA composition of the 16 species was compared, using GC coupled with flame ionization detection or mass spectrometry. A statistical hierarchical cluster analysis was used to determine the similarity between the FA profiles, based on total, saturated, unsaturated and branched-chain FAs (BCFAs). The BCFA profile allowed a distinction between the Stereocaulon species and the cyanolichens, in contrast to all the other types of FAs. A detailed characterization of eight FA subclasses, provided by the GC analysis, suggested a preferential pathway for the biogenesis of unsaturated FAs through desaturases, which was especially favoured in the cyanolichens.

Lectins are a diverse group of carbohydrate binding proteins often involved in cellular interactions. A lectin gene, lec-2, was identified in the mycobiont of the lichen Peltigera membranacea. Sequencing of lec-2 open reading frames from 21 individual samples showed an unexpectedly high level of polymorphism in the deduced protein (LEC-2), which was sorted into nine haplotypes based on amino acid sequence. Calculations showed that the rates of nonsynonymous versus synonymous nucleotide substitutions deviated significantly from the null hypothesis of neutrality, indicating strong positive selection. Molecular modeling revealed that most amino acid replacements were around the putative carbohydrate-binding pocket, indicating changes in ligand binding. Lectins have been thought to be involved in the recognition of photobiont partners in lichen symbioses, and the hypothesis that positive selection of LEC-2 is driven by variation in the Nostoc photobiont partner was tested by comparing mycobiont LEC-2 haplotypes and photobiont genotypes, as represented by the rbcLX region. It was not possible to pair up the two types of marker sequences without conflicts, suggesting that positive selection of LEC-2 was not due to variation in photobiont partners.

Mitochondrial genomes from the fungal partners of two terricolous foliose lichen symbioses, Peltigera membranacea and Peltigera malacea, have been determined using metagenomic approaches, including RNA-seq. The roughly 63 kb genomes show all the major features found in other Pezizomycotina, such as unidirectional transcription, 14 conserved protein genes, genes for the two subunit rRNAs and for a set of 26 tRNAs used in translating the 62 amino acid codons. In one of the tRNAs a CAU anticodon is proposed to be modified, via the action of the nuclear-encoded enzyme, tRNA Ile lysidine synthase, so that it recognizes the codon AUA (Ile) instead of AUG (Met). The overall arrangements and sequences of the two circular genomes are similar, the major difference being the inversion and deterioration of a gene encoding a type B DNA polymerase. Both genomes encode the RNA component of RNAse P, a feature seldom found in ascomycetes. The difference in genome size from the minimal ascomycete mitochondrial genomes is largely due to 17 and 20 group I introns, respectively, most associated with homing endonucleases and all found within protein-coding genes and the gene encoding the large subunit rRNA. One new intron insertion point was found, and an unusually small exon of seven nucleotides (nt) was identified and verified by RNA sequencing. Comparative analysis of mitochondrion-encoded proteins places the Peltigera spp., representatives of the class Lecanoromycetes, close to Leotiomycetes, Dothidiomycetes, and Sordariomycetes, in contrast to phylogenies found using nuclear genes.

Fifty-one species of lichenicolous fungi are reported from Kamchatka, all but three being new to the area. Epinephroma gen. nov., Endococcus peltigericola sp. nov. (on Peltigera membranacea), Epinephroma kamchatica sp. nov. (on Nephroma parile) and Stigmidium buelliae sp. nov. (on Buellia disciformis) are described as new to science. Phoma lobariicola is new to Asia and Russia, Epicladonia stenospora, Plectocarpon peltigerae, Sphaerellothecium propinquellum and Tremella cetrariicola are new to Asia, Zwackhiomyces sphinctrinoides is new to Russia.

