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[EN] The precise boundary delineations between taxa in symbiotic associations are very important for evolutionary and ecophysiological studies. Growing evidence indicates that in many cases, the use of either morphological characters or molecular markers results in diversity underestimation. In lichen symbioses. Trebouxia is the most common genus of lichen phycobionts, however, the diversity within this genus has been poorly studied and as such there is no clear species concept. This study constitutes a multifaceted approach incorporating aspects of ultrastructural characterization by TEM and phylogenomics to evaluate the morphological and genetic diversity of phycobionts within the sexually reproducing lichen Ramalina fraxinea in the context of Mediterranean and temperate populations. Results reveal an association with at least seven different Trebouxia lineages belonging to at least two species. T. decolorans and T. jamesii, and diverse combinations of such lineages coexisting within the same thallus depending on the analysed sample. Some of these lineages are shared by several other non-related lichen taxa. Our findings indicate the existence of a highly diverse assemblage of Trebouxia algae associating with R. fraxinea and suggest a possible incipient speciation within T. decolorans rendering a number of lineages or even actual species. This study stresses the importance of coordinated ultrastructural and molecular analyses to improve estimates of diversity and reveal the coexistence of more than one Trebouxia species within the same thallus. lt is also necessary to have clearer species delimitation criteria within the genus Trebouxia and microalgae in general., This study was funded by the Ministerio de Economia y Competitividad (MINECO CGL2012-40058-0O2-01/02), FEDER, the Generalitat Valenciana (PROMETEOII2013/021, GVA) and the Direccion General de Universidades e Investigacion de la Consejeria de Educacion de la Comunidad de Madrid - Universidad de Alcala (CCG10-UAH/GEN-5904). Drs. Arantxa Matins and Patricia Moya (Universitat de Valencia) made helpful comments on the manuscript.

Symbiosis is a close and often long-term interaction between two different biological organisms, i.e. plants or fungi and microorganisms. Two main types of plant–microorganism interactions, mutualistic and cooperative, have been categorized. Mutualistic interactions, including nitrogen-fixing and mycorrhizal symbioses, refer to mostly obligate relationships between a host plant and a symbiont microorganism. Cooperative interactions correspond to less obligate and specific relationships. They involve microorganisms, referred to as plant growth-promoting rhizobia (PGPR), able to colonize root surface or inner tissues. Lichens are symbiotic associations of host fungi and photosynthetic partners that may be Cyanobacteria or green algae. Increasing evidence has been reporting the presence of nitric oxide (NO) during symbiotic interactions. Most of the time, both the plant and the microorganism partners participate in NO production and catabolism. At early stage of the symbiosis, NO was shown to be potentially involved in the repression of plant defence reactions, favouring the establishment of the plant–microbe interaction. At later stages of the interactions, NO was shown to inhibit nitrogen fixation, but it was also demonstrated to have regulatory roles in nitrogen and carbon metabolisms, to play a beneficial metabolic function for the maintenance of the energy status under hypoxic conditions, to cross-react with hormone and reactive oxygen species pathways and to be potentially involved in the set-up of senescence processes. The present review provides an overview of NO production and many-faceted effects in symbiotic interactions and presents several tracks which appear to be particularly promising to decipher the roles of NO in plant–microbe symbioses.

The epiphytic lichen Ramalina farinacea is distributed throughout the northern hemisphere in which the same two algal Trebouxia species (provisionally named TR1 and TR9) coexist in every thallus. Ramalina farinacea symbionts were characterized based on the two fungal nuclear loci (nrITS and rpb2 ) along with the primary and secondary structures of nrITS from each Trebouxia species in the Iberian Peninsula and Canary Islands. The results indicated a noticeable genetic differentiation between mycobionts from these two geographic areas and also suggested concerted changes in the three partners of a lichen symbiosis toward two clearly distinguishable ‘holobiont’ lineages. Modeling of ITS2 RNA secondary structures suggested their temperature sensitivity in TR1 but not in TR9, which was consistent with the observed superior physiological performance of TR9 phycobionts under relatively high temperatures. Both TR1 and TR9 phycobionts have been also found in a variety of taxonomically distinct lichens with a preferably Mediterranean distribution, being TR1 much more widespread than TR9. Our observations support a model in which ecological diversification and speciation of lichen symbionts in different habitats could include a transient phase consisting of associations with more than one photobiont in individual thalli. Such diversification is likely to be promoted by different physiological backgrounds.

This study was funded by the Spanish Ministry of Education and Science (CGL2006-12917-C02-01/02), the Spanish Ministry of Science and Innovation (CGL2009-13429-C02-01/02), the AECID (PCI_A/024755/09) and the Generalitat Valenciana (PROMETEO 174/2008 GVA). We are grateful to Dr J. Gimeno-Romeu (University of California, Davis, USA) and to Dr P. J. G. de Nova (IREC, Ciudad Real, Spain), who were the first to isolate DNA from Ramalina farinacea thalli in our group. Wendy Ran revised the manuscript in English.

† Background and Aims Most lichens form associations with Trebouxia phycobionts and some of them simultaneously include genetically different algal lineages. In other symbiotic systems involving algae (e.g. reef corals), the relative abundances of different endosymbiotic algal clades may change over time. This process seems to provide a mechanism allowing the organism to respond to environmental stress. A similar mechanism may operate in lichens with more than one algal lineage, likewise protecting them against environmental stresses. Here, the physiological responses to oxidative stress of two distinct Trebouxia phycobionts (provisionally named TR1 and TR9) that coexist within the lichen Ramalina farinacea were analysed. † Methods Isolated phycobionts were exposed to oxidative stress through the reactive oxygen species propagator cumene hydroperoxide (CuHP). Photosynthetic pigments and proteins, photosynthesis (through modulated chlorophyll fluorescence), the antioxidant enzymes superoxide dismutase (SOD) and glutathione reductase (GR), and the stress-related protein HSP70 were analysed. † Key Results Photosynthetic performance was severely impaired by CuHP in phycobionts, as indicated by decreases in the maximal PSII photochemical efficiency (Fv/Fm), the quantum efficiency of PSII (FPSII) and the non-photochemical dissipation of energy (NPQ). However, the CuHP-dependent decay in photosynthesis was significantly more severe in TR1, which also showed a lower NPQ and a reduced ability to preserve chlorophyll a, carotenoids and D1 protein. Additionally, differences were observed in the capacities of the two phycobionts to modulate antioxidant activities and HPS70 levels when exposed to oxidative stress. In TR1, CuHP significantly diminished HSP70 and GR but did not change SOD activities. In contrast, in TR9 the levels of both antioxidant enzymes and those of HSP70 increased in response to CuHP. † Conclusions The better physiological performance of TR9 under oxidative conditions may reflect its greater capacity to undertake key metabolic adjustments, including increased non-photochemical quenching, higher antioxidant protection and the induction of repair mechanisms.

