Cyanobiont specificity in some Nostoc-containing lichens and in a Peltigera aphthosa photosymbiodeme

The cyanobacterial symbionts in some Nostoc -containing lichens were investigated using the nucleotide sequence of the highly variable cyanobacterial tRNA^Leu (UAA) intron. When comparing different Nostoc -containing lichens, identical intron sequences were found in different samples of the same lichen species collected from two remote areas. This was true for all species where this comparison was made ( Peltigera aphthosa (L.) Willd., P. canina (L.) Willd. and Nephroma arcticum (L.) Torss.). With one exception, a specific intron sequence was never found in more than one lichen species. However, for two of the species, Peltigera aphthosa and Nephroma arcticum , two different cyanobionts were found in different samples. By examining a P. aphthosa photosymbiodeme it could be shown that the same Nostoc is present in both bipartite and tripartite lobes of this lichen. It is thus possible for one cyanobiont/ Nostoc to form the physiologically different symbioses that are found in bipartite and tripartite lichens. The connection between photobiont identity and secondary chemistry is discussed, as a correlation between differences in secondary chemistry and different cyanobionts/ Nostoc s in the species Peltigera neopolydactyla (Gyeln.) Gyeln. was observed. It is concluded that more knowledge concerning the photobiont will give us valuable information on many aspects of lichen biology.     

The Nostoc-Nephroma symbiosis: localization, distribution pattern and levels of key proteins involved in nitrogen and carbon metabolism of the cyanobiont

The qualitative distribution and quantitative estimates of nitrogenase (EC 1.7.99.2), glutamine synthetase (EC 6.3.1.2), phycoerythrin and ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) were studied in the cyanobacterium Nostoc residing in internal cephalodia of the tripartite lichen Nephroma arcticum L. Polyclonal antisera, raised in rabbit against the proteins, and goat anti-rabbit IgG conjugated to 10 nm gold were used as probes to detect the antigens by transmission electron microscopy. Western blot analyses demonstrated the monospecificity of the antisera. Nitrogenase was localized in heterocysts, with vegetative cells showing a label intensity comparable to the background. Distribution of the antigen within the heterocysts was uniform. Glutamine synthetase labelling was very low, but appeared to be distributed in both cell types. An intense phycoerythrin labelling was associated with the thylakoid region of the vegetative cells, whereas a much lower labelling was observed in the heterocyst. No significant differences were found between cyanobionts in younger and older cephalodia except for the nitrogenase labelling, which was higher in heterocysts of the cyanobiont in younger cephalodia. Most of the ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) label was present in vegetative cells. The Rubisco label was pronounced in the carboxysomes, whereas the label in the cytoplasm, on a unit area basis, was much lower. Heterocysts showed a label intensity similar to that of the vegetative cell cytoplasm. In Nostoc of the bipartite lichen Peltigera canina L., the Rubisco protein showed a comparable distribution pattern, but the average number of carboxysomes per vegetative cell was about 4 times higher.     

Photosynthetic CO2-use efficiency in lichens and their isolated photobionts: the possible role of a CO2-concentrating mechanism

The CO₂ dependence of net CO₂ assimilation was examined in a number of green algal and cyanobacterial lichens with the aim of screening for the algal/cyanobacterial CO₂-concentrating mechanism (CCM) in these symbiotic organisms. For the lichens Peltigera aphthosa (L.) Willd., P. canina (L.) Willd. and P. neopolydactyla (Gyeln.) Gyeln., the photosynthetic performance was also compared between intact thalli and their respective photobionts, the green alga Coccomyxa PA, isolated from Peltigera aphthosa and the cyanobacterium Nostoc PC, isolated from Peltigera canina. More direct evidence for the operation of a CCM was obtained by monitoring the effects of the carbonic-anhydrase inhibitors acetazolamide and ethoxyzolamide on the photosynthetic CO₂use efficiency of the photobionts. The results strongly indicate the operation of a CCM in all cyanobacterial lichens investigated and in cultured cells of Nostoc PC, similar to that described for free-living species of cyanobacteria. The green algal lichens were divided into two groups, one with a low and the other with a higher CO₂-use efficiency, indicative of the absence of a CCM in the former. The absence of a CCM in the low-affinity lichens was related to the photobiont, because free-living cells of Coccomyxa PA also apparently lacked a CCM. As a result of the postulated CCM, cyanobacterial Peltigera lichens have higher rates of net photosynthesis at normal CO₂ compared with Peltigera aphthosa. It is proposed that this increased photosynthetic capacity may result in a higher production potential, provided that photosynthesis is limited by CO₂ under natural conditions.     

