Endogenous NO Is Involved in Dissimilar Responses to Rehydration and Pb(NO3)2 in Ramalina farinacea Thalli and Its Isolated Phycobionts.

Lichens undergo desiccation/rehydration cycles and are permeable to heavy metals, which induce free radicals. Nitrogen monoxide (NO) regulates important cellular functions, but the research on lichen NO is still very scarce. In Ramalina farinacea thalli, NO seems to be involved in the peroxidative damage caused by air pollution, antioxidant defence and regulation of lipid peroxidation and photosynthesis. Our hypothesis is that NO also has a critical role during the rehydration and in the responses to lead of its isolated phycobionts (Trebouxia sp. TR9 and Trebouxia jamesii). Therefore, we studied the intracellular reactive oxygen species (ROS) production, lipid peroxidation and chlorophyll autofluorescence during rehydration of thalli and isolated microalgae in the presence of a NO scavenger and Pb(NO3)2. During rehydration, NO scavenging modulates free radical release and chlorophyll autofluorescence but not lipid peroxidation in both thalli and phycobionts. Pb(NO3)2 reduced free radical release (hormetic effect) both in the whole thallus and in microalgae. However, only in TR9, the ROS production, chlorophyll autofluorescence and lipid peroxidation were dependent on NO. In conclusion, Pb hormetic effect seems to depend on NO solely in TR9, while is doubtful for T. jamesii and the whole thalli.

Effects of wood distillate (pyroligneous acid) on sensitive bioindicators (lichen and moss).

Wood distillate (pyroligneous acid) can be successfully applied in agriculture to increase crop quality and productivity with a lower risk for the environment respect to synthetic chemical herbicides, pesticides or fertilizers. However, the effects of wood distillate on the environment and biota are still under investigation, depending on biological attributes of potentially influenced organisms. The potential toxicological effects of wood distillate on sensitive non-target organisms, lichens and mosses, are studied for the first time. The physiological parameters (chlorophyll a fluorescence emission FV/FM and PI(ABS), chlorophyll content, spectral reflectance, antioxidant power, and dehydrogenase activity) and eventual bioaccumulation of selected elements (As, Ba, Cd, Cr, Cu, Fe, Ni, Pb, Zn) were investigated in the lichen Xanthoria parietina and the moss Hypnum cupressiforme after short-term treatments over a range of wood distillate solutions (1:300, 1:500, 1:700) to detect potential early stress responses. Overall, the lichen did not show changes after the treatments, while in the moss wood distillate caused only modest alterations in FV/FM and PI(ABS) and progressive increasing of antioxidant activity according to the dose supplied. The bioaccumulation of toxic elements was low and did not show any pattern of uptake with increasing concentrations of wood distillate.

Nitric oxide affects cadmium-induced changes in the lichen Ramalina farinacea.

Metabolic responses of epiphytic lichen Ramalina farinacea to cadmium (Cd) and/or nitric oxide (NO) scavenger (cPTIO) were studied. Accumulation of Cd and other metallic nutrients was not affected by cPTIO while total and absorbed amounts differed. Cd-induced NO formation was suppressed by cPTIO but ROS signal was synergistically enhanced, confirming that NO is essential to keep ROS under control. This excessive ROS generation could be a reason for depleted amount of all fatty acids, including SFAs, MUFAs and PUFAs. Total content of fatty acids reached 3.89 mg/g DW in control with linoleic (40%), palmitic (24%), oleic (12.8%) and stearic (8%) acids as major compounds: interestingly, shift in relative ratio of saturated (from 40 to 35% of total FAs) versus polyunsaturated fatty acids (from 42 to 48% of total FAs) was observed. Glutathione was suppressed by all treatments but Krebs acids were almost unaffected by cPTIO, indicating no regulatory role of NO in their accumulation. On the contrary, Cd-induced elevation in NO signal was related to increase in ascorbate and proline content while cPTIO suppressed it, indicating a tight relation between NO and these metabolites. Data are compared also with algae and vascular plants to show similarities between various life lineages.