For decades, lichenologists have developed numerous and varied methods to isolate lichen photobionts. Most procedures are tedious, slow, and require several months after the initial isolation to obtain clones. Furthermore, the purity of the isolated photobionts obtained by more rapid methods is not sufficient to establish phycobiont axenic cultures. We have developed a new method for isolating lichen photobionts from fruticose, foliose and crustose lichens. Basically, it involves homogenization of lichen thalli (from 15 mg to 2 g), a one-step centrifugation through Percoll ®, followed by washing with Tween 20 and sonication. With this simple and rapid method (which takes less than 2 h), photobiont cells are obtained in sufficient quantity and purity to obtain dozens of axenic algal cultures.

We have developed a simple and fast procedure for the purification of PCR-quality DNA from cultured lichen photobionts. This new one-step method uses the solvent dimethyl sulfoxide (DMSO) combined with heat treatment to chemically breakdown algal and plant tissues. The DMSO-DNA extracts may be directly precipitated and purified by standard techniques in a total time of approximately 30 min. Compared to other DNA extraction protocols, the DMSO-based method suppresses the need for liquid nitrogen, any extraction buffer, grinding, phenol, and long incubation or centrifugation steps, thereby considerably reducing the possibility of contamination. In addition, minimal amounts of starting material (5 × 10 6 -20 × 10 6 cells from liquid or agarized cultures) produce sufficient DNA for 200 PCR reactions approximately, making this protocol a practical option for colony screening. This method works well in a wide range of cultured lichen photobionts and reduces the amount of labor-intensive steps and time consumed by other multi-step procedures, allowing for efficient processing of an increased number of samples.

To date, species identification of lichen photobionts has been performed principally on the basis of microscopic examinations and molecular data from nuclear-encoded genes. In plants, the chloroplast genome has been more readily exploited than the nuclear genome for systematic investigations. At the present time, very little information is available about the chloroplast genome of lichen-forming algae. For this reason, we have sequenced a portion of the gene encoding for the chloroplast large sub-unit rRNA (LSU rDNA) as a new molecular marker. Sequencing of the chloroplast LSU rDNAs revealed the existence of an unusual diversity of group I introns (a total of 31) within 15 analyzed Trebouxia species. The number, sequence and insertion site of these introns were very different among species, contributing to their recognition. A relatively large intron-free portion of the chloroplast LSU rDNA and part of the nuclear ribosomal cistron (18S-5.8S-26S) between the nuclear internal transcribed spacers (nrITS) were subjected to phylogenetic analyses. The obtained results indicate that data combination from both nuclear and chloroplast sequences can improve phylogenetic accuracy. Herein, we propose the suitability of both intronic and exonic sequences of the chloroplast LSU rDNA for species recognition, and an exonic sequence spanning from position 879 to 1837 in the Escherichia coli 23S rDNA for phylogenetic analyses of Trebouxia phycobionts.

Chloroplasts contain a plastoquinone-NADH-oxidoreductase (Ndh) complex involved in protection against stress and the maintenance of cyclic electron flow. Inactivation of the Ndh complex delays the development of leaf senescence symptoms. Chlorophyll a fluorescence measurements, blue native gel electrophoresis, immunodetection and other techniques were employed to study tobacco (Nicotiana tabacum) Ndh-defective mutants (DeltandhF). The DeltandhF mutants compared with wild-type plants presented: (i) higher photosystem II : photosystem I (PSII : PSI) ratios; (ii) similar or higher levels of ascorbate, carotenoids, thylakoid peroxidase and superoxide dismutase, yield (Phi(PSII)) and maximal photochemical efficiency of PSII levels (F(v)/F(m)) than wild-type plant leaves of the same age; (iii) lower values of nonphotochemical quenching yield (Phi(NPQ)), but not at very high light intensities or during induced leaf senescence; (iv) a similar decrease of antioxidants during senescence; (v) no significant differences in the total foliar area and apical growth rate; and (vi) a production of viable seeds significantly higher than wild-type plants. These results suggest that the Ndh complex is involved in one of the redundant mechanisms that play a safety role in photosynthesis under stress, which has been conserved during evolution, but that its deletion increases fitness when plants are grown under favourable controlled conditions.

Resumen: Epigloea, es un genero de hongos de pequeno tamano asociados a algas libres, desconocido hasta el momento en la Peninsula Iberica. Durante los estudios llevados a cabo sobre los liquenes de la Reserva MAB de Muniellos (Asturias, NO Espana) se han descubierto siete especies de este genero. Se presentan los resultados del estudio y una clave para la identificacion de las especies presentes en la Peninsula Iberica.

Spinach (Spinacia oleracea L. cv. Clermont) leaves grown in open-top chambers and exposed to three different concentrations of ozone were measured for gas exchange, chlorophyll a fluorescence, antioxidant systems, and lipid peroxidation at the end of growing season. High O-3 concentration reduced F-v/F-m, indicating that the efficiency in the energy conversion of photosystem 2 (PS2) was altered. The rate of non-cyclic electron transport rate and the capacity to reduce the quinone pool were also affected. The development of non-photochemical quenching was not high enough to decrease the photon excess in the PS2. The limitation of photosynthetic activity was probably correlated with stomata closure and with an increase in intercellular CO2 concentration. Under oxidative stress, superoxide dismutase (SOD) activity was stimulated in parallel with lipid peroxidation. We did not find any differences in the ascorbate (AsA) pool and ascorbate peroxidase (APX) or glutathione reductase (GR) activities between air qualities. Small, but similar responses were observed in spinach leaves exposed to ambient ozone concentration.