Anti-oxidation activity of ethanol extracts from natural thalli of lichens

Screening test on anti-oxidation activity using 1,1-diphenyl-2-picrylhydrazyl(DPPH) was performed for 99 ethanol extracts of 85 species of natural thalli of lichens in order to find novel anti-oxidation compounds.The 17 extracts of natural thalli showed high anti-oxidation activity.Among them,the activities of extracts from Hypogymnia vittata,Peltigera aphthosa,Nephromopsis ornata,Pseudevernia furfuracea,Cladonia vulcani and Peltigera elizabethae were higher.Extracts of Peltigera spp.showed higher activity than those of other genera.The ethanol extract of P.aphthosa had been separated into ethyl acetate-soluble and water-soluble fractions.Two anti-oxidative spots were found only in the water-soluble fractions by thin-layer chromatography.The compound in the lower spot had the same Rf value,UV spectrum,and color as authentic solorinine that was previously found as a unique quaternary ammonium compound from Peltigera spp.We now report that the hydrophilic lichen substance,solorinine showed a nearly same anti-oxidation activity(EC50=120?mol/Lol/L) as standard antioxidant Trolox(EC50=150?mol/L).     

CYANOPHYTE CEPHALODIA IN THE LICHEN GENUS NEPHROMA

The lichens, Nephroma expallidum (Nyl.) Nyl. and N. arcticum (L.) Torss., consistently have at least two symbionts in a single thallus: a green alga in the algal layer and a blue-green alga in the internal cephalodia. The cephalodia originate from algal cells in contact with the lower surface of the lichen, in the zone of rhizine formation. The rhizines surround the epiphytic algal colony and form a second cortical layer; following dissociation of the original lower cortex, further growth of the two organisms results in the cyanophyte colony being enveloped by a compact layer of fungal tissue and positioned in the lichen medulla. The colony may eventually assume a superior or inferior position in relation to the lichen thallus, depending in part on the lichen species. Nephroma anticum may have two distinct morphological forms of blue-green algae in the same thallus and occasionally in the same cephalodium. It appears that the relationship that exists between the cephalodial algae and the lichen thallus is antagonistic and results, in some cases, in the exclusion of the green algal layer and death to the cephalodial cyanophytes.     

Preservation of secondary fungal metabolites in herbarium lichen specimens

Fresh picked and herbarium thalli of Cladonia stellaris, C. rangiferina, Allocetraria nivalis, A. cucullata, Cetraria islandica, Peltigera canina, and Nephroma articum epigene lichens were studied using the immune-enzyme analysis. No big difference was observed in the contents of mycotoxin secondary metabolites, i.e., deoxynivalenol, diacetoxyscirpenol, zearalenone, alternariol, citrinin, sterigmatocystin, cyclopiazonic acid, mycophenolic acid, emodin, and PR-toxin. The discovery of these substances in the specimens preserved for several decades shows that lichens have an effective system of conservation of metabolic exchange products.     

Three new genera of lichenicolous fungi

Three new genera of fungi occurring on lichens are described and illustrated. Clypeococcum D. Hawksworth (Pleosporales) is introduced for the species previously treated as Phaeodothis cladonema (Wedd.) Theiss. & Syd, which occurs in thalli of Cetrelia olivetorum sensu lato and Parmelia vermculifera. Doratomyces phillipsii , a species hitherto not recognized as lichenicolous, occurs on Steinia geophana and Thrombium epigaeum , both of which have Leptosira as the algal partner; this fungus is sufficiently different from other Doratomyces species to justify the new genus Leightoniomyces D. Hawksworth & Sutton (Dematiaceae). Refractohilum D. Hawksworth (Moniliaceae) is introduced for three species: Sporidesmium achromaticum Sutton (on Parmelia aff. sulcata and wood), R. galligenum sp. nov. (forming galls on Nephroma laevigatum) , and Ovularia peltigerae Keissl. (forming galls on Peltigera species).     

The species of Hemigrapha (lichenicolous Ascomycetes, Dothideales) on Peltigerales

The species of Hemigrapha (lichenicolous Ascomycetes, Dothideales) growing on Peltigerales are revised. H. atlantica sp. nov., on the cyanobacterial morph of Sticta canariensis from the Azores and the British Isles, H. nephromatis sp. nov., on Nephroma australe from Tasmania and New Zealand, and H. pseudocyphellariae sp. nov., on Pseudocyphellaria from Papua New Guinea are described and compared with the type species, H. asteriscus , which grows on species of Peltigera in the southern hemisphere and in Europe. Hemigrapha is shown to belong to the Microthyriaceae (Dothideales). Pycnothyrial anamorphs, morphologically identical to the thyriothecia, with hyaline, non-septate microconidia andor macroconidia, and conidiogenous cells arising from the upper conidiomatal wall, are known in H. asteriscus, H. atlantica and H. pseudocyphellariae. A key is given for the species of Hemigrapha on Peltigerales.     