Integrity of lichen cell membranes as an indicator of heavy-metal pollution levels in soil.

The epigeic lichens Cladonia rei and Diploschistes muscorum are effective heavy-metal-tolerant colonisers of highly polluted and disturbed sites. In this study we compare their bioaccumulation capacities, accumulation patterns, and responses to heavy-metal stress, as expressed in terms of cell membrane damage. We also aim at verifying the relationships between cell membrane damage and levels of soil pollution with heavy metals, and thereby to identify the bioindicative value of this physiological parameter. Total and intracellular concentrations of Zn, Pb, Cd, As, Cu, and Ni were measured in 140 samples of lichens and corresponding soil, collected from variously contaminated sites. Relative electrical conductivity (EC%) values were determined concurrently in the lichen samples. The studied lichens differ considerably in intracellular uptake susceptibility and the related reduction in membrane integrity. In C. rei thalli, more than half of Zn, Pb, Cd, and As loads are accumulated extracellularly, whereas D. muscorum exhibits a tendency towards intracellular accumulation of the same elements. This property is clearly reflected in cell membrane damage, which is considerably greater in the latter species irrespective of study site. This indicates that intracellular heavy-metal accumulation affects the level of cell membrane damage. Two soil pollution classes were distinguished for both lichens based on element contents in host-substrate samples. The losses of cell membrane integrity in lichen thalli are related to these classes. EC% values above 16 in C. rei and above 20 in D. muscorum suggest elevated levels of heavy metals in the soil. Consequently, this physiological parameter can serve as an early warning indicator for detection of elevated metal concentrations in soil. The biomonitoring method proposed here involves common and widespread lichen species and can be widely applied in post-industrial areas.

Lichen as a Biomonitor for Vehicular Emission of Metals: A Risk Assessment of Lichen Consumption by the Sichuan Snub-Nosed Monkey (Rhinopithecus roxellana).

Two lichen species, Usnea aciculifera and Usnea luridorufa, were used as biomonitors for the deposition of traffic-related metals in China's Shennongjia National Nature Reserve. The suitability of the two lichen species for use as biomonitors was compared. The health threat to the Sichuan snub-nosed (aka golden) monkey (Rhinopithecus roxellana) from consuming lichen with elevated metal concentrations due to vehicular traffic was then assessed. Lichens, with large surface areas and neither roots nor stomata, efficiently absorb both particulate and gaseous air pollutants. The resulting data was used to assess the effect of heavy metal accumulation on the lichens as well as the health risk imposed on the monkeys as lichen is a primary food source. Lichen samples were collected in the core area of the reserve at three locations of varying traffic intensity. A forth site in the reserve, with no proximate traffic, was used as the control. Results show: (1) lichen from high traffic sites has significantly higher concentrations of Fe, Cd, Pb Zn, and Cr than lichen collected from the control site; (2) vehicular traffic is the primary source of metals in lichen; (3) U. luridorufa collected at high traffic sites displayed decreased photosynthetic efficiency, an indication of stress; (4) intake of Cd and Pb from vehicle emissions in the Shennongjia National Nature Reserve could adversely affect snub-nosed monkey health. This research advances the science of biomonitoring, contributes to environmental protection efforts in China's nature reserves and helps improve food safety for Sichuan snub-nosed monkey, a national treasure of China.

Decolorization and detoxification of dye mixture and textile effluent by lichen Dermatocarpon vellereceum in fixed bed upflow bioreactor with subsequent oxidative stress study.