The photosynthesis response, antioxidant systems and lipid peroxidation were studied in leaves from spinach plants (Spinacia oleracea L.) in response to ozone fumigation, ambient air and charcoal filtered air treatments. The photosynthetic activity was tested through gas exchange and chlorophyll a fluorescence measurements. Ambient air and ozone fumigation caused a decrease in the photosynthetic rate (25% and 63%, respectively) mainly due to a reduced mesophyll activity, as evidenced by the increased intercellular CO, concentration. These data agree with a large reduction in the non-cyclic electron flow (7% and 16%), a lower capacity to reduce the quinone pool and a higher development of non-photochemical quenching upon high O-3 concentration. The results suggest that the oxidative stress produced, together with the stimulation of superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate peroxidase (APX, EC 1.11.1.11) activities and the increase in lipid peroxidation (20% and 36%, respectively), generated an alteration of the membrane properties. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

Lichenostigma radicans Calat. & Barreno, a lichenicolous fungus growing on vagrant Aspicilia species, is described as new to science from Spain. It is superficially similar to L. maureri, with cushion-like ascomata, but differing mainly by its ascomata connected at their base to immersed, brown, vegetative hyphae. Comments on the variability of iodine reactions of asci and ascospore walls, and on an associated conidial fungus with small bacilliform conidia, are given for L. maureri.

The effects of ozone exposure on lettuce leaves were investigated by means of gas exchange, modulated chlorophyll a fluorescence and antioxidants systems. High ozone concentration decreased the rate of photosynthesis at light saturation level and changes in stomatal conductance and transpiration rate. However, an increase in intercellular CO 2 concentration indicated a decrease in the carboxylation efficiency. These data agreed with a reduction of non-cyclic electron flow and lower capacity to reduce the quinone pool. Ozone affected the ascorbate pool and decreased ascorbate peroxidase activity, increased lipid peroxidation, altered membrane properties and reduced the development of non-photochemical quenching.

Ozone is the major phytotoxic air pollutant that reduces the yield of several agricultural crops in the Spanish Mediterranean area. We studied four lettuce cultivars (Lactuca sativa L.) for the effects of different O3 concentrations during the winter on chlorophyll (Chl) a fluorescence, lipid peroxidation, and root length in outdoor open-top chambers. Under O3 the photosynthetic quantum conversion declined while heat emissions increased in all cultivars; these results provide more evidence of non-filtered air with additional ozone (NFA+O3) treatment compared with non-filtered air (NFA) and charcoal filtered ozone-free air (CFA). Changes in the Chl a fluorescence may be associated with an increase in membrane lipid peroxidation as well as with observed reduction of root length under O3 stress.

Summary The effects of air quality with three levels of ozone (O 3 ) were studied on three cabbage varieties during a one month exposure period in the Valencia area by means of modulated chlorophyll (Chl) a fluorescence, lipid peroxidation and solute leakage. Increasing O 3 exposure reduced the maximum quantum yield of PSII photochemistry (F v /F m ) in line with a reduction in non-cyclic electron flow (ϕ PSII ), lower capacity to reduce the quinone pool (q P ) and a decrease in the potential phothosynthetic quantum conversion, Rfd ratio. These reductions were more evident in Caramba and Sentinel varieties but lower in Othelo. Ozone and its oxidative derivates weakened the plasmalemma, which caused a leakage of cellular liquids into intercellular spaces. In our results, the ion leakage increased with O 3 concentration. Moreover, data from thiobarbituric acid reactive (TBARS) analysis confirmed the hypothesis that O 3 modified the membrane structure by increasing the lipid peroxidation more in Caramba and Sentinel varieties. These alterations in the membranes reduced the ability to develop non-photochemical quenching (NPQ). The O 3 effects on solute leakage and lipid peroxidation in the Othelo variety were lower than in the other two varieties, which allowed non-photochemical quenching to develop in this variety. These results suggest the possibility adverse effects of ozone on winter crops grown in the Valencia region.

The current literature reveals that the intrathalline coexistence of multiple microalgal taxa in lichens is more common than previously thought, and additional complexity is supported by the coexistence of bacteria and basidiomycete yeasts in lichen thalli. This replaces the old paradigm that lichen symbiosis occurs between a fungus and a single photobiont. The lichen Ramalina farinacea has proven to be a suitable model to study the multiplicity of microalgae in lichen thalli due to the constant coexistence of Trebouxia sp. TR9 and T. jamesii in long-distance populations. To date, studies involving phycobiont diversity within entire thalli are based on Sanger sequencing, but this method seems to underestimate the diversity. Here, we aim to analyze both the microalgal diversity and its community structure in a single thallus of the lichen R. farinacea by applying a 454 pyrosequencing approach coupled with a careful ad hoc-performed protocol for lichen sample processing prior to DNA extraction. To ascertain the reliability of the pyrosequencing results and the applied bioinformatics pipeline results, the thalli were divided into three sections (apical, middle and basal zones), and a mock community sample was used. The developed methodology allowed 40448 filtered algal reads to be obtained from a single lichen thallus, which encompassed 31 OTUs representative of different microalgae genera. In addition to corroborating the coexistence of the two Trebouxia sp. TR9 and T. jamesii taxa in the same thallus, this study showed a much higher microalgal diversity associated with the lichen. Along the thallus ramifications, we also detected variations in phycobiont distribution that might correlate with different microenvironmental conditions. These results highlight R. farinacea as a suitable material for studying microalgal diversity and further strengthen the concept of lichens as multispecies microecosystems. Future analyses will be relevant to ecophysiological and evolutionary studies to understand the roles of the multiple photobionts in lichen symbioses.

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.

Bioassays constitute a tool for pollution analysis providing a holistic approach and high-quality indication of the toxicity. Microbioassays allow evaluating the toxicity of many samples, implying lower costs and enabling routine monitoring and pollution control. But tests conducted so far are limited to the use of a small number of taxa. Lichens are excellent bioindicators of pollution with great ecological significance. Studies show that the phycobiont is more sensitive to pollutants than the mycobiont. Phycobiont have features such as adaptation to anhydrobiosis and relatively rapid growth in vitro, making them suitable for microbioassays. Our aim is to determine the sensitivity of phycobionts to the pharmaceutical micropollutants carbamazepine and diclofenac as a preliminary step for the development of a toxicity microbioassay based on phycobionts. Optical dispersion and chlorophyll autofluorescence were used as endpoints of toxicity on two algal species showing that suspensions present cyclic and taxon specific patterns of aggregation. Trebouxia TR9 suspensions present a very high grade of aggregation while Asterochloris erici cells do not. Both micropollutants alter optical properties of the suspensions of both species. No significant alteration of chlorophyll autofluorescence by carbamazepine is observed. A. erici chlorophyll autofluorescence is extremely sensitive to diclofenac but the effect is not dependent on the drug concentration or on the time of exposure. Differently, TR9 only shows punctual chlorophyll alterations. Fluctuations in optical dispersion may indicate changes in the population structure of the species, including reproductive strategy. A. erici seems more sensitive to micropollutants, is better characterized and is available from commercial collections.