Laccases produced by lichens of the order Peltigerales

'Large' and 'small' fractions of laccase were found in the thalli of lichens Solorina crocea and Peltigera aphthosa. In both lichens, 'large', possibly dimeric, laccases were determined as 175 and 165 kDa (based on the gel filtration data), and 'small' ones were 76 and 97 kDa (according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis data), respectively. By their substrate specificity, pH optima, and thermostability, they were typical laccases. The fractions of 'small' laccases of 45 kDa from S. crocea and 55 kDa from P. aphthosa consisted of two enzymes.     

Contribución al conocimiento de los líquenes epifíticos del Valle de Sanabria (Zamora,España)

A catalogue of epiphytic lichens from the Sanabria valley (Zamora) is presented in this study. A total of 145 taxa are reported. According to our data, 137 taxa are new records for Zamora Province. For each species, ecological and chorological remarks are included. Chaenotheca furfuracea, Chromatochlamys muscorum, Cladina arbuscula subsp. mitis, Degelia atlantica, Julella lactea, Mycobilimbia hypnorum, Nephroma parile, Pannaria ignobilis, Pannaria pezizoides, Peltigera britannica, P. hymenina, Pertusaria ophthalmiza, Rinodina archaea, R. conradii, Thelenella modesta and Xylographa abietina are specially considered, due to their chorological implications in the Iberian Peninsula. We highlight the area “arroyo de las Truchas”, in Cobreros, because of the particularly wide diversity of epiphytic lichens.     

Chitin-glucan complex in cell walls of the Peltigera aphthosa lichen

Cell walls and chitin-glucan complexes isolated from uneven-aged components of the thallus of the Peltigera aphthosa lichen were studied. The mass fraction of the cell wall and chitin-glucan complexes increased with age, but the content of nitrogen in these structures decreased with age. The basal area of the thallus was characterized by the largest mass fraction of the chitin-glucan complex from the dry mass of the thallus; the apical area, by the largest mass fraction of chitin in the complex. It was demonstrated that in P. aphthosa, the degree of deacetylation of chitin in the complex (depending on the age) was 33 and 54% in the apical and basal areas, respectively. The suggested method of functional analysis of chitin-glucan complexes for the presence of free amino groups in them can be used for studying other lichenified fungi.     

Characterization and identification of mycosporines-like compounds in cyanolichens. Isolation of mycosporine hydroxyglutamicol from Nephroma laevigatum Ach

Mycosporine-like compounds, comprising mycosporines and mycosporine-like amino acids (MAAs) are UV protecting secondary metabolites described in organisms such as fungi, algae, cyanobacteria or animals. Lichens however, were only poorly investigated for such constituents so far. Here, a method for the characterization of mycosporines and MAAs in purified aqueous extracts, involving HPTLC coupled to spectrophotodensitometry, HPLC-DAD-MS(n) and UPLC-HRMS analysis, is described. This optimized protocol was validated on three algae and one cyanolichen containing known MAAs and mycosporines, and then applied to 18 cyanolichen species. Analyses revealed the presence of five already described mycosporine-like compounds in the investigated species, including mycosporine serinol in Lichina and Peltigera species and mycosporine glutamicol in Degelia plumbea. Apart from that, eight unknown mycosporine-like compounds were detected and tentatively characterized on the basis of their DAD spectra and their MS(n) and HRMS data: two in the alga Porphyra dioica and six in cyanolichen species belonging to the genera Degelia, Nephroma and Stereocaulon. From Nephroma laevigatum, the mycosporine hydroxyglutamicol was preparatively isolated and identified through HRMS, 1D and 2D NMR spectroscopic data. The optimized analytical protocol allowed the characterization of mycosporine-like compounds in small amounts of material and confirmed the potential of cyanolichens as a source of mycosporine compounds. It should also be applicable to investigate lichen species with green algae photobionts for mycosporine-like compounds.     

Apothecia and ascospores of Lobaria oregana and Lobaria pulmonaria investigated

Apothecia of Lobaria oregana and L. pulmonaria emerge in late spring and discharge ascospores throughout the year. Most populations have a few fertile thalli, although the proportion of fertile thalli usually is less than 25 percent. Ascospores fail to germinate in water or on water agar but do germinate on agar containing an adsorbant and either a sugar or the sugar-alcohol ribitol. It is postulated that the ascospores of these species contain an autoinhibitor that must be removed before germination. Widespread ascospore germination in the presence of an adsorbant in Peltigera aphthosa, P. membranacea, and Pseudocyphellaria anthraspis, as well as in Lobaria, suggest that this phenomenon might be widespread in the Peltigerales.     

