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Lichens are a mutualistic symbiosis between a fungus and one or more photosynthetic partners. They are photosynthetically active during desiccation down to relative water contents (RWCs) as low as 30% (on dry mass). Experimental evidence suggests that during desiccation, the photobionts have a higher hydration level than the surrounding fungal pseudo-tissues. Explosive cavitation events in the hyphae might cause water movements towards the photobionts. This hypothesis was tested in two foliose lichens by measurements of ultrasonic acoustic emissions (UAEs), a method commonly used in vascular plants but never in lichens, and by measurements of PSII efficiency, water potential, and RWC. Thallus structural changes were characterized by low-temperature scanning electron microscopy. The thalli were silent between 380% and 30% RWCs, when explosive cavitation events should cause movements of liquid water. Nevertheless, the thalli emitted UAEs at ~5% RWC. Accordingly, the medullary hyphae were partially shrunken at ~15% RWC, whereas they were completely shrunken at <5% RWC. These results do not support the hypothesis of hyphal cavitation and suggest that the UAEs originate from structural changes at hyphal level. The shrinking of hyphae is proposed as an adaptation to avoid cell damage at very low RWCs., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Two-dimensional (2D) materials have attracted tremendous interest ever since the isolation of atomically thin sheets of graphene in 2004 due to the specific and versatile properties of these materials. However, the increasing production and use of 2D materials necessitate a thorough evaluation of the potential impact on human health and the environment. Furthermore, harmonized test protocols are needed with which to assess the safety of 2D materials. The Graphene Flagship project (2013-2023), funded by the European Commission, addressed the identification of the possible hazard of graphene-based materials as well as emerging 2D materials including transition metal dichalcogenides, hexagonal boron nitride, and others. Additionally, so-called green chemistry approaches were explored to achieve the goal of a safe and sustainable production and use of this fascinating family of nanomaterials. The present review provides a compact survey of the findings and the lessons learned in the Graphene Flagship.

Background: The "Herbarium Universitatis Tergestinae" (TSB), with a total of ca. 50,000 specimens, includes the largest modern collection of lichens in Italy, with 25,796 samples collected from all over the country since 1984, representing 74% of all taxa known to occur in Italy. Almost all specimens have been georeferenced "a posteriori". The dataset is available through GBIF, as well as in ITALIC, the Information System of Italian Lichens., New Information: The TSB Herbarium hosts the largest modern lichen collection in Italy, with a total of ca. 50,000 specimens. This dataset contains all of the 25,796 specimens collected within the administrative borders of Italy. Amongst them, 98% are georeferenced and 87% have the date of collection. The dataset includes several type specimens (isotypes and holotypes) and exsiccata., (Matteo Conti, Pier Luigi Nimis, Mauro Tretiach, Lucia Muggia, Andrea Moro, Stefano Martellos.)

The increasing use of graphene-related materials (GRMs) in everyday-life products raises concerns for their possible release into the environment and consequent impact on organisms. GRMs have widely varying effects on plants and, according to recent evidences, graphene oxide (GO) has the potential to interfere with the sexual reproduction owing to its acidic properties and production residues. Here, stigmas of the model plant Cucurbita pepo (summer squash) were subjected to simulated dry depositions of GO and GO purified from production residues (PGO). Stigmas were then hand-pollinated and GRM deposition was checked by ESEM and confocal microscopy. Analysis of stigma integrity, pH homeostasis and pollen-stigma interactions did not reveal negative effects. Fruit and seed production were not affected, but GO depositions of 22.1 ± 7.2 ng mm -2 affected the normal development of seeds, decreasing seed dimensions, seed germination and germination speed. The elemental analysis revealed that GO has significant quantities of production residues, such as strong acids and oxidants, while PGO has only traces, which justifies the differences observed in the effects caused by the two materials. Our results show that GO depositions of up to 11.1 ± 3.6 ng mm -2 , which fall within the variation range of total dry particulate matter depositions reported in the literature, are safe for reproduction of C. pepo. This is the first "safety" limit ever recorded for depositions of "out-of-the-box" GO concerning the reproduction of a seed plant. If confirmed for wind-pollinated species, it might be considered for policymaking of GRMs emissions in the air., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Products containing graphene-related materials (GRMs) are becoming increasingly common, allowing GRM nanoparticles (NPs) to enter the environment during their life cycle. Thanks to their lightness and bidimensional geometry, GRM NPs can be easily dispersed in the air and travel very long distances. The flowers of wind-pollinated plants may be exposed to airborne GRMs, being apt to intercept pollen from the air and, inevitably, other airborne particles. Here, stigmas of four wind-pollinated plants (Corylus avellana, common hazel; Juglans regia, walnut; Quercus ilex, holm oak; Zea mays, maize) were exposed to airborne graphene oxide (GO) and GO purified from production residues (PGO) at a concentration of 3.7 ng m -3 . Subsequently, the stigmas were pollinated and the adhesion of GOs and their effects on stigma integrity and pollen-stigma interaction were examined. The effect of GO NPs in presence of liquid water on the stigma of C. avellana was also investigated. GOs NPs were intercepted by all species, but their effect varied among them. GO reduced pollen adhesion in J. regia and Q. ilex, whereas pollen germination was unaffected in all four species. The presence of a film of water neither completely removed GO NPs from the stigma, nor it enhanced the toxic effect of GO acidity. PGO never affected pollen-stigma interaction, indicating that the phytotoxic substances used for the production of GO, still in traces in commercial GO, are the main cause of GO toxicity. These results reconfirm the need to verify GRMs effects also on key biological processes beside single model organisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Fungal-algal relationships-both across evolutionary and ecological scales-are finely modulated by the presence of the symbionts in the environments and by the degree of selectivity and specificity that either symbiont develop reciprocally. In lichens, the green algal genus Trebouxia Puymaly is one of the most frequently recovered chlorobionts. Trebouxia species-level lineages have been recognized on the basis of their morphological and phylogenetic diversity, while their ecological preferences and distribution are still only partially unknown. We selected two cosmopolitan species complexes of lichen-forming fungi as reference models, i.e., Rhizoplaca melanophthalma and Tephromela atra , to investigate the diversity of their associated Trebouxia spp. in montane habitats across their distributional range worldwide. The greatest diversity of Trebouxia species-level lineages was recovered in the altitudinal range 1,000-2,500 m a.s.l. A total of 10 distinct Trebouxia species-level lineages were found to associate with either mycobiont, for which new photobionts are reported. One previously unrecognized Trebouxia species-level lineage was identified and is here provisionally named Trebouxia "A52." Analyses of cell morphology and ultrastructure were performed on axenically isolated strains to fully characterize the new Trebouxia "A52" and three other previously recognized lineages, i.e., Trebouxia "A02," T. vagua "A04," and T. vagua "A10," which were successfully isolated in culture during this study. The species-level diversity of Trebouxia associating with the two lichen-forming fungi in extreme habitats helps elucidate the evolutionary pathways that this lichen photobiont genus traversed to occupy varied climatic and vegetative regimes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 De Carolis, Cometto, Moya, Barreno, Grube, Tretiach, Leavitt and Muggia.)