Ozone is a widely distributed phytotoxic air pollutant and is known to reduce the yield of several important agricultural crops in Spain. However, benomyl has been found to lessen the adverse impact of ozone on plants. We studied the effects of ozone and benomyl on chlorophyll a fluorescence, antioxidant enzymes, and lipid peroxidation in tomato (Lycopersicon esculentum Mill. cv. Tiny Tim) grown in open-top chambers in the field. Our results indicate that benomyl prevented the peroxidation of membrane lipids and increased protection of PSII from ozone. There was also a significant reduction in the activity of the antioxidant enzyme superoxide dismutase in ozone-exposed plants that had not been treated with benomyl. Comparing plants treated with benomyl to untreated plants we found that, on exposure to ozone, a greater fraction of light absorption energy was cycled through the photosynthetic system in benomyl-treated plants, as shown by the higher PSII-mediated electron flow and the higher fraction of open PSII reaction centers. The values analyzed in the fluorescence parameters and lipid peroxidation were similar for plants without benomyl grown in a charcoal-filtered environment and benomyl-treated plants exposed to ozone.

Its high oxidant capacity and ability to generate reactive oxygen species cause ozone toxicity. We studied the effect of ambient ozone on chlorophyll (Chl) a fluorescence, antioxidant enzymes, ascorbate contents, and lipid peroxidation in potatoes grown in open-top chambers in the field. In plants grown in non-filtered air (NFA), the development of non-photochemical quenching brought about a decrease in photosystem 2 (PS2) photochemical efficiency. Also the ability of PS2 to reduce the primary acceptor QA was lower than in charcoal-filtered, ozone-free air (CFA). Changes in Chl fluorescence yield were associated with changes in the thylakoid membrane. Ozone altered chloroplast membrane properties, as indicated by an increase in membrane lipid peroxidation in FNA-leaves compared to CFA plants. The ascorbate pool and activities of antioxidant enzymes were used for an indication of the detoxification system state in NFA and CFA leaves, whereby ozone affects the ascorbate concentration and decreases the antioxidant enzymes activities. The capacity of both detoxifying systems together was not high enough to protect potato plants against ambient ozone concentrations which reduced the photosynthetic yield in this potato cultivar.

A rapid method to isolate intact functional algae from the lichens Evemia prunastri and Ramalina farinacea has been developed. This method is based on the use of Percoll® gradients after mechanical disruption of lichen thalli. Results obtained show that the algal preparations were virtually free of contamination by fungal hyphae. The purified algal cells were photosynthetically active and without symptoms of photoinhibition, which indicates their functional integrity. This method may be used for the isolation of intact algae from a broad range of lichen species.

Lecanora herteliana is described from several collections from hard siliceous sandstone, in typically Mediterranean habitats in eastern Spain. The new species has a whitish thallus containing atranorin and norstictic acid. Its apothecia are mostly black, with a thalline exciple, which is finally excluded, and with a well developed true exciple.Lecanora herteliana is most closely related to L. subcoarctata, and it is discussed in relation to this species and other members of the L. marginata group.

The releasing rates of inorganic ions in lichens when a thallus sample is sonicated under water, were studied. Ramalina farinacea samples taken from two locations exposed to atmospheric pollution sources at Els Ports (Castellon, Spain), and from three control locations, were used. Samples without visual symptoms of injuries were sonicated from 3 to 6 successive 10-min periods while renewing the water layer in between. Experiments with successive 2-min-sonication periods were also performed. The extracts were analysed by capillary zone electrophoresis, and K + , NH 4 + , Na + , Ca 2+ , Mg 2+ , Cl − , SO 4 2− and NO 3 − were detected. The apparently undamaged lichens sampled at locations exposed to SO 2 and ozone sources released high amounts of K + during the first sonication treatment and much lower amounts afterwards, whereas samples taken from control locations released approximately constant or increasing amounts of K + along the successive sonication treatments. The initial releasing rates of other ions showed no significant differences between samples from control and polluted locations. The initial releasing rates of K + and the SO 4 2− /Ca 2+ molar ratio were found to be useful as pollution indicators. The data were correlated with chlorophyll fluorescence quantum yield measurements, and with data obtained by visual evaluation of injury symptoms produced by pollution on representative lichen samples taken from the same locations.

The acclimation potential to high irradiance of two cultivars of watermelon, Reina de Corazones and Toro, calculated as the ratio of sun vs. shade activities of O2− and H2O2 scavenging enzymes and non-radiative energy dissipation, was similar. However, Reina de Corazones exhibited a higher capacity in absolute terms for photoprotection (harmless dissipation of absorbed light energy at PS 2 and ascorbate and O2− and H2O2 scavenging enzymes) suggesting a larger resistance of this cultivar to high irradiance. This could be seen as smaller decreases in fruit productivity and in lower oxidative injury as probed by malondialdehyde content in sun plants of Reina de Corazones than in Toro plants. Additionally, the results show that shading might be beneficial to both cultivars, presumably because it reduces the susceptibility of high irradiance-induced stress.

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 QA, 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.

The effects of foliar spraying of the dithiocarbamate zineb on two cultivars of tomato grown in the field in a site with high ozone concentrations were studied by means of biomass assessment, antioxidant enzyme assays, lipid peroxidation, and chlorophyll fluorescence measurements. Zineb prevented the peroxidation of membrane lipids and decreased the activity of scavenging enzymes, which suggests that plants sprayed with zineb are subjected to lower oxidative stress than controls. The beneficial effects of zineb protection is the utilization of a larger fraction of absorbed radiant energy in photosynthesis and a larger fruit yield in plants of both cultivars.