Many analytical methods are known to discriminate natural products based on the set of chemical compounds they contain. Here, we evaluated the ability of Fibre Evanescent Wave Spectroscopy (FEWS) to discriminate lichen natural extracts. Ten species of lichens including four chlorolichens (three Cladonia species and Stereocaulon scutelligerum), three tripartite lichens (Stereocaulon species) and three cyanolichens (Lichina pygmaea, Collema cristatum, Peltigera membranacea) belonging to six genera and six families were studied. One macroscopic cyanobacterium (Nostoc sp.), corresponding to the photobiont partner of Peltigera and Collema cyanolichens, was included for comparison. MIR spectra were acquired from lichen organic extracts between 3800-950 cm-1 using a chalcogenide infrared fibre optical device. Principal Component Analysis (PCA) and PCA-LDA (PCA-Linear Discriminant Analysis) based on the concept of variable clustering has been applied to absorbance MIR second derivative spectra for classification of the 11 species. The method succeeded in separating all lichen species and in identifying the major compounds. Moreover, the signature of the sample was specific of each lichen suggesting that minor compounds played a role. Our data showed that FEWS optical sensor profiling is a rapid, efficient and convenient tool for metabolic profiling and suggest that it can be used: (i) in chemotaxonomic approaches, (ii) in processes for certification requirements in active compounds availability, and (iii) in identifying new biosourcings in so far non-investigated lichen species. © The Royal Society of Chemistry 2016.

Differences in the soluble protein fraction between the freshly isolated cyanobiont of lichen Peltigera membranacea , the corresponding free-living strain, and Nostoc punctiforme were analyzed. One protein, which was among the most prominent proteins of the freshly isolated cyanobiont, was expressed at a lower level in the corresponding free-living strain and was not detected at all on the two-dimensional gels of N. punctiforme . This protein was partially sequenced, and the corresponding open reading frame (ORF) in the N. punctiforme genome was identified. This ORF contains a fasciclin domain typical of a class of surface-associated proteins involved in cell adhesion. Similar fasciclin motif-containing genes have previously been shown to be symbiotically induced in other symbiotic systems.

Patterns of photobiont diversity were examined in some Nostoc-containing lichens using the nucleotide sequence of the cyanobacterial tRNALeu (UAA) intron. Lichen specimens collected in northwestern USA were analysed and the sequence data were compared with tRNALeu(UAA) intron sequences previously obtained from lichens in northern Europe. Generally, it is the species identity of a lichen rather than the geographical origin of the specimen that determines the identity of the cyanobiont. Identical intron sequences were found in Peltigera membranacea specimens collected in Oregon (USA) and in Sweden, and very similar sequences were also found in Nephroma resupinatum thalli collected in Oregon and Finland. Furthermore, in mixed assemblages where two Peltigera species grew in physical contact with each other, the different lichen species housed different photobiont strains. There is however not a one-to-one relation between mycobiont and photobiont as some intron sequences were found in more than one lichen species, and different intron sequences were found in different samples of some lichen taxa. Peltigera venosa exhibited a higher level of photobiont diversity than any other lichen species studied, and several intron sequences could for the first time be obtained from a single thallus. It is not clear whether this is evidence of lower cyanobiont specificity, or reflects an ability to exhibit different degrees of lichenization with different Nostoc strains. In one specimen of P. venosa, which contained bipartite cyanosymbiodemes and tripartite, cephalodiate thalli, both thallus types contained the same intron sequence.

A molecular study was undertaken to clarify the identity of the photobiont in colourmorphs of the lichen, Peltigera membranacea. Two strains of cyanobacteria, identified as Nosroc sp. by morphology, were cultivated from each of two lichen specimens. Prokaryotic (16S) ribosomal RNA gene fragments were amplified by the polymerase chain reaction (PCR) from DNA extracted from the isolated strains and the lichens, and sequenced directly. Sequences were 98 1% identical between lichen specimens, TDI#AR94 and TDI#AR95, and highly similar to sequences published, or generated in this study from a type culture, for Nostoc. The 16S ribosomal RNA gene sequences (‘ 16S rDNA’) of all four lichen-derived cyanobacteria appeared the same, even though the lichen specimens from which they originated had different sequences. The 16S rDXA from strains 9A and 9B were different from that of specimen TDI#AR94, the thallus from which they were isolated, and instead were the same as that of strains 10A and 10B, and their source, specimen TDI#AR95. When primers selective for the strain 9A sequence were used, however, a small amount of PCR product corresponding to the 16S rDNA of strain 9A was obtained from lichen TDI#AR94. The results confirm that the photobionts of P. membranacea belong to Nostoc, and suggest that genetic differences in the photobiont may be a factor in the occurrence of colourmorphs among cyanolichens.