Navy Blue HE22 (NBHE22), dye mixture and real textile effluent were decolorized and degraded by lichen Dermatocarpon vellereceum. Up-flow bioreactor showed about 80%, 70%, 80% and 65% removal of American dye manufacturer index (ADMI), biological oxygen demand (BOD), total suspended solids (TSS) and total dissolved solids (TDS), respectively of dye mixture at flow rate of 25mlh-1. The removal of ADMI, BOD, TSS and TDS of real textile effluent were 75%, 65%, 82% and 70%, respectively at flow rate of 30mlh-1. Significant induction of extracellular enzymes such as manganese peroxidase and lignin peroxidase was observed up to 46% and 36% during decolorization of dye mixture, while 43% and 24% during effluent treatment, respectively. Exponential enhancement in the activities of stress enzymes such as catalase (CAT) and guaiacol peroxidase (GPX) was observed after exposure to NBHE22 (116% and 125%, respectively), dye mixture (150% and 300%, respectively) and effluent (400% and 350%, respectively) endorsing the stress tolerance ability of model lichen. Phytotoxicity and genotoxicity studies demonstrated less toxic nature of metabolites resulted from biodegradation.

Toxicity of Diclofenac in the Fern Azolla filiculoides and the Lichen Xanthoria parietina.

This study investigated the occurrence of toxicity, expressed as damage to the photosynthetic apparatus, in the aquatic fern Azolla filiculoides and the lichen Xanthoria parietina following treatments with diclofenac at different concentrations (0.1, 1, 10 and 100 mg/L) and different exposure times (24, 48, 72 and 240 h). Measurements of photosynthetic efficiency, chlorophyll content and chlorophyll degradation indicated dose- and time-dependent toxicity, since significant differences with control samples as well as among treatments, emerged mainly for the highest concentration (100 mg/L) and the longest time (240 h). In addition, also the mycobiont of the lichen X. parietina showed similar toxic effects, expressed as ergosterol content. The absence of relevant alterations at the lowest concentration (0.1 mg/L) suggested a very limited susceptibility of these species to environmentally relevant levels of this pharmaceutical.

Biological nitrogen fixation by alternative nitrogenases in boreal cyanolichens: importance of molybdenum availability and implications for current biological nitrogen fixation estimates.

Cryptogamic species and their associated cyanobacteria have attracted the attention of biogeochemists because of their critical roles in the nitrogen cycle through symbiotic and asymbiotic biological fixation of nitrogen (BNF). BNF is mediated by the nitrogenase enzyme, which, in its most common form, requires molybdenum at its active site. Molybdenum has been reported as a limiting nutrient for BNF in many ecosystems, including tropical and temperate forests. Recent studies have suggested that alternative nitrogenases, which use vanadium or iron in place of molybdenum at their active site, might play a more prominent role in natural ecosystems than previously recognized. Here, we studied the occurrence of vanadium, the role of molybdenum availability on vanadium acquisition and the contribution of alternative nitrogenases to BNF in the ubiquitous cyanolichen Peltigera aphthosa s.l. We confirmed the use of the alternative vanadium-based nitrogenase in the Nostoc cyanobiont of these lichens and its substantial contribution to BNF in this organism. We also showed that the acquisition of vanadium is strongly regulated by the abundance of molybdenum. These findings show that alternative nitrogenase can no longer be neglected in natural ecosystems, particularly in molybdenum-limited habitats.

Mapping Exposure to Multi-Pollutants Using Environmental Biomonitors-A Multi-Exposure Index.

Atmosphere is a major pathway for transport and deposition of pollutants in the environment. In industrial areas, organic compounds are released or formed as by-products, such as polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F's). Inorganic chemical elements, including lead and arsenic, are also part of the pollutants mixture, and even in low concentrations may potentially be toxic and carcinogenic. However, assessing the spatial pattern of their deposition is difficult due to high spatial and temporal heterogeneity. Lichens have been used as biomonitors of atmospheric deposition, because these organisms encompass greater spatial detail than air monitoring stations and provide an integration of overall pollution. Based upon the ability of lichens to concentrate pollutants such as PCDD/F and chemical elements, the main objectives of this study were to develop a new semi-quantitative multi-pollutant toxicity exposure index (TEQ-like), derived from risk estimates, in an attempt to correlate several atmospheric pollutants to human exposure levels. The actual pollutant concentrations were measured in the environment, from biomonitors (organisms that integrate multi-pollutants), enabling interpolation and mapping of contaminant deposition within the region. Thus, the TEQ-like index provides a spatial representation not from absolute accumulation of the different pollutants, but from the accumulation weighted by their relative risk. The assessment of environmental human exposure to multi-pollutants through atmospheric deposition may be applied to industries to improve mitigation processes or to health stakeholders to target populations for a comprehensive risk assessment, epidemiological studies, and health recommendations.