The environmental biodegradability profile of graphene related materials (GRMs) is important to know in order to predict whether these materials will accumulate in soil or will be transformed by primary decomposers. In this study, few-layer graphene (FLG) was exposed to living and devitalized axenic cultures of two white-rot basidiomycetes (Bjerkandera adusta and Phanerochaete chrysosporium) and one soil saprotrophic ascomycete (Morchella esculenta) with or without lignin, for a period of four months. Over this time, the increase of fungal biomass and presence of H 2 O 2 and oxidizing enzymes [laccase/peroxidase and lignin peroxidase (LiP)] in growth media was assessed by gravimetric and spectrophotometric measurements, respectively. Raman spectroscopy and transmission electron microscopy (TEM) were used to compare the structure of FLG before and after incubation. All of the test fungi decreased pH in growth media and released H 2 O 2 and laccase/peroxidase, but only basidiomycetes released LiP. Independent of growth media composition all fungi were found to be capable to oxidize FLG to a graphene oxide-like material, including M. esculenta, which released only laccase/peroxidase, i.e. the most common enzymes among primary decomposers. These findings suggest that FLG involuntarily released into terrestrial environments would likely be oxidized by soil microflora., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

In bioaccumulation studies, sample devitalization through acid washing or oven drying is commonly applied to enhance the element accumulation efficiency of moss sample. Such aspect, however, has never been considered in biomonitoring surveys using lichens. In this study, the trace element accumulation performance of living (L) and dead (D) samples of the lichen Pseudevernia furfuracea was compared by a side-by-side transplanting at 40 sites in a large, mixed land use area of NE Italy for 8 weeks. Devitalization was achieved without any physico-chemical treatments, by storing lichen samples in a dark cool room for 18 months. Health status of lichens was assessed before and after the sample exposure by chlorophyll fluorescence emission. Although elemental analysis of the two exposed sample sets revealed a similar trace element pollution scenario, the content of 13 out of the 24 selected elements was higher in D samples. By expressing results as exposed-to-unexposed (EU) ratio, D samples show a higher bioaccumulation signal in 80% of transplant sites for Al, Ca, Fe, Hg, Pb and Ti. Overall, the health status of lichen samples might lead to interpretational discrepancies when EU ratio is classified according to the recently proposed bioaccumulation scale.

The botanical exploration of the Majella National Park has a long tradition dating back to the eighteenth century. However, the lichen biota of this area is still poorly investigated. To provide a baseline for future investigations, in this annotated checklist, we summarised all available information on the occurrence of lichens in the Majella National Park, retrieved from previous literature, herbarium material and original data produced by recent research. The checklist includes 342 infrageneric taxa. However, seven taxa are considered as dubious, thus setting the number of accepted taxa at 335, i.e. 45.8% of those currently known to occur in the Abruzzo Region. This checklist provides a baseline of the lichens known to occur in the Majella National Park, highlighting the potential of this area as a hotspot of lichen biodiversity, especially from a biogeographical point of view as indicated by the occurrence of several arctic-alpine species that form disjunct populations in the summit area of the massif., (Gabriele Gheza, Luca Di Nuzzo, Chiara Vallese, Renato Benesperi, Elisabetta Bianchi, Valter Di Cecco, Luciano Di Martino, Paolo Giordani, Josef Hafellner, Helmut Mayrhofer, Pier Luigi Nimis, Mauro Tretiach, Juri Nascimbene.)