Simultaneous determination of ascorbic acid (AA), glutathione (GSH), and oxidized glutathione (GSSG) in vascular plants has been achieved by capillary zone electrophoresis. A background electrolyte containing a phosphate buffer of pH 7 and hexadimethrin bromide for dynamic control of electroosmotic flow was used. Separation was performed in less than 5 min. The signal was simultaneously monitored at 265 and 200 nm. Dehydroascorbic acid (DHA) was determined in another aliquot of the same plant extract upon reduction with dl-homocysteine. Limits of detection of AA, GSH, and GSSG ranged from 0.2 to 0.8 μg mL-1, which corresponded from 0.04 to 0.16 mg per 100 g fresh weight of tomato, watermelon, and potato leaves. Plants exposed to a high ozone concentration (1600 μg m-3) in a laboratory fumigation system during 1, 2, 3, and 5 h showed progressive ozone-induced oxidative stress. This could be seen as a simultaneous decrease of the AA/(AA+DHA) and GSH/(GSH+GSSG) ratios; however, the decline rate was higher fo...

Pinus canariensis Chr, Sm. ex DC. samples showing severe damage produced by atmospheric pollution were taken from three locations under the possible influence of the emissions of a fuel-fired power plant in Tenerife. Other samples were taken at four control locations in Tenerife, La Palma and El Hierro (Canary Islands, Spain), distant from local sources of pollution. Injury symptoms were evaluated by visual examination of the needles, and the data were correlated with the releasing rates of inorganic anions and cations from the needles when sonicated under water. For this purpose, the samples were sonicated 4-times during 10 min successive periods while renewing the water layer in between, and the leachates were analyzed by capillary zone electrophoresis. Measurable amounts of K+, Na+, Ca2+, Mg2+, Cl- and SO4 2- were detected. Potassium ions were released in approximately constant or increasing amounts along the sonication series in the control samples, whereas the samples damaged by pollution released larger amounts during the first sonication treatment, and much lower amounts afterwards. Indices which estimated the initial releasing rate of the cations were examinated as possible indicators to evaluate atmospheric pollution. Only the K+ index showed a large positive correlation with the brown-reddish decoloration and tip necrosis of the needles. The correlation of the K+ index with flecking and steepling of the needles was much lower. After principal component analysis of the autoscaled data, the sampling sites and the variables were projected on the plane of the first and second eigenvectors. Using the visual examination parameters as variables, both without and with addition of the cation indices, the sampling sites appeared in the order of increasing damage along the first eigenvector. Evidence of the effects of long-range transported pollutants was obtained, and no relationship was demonstrated between damage of pine needles and emissions from the power plant.

The effects of elevated gaseous SO2 concentrations in the lichens Evernia prunastri (L.) Ach. and Ramalina farinacea (L.) Ach. were investigated by means of gas exchange, modulated chlorophyll fluorescence analysis and antioxidant enzyme assays. The response to SO2 of the studied species differed markedly. Net photosynthetic rates were more adversely affected in E. prunastri than in R. farinacea. In addition, processes dependent on thylakoid membrane integrity such as PSII-mediated electron flow and nonphotochemical quenching were reduced to a greater extent by exposure to SO2 in E. prunastri. Moreover, the ability to reoxidize the quinone pool was lower in this species. Finally, the activity of chloroplastidic and cytoplasmic antioxidant enzymes was decreased in E. prunastri in response to fumigations but increased in R. farinacea. The results suggest that the ability to process and deal with the SO2 once it has been absorbed must play a role in determining the sensitivity of these lichen species to this air pollutant.

An automatic system for the stepwise treatment of solid samples is described. The system is controlled by a PC computer that is connected to a burette equipped with a three-way valve, to a peristaltic pump, and to a robot arm through two serial interfaces. In a cycle, the burette introduces a solvent volume in the sample cell, sonication is applied, and, after a fixed time, the pump carries the solvent to the vial selected by the robot arm. Measurements can be performed both in-line and off-line by analyzing the vial series. The program was written in Visual Basic 3. Lixiviation rates of several cations from the lichen Ramalina farinacea were measured by capillary zone electrophoresis using the vial series obtained in several experimental conditions. The automatic system gave better reproducibility than the manual procedure. Initial lixiviation rates were higher in the presence of ultrasounds. © 1999 John Wiley & Sons, Inc. Lab Robotics and Automation 11: 121–126, 1999

The interactions among water content, chlorophyll a fluorescence emission, xanthophyll interconversions and net photosynthesis were analyzed during dehydration in desiccation-tolerant Frullania dilatata (L.) Dum. and desiccation-intolerant Pellia endiviifolia (Dicks) Dum. Water loss led to a progressive suppression of photosynthetic carbon assimilation in both species. Their chlorophyll fluorescence characteristics at low water content were: low photosynthetic quantum conversion efficiency, high excitation pressure on photosystem II and strong non-photochemical quenching. However, dissipation activity was lower in P. endiviifolia and was not accompanied by a rise in the concentration of de-epoxidised xanthophylls as F. dilatata. The photosynthetic apparatus of F. dilatata remained fully and speedily recuperable after desiccation in as indicated by the restoration of chlorophyll fluorescence parameters to pre-desiccation values upon rehydration. A lack of recovery upon remoistening of P. endiviifolia indicated permanent and irreversible damage to photosystem II. The results suggest that F. dilatata possesses a desiccation-induced zeaxanthin-mediated photoprotective mechanism which might aid photosynthesis recovery when favourable conditions are restored by alleviating photoinhibitory damage during desiccation. This avoidance mechanism might have evolved as an adaptative response to repeated cycles of desiccation and rehydration that represent a real threat to photosynthetic viability.

The interactions among water content, chlorophyll a fluorescence emission, and potassium leakage were analyzed during dehydration in desiccation-tolerant bryophytes from xeric habitats (Hedwigia ciliata (Hedw.) P. Beauv., Hypnum cupressiforme Hedw., Leucodon sciuroides (Hedw.) Schwaegr., Orthotrichum cupulatum Brid., Pleurochaete squarrosa (Brid.) Lindb., Porella platyphylla (L.) Pfeiff., and Tortula ruralis (Hedw.) Gaertn., Meyer & Scherb.) and desiccation-intolerant bryophytes from mesic and hydric environments (Barbula ehrenbergii (Lor.) Fleisch., Cinclidotus aquaticus (Hedw.) B. & S., Conocephalum conicum (L.) Underw., Lunularia cruciata (L.) Dum. ex Lindb., Palustriella commutata (Hedw.) Ochyra, Philonotis calcarea (B. & S.) Schimp., and Rhynchostegium riparioides (Hedw.) Card.). Their fluorescence characteristics at low water content were low efficiency of photosynthetic quantum conversion, closed photosystem II reaction centers, and strong nonphotochemical quenching only in desiccation-tolerant species. Full restoration of fluorescence parameters upon rewatering in species from xeric environments indicated that the photosynthetic apparatus was fully functional after desiccation. Species from hydric and mesic habitats were unable to restore photochemical activity. This might be a consequence of photoinhibition but also of membrane damage, as indicated by the large leakage of potassium. It is suggested that the capacity to enhance thermal energy dissipation during dehydration might have evolved in species from xeric environments as an adaptation to the utilization of an erratic supply of water. This protective strategy would lower the probability of photodamage during water loss and thus maintain the photosynthetic apparatus in a quickly recuperable state.Key words: bryophytes, chlorophyll fluorescence, dehydration, desiccation tolerance, thermal energy dissipation.