Short-term influence of Cu, Zn, Ni and Cd excess on metabolism, ultrastructure and distribution of elements in lichen Xanthoria parietina (L.) Th. Fr.

Lichens are symbiotic organisms that are very sensitive to heavy metal pollution. However, there is little evidence of how heavy metal pollution affects the physiological status, ultrastructural changes and distribution of elements in the layers of lichen thalli. For this purpose we simulated metal pollution to lichens and studied its impact on Xanthoria parietina. Thalli were treated with the heavy metals Cu, Zn, Ni, Cd in the form of sulfates at concentrations of 100µM and 500µM during 24, 48 and 72h. Untreated lichens served as controls. We assessed the status of physiological parameters (fluorescence and integrity of chlorophyll a, content of soluble proteins and ergosterol), ultrastructural changes, especially to the photobiont, and the distribution of elements in the layers of thalli in relation to treatment with heavy metals. We found positive correlations between the content of all tested heavy metals and the physiological response. We assessed the toxicity of the selected metals as follows: Cd >= Cu >= Ni > Zn, based on the effects on the photobiont layer in the lichen thallus and physiological measurements.

Lichen microalgae are sensitive to environmental concentrations of atrazine.

The identification of new organisms for environmental toxicology bioassays is currently a priority, since these tools are strongly limited by the ecological relevance of taxa used to study global change. Lichens are sensitive bioindicators of air quality and their microalgae are an untapped source for new low-cost miniaturized bioassays with ecological importance. In order to increase the availability of a wider range of taxa for bioassays, the sensitivity of two symbiotic lichen microalgae, Asterochloris erici and Trebouxia sp. TR9, to atrazine was evaluated. To achieve this goal, axenic cultures of these phycobionts in suspension were exposed to a range of environmental concentrations of the herbicide atrazine, a common water pollutant. Optical density and chlorophyll autofluorescence were used as endpoints of ecotoxicity and ecophysiology on cell suspensions. Results show that lichen microalgae show high sensitivity to very low doses of atrazine, being higher in Asterochloris erici than in Trebouxia sp. TR9. We conclude that environmental concentrations of atrazine could modify population dynamics probably through a shift in reproduction strategies of these organisms. This seminal work is a breakthrough in the use of lichen microalgae in the assessment of micropollution effects on biodiversity.

Lichen rehydration in heavy metal-polluted environments: Pb modulates the oxidative response of both Ramalina farinacea thalli and its isolated microalgae.

Lichens are adapted to desiccation/rehydration and accumulate heavy metals, which induce ROS especially from the photobiont photosynthetic pigments. Although their mechanisms of abiotic stress tolerance are still to be unravelled, they seem related to symbionts' reciprocal upregulation of antioxidant systems. With the aim to study the effect of Pb on oxidative status during rehydration, the kinetics of intracellular ROS, lipid peroxidation and chlorophyll autofluorescence of whole Ramalina farinacea thalli and its isolated microalgae (Trebouxia TR1 and T. TR9) was recorded. A genetic characterization of the microalgae present in the thalli used was also carried out in order to assess possible correlations among the relative abundance of each phycobiont, their individual physiological responses and that of the entire thallus. Unexpectedly, Pb decreased ROS and lipid peroxidation in thalli and its phycobionts, associated with a lower chlorophyll autofluorescence. Each phycobiont showed a particular pattern, but the oxidative response of the thallus paralleled the TR1's, agreeing with the genetic identification of this strain as the predominant phycobiont. We conclude that: (1) the lichen oxidative behaviour seems to be modulated by the predominant phycobiont and (2) Pb evokes in R. farinacea and its phycobionts strong mechanisms to neutralize its own oxidant effects along with those of rehydration.

Cyanobacteria produce a high variety of hepatotoxic peptides in lichen symbiosis.