Lichens can withstand extreme desiccation to water contents of ≤ 0.1 g H2O g-1 DW, and in the desiccated state are among the most extremotolerant organisms known. Desiccation-tolerant life-forms such as seeds, mosses and lichens survive 'vitrification', that is the transition of their cytoplasm to a 'glassy' state, which causes metabolism to cease. However, our understanding of the mechanisms of desiccation tolerance is hindered by poor knowledge of what reactions occur in the desiccated state. Using Flavoparmelia caperata as a model lichen, we determined at what water contents vitrification occurred upon desiccation. Molecular mobility was assessed by dynamic mechanical thermal analysis, and the de- and re-epoxidation of the xanthophyll cycle pigments (measured by HPLC) was used as a proxy to assess enzyme activity. At 20 °C vitrification occurred between 0.12-0.08 g H2O g-1 DW and enzymes were active in a 'rubbery' state (0.17 g H2O g-1 DW) but not in a glassy state (0.03 g H2O g-1 DW). Therefore, desiccated tissues may appear to be 'dry' in the conventional sense, but subtle differences in water content will have substantial consequences on the types of (bio)chemical reactions that can occur, with downstream effects on longevity in the desiccated state., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Despite the broad use of lichens as biomonitors of airborne trace elements, the surface chemistry and metal adsorption parameters of these organisms are still poorly known. The current investigation is aimed at (i) quantifying the acid-base surface properties and the first-order physical-chemical parameters of Cu 2+ and Zn 2+ adsorption of devitalized Pseudevernia furfuracea, a lichen commonly used in biomonitoring of airborne trace elements, and (ii) comparing the results with those available for moss biomonitors. Equilibrium constants and metal-binding site concentrations were calculated with a thermodynamic model by taking into account the presence/absence of ancillary extracellular cell wall compounds, namely melanin and acetone-soluble lichen substances. An acid-base titration experiment performed in the pH range of 3-10 showed that melanised and non-melanised P. furfuracea samples have lower pH PZC (3.53-3.99) and higher metal-binding site concentrations (0.96-1.20 mmol g -1 ) compared to that of the mosses investigated so far at the same experimental conditions. Melanin biosynthesis increased the content of carboxyl and phosphoryl groups and reduces that of amine/polyphenols. Cu 2+ and Zn 2+ adsorption was unaffected by the degree of melanisation while the removal of extracellular lichen substances slightly decreased Zn 2+ adsorption. Although Cu 2+ and Zn 2+ adsorption parameters related to P. furfuracea surfaces were 3 times lower than in the mosses, lichen samples adsorbed the same amount of Cu 2+ and 30% more Zn 2+ . The present study contributes in understanding the role of ancillary cell wall compounds in Cu 2+ and Zn 2+ adsorption in a model lichen. It also provides a first comparison between the surface physico-chemical characteristics of lichens and mosses frequently used as biomonitors of trace elements., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Lichens and their isolated symbionts are potentially valuable resources for biotechnological approaches. Especially mycobiont cultures that produce secondary lichen products are receiving increasing attention, but lichen mycobionts are notoriously slow-growing organisms. Sufficient biomass production often represents a limiting factor for scientific and biotechnological investigations, requiring improvement of existing culturing techniques as well as methods for non-invasive assessment of growth. Here, the effects of pH and the supplement of growth media with either D-glucose or three different sugar alcohols that commonly occur in lichens, D-arabitol, D-mannitol and ribitol, on the growth of the axenically cultured mycobiont isolated from the lichen Xanthoria parietina  were tested. Either D-glucose or different sugar alcohols were offered to the fungus at different concentrations, and cumulative growth and growth rates were assessed using two-dimensional image analysis over a period of 8 weeks. The mycobiont grew at a pH range from 4.0 to 7.0, whereas no growth was observed at higher pH values. Varying the carbon source in Lilly-Barnett medium (LBM) by replacing 1% D-glucose used in the originally described LBM by either 1%, 2% or 3% of D-mannitol, or 3% of D-glucose increased fungal biomass production by up to 26%, with an exponential growth phase between 2 and 6 weeks after inoculation. In summary, we present protocols for enhanced culture conditions and non-invasive assessment of growth of axenically cultured lichen mycobionts using image analysis, which may be useful for scientific and biotechnological approaches requiring cultured lichen mycobionts., Supplementary Information: The online version contains supplementary material available at 10.1007/s11557-021-01707-7., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2021.)

Environmental DNA (eDNA) metabarcoding and metagenomics analyses can improve taxonomic resolution in biodiversity studies. Only recently, these techniques have been applied in aerobiology, to target bacteria, fungi and plants in airborne samples. Here, we present a nine-month aerobiological study applying eDNA metabarcoding in which we analyzed simultaneously airborne diversity and variation of fungi and plants across five locations in North and Central Italy. We correlated species composition with the ecological characteristics of the sites and the seasons. The most abundant taxa among all sites and seasons were the fungal genera Cladosporium, Alternaria, and Epicoccum and the plant genera Brassica, Corylus, Cupressus and Linum, the latter being much more variable among sites. PERMANOVA and indicator species analyses showed that the plant diversity from air samples is significantly correlated with seasons, while that of fungi varied according to the interaction between seasons and sites. The results consolidate the performance of a new eDNA metabarcoding pipeline for the simultaneous amplification and analysis of airborne plant and fungal particles. They also highlight the promising complementarity of this approach with more traditional biomonitoring frameworks and routine reports of air quality provided by environmental agencies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Phytohormones are pivotal signaling compounds in higher plants, in which they exert their roles intracellularly, but are also released for cell-to-cell communication. In unicellular organisms, extracellularly released phytohormones can be involved in chemical crosstalk with other organisms. However, compared to higher plants, hardly any knowledge is available on the roles of phytohormones in green algae. Here, we studied phytohormone composition and extracellular release in aero-terrestrial Trebouxiophyceae. We investigated (a) which phytohormones are produced and if they are released extracellularly, and if extracellular phytohormone levels are (b) affected by environmental stimuli, and (c) differ between lichen-forming and non-lichen-forming species. Three free-living microalgae (Apatococcus lobatus, Chloroidium ellipsoideum, and Myrmecia bisecta) and three lichen-forming microalgae (Asterochloris glomerata, Trebouxia decolorans, and Trebouxia sp.) were studied. Algae were grown on solid media and the following cellular phytohormones were identified by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS): indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), abscisic acid (ABA), gibberellin A 4 (GA 4 ), and zeatin (ZT). Furthermore, IAA, IBA, ABA, jasmonic acid (JA), gibberellin A 3 (GA 3 ), and GA 4 were found to be released extracellularly. IAA and ABA were released by all six species, and IAA was the most concentrated. Phytohormone release was affected by light and water availability, especially IAA in A. glomerata, Trebouxia sp., and C. ellipsoideum. No clear patterns were observed between lichen-forming and non-lichen-forming species. The results are envisaged to contribute valuable baseline information for further studies into the roles of phytohormones in microalgae., (© 2020 The Authors. Journal of Phycology published by Wiley Periodicals LLC on behalf of Phycological Society of America.)

Products containing graphene-related materials (GRMs) are becoming quite common, raising concerns for environmental safety. GRMs have varying effects on plants, but their impact on the sexual reproduction process is largely unknown. In this study, the effects of few-layer graphene (FLG) and a similarly layered phyllosilicate, muscovite mica (MICA), were tested in vivo on the reproductive structures, i.e., pollen and stigma, of Cucurbita pepo L. ssp. pepo ' greyzini ' (summer squash, zucchini). Pollen was exposed to FLG or MICA, after careful physical-chemical characterization, at concentrations of 0.5 and 2 mg of nanomaterial (NM) per g of pollen for up to six hours. Following this, pollen viability was tested. Stigmas were exposed to FLG or MICA for three hours and then analyzed by environmental scanning electron microscopy to verify possible alterations to their surface. Stigmas were then hand-pollinated to verify the effects of the two NMs on pollen adhesion and in vivo pollen germination. FLG and MICA altered neither pollen viability nor the stigmatic surface. However, both NMs equivalently decreased pollen adhesion and in vivo germination compared with untreated stigmas. These effects deserve further attention as they could impact on production of fruits and seeds. Importantly, it was shown that FLG is as safe as a naturally occurring nanomaterial.

