The effect of single and combined pollutants on lichen water content

The effects of lead, sodium chloride and sulphur dioxide, single and combined, on water content ofHypogymnia physodes (L.)Nyl. andXanthoria parietina (L.) Th. Fr. were studied. Lead, sodium chloride and their combinations, reduced water content in lichens, this reduction being partially correlated with the decrease of pollutant-influenced photosynthesis.     

Do lichens have “memory” of their native nitrogen environment?

This study aimed to deepen the knowledge about intraspecific mechanisms regulating nitrogen tolerance in lichens to wet nitrogen deposition. Thalli of the nitrophilous lichen Xanthoria parietina were collected from environments with different nitrogen availabilities and immersed in 80 mL of ammonium sulphate (NH₄)₂SO₄ solutions with distinct concentrations (0, 0.025, 0.05 and 0.25 M) for 5 h per day during 3 days in a week. After each soaking event, lichens were air dried. After each treatment, maximal PSII efficiency, localization of ammonium ions, concentrations of K⁺ and Mg²⁺ and thalli buffer capacity were determined. Our results show that lichens are marked by their native nitrogen environment, since there were important differences between the physiological responses of X. parietina thalli previously grown in an area with high nitrogen deposition (nitrogen emissions of ca. 13,000 t/year) and those previously grown in an unpolluted area (nitrogen emissions of ca. 500 t/year). Greater N availability seems to enable X. parietina to cope better with the effects of nitrogen pollution.     

Host use of a specialist lichen-feeder: dealing with lichen secondary metabolites

Host use by herbivores is largely determined by host properties such as nutrient content and chemical defence against foragers. The impacts of these attributes on a herbivore may largely depend on its life cycle stage. Lichen species are known to differ in nutritional quality and level of chemical defence and, consequently, vary as fodder for herbivores. The aim of this study was to explore the impact of several lichen species and the presence of their secondary metabolites on their use as hosts by a specialist lichen-feeder, Cleorodes lichenaria. This study also addressed, for the first time, how a specialist lichen-feeder deals with different lichen secondary metabolites. In the beginning of their development, larvae grew better on Xanthoria parietina than on the other host lichens, whereas older larvae grew best on Ramalina fraxinea. Lichen secondary chemicals in R. fraxinea and Parmelia sulcata hindered larval growth in the beginning but after 75 days lichen secondary chemicals had no impact on the mass of larvae. Physodic acids in Hypogymnia physodes were lethal to larvae. In general, larvae metabolized 70–95% of ingested lichen secondary chemicals and the rest of these were excreted in frass. Lichen secondary metabolites in P. sulcata restrict and in H. physodes prevent their use as a host for C. lichenaria larvae. Our main finding, the ability of larvae to metabolize several lichen secondary metabolites, indicates digestive adaptation to these chemicals. No signs of sequestration of these chemicals were found.     

Chromatic adaptation in lichen phyco- and photobionts

The effect of light quality on the photosynthetic pigments as chromatic adaptation in 8 species of lichens were examined. The chlorophylls, carotenoids in 5 species with green algae as phycobionts (Cladonia mitis, Hypogymnia physodes, H. tubulosa var. tubulosa and subtilis, Flavoparmelia caperata, Xanthoria parietina) and the chlorophyll a, carotenoids and phycobiliprotein pigments in 3 species with cyanobacteria as photobionts (Peltigera canina, P. polydactyla, P. rufescens) were determined. The total content of photosynthetic pigments was calculated according to the formule and particular pigments were determined by means CC, TLC, HPLC and IEC chromatography. The total content of the photosynthetic pigments (chlorophylls, carotenoids) in the thalli was highest in red light (genus Peltigera), yellow light (Xanthoria parietina), green light (Cladonia mitis) and at blue light (Flavoparmelia caperata and both species of Hypogymnia). The biggest content of the biliprotein pigments at red and blue lights was observed. The concentration of C-phycocyanin increased at red light, whereas C-phycoerythrin at green light.     