Lichens are symbiotic associations between fungi and photosynthetic algae or cyanobacteria. Microcystins are potent toxins that are responsible for the poisoning of both humans and animals. These toxins are mainly associated with aquatic cyanobacterial blooms, but here we show that the cyanobacterial symbionts of terrestrial lichens from all over the world commonly produce microcystins. We screened 803 lichen specimens from five different continents for cyanobacterial toxins by amplifying a part of the gene cluster encoding the enzyme complex responsible for microcystin production and detecting toxins directly from lichen thalli. We found either the biosynthetic genes for making microcystins or the toxin itself in 12% of all analyzed lichen specimens. A plethora of different microcystins was found with over 50 chemical variants, and many of the variants detected have only rarely been reported from free-living cyanobacteria. In addition, high amounts of nodularin, up to 60 µg g(-1), were detected from some lichen thalli. This microcystin analog and potent hepatotoxin has previously been known only from the aquatic bloom-forming genus Nodularia. Our results demonstrate that the production of cyanobacterial hepatotoxins in lichen symbiosis is a global phenomenon and occurs in many different lichen lineages. The very high genetic diversity of the mcyE gene and the chemical diversity of microcystins suggest that lichen symbioses may have been an important environment for diversification of these cyanobacteria.

Effects of Cu on the content of chlorophylls and secondary metabolites in the Cu-hyperaccumulator lichen Stereocaulon japonicum.

Understanding the relationship between Cu and Cu-hyperaccumulator lichens is important for their application in monitoring and assessing heavy metal pollution. We investigated the Cu-hyperaccumulator lichen Stereocaulon japonicum at several Cu-polluted and control sites in Japan, and found the lichen to be widely distributed. Its concentrations of Cu, chlorophylls, and secondary metabolites, chlorophyll-related indices, and absorption spectra were measured, and we observed negative effects of Cu on these concentrations and indices. For highly Cu-polluted samples (>100ppm dry weight), however, we found significant linear correlations between Cu and chlorophyll concentrations. This can be considered as the response of the photobiont in S. japonicum to Cu stress. In highly Cu-polluted samples the chlorophyll-related indices and concentration of total secondary metabolites were almost constant regardless of Cu concentration. This suggests that the increase in chlorophyll concentration with the increase in Cu concentration enhances photosynthetic productivity per unit biomass, which will allow the production of extra structure and energy for maintaining the chlorophyll-related indices under Cu stress. The relationship between the increase in chlorophyll concentration of S. japonicum and the decrease in secondary metabolite concentration of the lichen can be explained by considering the balance of carbohydrates in the lichen. We found that a spectral index A372-A394 can be a useful index of the concentrations of Cu and total secondary metabolites in S. japonicum. These findings show the adjustment of the content of chlorophylls and secondary metabolites in S. japonicum to Cu stress, and provide a better understanding of the relationship between Cu and the Cu-hyperaccumulator lichen.

Physiological changes of the lichen Parmotrema tinctorum as result of carbon nanotubes exposition.

Carbon nanotubes (CNT) is one of the more abundant nanomaterial produced in the world. Therefore, it is desirable to access its effects in all environment compartments, in order to mitigate environmental distress. This study aims to verify the potential use of lichens - classical atmospheric pollution indicators - as biomonitors of carbon nanotubes aerosols. To examine cause-effect relationships, preserving environmental microclimatic parameters, the lichen Parmotrema tinctorum (Nyl.) Hale was transplanted to open top chambers where aerosols of CNT were daily added. Physiological parameters such as cell viability, photosynthetic efficiency, cell permeability as well as nanoparticle internalization were assessed. Carbon nanotubes exposure led to reduction on the cell viability of P. tinctorum. The treatment with 100µg/mL of MWCNT-COOH resulted in intracellular ion leakage, probably due to changes in membrane permeability. No alterations on photosynthetic efficiency were detected. Carbon nanotubes entrapment and internalization into the lichen thallus were observed. Short term exposition of CNT produced measurable physiological changes in P. tinctorum lichen. This suggests the possibility of use of lichens as models to assess the environmental impact (air related) of engineered nanomaterials.