Graphene related materials (GRMs) are currently being used in products and devices of everyday life and this strongly increases the possibility of their ultimate release into the environment as waste items. GRMs have several effects on plants, and graphene oxide (GO) in particular, can affect pollen germination and tube growth due to its acidic properties. Despite the socio-economic importance of sexual reproduction in seed plants, the effect of GRMs on this process is still largely unknown. Here, Corylus avellana L. (common Hazel) pollen was germinated in-vitro with and without 1-100 μg mL -1 few-layer graphene (FLG), GO and reduced GO (rGO) to identify GRMs effects alternative to the acidification damage caused by GO. At 100 μg mL -1 both FLG and GO decreased pollen germination, however only GO negatively affected pollen tube growth. Furthermore, GO adsorbed about 10 % of the initial Ca 2+ from germination media accounting for a further decrease in germination of 13 % at the pH created by GO. In addition, both FLG and GO altered the normal tip-focused reactive oxygen species (ROS) distribution along the pollen tube. The results provided here help to understand GRMs effect on the sexual reproduction of seed plants and to address future in-vivo studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Atmospheric dispersion model (ADM) simulations are increasingly used as management tools in air pollution monitoring programs, even in the absence of proper validation. Biomonitors can provide important information for ADM validation, but an open question is their temporal frame of application, particularly when native organisms are used. In this study, we tested two alternative ADM simulating the total suspended particulate (TSP) released by a coal power station, against the element content of two native lichens collected at 40 sites, integrated by soil samples. The ADM simulations differed by the time references: the 6-month period preceding lichen sampling, approximately corresponding to the estimated age of the samples (Mod. A), and the whole year 2005, representative of the local average conditions and used in the plant authorization processes (Mod. B). A generalized regression model analysis clearly showed that the Cr, Pb and V content of lichen samples was spatially associated to the outcomes of Mod. A, but not with Mod. B. Interestingly, the Cr content of lichen samples consistently correlated to TSP concentration predicted by Mod. A along two transects placed downwind from the coal power station. This result was corroborated by an air particulate matter sampling which pointed out that air Cr concentrations increased during the operative period of the source. Overall, our results suggest that lichen bioaccumulation data can proficiently be used to validate ADM simulations if the exposure time of the biological samples is consistent with the temporal domain of the ADM simulations.

Evidence is emerging that phytohormones represent key inter-kingdom signalling compounds supporting chemical communication between plants, fungi and bacteria. The roles of phytohormones for the lichen symbiosis are poorly understood, particularly in the process of lichenization, i.e. the key events which lead free-living microalgae and fungi to recognize each other, make physical contact and start developing a lichen thallus. Here, we studied cellular and extracellularly released phytohormones in three lichen mycobionts, Cladonia grayi , Xanthoria parietina and  Tephromela atra , grown on solid medium, and the effects of indole-3-acetic acid (IAA) on their respective photobionts, Asterochloris glomerata , Trebouxia decolorans , Trebouxia sp. Using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) we found that mycobionts produced IAA, salicylic acid (SA) and jasmonic acid (JA). IAA represented the most abundant phytohormone produced and released by all mycobionts, whereas SA was released by X. parietina and T. atra , and JA was released by C. grayi only. With a half-life of 5.2 days, IAA degraded exponentially in solid BBM in dim light. When IAA was exogenously offered to the mycobionts' compatible photobionts at "physiological" concentrations (as released by their respective mycobionts and accumulated in the medium over seven days), the photobionts' water contents increased up to 4.4%. Treatment with IAA had no effects on the maximum quantum yield of photosystem II, dry mass, and the contents of photosynthetic pigments and α-tocopherol of the photobionts. The data presented may be useful for designing studies aimed at elucidating the roles of phytohormones in lichens., (© The Author(s) 2020.)

The effects of two graphene-based materials (GBMs), few-layers graphene (FLG) and graphene oxide (GO), were studied in the aeroterrestrial green microalga Trebouxia gelatinosa . Algae were subjected to short- and long-term exposure to GBMs at 0.01, 1 and 50 μg mL - 1 . GBMs internalization after short-term exposures was investigated with confocal microscopy, Raman spectroscopy and TEM. Potential negative effects of GBMs, compared to the oxidative stress induced by H 2 O 2 , were verified by analyzing chlorophyl a fluorescence (Chl a F), expression of stress-related genes and membrane integrity. Effects of up to 4-week-long exposures were assessed analyzing growth dynamics, Chl a F and photosynthetic pigments. GBMs were not observed in cells but FLG was detected at the interface between the cell wall and plasma membrane, whereas GO was observed adherent to the external wall surface. FLG caused the down-regulation of the HSP70-1 gene, with the protein levels remaining stable, whereas GO had no effect. In comparison, H 2 O 2 produced dose- and time-dependent effects on Chl a F, gene expression and HSP70 protein level. Long-term exposures to GBMs did not affect growth dynamics, Chl a F or photosynthetic pigment contents, indicating that the few observed short-term effects were not dangerous on the long-term. Results suggest that interactions between FLG and plasma membrane were harmless, activating a down-regulation of the HSP70-1 gene similar to that induced by H 2 O 2 . Our work shows that studying GBMs effects on non-model organisms is important since the results of model green microalgae are not representative of the whole taxonomic group.

In bioaccumulation studies, the interpretation of pollutant contents in the target biomonitor has to be performed by assessing a deviation from an unaltered reference condition. A common strategy consists in the comparison with background element content (BEC) values, often built up by uncritically merging methodologically heterogeneous data. In this respect, the acid digestion of samples was identified as a major step affecting BEC data. Here, the analytical outcomes of two acid mixtures were compared on a set of matched paired samples of the lichen Pseudevernia furfuracea, a widely used biomonitor for which BEC values based on partial digestion were previously provided. The standard reference material BCR 482 (P. furfuracea) was used to validate analytical procedures consisting of either a HF total mineralization or an aqua regia partial one, both associated to ICP-MS multi-element analysis. In particular, the performance of the procedures was evaluated by comparing analytical results of field samples with the accuracy obtained on BCR aliquots (measured-to-expected percentage ratio). The total digestion showed a better performance for Al, As, Ba, Ca, Cd, Cu, Fe, Mn, Ni, Se, Sn, and Zn, whereas the opposite was found for Cr, Co, P, and S. Moreover, new BEC values were provided for P. furfuracea using a consolidated statistical approach, after a total sample digestion with hydrofluoric acid. The multivariate investigation of the background variability of 43 elements in 57 remote Italian sites led to the identification of geographically homogeneous areas for which BEC values are provided for use as reference in biomonitoring applications.