Nitrogenase in free-living and symbiotic cyanobacteria: Immunoelectron microscopic localization

Abstract Nitrogenase (Fe-protein) was localized in the free-living cyanobacterium Anabaena cylindrica and in the cyanobionts of Cycas revoluta and Peltigera aphthosa , using colloidal gold as an immunocytochemical marker. The Fe-protein was found to be evenly distributed throughout the heterocyst cytoplasma in A. cylindrica and in both the cyanobionts, including multiple heterocysts of the C. revoluta cyanobiont. No label was observed in the vegetative cells of free-living A. cylindrica or of the cyanobionts, although the cyanobionts apparently live under microaerobic conditions.     

Water-soluble phenolic compounds in lichens

The quantity and the qualitative composition (for some species) of phenolic compounds (PC) washed out of the intact thalli of lichens of the orders Peltigerales (the genera Peltigera, Solorina, and Nephroma) and Lecanorales (the genera Cladonia, Alectoria, and Cetraria) were studied. It was shown that the quantity of leachable PCs in Peltigerales was on average 2–3 times higher than in Lecanorales. At the same time, the extractability of PC from intact thalli by water was higher in Lecanorales than in Peltigerales: 48–88% and 34–70%, respectively, of the PC content in ethanol extracts from crushed thalli (i.e., of the total content of soluble PC). Water-soluble PC in the lichens Peltigera aphthosa, Solorina crocea, Cetraria islandica, Flavocetraria nivalis, Cladonia uncialis, and Cladonia arbuscula were represented by 7–12 phenolic compounds with similar qualitative composition in the species of the same order. The most part of water soluble PC were phenylpropanoids. All of the studied species showed the presence of p-hydroxybenzoic acid derivatives; vanillic and protocatechuic acid derivatives were found in Cetraria and Cladonia species, respectively.     

Spatial patterns of photobiont diversity in some Nostoc-containing lichens

Patterns of photobiont diversity were examined in some Nostoc -containing lichens using the nucleotide sequence of the cyanobacterial tRNA^Leu (UAA) intron. Lichen specimens collected in northwestern USA were analysed and the sequence data were compared with tRNA^Leu(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.     

Effect of acute gamma radiation on some physiological features of lichens

The influence of acute gamma radiation on four physiological characteristics of lichens was investigated. Membrane permeability in irradiated thalli of Cladonia arbuscula, Cetraria islandica and Hypogymnia physodes increased considerably compared to controls. The nitrogen content in Peltigera aphthosa appeared to be stable to acute gamma radiation. Up to 1000 Gy there was no essential decrease in respiration intensity in H. physodes and P. aphthosa or of peroxidase activity in P. aphthosa.     

Water Status of Green and Blue-green Phycobionts in Lichen Thalli after Hydration by Water Vapor Uptake: Do They Become Turgid?

It is known from previous investigations that dry lichens with green algae are able to recover net photosynthesis through rehydration with water vapor, whereas all blue-green lichens tested so far lack this ability. The REM micrographs of the present study show that the green phycobionts (Trebouxia spec.) of Ramalina maciformis become turgid only after water vapor uptake. In contrast, the blue-green phycobionts (Nostoc spec.) of Peltigera rufescens do not differ in appearance from the dry state, even when the thallus has reached equilibrium with the water vapor-saturated air; they require liquid water for turgidity. It is hypothesized that, after humidity hydration, water content is not sufficient for reestablishment of a functioning osmotic cell system in the blue-green phycobiont.     

犬地卷[Peltigera canina(L.)Willd.]叶状体的光合速率与温度和湿度的关系

利用CID-310便携式光合作用测定系统,测定了叶状体地衣犬地卷[Peltigera canina(L.)Willd.]的光合速率及其与气候要素的关系,结果表明叶状体地衣的生长与湿度(水分)关系密切。犬地卷在长期进化的过程中,每日在日出后湿度最高的时刻,积极地进行光合作用;一天内最高光合速率在上午9:00~10.00时之间(东经87°地方时间为准),其余时间处于休眠状态,进行光合作用时温度的变化范围不大。     

The Role of Calcium in Intracellular Cadmium Uptake by the Lichen Peltigera membranacea

The possible role of calcium in the intracellular uptake ofcadmium by the lichen Peltigera membranacea was investigated.Even when calcium was removed from extracellular sites by potassiumpretreatment, kinetic studies failed to provide evidence insupport of added calcium being a competitive inhibitor of cadmiumuptake. Calcium and cadmium uptake responded differently tothe presence of lanthanum, vanadate or verapamil. Except forthe effect of verapamil, intracellular of cadmium mostly reflectedthe quantity of cadmium bound to extracellular exchange sites.Copyright1993, 1999 Academic Press Peltigera membranacea (Ach.) Nyl., intracellular cadmium uptake, calcium uptake inhibitors, lichen