Uptake and toxicity of glyphosate in the lichen Xanthoria parietina (L.) Th. Fr.

This study investigated if treatment of the lichen Xanthoria parietina (L.) Th. Fr. with glyphosate caused uptake of this herbicide as well as physiological alterations. Samples were treated with Glifene SL®, a common commercial glyphosate-based herbicide, at the lowest recommended doses (3.6g/L) as well as with doses slightly higher than the highest suggested (36 g/L). The results clearly showed glyphosate uptake in X. parietina proportionally to the dose provided. Adverse physiological effects were evident on the photosynthetic apparatus (photosynthetic efficiency, chlorophyll a content, chlorophyll degradation) as well as on the fungal respiration rates and cell membrane integrity (ergosterol content, dehydrogenase activity) already after 24h from treatment, also at the low application dose. It is concluded that lichens are suitable organisms for monitoring unwanted biological effects from the application of glyphosate-based herbicides, as well as for detecting the accumulation of this compound in the biota, thus screening for its environmental fate.

Effects of acute NH3 air pollution on N-sensitive and N-tolerant lichen species.

Lichens are sensitive to the presence of ammonia (NH3) in the environment. However, in order to use them as reliable indicators in biomonitoring studies, it is necessary to establish unequivocally the occurrence of certain symptoms following the exposure to NH3 in the environment. In this paper, we simulated an episode of acute air pollution due to the release of NH3. The biological effects of acute air pollution by atmospheric NH3 have been investigated using N-sensitive (Flavoparmelia caperata) and N-tolerant (Xanthoria parietina) species. Lichen samples were exposed to ecologically relevant NH3 concentrations for 8 weeks, simulating three areas of impact: a control area (2 µg/m(3)), an area of intermediate impact (2-35 µg/m(3)) and an area of high impact (10-315 µg/m(3)), with a peak of pollution reached between the fourth and fifth week. Ammonia affected both the photobiont and the mycobiont in F. caperata, while in X. parietina only the photosynthetic performance of the photobiont was altered after exposure to the highest concentration. In the photobiont of F. caperata we recorded chlorophyll degradation as indicated by OD435/415 ratio, decrease of the photosynthetic performance (as reflected by the maximum quantum yield of primary photochemistry FV/FM and the performance index PIABS); in the mycobiont, ergosterol reduction, membrane lipid peroxidation (as reflected by the increase of thiobarbituric acid reactive substances), alteration (decrease) of the secondary metabolite usnic acid. No effects were detected on caperatic acid and dehydrogenase activity. In X. parietina, the only signal determined by NH3 was the alteration of FV/FM and the performance index PIABS. The results suggest that physiological parameters in N-sensitive lichens well reflect the effects of NH3 exposure and can be applied as early indicators in monitoring studies.

Application of molecular markers to detect DNA damage caused by environmental pollutants in lichen species.

Pseudevernia furfuracea L. (Zopf), Peltigera praetextata (Flörke ex Sommerf.) Zopf, Lobaria pulmonaria (L.) Hoffm., and Usnea longissima Ach. lichen species were used as bioindicators to assess the genotoxicity of air pollutants. In the present study, we examined significant environmetal pollutants and investigate how changes may lead to damage in DNA structure using RAPD markers. In the study area (Erzurum, Turkey), poor-quality lignite, which generates a large amount of sulfur dioxide, nitrogen oxides, and particle matter, is used for domestic heating, and vehicles also contribute to air pollution. Control lichen samples were collected far from large urban and industrial settlements and transplanted to four polluted sites for 4, 8, or 12 months. The total soluble protein content of the examined four lichen species did not significantly change with exposure time (P < 0.05). The four lichen samples exposed to the pollutants for 8 months had the highest ratio of DNA changes. The ratio of band differences in P. praetextata was higher than that in the other three lichen species, possibly because it has broad leaves that accumulated more pollutants. The average incidences of polymorphism were 64.14, 54.58, 65.76, and 43.06% for P. furfuracea, P. praetextata, L. pulmonaria, and U. longissima, respectively. The genomic template stability (GTS) significantly decreased following exposure to pollutants. GTS ratios revealed that the highest value (98.36%) belonged to U. longissima samples from Site 1 (10 m) after 4 months of exposure, and the lowest values belonged to P. praetextata (73.58%) from Site 3 (100 m) after 8 months of exposure. Based on our findings, we recommend the use of P. praetextata as an indicator of genotoxicity.