The protocols commonly applied in surveys with lichens as biomonitors of airborne trace elements require analyses of samples derived from thalli or parts of thalli grown in the last year before sampling, under the postulation that samples of the same size are of the same age. Unfortunately, the influence of ecological site-specific factors on lichen growth is still largely ignored, so that samples of the same size collected in environmentally and climatically diverse sites might actually differ in age. This work aims at quantifying the influence of climatic conditions on the radial growth rates (RaGRs) of Xanthoria parietina, one of the most popular lichen biomonitors. RaGR was monitored in seven populations distributed along an altitudinal transect of 30 km in the Classical Karst (NE Italy), from 20 to 500 m above sea level. For c. 17 months, lobe growth was measured seasonally with a digital calliper, and site-specific climatic variables were monitored by means of thermo-hygrometric sensors and implemented by meteorological data. Finally, the lobe growth of X. parietina was modelled as a function of 18 environmental variables. Results revealed that thalli of relatively dry sites had significantly lower seasonal RaGR with respect to moister ones. Considering that cumulative precipitations were equally distributed along the transect, it was concluded that RaGR of X. parietina is affected negatively by high air temperatures and positively by high relative humidity. The importance of RaGR variation in lichen bioaccumulation studies is critically discussed.

Graphene and its derivatives are heralded as "miracle" materials with manifold applications in different sectors of society from electronics to energy storage to medicine. The increasing exploitation of graphene-based materials (GBMs) necessitates a comprehensive evaluation of the potential impact of these materials on human health and the environment. Here, we discuss synthesis and characterization of GBMs as well as human and environmental hazard assessment of GBMs using in vitro and in vivo model systems with the aim to understand the properties that underlie the biological effects of these materials; not all GBMs are alike, and it is essential that we disentangle the structure-activity relationships for this class of materials.

The relation between water status and expression profiles of desiccation -related genes has been studied in the desiccation tolerant (DT) aeroterrestrial green microalga Trebouxia gelatinosa, a common lichen photobiont. Algal colonies were desiccated in controlled conditions and during desiccation water content (WC) and water potential (Ψ) were measured to find the turgor loss point (Ψ tlp ). Quantitative real-time PCR was performed to measure the expression of ten genes related to photosynthesis, antioxidant defense, expansins, heat shock proteins (HSPs), and desiccation related proteins in algal colonies collected during desiccation when still at full turgor (WC > 6 g H 2 O g -1 dry weight), immediately before and after Ψ tlp (-4 MPa; WC ∼ 1 g H 2 O g -1 dry weight) and before and after complete desiccation (WC < 0.01 g H 2 O g -1 dry weight), quantifying the HSP70 protein levels by immunodetection. Our analysis showed that the expression of eight out of ten genes changed immediately before and after Ψ tlp . Interestingly, the expression of five out of ten genes changed also before complete desiccation, i.e. between 0.2 and 0.01 g H 2 O g -1 dry weight. However, the HSP70 protein levels were not affected by changes in water status. The study provides new evidences of the link between the loss of turgor and the expression of genes related to the desiccation tolerance of T. gelatinosa, suggesting the former as a signal triggering inducible mechanisms., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

In biomonitoring, the knowledge of background element content (BEC) values is an essential pre-requisite for the correct assessment of pollution levels. Here, we estimated the BEC values of a highly performing biomonitor, the epiphytic lichen Pseudevernia furfuracea, by means of a careful review of literature data, integrated by an extensive field survey. Methodologically homogeneous element content datasets, reflecting different exposure conditions across European and extra-European countries, were compiled and comparatively analysed. Element content in samples collected in remote areas was compared to that of potentially enriched samples, testing differences between medians for 25 elements. This analysis confirmed that the former samples were substantially unaffected by anthropogenic contributions, and their metrics were therefore proposed as a first overview at supra-national background level. We also showed that bioaccumulation studies suffer a huge methodological variability. Limited to original field data, we investigated the background variability of 43 elements in 62 remote Italian sites, characterized in GIS environment for anthropization, land use, climate and lithology at different scale resolution. The relationships between selected environmental descriptors and BEC were tested using Principal Component Regression (PCR) modelling. Elemental composition resulted significantly dependent on land use, climate and lithology. In the case of lithogenic elements, regression models correctly reproduced the lichen content throughout the country at randomly selected sites. Further descriptors should be identified only for As, Co, and V. Through a multivariate approach we also identified three geographically homogeneous macro-regions for which specific BECs were provided for use as reference in biomonitoring applications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Fungal spores and mycelium fragments are particles which become and remain airborne and have been subjects of aerobiological studies. The presence and the abundance of taxa in aerobiological samples can be very variable and impaired by changeable climatic conditions. Because many fungi produce mycotoxins and both their mycelium fragments and spores are potential allergens, monitoring the presence of these taxa is of key importance. So far data on exposure and sensitization to fungal allergens are mainly based on the assessment of few, easily identifiable taxa and focused only on certain environments. The microscopic method used to analyze aerobiological samples and the inconspicuous fungal characters do not allow a in depth taxonomical identification. Here, we present a first assessment of fungal diversity from airborne samples using a DNA metabarcoding analysis. The nuclear ITS2 region was selected as barcode to catch fungal diversity in mixed airborne samples gathered during two weeks in four sites of North-Eastern and Central Italy. We assessed the taxonomic composition and diversity within and among the sampled sites and compared the molecular data with those obtained by traditional microscopy. The molecular analyses provide a tenfold more comprehensive determination of the taxa than the traditional morphological inspections. Our results prove that the metabarcoding analysis is a promising approach to increases quality and sensitivity of the aerobiological monitoring. The laboratory and bioinformatic workflow implemented here is now suitable for routine, high-throughput, regional analyses of airborne fungi.