Iron and phosphate uptake in epiphytic and saxicolous lichens differing in their pH requirements

The hypothesis is tested that pH-dependent Fe and P uptake influence the preference of epiphytic and saxicolous lichens for certain ranges of ambient pH. Five species from acidic substrata (Hypogymnia physodes, Parmeliopsis ambigua, and Platismatia glauca) or covering the range from weakly acidic to alkaline substrata (Lecanora muralis and Phaeophyscia orbicularis) were exposed to solutions of FeCl₂, FeCl₃, or KH₂PO₄ at pH 3 and 8 in the laboratory. Avoidance of alkaline substrata is explainable by low Fe³⁺ uptake at pH 8 in the case of H. physodes and the inability for net P uptake and membrane damage in P. ambigua at this pH. Preference for acidic substrata in Pl. glauca, however, is neither related to Fe nor P uptake. Efficient Fe³⁺ and P uptake at pH 8 explains the tolerance of L. muralis and Ph. orbicularis to alkaline conditions. Intracellular accumulation of Fe²⁺ in probably toxic amounts at pH 3 in Ph. orbicularis is correlated with the absence of this lichen from strongly acidic substrata. Avoidance of acidic sites by L. muralis is not attributable to Fe or P uptake. In summary, the results suggest that pH-dependent Fe and P uptake characteristics are involved in the determination of pH preferences of epiphytic and saxicolous lichens, but are not the only relevant factor.     

Antimicrobial activity of extracts of the lichens <Emphasis Type="Italic">Cladonia furcata,Parmelia caperata,Parmelia pertusa,Hypogymnia physodes</Emphasis> and <Emphasis Type="Italic">Umbilicaria polyphylla</Emphasis>

Antimicrobial features of acetone, methanol and aqueous extracts of lichens of Cladonia furcata, Parmelia caperata, Parmelia pertusa, Hypogymnia physodes and Umbilicaria polyphylla were investigated by two different methods at the same time. Testing of antimicrobial activities of extracts from five species of lichens was performed by disc diffusion test in relation to Gram-positive and Gram-negative bacteria and fungal organisms, and through determination of minimal inhibitory concentration (MIC) by Broth Tube Dilution method. The obtained results indicated that acetone and methanol extracts of all investigated lichens in different concentrations manifested selective antibacterial and antifungal activity. That activity was more evident in relation to Gram-positive, than Gram-negative bacteria and fungal organisms. Acetone and methanol extracts of lichens Parmelia pertusa, Hypogymnia physodes and Umbilicaria polyphylla inhibited the growth of all tested microorganisms, most of all of lichens Cladonia furcata and Parmelia caperata. Although, the methanol extracts were generally the most active against the test organisms, the lowest MIC value was measured for acetone extract of species Cladonia furcata 0.39 mg/mL in relation to bacterium Bacillus subtilis. Aqueous extracts of investigated lichens were inactive against all tested organisms.     

Effects of manganese on chlorophyll fluorescence in epiphytic cyano- and chlorolichens

Incubation with 10 mM MnCl₂ for 1 h decreased the effective quantum yield of photochemical energy conversion in photosystem 2 in the bipartite chlorolichen Hypogymnia physodes as well as in the bipartite cyanolichens Leptogium saturninum and Nephroma helveticum, but not in the tripartite lichen Lobaria pulmonaria. Among the bipartite species, Mn sensitivity increased in the order H. physodes < N. helveticum < L. saturninum. This equals the sequence heteromerous chlorolichen < heteromerous cyanolichen < homoiomerous (gelatinous) cyanolichen. MnCl₂ reduced non-photochemical quenching of chlorophyll fluorescence in the bipartite cyanolichens and in H. physodes; in the latter, however, this decrease was limited to light intensities above the adapted growth light intensity. Photochemical quenching was increased in H. physodes, but reduced in the bipartite cyanolichens. The results indicate that the bipartite cyanolichens L. saturninum and N. helveticum are even more sensitive to high Mn concentrations than the chlorolichen H. physodes, the low Mn tolerance of which has been already demonstrated. This agrees with results of field studies from western North America, where conifer bark under cyanolichens (including L. saturninum and N. helveticum) was found to contain less Mn than bark which only supported chlorolichens. The high sensitivity of the bipartite cyanolichens probably results from high sensitivity of the Nostoc photobiont. The high Mn tolerance of L. pulmonaria is probably not due to its being a tripartite lichen, but might be caused by high tolerance of the green-algal primary photobiont Dictyochloropsis, which is, however, not experimentally proven. The high Mn tolerance of the highly SO₂-sensitive L. pulmonaria shows that different mechanisms are responsible for Mn and SO₂ toxicity in lichens.     