Spatial variation of eco-physiological parameters in the lichen Pseudevernia furfuracea transplanted in an area surrounding a cement plant (S Italy).

Thalli of the lichen Pseudevernia furfuracea were transplanted for 3 months (November 2010-January 2011) at 61 monitoring sites around a cement plant near Castrovillari (Calabria, southern Italy). NH3, NO x and SO2 concentrations were monitored monthly in a subarea of 10 sites (SA10) where the cement plant was located. At the end of the exposure period, the integrity of cell membranes; membrane lipid peroxidation (thiobarbituric acid reactive substances, TBARS level); vitality (cell respiration); chlorophyll a; chlorophyll b; carotenoids; phaeophytization quotient; photosynthetic efficiency and thalli concentrations of Al, Ca, Mg, V and Fe were measured. NO x concentrations correlated with the site distance from the cement plant while NH3 concentrations correlated with lichen vitality within SA10. For the monitoring area as a whole, only Fe and Mg concentrations correlated with membrane lipid peroxidation, while TBARS levels showed a significant increase and chlorophyll a, chlorophyll b and carotenoids a significant decrease with respect to the lichen origin area. Multivariate analysis (detrended correspondence analysis, cluster analysis and multi-response permutation procedure) of the eco-physiological parameters × monitoring sites data set resulted in four clusters termed C1, C2, C3 and C4. The eco-physiological parameters were compared among the four clusters and lichen origin area by one-way ANOVA. An index of environmental favourableness (IEF) to lichens was calculated to evaluate the spatial recovery of impaired values of TBARS, chlorophyll a, chlorophyll b, xanthophylls + carotenoids and phaeophytization quotient. The results indicate that there is no clear spatial trend in mycobiont impairment even though the IEF values suggest a higher number of sites with low levels of membrane lipid peroxidation in the 2--3-km distance band from the cement plant (the outermost) than in the two other distance bands (0-1 and 1-2 km). The photobiont seems to be damaged mainly in the inner distance band of the study area as suggested by the gradual but significant recovery trend of pigment levels and phaeophytization quotient from the inner distance band to the outer one (as shown by the IEF values). Conversion of chlorophyll to phaeophytin probably is not the only process affecting pigment levels.

Is vanadium a biometal for boreal cyanolichens?

Molybdenum (Mo) nitrogenase has long been considered the predominant isoenzyme responsible for dinitrogen fixation worldwide. Recent findings have challenged the paradigm of Mo hegemony, and highlighted the role of alternative nitrogenases, such as the vanadium-nitrogenase. Here, we first characterized homeostasis of vanadium (V) along with other metals in situ in the dinitrogen fixing cyanolichen Peltigera aphthosa. These lichens were sampled in natural sites exposed to various levels of atmospheric metal deposition. These results were compared with laboratory experiments where Anabaena variabilis, which is also hosting the V-nitrogenase, and a relatively close relative of the lichen cyanobiont Nostoc, was subjected to various levels of V. We report here that V is preferentially allocated to cephalodia, specialized structures where dinitrogen fixation occurs in tri-membered lichens. This specific allocation is biologically controlled and tightly regulated. Vanadium homeostasis in lichen cephalodia exposed to various V concentrations is comparable to the one observed in Anabaena variabilis and other dinitrogen fixing organisms using V-nitrogenase. Overall, our findings support current hypotheses that V could be a more important factor in mediating nitrogen input in high latitude ecosystems than previously recognized. They invite the reassessment of current theoretical models linking metal dynamics and dinitrogen fixation in boreal and subarctic ecosystems.