Tropospheric ozone (O 3 ) causes severe damage to many vascular plants but not to lichens. It was recently suggested that this may be due to their high levels of natural defences against the oxidative bursts associated to their fluctuating water content. In this study, the combined effects of watering regime (with or without a daily spray of distilled water), air relative humidity (20 ± 5 vs. 80 ± 5% RH) and O 3 (250 vs. 0 ppb, 5 h day -1 for 2 weeks) were monitored in two chlorolichens with different ecology, Parmotrema perlatum and Xanthoria parietina. Modulated chlorophyll a fluorescence (Chl a F), superoxide anion radical (O 2 •- ) and hydrogen peroxide (H 2 O 2 ) production, antioxidant content and enzyme activity of the ascorbate/glutathione cycle were measured after exposure and, for Chl a F, after 1 and 2 days of recovery. The species differed in the antioxidant profile (ascorbate was higher in X. parietina, glutathione in P. perlatum), and in the activity of ROS-scavenging enzymes, more intense in the hygrophilous P. perlatum than in the meso-xerophilous X. parietina. O 3 slightly modified Chl a F parameters related to the controlled dissipation, with reduction of F m , F v /F m (both species) and ETR (in P. perlatum), and increase in NPQ and qN (in X. parietina). It also influenced, particularly in P. perlatum, the content of H 2 O 2 , glutathione (GSH) and oxidized glutathione (GSSG) (but not that of O 2 •- and AsA + DHA) and the activity of superoxide dismutase, ascorbate peroxidase and dehydroascorbate reductase. These parameters, however, were more heavily affected by water availability. The hypothesis that lichens are O 3 -tolerant thanks to the constitutive antioxidant systems, intimately related to their poikilohydric life-style, is thus confirmed.

Main Conclusion: Drought tolerance was greater in the whole lichen than in its isolated photobiont. Cell turgor state has an influence on the functionality of photosynthetic process in lichens. Irreversible thermodynamics is widely used to describe the water relations of vascular plants. However, poikilohydrous organisms like lichens and aeroterrestrial microalgae have seldom been studied using this approach. Water relations of lichens are generally addressed without separate analysis of the mycobiont and photobiont, and only few studies have correlated changes in photosynthetic efficiency of dehydrating lichens to accurate measurements of their water potential components. We measured water potential isotherms and chlorophyll a fluorescence in the lichen Flavoparmelia caperata harvested in different seasons, as well as in its isolated photobiont, the green alga Trebouxia gelatinosa, either exposed to water stress cycles or fully hydrated. No significant seasonal trends were observed in lichen water relations parameters. Turgor loss point and osmotic potential of the whole thallus were significantly lower than those measured in the photobiont, while differences between the water stressed photobiont and controls were not significant. Dehydration-induced drop of F v /F m was correlated with turgor loss, revealing that the photosynthetic activity of lichens partly depends on their turgor level. We provided one of the first quantitative evidences of the influence that turgor status could exert on the functionality of photosynthetic processes in lichens.

Lichens belonging to Parmeliaceae are highly diversified, but most of them share an extremely conserved morpho-chemical trait: the lower cortex is heavily melanized. The adaptive value of this character is still uncertain. Melanins are ubiquitous compounds found in most organisms since they fulfil several biological functions including defense against UV radiation, oxidizing agents, microbial stress, and metal complexation. This work aims to establish whether melanization can affect the elemental content of lichen thalli. The relative abundance of macro- (Ca, K and S) and micro- (Fe, Mn and Zn) nutrients in melanized and non-melanized pseudotissues of nine species was first evaluated by a non-destructive micro-X-ray fluorescence elemental analysis on either the upper and lower cortex, and on the internal medulla, which was artificially exposed to the mechanical removal of the lower cortex. Afterwards, the total concentration of the same elements was measured in composite samples by inductively coupled plasma atomic emission spectroscopy after acidic digestion. In order to verify whether Fe and Zn are chemically bound to the melanized pseudotissues, a sequential elution experiment was performed on two species: the two-side heavily melanized Melanelixia glabratula and the one-side lightly melanized Punctelia subrudecta. The content of Fe and Zn was higher in the melanized species than in the non-melanized ones. Species deprived of their melanized lower cortex showed a sharp decrease in Fe but not in Zn, suggesting that the melanized lower cortex is involved in Fe complexation, whereas Zn is homogeneously distributed throughout the thallus.

The aim of this study was to verify whether it is possible to discriminate between the different pollution sources present in a mixed land use area of NE Italy on the basis of the magnetic properties and the element content of lichen transplants. Thalli of Pseudevernia furfuracea were collected in a pristine area of the South-Eastern Alps and exposed for 2months in 40 sites located at the knots of a 700m step grid covering ca. 40km 2 of a mosaic of agricultural, forested, industrial and urban areas. In this way, the samples could be analyzed after a defined period of time, and compared to pre-exposure conditions. The post-exposure element content and the magnetic data substantially agreed, revealing a rather modest anthropogenic impact on the territory, mostly limited to an industrial park. Since the magnetic mineralogy was homogeneous throughout the entire set of samples, with magnetite-like minerals as the main magnetic carriers, it was not possible to discriminate between PM originating from the different pollution sources. The contribution given by the industrial park could be confirmed by the multivariate analysis of the element data set. Conversely, it was possible to assess the low environmental impact of the largest local industry, a cement plant, located outside the industrial park. Notwithstanding the relatively short time of the survey, P. furfuracea was proven to be an effective accumulator for biomagnetic monitoring studies, its magnetic properties being excellent proxies for heavy metal pollution even when the anthropogenic impact on the territory is low., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The epiphytic lichen Pseudevernia furfuracea is widely used as biomonitor of airborne trace elements and other contaminants and consists of two taxonomic varieties (var. furfuracea and var. ceratea). Here, we assessed the occurrence of inter-varietal differences in the elemental composition of paired samples of var. furfuracea and var. ceratea collected in 20 remote sites of Italian mountains. The concentration of 40 elements was measured by inductively coupled plasma mass spectroscopy, after digestion with HNO 3 and aqua regia. The magnitude of inter-varietal differences compared to the effect of large-scale site-dependent environmental factors (i.e., lithological substrate, host tree species, and altitude) on overall element content was explored by multivariate analysis techniques and tested by generalized linear mixed modeling (GLMM). Further GLMMs were separately fitted for each element testing taxonomic-related variability against uncertainty associated to the analytical procedure. Inter-varietal differences were statistically significant only for Hg and P, with higher content in var. ceratea at most sites, and for Mg and Zn, showing the opposite pattern. Since the elemental composition of P. furfuracea in remote sites was mostly affected by local lithology and climatic conditions, our results confirm that lichen material for active biomonitoring should be collected in a single ecologically homogeneous remote area. We also indicate sites in the Eastern Alps where P. furfuracea showed the minimum content of most elements, which are suggested as locations to collect lichen material for transplants. Besides the context-dependency at large spatial scale, variations of elemental composition apparently related to taxonomy, could possibly be due to unequal incidence of morphological traits of the collected material. Further research is needed to clarify this issue, and how it affects bioaccumulation phenomena.