The function of photosystem II (PSII) during desiccation was investigated via analysis of Chl a fluorescence emission in thalli from Parmelia quercina (Willd.) Vainio, Parmelia acetabulum (Necker) Duby, Ramalina farinacea (L.) Ach., Pseudevernia furfuracea (L.) Zopf., and Evernia prunastri (L.) Ach. Water loss followed the same exponential pattern in all these species, the half time being dependent on species. Desiccation affected the fluorescence parameters. Dark-adapted maximum fluorescence (F m ), instantaneous fluorescence (F o ) and the ratio of variable (F m -F o ) to F m were dependent on water content and decreased in two distinct phases: a slow and apparently linear phase, followed by a more steep decline at low water content. Actual PSII photochemical yield (Φpsu), non-photochemical quenching (NPQ), efficiency of photon capture (Φ exc ), and photochemical quenching (q p ) remained nearly constant until 30% relative water content (RWC), decreasing rapidly thereafter. In contrast, increased NPQ appeared to occur only at water content values lower than 20%. Treatment of thalli with dithiothreitol (DTT) effectively reduced NPQ during desiccation and increased susceptibility to photoinhibition caused by exposure to high light as measured by dark recovery of the F v /F m ratio. HPLC analysis showed that the level of the de-epoxidized xanthophyll cycle pigments antheraxanthin (Anth) and zeaxanthin (Zea) increased during lichen desiccation. The results point towards the existence of a photoprotective mechanism with the involvement of Zea and Anth in non-radiative dissipation of the desiccation-induced excess of energy.

Completely sequenced plastomes provide a valuable source of information about the duplication, loss, and transfer events of chloroplast genes and phylogenetic data for resolving relationships among major groups of plants. Moreover, they can also be useful for exploiting chloroplast genetic engineering technology. Ericales account for approximately six per cent of eudicot diversity with 11,545 species from which only three complete plastome sequences are currently available. With the aim of increasing the number of ericalean complete plastome sequences, and to open new perspectives in understanding Mediterranean plant adaptations, a genomic study on the basis of the complete chloroplast genome sequencing of Arbutus unedo and an updated phylogenomic analysis of Asteridae was implemented. The chloroplast genome of A. unedo shows extensive rearrangements but a medium size (150,897 nt) in comparison to most of angiosperms. A number of remarkable distinct features characterize the plastome of A. unedo: five-fold dismissing of the SSC region in relation to most angiosperms; complete loss or pseudogenization of a number of essential genes; duplication of the ndhH-D operon and its location within the two IRs; presence of large tandem repeats located near highly re-arranged regions and pseudogenes. All these features outline the primary evolutionary split between Ericaceae and other ericalean families. The newly sequenced plastome of A. unedo with the available asterid sequences allowed the resolution of some uncertainties in previous phylogenies of Asteridae.

Thelopsis foveolata Renob. & Barreno sp. nov., is described from Northern Spain. It appears to be very rare and easily overlooked, but has a well-defined ecology. It grows on limestone within sciaphilous and humidity-loving communities, such as Gyalectetum leucaspidis and Petractinetum hypoleucae. The pycnidia and conidiogenous cells are described and illustrated, this being the first detailed account of these structures in the genus Thelopsis.

Measurements of modulated chlorophyll a fluorescence emission and chlorophyll content were made in thalli of Parmelia quercina collected from northern Castellon(Spain). Althoug high air pollution concentrations have been measured in this area, the ratio of variable to maximal fluorescence is unaffected. Following dark-adaptation, the response to irradiance of chlorophyll fluorescence was examined. With respect to control samples, thalli collected in northern Castellon consistently showed differences in fluorescence quenching, the efficiency of excitation energy capture and quenching of basal fluorescence. However, the quantum ield of photochemistry and non-cyclic electron flow were not affected. The changes in chlorophyll fluorescence parameters were observed in the absence of both visual injuries and decrease in chlorophyll content. The results are discussed with regard to the possible role of environmental stress in chlorophyll fluorescence yield.

† Background and Aims The aquatic moss Fontinalis antipyretica requires a slow rate of dehydration to survive a desiccation event. The present work examined whether differences in the dehydration rate resulted in corresponding differences in the production of reactive oxygen species (ROS) and therefore in the amount of cell damage. † Methods Intracellular ROS production by the aquatic moss was assessed with confocal laser microscopy and the ROS-specific chemical probe 2,7-dichlorodihydrofluorescein diacetate. The production of hydrogen peroxide was also quantified and its cellular location was assessed. † Key Results The rehydration of slowly dried cells was associated with lower ROS production, thereby reducing the amount of cellular damage and increasing cell survival. A high oxygen consumption burst accompanied the initial stages of rehydration, perhaps due to the burst of ROS production. † Conclusions A slow dehydration rate may induce cell protection mechanisms that serve to limit ROS production and reduce the oxidative burst, decreasing the number of damaged and dead cells due upon rehydration.