SUMMARY Cadmium uptake by photosynthetically Cd-tolerant and intolerant populations of the moss Rhytidiadelphus squarrosus (Hedw.) Warnst and the lichen Peltigera membranacea (Ach.) Nyl. was investigated. Intolerant plants showed increased intra cellular Cd uptake compared to Cd-tolerant material. Kinetic analysis indicated that reduced intracellular Cd uptake by Cd-tolerant plant material was reflected in an increased Km or decreased Vmax for Cd. Pretreatment with KNO3 modified the extra cellular ion content of plant material, significantly reduced the apparent Km and could increase or decrease the Vmax for intracellular Cd uptake. Changes in kinetic constants caused by K pretreatment could be related to altered chemical equilibria when plant material was incubated in Cd solutions. Selective exclusion of Cd from intracellular locations was excluded as a tolerance mechanism in R. squarrosus and P. membranacea. Contrasting kinetic constants for intracellular Cd uptake in intolerant and Cd-tolerant plant material were due to the prevailing extra cellular ion content when collected from the field. In R. squarrosus, K pretreatment resulted in increased uptake capacities and cell-wall binding affinities for Cd in field-grown moss apices. Laboratory-grown apices, following K pretreatment, displayed increased uptake capacities but reduced affinities for Cd. Increased extra cellular Cd uptake in K-pretreated moss apices was interpreted as additional weak electrostatic binding. Kinetic analysis indicated that electrostatic binding by extra cellular sites was less influential than the available supply of wall-bounds ions on intracellular Cd uptake by moss apices.

The possible role of calcium in the intracellular uptake of cadmium by the lichen Peltigera membranacea was investigated. Even when calcium was removed from extracellular sites by potassium pretreatment, kinetic studies failed to provide evidence in support of added calcium being a competitive inhibitor of cadmium uptake. Calcium and cadmium uptake responded differently to the presence of lanthanum, vanadate or verapamil. Except for the effect of verapamil, intracellular of cadmium mostly reflected the quantity of cadmium bound to extracellular exchange sites.

The effect of desiccation stress on Cd2 + uptake by the lichen Peltigera was investigated. Results showed that even a severe desiccation stress does not damage the membranes or reduce the ability of Peltigera to take up Cd2 +.

Intra- and extracellular Cd uptake in the lichen genus Peltigera were investigated, and intracellular uptake found to display Michaelis-Menten kinetics. Compared with values reported for Zn uptake in free-living algae and fungi, Peltigera had low affinities for Cd and low maximum uptake rates. Intra-, and to a lesser extent extracellular uptake rates were temperature dependent. When lichens were incubated concurrently with Cd and equimolar concentrations of a range of other cations, most were found to reduce both extra- and intracellular Cd uptake, implying that the Cd uptake systems had low specificities. Mg, though not a strong competitor for extracellular Cd uptake, inhibited intracellular Cd uptake to a similar extent to borderline elements, and it is postulated that intracellular Cd uptake occurs by a system which normally transports Mg. Although concurrently-supplied cations reduced Cd-induced inhibitions of photosynthesis, the reductions were not proportional to the effect of the cations on intracellular Cd uptake. This indicated that other cations affected the toxicity of Cd to photosynthesis by some means in addition to reducing Cd uptake. Intracellular Cd uptake was light-stimulated, suggesting that a close relationship existed between metal uptake and metabolism. The rate of intracellular Cd uptake in the dark was probably not directly linked to the supply of respirable reserves, as it was unaffected by prolonged storage in the dark, and was not increased by adding glucose. It is hypothesized that light-stimulated Cd uptake represents active entry into algal cells, but with uptake in the dark it is not clear which symbiont is involved, and whether energy is required.

SUMMARY The in vivo evolution of hydrogen by three nitrogen fixing lichens, Peltigera membranacea, Peltigera polydactyla and Lobaria pulmonaria has been studied. The hydrogen evolved concomitant with nitrogen fixation was recycled by means of an uptake hydrogenase and in general the net evolution was zero or small at 5 and 15 °C. At 25 °C there was appreciable net evolution of hydrogen in the Petigera spp. but Lobaria showed no net evolution. Direct comparison of acetylene reduction and nitrogen fixation using 15N2 gave ratios of six and four for Peltigera and Lobaria; these figures imply a significant production of hydrogen and indicate an effective mechanism for countering energy loss under normal in vivo conditions.