Chlorophyll a Fluorescence Emission,Xanthophyll Cycle Activity,and Net Photosynthetic Rate Responses to Ozone in Some Foliose and Fruticose Lichen Species

The lichens Parmelia quercina, Parmelia sulcata, Evernia prunastri, Hypogymnia physodes, and Anaptychia ciliaris were exposed to ozone (O₃) in controlled environment cuvettes designed to maintain the lichens at optimal physiological activity during exposure. Measurements of gas exchange, modulated chlorophyll (Chl) fluorescence, and pigment analysis were conducted before and after exposure to 300 mm³ (O₃) m^–3, 4 h per d for 14 d. No changes in the efficiency of photosystem 2 (PS2) photochemistry, the reduction state of Q_A, or the electron flow through PS2, measured by Chl fluorescence, were detected in any of the five lichen species studied. Additionally, neither photosynthetic CO₂ assimilation nor xanthophyll cycle activity or photosynthetic pigment concentration were affected by high O₃ concentrations. Thus the studied lichen species have significant capacities to withstand oxidative stresses induced by high concentration of O₃.     

Biosorption of Co<Superscript>2+</Superscript> ions by lichen <Emphasis Type="Italic">Hypogymnia physodes</Emphasis> from aqueous solutions

Cobalt is one of the possible contaminants originating from radioactive wastes or from metal mines and refineries. This paper describes sorption of cobalt by the foliose lichen Hypogymnia physodes from CoCl₂ solutions spiked with ⁶⁰Co²⁺ in laboratory experiments. Maximum uptake was reached within 1 hour; the biosorption after 24 hours is not pH-dependent within the range of pH 4–7, negligible at pH 2 and is not dependent on metabolic activity. The process can be described by the Freundlich adsorption isotherm with ln k = 2.77, 1/n = 0.22 and R ² = 0.94. Bivalent metal ions showed a concentration-dependent competitive effect on cobalt biosorption, decreasing in the order: Cu > Ni > Ca > Mg. Monovalent ions, such as K⁺ and Na⁺, showed only very weak competitive effect. Up to 98% of Co taken up by lichen can be removed by washing with 0.1 M NiCl₂ at 20°C. This means that only a small fraction of the cobalt is localized intracellularly. These results can be used for elucidating the behaviour of lichens as bioindicators of cobalt pollution in water systems, including the risk of cobalt leakage from lichen probes under the influence of rain, snow and atmospheric humidity.     

Nitrogen and carbon isotope variability in the green‐algal lichen Xanthoria parietina and their implications on mycobiont–photobiont interactions

Stable isotope patterns in lichens are known to vary largely, but effects of substrate on carbon and nitrogen stable isotope signatures of lichens were previously not investigated systematically. N and C contents and stable isotope (δ¹⁵N, δ¹³C) patterns have been measured in 92 lichen specimens of Xanthoria parietina from southern Bavaria growing on different substrates (bark and stone). Photobiont and mycobiont were isolated from selected populations and isotopically analyzed. Molecular investigations of the internal transcribed spacer of the nuclear ribosomal DNA (ITS nrDNA) region have been conducted on a subset of the specimens of X. parietina. Phylogenetic analysis showed no correlation between the symbionts X. parietina and Trebouxia decolorans and the substrate, isotope composition, or geographic origin. Instead specimens grown on organic substrate significantly differ in isotope values from those on minerogenic substrate. This study documents that the lichens growing on bark use additional or different N sources than the lichens growing on stone. δ¹⁵N variation of X. parietina apparently is controlled predominantly by the mass fraction of the mycobiont and its nitrogen isotope composition. In contrast with mycobionts, photobionts of X. parietina are much more ¹⁵N‐depleted and show less isotopic variability than mycobionts, probably indicating a mycobiont‐independent nitrogen acquisition by uptake of atmospheric ammonia.     