Green roofs are gaining momentum in the arid and semi-arid regions due to their multiple benefits as compared with conventional roofs. One of the most critical steps in green roof installation is the selection of drought and heat tolerant species that can thrive under extreme microclimate conditions. We monitored the water status, growth and survival of 11 drought-adapted shrub species grown on shallow green roof modules (10 and 13cm deep substrate) and analyzed traits enabling plants to cope with drought (symplastic and apoplastic resistance) and heat stress (root membrane stability). The physiological traits conferring efficiency/safety to the water transport system under severe drought influenced plant water status and represent good predictors of both plant water use and growth rates over green roofs. Moreover, our data suggest that high substrate temperature represents a stress factor affecting plant survival to a larger extent than drought per se. In fact, the major cause influencing seedling survival on shallow substrates was the species-specific root resistance to heat, a single and easy measurable trait that should be integrated into the methodological framework for screening and selection of suitable shrub species for roof greening in the Mediterranean., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Trebouxia is the most common lichen-forming genus of aero-terrestrial green algae and all its species are desiccation tolerant (DT). The molecular bases of this remarkable adaptation are, however, still largely unknown. We applied a transcriptomic approach to a common member of the genus, T. gelatinosa, to investigate the alteration of gene expression occurring after dehydration and subsequent rehydration in comparison to cells kept constantly hydrated. We sequenced, de novo assembled and annotated the transcriptome of axenically cultured T. gelatinosa by using Illumina sequencing technology. We tracked the expression profiles of over 13,000 protein-coding transcripts. During the dehydration/rehydration cycle c. 92 % of the total protein-coding transcripts displayed a stable expression, suggesting that the desiccation tolerance of T. gelatinosa mostly relies on constitutive mechanisms. Dehydration and rehydration affected mainly the gene expression for components of the photosynthetic apparatus, the ROS-scavenging system, Heat Shock Proteins, aquaporins, expansins, and desiccation related proteins (DRPs), which are highly diversified in T. gelatinosa, whereas Late Embryogenesis Abundant Proteins were not affected. Only some of these phenomena were previously observed in other DT green algae, bryophytes and resurrection plants, other traits being distinctive of T. gelatinosa, and perhaps related to its symbiotic lifestyle. Finally, the phylogenetic inference extended to DRPs of other chlorophytes, embryophytes and bacteria clearly pointed out that DRPs of chlorophytes are not orthologous to those of embryophytes: some of them were likely acquired through horizontal gene transfer from extremophile bacteria which live in symbiosis within the lichen thallus.

Main Conclusions: A comparative study of isolated vs. lichenized Trebouxia sp. showed that lichenization does not influence the survival capability of the alga to the photo-oxidative stress derived from prolonged desiccation. Coccoid algae in the Trebouxia genus are the most common photobionts of chlorolichens but are only sporadically found in soil or bark outside of a lichen. They all appear to be desiccation tolerant, i.e. they can survive drying to water contents of below 10%. However, little is known about their longevity in the dry state and to which extent lichenization can influence it. Here, we studied the longevity in the dry state of the lichenized alga (LT) Trebouxia sp. in the lichen Parmotrema perlatum, in comparison with axenically grown cultures (CT) isolated from the same lichen. We report on chlorophyll fluorescence emission and reactive oxygen species (ROS) production before desiccation, after 15-45 days in the dry state under different combinations of light and air humidity and after recovery for 1 or 3 days in fully hydrated conditions. Both the CT and the LT were able to withstand desiccation under high light (120 µmol photons m(-2) s(-1) for 14 h per day), but upon recovery after 45 days in the dry state the performance of the CT was better than that of the LT. By contrast, the quenching of excess light energy was more efficient in the LT, at high relative humidities especially. ROS production in the LT was influenced mostly by light exposure, whereas the CT showed an oxidative burst independent of the light conditions. Although lichenization provides benefits that are essential for the survival of the photobiont in high-light habitats, Trebouxia sp. can withstand protracted periods of photo-oxidative stress even outside of a lichen thallus.

Urban trees help towns to cope with climate warming by cooling both air and surfaces. The challenges imposed by the urban environment, with special reference to low water availability due to the presence of extensive pavements, result in high rates of mortality of street trees, that can be increased by climatic extremes. We investigated the water relations and xylem hydraulic safety/efficiency of Quercus ilex trees growing at urban sites with different percentages of surrounding impervious pavements. Seasonal changes of plant water potential and gas exchange, vulnerability to cavitation and embolism level, and morpho-anatomical traits were measured. We found patterns of increasing water stress and vulnerability to drought at increasing percentages of impervious pavement cover, with a consequent reduction in gas exchange rates, decreased safety margins toward embolism development, and increased vulnerability to cavitation, suggesting the occurrence of stress-induced hydraulic deterioration. The amount of impermeable surface and chronic exposure to water stress influence the site-specific risk of drought-induced dieback of urban trees under extreme drought. Besides providing directions for management of green spaces in towns, our data suggest that xylem hydraulics is key to a full understanding of the responses of urban trees to global change., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)