The RPL10A gene encodes the RPL10 protein, required for joining 40S and 60S subunits into a functional 80S ribosome. This highly conserved gene, ubiquitous across all eukaryotic super-groups, is characterized by a variable number of spliceosomal introns, present in most organisms. These properties facilitate the recognition of orthologs among distant taxa and thus comparative studies of sequences as well as the distribution and properties of introns in taxonomically distant groups of eukaryotes. The present study examined the multiple ways in which RPL10A conservation vs. sequence changes in the gene over the course of evolution, including in exons, introns, and the encoded proteins, can be exploited for evolutionary analysis at different taxonomic levels. At least 25 different positions harboring introns within the RPL10A gene were determined in different taxa, including animals, plants, fungi, and alveolates. Generally, intron positions were found to be well conserved even across different kingdoms. However, certain introns seemed to be restricted to specific groups of organisms. Analyses of several properties of introns, including insertion site, phase, and length, along with exon and intron GC content and exon–intron boundaries, suggested biases within different groups of organisms. The use of a standard primer pair to analyze a portion of the intron-containing RPL10A gene in 12 genera of green algae within Chlorophyta is presented as a case study for evolutionary analyses of introns at intermediate and low taxonomic levels. Our study shows that phylogenetic reconstructions at different depths can be achieved using RPL10A nucleotide sequences from both exons and introns as well as the amino acid sequences of the encoded protein.

Lichen thalli are permeable to airborne substances, including heavy metals, which are harmful to cell metabolism. Ramalina farinacea shows a moderate tolerance to Pb. This lichen comprises two Trebouxia phycobionts, provisionally referred to as TR1 and TR9, with distinct physiological responses to acute oxidative stress. Thus, there is a more severe decay in photosynthesis and photosynthetic pigments in TR1 than in TR9. Similarly, under oxidative stress, antioxidant enzymes and HSP70 protein decrease in TR1 but increase in TR9. Since Pb toxicity is associated with increased ROS formation, we hypothesized greater Pb tolerance in this phycobiont. Accordingly, the aim of the present study was to characterize the physiological differences in the responses of TR1 and TR9 to Pb exposure. Liquid cultures of isolated phycobionts were incubated for 7 days in the presence of Pb(NO3)2. Thereafter, extracellular and intracellular Pb accumulation, photosynthetic pigments, and photosynthesis (as modulated chlorophyll fluorescence) were analyzed along with the antioxidant enzymes glutathione reductase (GR), superoxide dismutase (SOD), ascorbate peroxidase (APx), and catalase (CAT), and the stress-related protein HSP70. Pb uptake increased with the amount of supplied Pb in both algae. However, while significantly more metal was immobilized extracellularly by TR9, the amount of intracellular Pb accumulation was three times higher in TR1. In neither of the phycobionts were significant effects on photosynthetic pigments or photosynthetic electron transport observed. While under control conditions GR, SOD, and APx levels were significantly higher in TR1 than in TR9, only in the latter were these enzymes induced by Pb. This resulted in quantitatively similar antioxidant activities in the two algae when exposed to Pb. In conclusion, the phycobionts of R. farinacea make use of two different strategies against stress, in which the integration of distinct anatomical and physiological features affords similar levels of Pb tolerance., This study was supported by the Spanish Ministry of Science and Innovation (CGL2009-13429-C02-01/02) and the Generalitat Valenciana (PROMETEO 174/2008 and GVACOMP2011-205). We are grateful to the Central Support Service in Experimental Research (SCSIE), University of Valencia (Spain), for the TEM studies. We acknowledge Dr. Said Agouram (SCSIE, University of Valencia, Spain) for TEM-EDXS characterizations and for helpful discussions during the TEM investigations. We thank Wendy Ran and Daniel Sheerin for the English revision of the manuscript.

The study of desiccation tolerance of lichens, and of their chlorobionts in particular, has frequently focused on the antioxidant system that protects the cell against photo-oxidative stress during dehydration/rehydration cycles. In this study, we used proteomic and transcript analyses to assess the changes associated with desiccation in the isolated phycobiont Asterochloris erici. Algae were dried either slowly (5-6 h) or rapidly (60 min), and rehydrated after 24 h in the desiccated state. To identify proteins that accumulated during the drying or rehydration processes, we employed two-dimensional (2D) difference gel electrophoresis (DIGE) coupled with individual protein identification using trypsin digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteomic analyses revealed that desiccation caused an increase in relative abundance of only 11-13 proteins, regardless of drying rate, involved in glycolysis, cellular protection, cytoskeleton, cell cycle, and targeting and degradation. Transcripts of five Hsp90 and two β-tubulin genes accumulated primarily at the end of the dehydration process. In addition, transmission electron microscopy (TEM) images indicate that ultrastructural cell injuries, perhaps resulting from physical or mechanical stress rather than metabolic damage, were more intense after rapid dehydration. This occurred with no major change in the proteome. These results suggest that desiccation tolerance of A. erici is achieved by constitutive mechanisms.

Lichens are symbiotic associations (holobionts) established between fungi (mycobionts) and certain groups of cyanobacteria or unicellular green algae (photobionts). This symbiotic association has been essential in establishing the colonization of terrestrial and consequently dry habitats. About 44 genera of algae and cyanobacteria have been reported as lichen photobionts. Due to the uncertain taxonomy of many of these photobionts, these numbers were considered as approximations only. Ahmadjian (1993) estimates that only 25 genera were typical lichen photobionts. The most common cyanobionts are Nostoc, Scytonema, Stigonema, Gloeocapsa, and Calothrix, in order of frequency (Budel, 1992). Green algal photobionts include Asterochloris, Trebouxia, Trentepohlia, Coccomyxa, and Dictyochloropsis (Gartner, 1992). These authors assessed that more than 50% of all lichen species are associated with Trebouxia and Asterochloris species. However, this is just estimation since in only 2% of the described lichen species the photobiont genus is reported (Tschermak-Woess, 1989), mostly by the difficulties to isolate and then characterize the algae from the lichen thalli. Lichens are well known for their slow growth and longevity. Their radial growth is measured in millimetres per year (Hale, 1973), while individual lichens live for hundreds or even thousands of years. It is assumed that in lichens the photobiont population is under mycobiont control. Lichenologists have proposed some control mechanisms such as, cell division inhibitors (Honegger, 1987), phytohormones (Backor & Hudak, 1999) or nutrients competition (Crittenden et al., 1994; Schofield et al., 2003). Similar to plants, all lichens photosynthesise. They need light to provide energy to make their own matter. More specifically, the algae in the lichen produce carbohydrates and the fungi take those carbohydrates to grow and reproduce. The amount of light intensity needed for optimal lichen growth varies widely among species. The optimum light intensity range of most algal photobionts in axenic cultures is very low, between 16-27 μmol m-2 s-1. If the response of cultured photobionts to light is similar to that of the natural forms (lichen), then there must be additional mechanisms protecting the algae in the lichen that are not developed under culture conditions. Pigments and crystal of secondary metabolites in the