THE EFFECT OF AGRONOMIC PHOTOSYSTEM-II HERBICIDES ON LICHENS

The sensitivity ofHypogymnia physodes,Lobaria pulmonariaandPeltigera aphthosaH. physodesto six photosystem II herbicides and to DBMIB was tested in the laboratory by chlorophyll flouresence and oxygen-exchange measurements. in addition, experiments with freshly isolated photobiont cells fromH. physodesandL. pulmonariawere performed. Generally, the lichens were most sensitive to the urea herbicides diuron and isoproturon, whereas the triazines atrazine, terbuthylazine, and simazine and the triazinone metamitron wre less inhibitory. Among the three lichen species invesigated,H. physodeswas the most sensitive to the urea herbicides. For the other agents, no signifiant differences between lichen species could be found. The highest pI₅₀values obtained from dose response curves were around 6.5 for isolated photobionts, but most values for lichen thalli were in the range 5-6. Thus, there is no particular sensitivity of green algal lichen photobionts to photosytem II herbicides as compared to other algae, higher plant chloroplasts or protoplasts. In nature, we observed recovery from (damaging) treatment with 10⁻⁵mol diuron 1⁻¹forH. physodeswithin weeks. Therefore, damage to lichens fromt he use of photosystem-II herbicides in agriculture is probably only of very local occurence.     

Reducing power and radical scavenging activity of four Parmeliaceae species

The methanol extracts of four Parmeliaceae lichens (Hypogymnia physodes, Evernia prunastri, Flavoparmelia caperata and Parmelia sulcata) were screened for antioxidant properties and total phenol content. The H. physodes extract was the most effective at reducing iron(III) and scavenging 1,1-diphenyl-2-picrylhydrazyl radicals, while the P. sulcata extract was the most effective in reducing molybdenum(VI) in an acidic medium. The E. prunastri and H. physodes extracts contained more Folin-Ciocalteu reagent reactive substances than the F. caperata and P. sulcata extracts. Significant activity of the H. physodes extract in DPPH and reducing Fe(III) assays suggest that this lichen can be considered as a potential source of antioxidants.     

Parietin,a photoprotective secondary product of the lichen Xanthoria parietina

Secondary lichen products can be extracted from air-dry thalli of Xanthoria parietina, Xanthoparmelia conspersa and Parmelina tiliacea with 100% acetone without affecting either short-or long-term viability. In Xanthoria parientina damage by acetone started to occur as water content reached the critical lower limit for photosystem II (PSII) activity. Extraction of the blue-light absorbing cortical pigment parietin increased the susceptibility of both air-dry and hydrated thalli to high light. Damage by high light levels caused a permanent reduction in F _v/F_m, quantum yield for photosynthetic O₂ production and photosynthetic capacity measured after a 2-day recovery period at low light levels (20 mol photons m^-2 s^-1). Parietin therefore protects the photosynthetic apparatus of Xanthoria parietina against damage by high light levels. Extraction of UV-absorbing pigments from Xanthoparmelia conspersa and Parmelina tiliacea did not increase photoinhibition after 24 h exposure to high light.     