Thalli of Peltigera membranacea, Peltigera polydactyla and Lobaria pulmonaria were labelled with '5N and subjected to simulated episodes of heavy rainfall after periods of drying in a lighted air-conditioned incubator. Inorganic and total nitrogen was estimated in the eluates. The drying-wetting cycle was repeated three times. From the level of "5N labelling of the eluates, it was evident that recently fixed nitrogen was released, the inorganic fraction (NH4+) being up to three times more heavily labelled than the organic. The amounts of nitrogen lost in successive episodes generally diminished in logarithmic fashion, the level of labelling remaining approximately constant, implying little or no replenishment of mobile material from insoluble reserves. Up to three times the total nitrogen fixed per day could be lost in one rainfall episode. A preliminary trial showed that nitrogen losses from thalli under less rigorous regimes could be of nutritional benefit to the underlying moss substratum.

Summary Intra- and extra-cellular Cd uptake were investigated in members of the lichen genus Peltigera sampled from sites with contrasting metal status. Peltigera membranacea (Ach.) Nyl. from a disused Zn mine, previously shown to be tolerant to Zn-induced inhibitions of photosynthesis, displayed a reduced rate of Cd uptake into the cell compared with Peltigera spp. from unpolluted sites, suggesting metal exclusion was a tolerance mechanism. Although some differences in extracellular Cd uptake were found, these appeared to be a reflection of inter-specific variation rather than environmentally-induced alterations in Cd-binding capacity. Exposure to a mixture of Cd and Mg showed that intracellular Cd uptake was less inhibited by Mg when lichens originated from the Zn mine, also implying that mine-site plants had modified uptake systems. Although intracellular Cd uptake is normally light-stimulated in Peltigera, lichens from the Zn mine lacked this response, suggesting that algal uptake was reduced more than fungal uptake. Photosynthetically Zn-tolerant P. hymenina (Ach.) Delise from a disused Pb mine, more moderately polluted than the Zn mine, had intracellular Cd uptake rates comparable to those found in lichens from background localities. In addition, treatment of lichens for 1 week with low concentrations of Zn, previously shown to induce photosynthetic Zn and Cd tolerance, had no effect on intracellular Cd uptake. Tolerance in these instances must have been achieved by some other means. It is suggested that a severe metal stress may be required before metal tolerance by exclusion occurs.

SUMMARY Thalli of Peltigera membranacea, Peltigera polydactyla and Lobaria pulmonaria were labelled with 15N and subjected to simulated episodes of heavy rainfall after periods of drying in a lighted air-conditioned incubator. Inorganic and total nitrogen was estimated in the eluates. The drying-wetting cycle was repeated three times. From the level of 15N labelling of the eluates, it was evident that recently fixed nitrogen was released, the inorganic fraction (NH4+) being up to three times more heavily labelled than the organic. The amounts of nitrogen lost in successive episodes generally diminished in logarithmic fashion, the level of labelling remaining approximately constant, implying little or no replenishment of mobile material from insoluble reserves. Up to three times the total nitrogen fixed per day could be lost in one rainfall episode. A preliminary trial showed that nitrogen losses from thalli under less rigorous regimes could be of nutritional benefit to the underlying moss substratum.

Summary Phytase activity was investigated in 13 lichen species using a novel assay method. The work tested the hypothesis that phytase is a component of the suite of surface-bound lichen enzymes that hydrolyse simple organic forms of phosphorus (P) and nitrogen (N) deposited onto the thallus surface. Hydrolysis of inositol hexaphosphate (InsP6, the substrate for phytase) and appearance of lower-order inositol phosphates (InsP5–InsP1), the hydrolysis products, were measured by ion chromatography. Phytase activity in Evernia prunastri was compared among locations with contrasting rates of N deposition. Phytase activity was readily measurable in epiphytic lichens (e.g. 11.3 μmol InsP6 hydrolysed g−1 h−1 in Bryoria fuscescens) but low in two terricolous species tested (Cladonia portentosa and Peltigera membranacea). Phytase and phosphomonoesterase activities were positively correlated amongst species. In E. prunastri both enzyme activities were promoted by N enrichment and phytase activity was readily released into thallus washings. InsP6 was not detected in tree canopy throughfall but was present in pollen leachate. Capacity to hydrolyse InsP6 appears widespread amongst lichens potentially promoting P capture from atmospheric deposits and plant leachates, and P cycling in forest canopies. The enzyme assay used here might find wider application in studies on plant root–fungal–soil systems.