Ramalina farinacea is an epiphytic fruticose lichen that is relatively abundant in areas with Mediterranean, subtropical or temperate climates. Little is known about photobiont diversity in different lichen populations. The present study examines the phycobiont composition of several geographically distant populations of R. farinacea from the Iberian Peninsula, Canary Islands and California as well as the physiological performance of isolated phycobionts. Based on anatomical observations and molecular analyses, the coexistence of two different taxa of Trebouxia (working names, TR1 and TR9) was determined within each thallus of R. farinacea in all of the analysed populations. Examination of the effects of temperature and light on growth and photosynthesis indicated a superior performance of TR9 under relatively high temperatures and irradiances while TR1 thrived at moderate temperature and irradiance. Ramalina farinacea thalli apparently represent a specific and selective form of symbiotic association involving the same two Trebouxia phycobionts. Strict preservation of this pattern of algal coexistence is likely favoured by the different and probably complementary ecophysiological responses of each phycobiont, thus facilitating the proliferation of this lichen in a wide range of habitats and geographic areas.

The scarcity of universally applied molecular markers for algae has resulted in the development of multiple, independent and not easily comparable systems. The goal of this work is to increase the number of available molecular markers and to generate easily comparable systems. Thereby, we have designed a primer pair capable of amplifying a broad range of organisms: Cyanobacteria, Chlorophyta, Chlorarachniophyta, Cryptophyta, Euglenida, Glaucophyta, Rhodophyta, Stramenopiles and Streptophyta including plants. This primer pair can amplify a portion of the 23S rRNA gene with sufficient variability to identify reference material form collections across a broad range of taxa and perform phylogenetic studies alongside other available markers.

[EN] Little is currently known about the population structure of lichen photobionts. Accordingly, in this study we analyzed the nature and distribution of phycobionts in different Spanish populations of Ramalina farinacea. Two different Trebouxia algae were isolated and identified on the basis of anatomical and chloroplast LSU rDNA sequence studies. Interestingly, most of the thalli contained the same two algae. To search for possible genetic variability among these algae, the internal transcribed sequences (ITS) of nuclear rDNA were compared. Surprisingly, only one ITS sequence was found in each thallus, even in those exhibiting multiple morphologically differentiated algae observed in thallus TEM sections. Therefore, ITS sequence analyses are probably only indicative of the predominant photobiont while the other photobiont remains undetected. One photobiont seemed to predominate in the Canary Islands populations and the other in peninsular ones. Additionally, here we describe a new potential DNA marker, based on plastid LSU rDNA, which may allow rapid identification of coexisting algae within the same thallus., This project was funded by the Spanish Ministry of Education and Science [CGL2006-12917-C00, CGL2009-13429-C02-00], and Generalitat Valenciana [PROMETEO 174/2008 GVA]. Dr. A. BECK (Germany) and reviewers are thanked for helpful comments. Wendy Ran revised the manuscript in English.

The chloroplast-encoded large subunit ribosomal RNA gene of several free-living green algae contains group I introns at Escherichia coli genic positions 1917, 1931, 1951, and 2449. Herein we report the presence of group I introns at these positions within the chloroplast-encoded large subunit ribosomal RNA gene of several lichen-forming green algae belonging to the Trebouxia genus. In contrast to the introns inserted at position 2449, all introns inserted at positions 1917, 1931, and 1951 contained LAGLIDADG homing endonuclease genes. Phylogenetic analyses show that: (i) introns inserted at positions 1917, 1931, and 1951 are closely related to introns located at homologous insertion sites in bacterial rDNA genes; and (ii) introns inserted at position 2449 are closely related to fungal introns located at homologous insertion sites in mitochondrial rDNA genes. The symbiogenetic thalli of some lichens are proposed as the likely setting of horizontal transfer of genetic material among distantly related organisms such as bacteria, fungi, and green algae.

The present monitoring study was undertaken to analyze the impact of atmospheric pollution on the lichens found near a power station in northern Spain. Data from 565 releves gathered on 113 sites were analyzed. In areas near the power station the lichen species belong to the Xanthorion community. In areas away from the power station and that are not under the influence of dominant winds, the lichenic diversity increases and the species present belong to the Parmelion community. In intermediate areas between the two, there is a great increase in lichen diversity, in these areas there is a replacement of the species that typically belong to the Xanthorion community, by species typical of the Parmelion community. Because of this we propose the analysis of the number of lichen species together with the distribution of bioindicator taxa as a complement to lichen diversity analysis in biomonitoring studies.

Three-years-old trees of Satsuma mandarin (Citrus unshiu [Mak.] Marc.) cv. Okitsu were exposed to O-3 fumigation during long term (one year) in open-top chambers. As a result of the treatment, chlorophyll a fluorescence and gas exchange parameters were modified with respect to trees growing in O-3-free conditions. Net photosynthetic rate and stomatal conductance decreased and intercellular CO2 concentration increased according to a reduction of the non-cyclic electron flow and a lower capacity to reduce the quinone pool. O-3 also reduced the development of non-photochemical quenching preventing the dissipation of excess excitation energy and, therefore, generated several alterations in photosynthetic apparatus. All these effects were obtained in long-term exposure and higher O-3 concentration. In O-3 ambient conditions, the effects were minor.

Watermelon (Citrillus lanants) plants were grown for two consecutive years in open-top chambers with three different ozone concentrations (O-3-free air, O-3 ambient, and air with additional O-3; CFA, NFA, and NFA+O-3) and three nitrogen fertilizer concentrations [0, 14.0, and 29.6 g N per pot; N0, N1, and N2). There was an interaction between ozone and N fertilizer for the major parameters studied. O-3 and N2 treatments led to a significant decrease in maximum efficiency of photosystem 2 (PS2) photochemistry (F-v/F-m), and induced a significant decrease in the actual quantum yield of PS2 (Phi(PS2)), due mainly to the increased closure of PS2 reaction centres (q(P)) and to an increase in the non-photochemical quenching, (NPQ). On the other hand, these plants exhibited an increased susceptibility to photo inhibition, which could be associated with an increased fraction of reduced Q(A). An increase in lipid peroxidation indicated that damage was occurring at the membrane levels. High N concentration enhanced the detrimental effects of ozone on the fluorescence parameter induction and lipid peroxidation. All these negative alterations led to a decreased yield.