Evidence for the functioning of photosynthetic CO2-concentrating mechanisms in lichens containing green algal and cyanobacterial photobionts

The photosynthetic properties of a range of lichens containing both green algal (11 species) and cyanobacterial (6 species) photobionts were examined with the aim of determining if there was clear evidence for the operation of a CO₂-concentrating mechanism (CCM) within the photobionts. Using a CO₂-gas-exchange system, which allowed resolution of fast transients, evidence was obtained for the existence of an inorganic carbon pool which accumulated in the light and was released in the dark. The pool was large (500–1000 nmol · mg Chl) in cyanobacterial lichens and about tenfold smaller in green algal lichens. In Hypogymnia physodes (L.) Nyl., which contains the green alga Trebouxia jamesii, a small inorganic carbon pool was rapidly formed in the light. Carbon dioxide was released from this pool into the gas phase upon darkening within about 20 s when photosynthesis was inhibited by the carbon-reduction-cycle inhibitor glycolaldehyde. In the absence of this inhibitor, release appeared to be obscured by carboxylation of ribulose bisphosphate. The kinetics of CO₂ uptake and release were monophasic. The operation of an active CCM could be distinguished from passive accumulation and release accompanying the reversible light-dependent alkalization of the stroma by the presence of saturation characteristics with respect to external CO₂. In Peltigera canina (L.) Willd., which contains the cyanobacterium Nostoc sp., a larger CO₂ pool was taken up over a longer period in the light and the release of this pool in the dark was slow, lasting 3–5 min. This pool also accumulated in the presence of glycolaldehyde, and under these conditions the CO₂ release was biphasic. In both species, photosynthesis at low CO₂ was inhibited by the carbonic-anhydrase inhibitor ethoxyzolamide (EZ). Inhibition could be reversed fully or to a considerable extent by high CO₂. In Peltigera, EZ decreased both the accumulation of the CO₂ pool by the CCM and the rate of photosynthesis. Free-living cultures of Nostoc sp. showed a similar effect of EZ on photosynthesis, although it was more dramatic than that seen with the lichen thalli. In contrast, in Hypogymnia, EZ actually increased the size of the CO₂ pool, although it inhibited photosynthesis. This effect was also seen when glycolaldehyde was present together with EZ. Surprisingly, EZ did not alter the kinetics of either CO₂ uptake or release. Taken together, the evidence indicates the operation in cyanobacterial lichens of a CCM which is capable of considerable elevation of internal CO₂ and is similar to that reported for free-living cyanobacteria. The CCM of green algal lichens accumulates much less CO₂ and is probably less effective than that which operates in cyanobacterial lichens.     

Proteins from the lichenXanthoria parietina which bind to phycobiont cell walls. Correlation between binding patterns and cell wall cytochemistry

Summary A protein fraction was isolated from the lichenXanthoria parietina which bound to the appropriate cultured phycobiont, but not to the freshly isolated symbiotic alga. The protein also appeared to discriminate between five other strains of cultured phycobionts from different lichens; phycobionts isolated from lichens in the familyTeloschistaceae bound the protein whereas phycobionts isolated from lichens in other families did not. Using cytochemical techniques, it was shown that protein binding ability was correlated with high levels of acidic polysaccharide in the cell wall, and the presence of a protein coat on the cell wall surface of the phycobiont. The possible role of this protein in recognition between lichen symbionts is briefly discussed.     

COMPARATIVE STUDIES ONXANTHORIA PARIETINA,A POLLUTION-RESISTANT LICHEN,ANDRAMALINA DURIAEI,A SENSITIVE SPECIES. II. EVALUATION OF POSSIBLE AIR POLLUTION-PROTECTION MECHANISMS

Surveys of the distribution of the lichensXanthoria parietinaandRamalina duriaeiin Israel showed that environments with air pollution had no damaging effects onX. parietina, whereasR. duriaeihad disappeared from polluted environments: physiological studies supported this relative sensitivity. Investigations of possible defence mechanisms protectingX. parietinafrom the damaging effects of air pollution showed a multitude of possible protective systems. These included constitutive avoidance such as: efficient buffering capacity; a relatively high potassium content; and antioxidation by parietin, and induced tolerance such as: SO₂oxidation to non-toxic sulphate; increased glutathione content; induced proline and arginine synthesis; and increased detoxification of active oxygen forms.     

Nitrogen uptake in relation to excess supply and its effects on the lichens<Emphasis Type="Italic"> Evernia prunastri</Emphasis> (L.) Ach and<Emphasis Type="Italic"> Xanthoria parietina</Emphasis> (L.) Th. Fr.

The aim of this study was to compare the physiological responses to increased nitrogen (N) supply between the nitrophytic lichen Xanthoria parietina (L.) Th. Fr. and the acidophytic lichen Evernia prunastri (L.) Ach. The two lichens were exposed to a weekly dosage of 0.05, 0.1, 0.2, 0.6 or 2.4 g N m^–2 for 2 months, administered as NH₄NO₃ dissolved in artificial rainwater (1 l m^–2). After the treatments, in vivo chlorophyll a fluorescence was determined to assess vitality; concentrations of total N, ammonium, nitrate and dominant amino acids, including glutamate, glutamine and arginine, were quantified in order to follow changes in N status; and the polyols ribitol, arabitol and mannitol were quantified to follow changes in the lichens carbon (C) status. The uptake of N was quantified by labelling the fertiliser with ¹⁵N in the ammonium position; chlorophyll a was used as an indirect marker for algal activity, and ergosterol as an indirect marker of fungal activity. Nitrogen uptake was higher in E. prunastri than in X. parietina, although the latter species may have used the mannitol reserves to obtain C skeletons and energy for N assimilation. Chlorophyll a and ergosterol concentrations remained unaltered in X. parietina irrespective of N dosage while ergosterol decreased with increasing N uptake in E. prunastri. The latter species had accumulated a large pool of ammonium at the highest N dosage, whilst in X. parietina a significant nitrate pool was instead observed. Taken together, these short-term responses to high N supply observed in the two lichens, and the differences between them, can partly explain the higher tolerance of X. parietina towards increased atmospheric N levels.     

The significance of stemflow chemistry for epiphytic lichen diversity in a dieback-affected spruce forest on Mt Brocken,northern Germany

Epiphytic lichen diversity in a boggy stand of Norway spruce (Picea abies) was studied in the eastern Harz Mountains, northern Germany. Spruce trees at wet sites were affected by forest dieback, whereas trees on drier sites remained unaffected. Lichen diversity was higher on dieback-affected trees than on healthy ones. The foliose lichen Hypogymnia physodes was significantly more frequent on dead trees, whereas the crustose, extremely toxitolerant Lecanora conizaeoides occurred more frequently on healthy trees. Stemflow concentrations of NH₄⁺, NO₃⁻, PO₃⁻, and SO₄²⁻ were lower on affected trees. This is attributed to reduced interception from the atmosphere due to needle loss. Cover of H. physodes decreased with increasing mean SO₄²⁻ concentration in stemflow. The total of lichen species per sample tree also decreased with increasing SO₄²⁻ concentration in stemflow, indicating that most species reacted in a similar way as H. physodes. Cover of L. conizaeoides increased with increasing SO₄²⁻ concentration, but decreased at higher SO₄²⁻ concentrations. Bark chemistry had a minor influence on lichen diversity.     

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.     

Characterization of two novel non-reducing polyketide synthase genes from the lichen-forming fungus Hypogymnia physodes

Lichens are known to produce a variety of secondary metabolites including polyketides, which have valuable biological activities. Some polyketides are produced solely by lichens. The biosynthesis of these compounds is primarily governed by iterative type I polyketide synthases. Hypogymnia physodes synthesize polyketides such as physodic, physodalic and hydroxyphysodic acid and atranorin, which are non-reducing polyketides. Two novel non-reducing polyketide synthase (PKS) genes were isolated from a fosmid genomic library of a mycobiont of H. physodes using a 409bp fragment corresponding to part of the reductase (R) domain as a probe. H. physodes PKS1 (Hyopks1) and PKS2 (Hypopks2) contain keto synthase (KS), acyl transferase (AT), acyl carrier protein (ACP), methyl transferase (ME) and R domains. Classification based on phylogeny analysis using the translated KS and AT domains demonstrated that Hypopks1 and Hypopks2 are members of the fungal non-reducing PKSs clade III. This is the first report of non-reducing PKSs containing the R domain-mediated release mechanisms in lichens, which are also rare fungal type I PKS in non-lichenized filamentous fungi.