Lichens are among the best biomonitors of airborne pollutants, but surprisingly they reveal high tolerance to ozone (O3). It was recently suggested that this might be due to the high levels of natural defences against oxidative stress, related to their poikilohydric life strategy. The objective of this work is to give a thorough description of the biochemical and physiological mechanisms that are at the basis of the O3-tolerance of lichens. Chlorophyll a fluorescence (ChlaF) emission, histochemical ROS localization in the lichen thallus, and biochemical markers [enzymes and antioxidants involved in the ascorbate/glutathione (AsA/GSH) cycle; hydrogen peroxide (H2O2) and superoxide anion (O2(-))] were used to characterize the response of the epiphytic lichen Flavoparmelia caperata (L.) Hale exposed to O3 (250 ppb, 5 hd(-1), 2 weeks) at different watering regimes and air relative humidity (RH) in a fumigation chamber. After two-week exposure ChlaF was affected by the watering regime but not by O3. The watering regime influenced also the superoxide dismutase activity and the production of ROS. By contrast O3 strongly influenced the AsA/GSH biochemical pathway, decreasing the reduced ascorbate (AsA) content and increasing the enzymatic activity of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) independently from the watering regime and the relative humidity applied. This study highlights that F. caperata can face the O3-induced oxidative stress thanks to high levels of constitutive enzymatic and non-enzymatic defences against ROS formed naturally during the dehydration-rehydration cycles to which lichens are frequently exposed., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Graphing graphene: Because the naming of graphene-based materials (GBMs) has led to confusion and inconsistency, a classification approach is necessary. Three physical-chemical properties of GBMs have been defined by the GRAPHENE Flagship Project of the European Union for the unequivocal classification of these materials (see grid)., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In mountain areas of touristic interest the evaluation of the impact of human activities is crucial for ensuring long-term conservation of ecosystem biodiversity, functions and services. This study aimed at verifying the biological impact of polycyclic aromatic hydrocarbon (PAH) emissions due to traffic along the roads leading to seven passes of the Dolomites (SE Alps), which were recently declared a UNESCO World Heritage Site. Thalli of the epiphytic lichen Pseudevernia furfuracea, collected at increasing distances from the roads, were used as biomonitors. Our study revealed a gradient of decreasing PAH pollution within 300 m from the roads. Differences among passes were evident mainly for samples collected nearest to the roads, but PAH concentrations at 300 m were almost always higher than those of undisturbed reference sites, indicating that traffic PAH pollution may impact natural ecosystems and lichen diversity at relatively long distances from the emission source., (Copyright © 2014 Elsevier B.V. All rights reserved.)

CO2 gas exchange, radial growth, chlorophyll (Chl) content and photobiont density of an epiphytic population of Parmelia sulcata were monitored every 2 months during 1 year in a temperate deciduous forest of Central Italy, to verify possible seasonal variations. Light response curves of south-exposed thalli, built up in the laboratory at 6 and 27°C at optimal thallus hydration, showed that CO2 gas exchange changed significantly during the year, with a maximum for gross photosynthesis in December at both temperatures. Photoinhibition phenomena occurred in early spring, immediately before tree leaves sprouted. The principal component analysis of CO2 gas exchange parameters clearly separated the months with from the months without tree canopy cover. Radial growth, measured on marginal lobes of north- and south-exposed thalli, was the highest in December, and the lowest in April. Photobiont density, measured in lobes of south- and north-exposed thalli with a sedimentation chamber, also changed during the year: the number of photobionts was highest in June and December, and lowest in April, although no significant change in cell size and Chl content per cell was evident throughout the year. South-exposed thalli had slightly, but constantly higher photobiont density both on a weight and an area basis. The acclimation of lichen photosynthesis and Chl content to seasonal temperature and light changes should partially be re-visited on the basis of the significant variation in photobiont population density. This phenomenon still awaits, however, a satisfactory explanation, although it is probably related to the seasonal change in nutrient availability.

The control of lichen growth, particularly important in the field of stone conservation of outdoor monuments, largely depends on the use of biocides, that may be dangerous for the users, the environment and the substratum. A new, alternative approach is proposed, which makes the most of a poorly known peculiarity of poikilohydrous organisms: they are thermo-tolerant (up to 65-70 °C) when dry, but thermo-sensitive when wet. The efficacy of thermal treatments (range: 20-55 °C), in parallel to the application of three biocides, was verified in the laboratory with six epi- and endolithic lichens. Chlorophyll a fluorescence emission was checked in treated and nontreated samples of all the species, whereas histochemical observations with a dead cell stain were carried out on one of them. The feasibility of the thermal treatments in the field was verified with a seventh species. The results confirm that a 6 h treatment at 55 °C is sufficient to kill the lichens if they are kept fully hydrated. At 40 °C the organisms are damaged: in this case biocides at concentrations 10× lower than in normal applications can profitably be used. The new protocol is simple, the field equipment cheap, and the negative effects associated with standard biocide treatments are absent.

The hypothesis that a daily water supply allows a lichen to endure the negative effects of environmental concentrations of NO(x) and O(3) was tested with a transplant experiment. Five groups (0, A-D) of Flavoparmelia caperata samples derived from the same thalli were used for destructive, pre-exposure measurements (0), or exposed for 5 weeks in the rural collection site (A), and in a urban site with high levels of NO(x) and O(3) (B-D). Two groups (C, D) were daily watered half an hour before the daily peak of NO(x) (C), and O(3) (D). The comparison between pre- and post-exposure measurements of stress biomarkers revealed that the different thallus hydration regime modified the pollution tolerance as well as the physiology of the exposed samples. The non-watered group B suffered an evident decrease in F(v)/F(m) and reduced glutathione, but increased ion leakage, whereas the watered groups C and D showed only decreased non-photosynthetic-quenching, possibly derived from NO(x) exposure. Ozone, which was higher in the rural than in the urban site, did not significantly affect the lichen metabolism. Our results re-open the discussion on the so-called "drought hypothesis", which suggests that the lichen desert observed in urban areas of central and eastern Europe is more a matter of dry microclimate than of air pollution.

A lichen transplant study aimed at investigating a strong increase in mercury concentrations in lichens was run in a territory of NE Italy where background values were very low only 8 years before. Thalli of the lichen Pseudevernia furfuracea collected in a pristine area were exposed for 1.5, 3 and 6 months at 31 sites selected according to the observed pattern of Hg concentrations, location of the suspected source (a new waste incinerator) and prevailing wind direction. Hg strongly increased at eight sites after 1.5 months, at 12 after 3 months and at 20 after 6 months. The highest values were always located SW and S of the incinerator, in good agreement with the prevailing night wind direction. It was concluded that, although the immediate risk for the population living close to the incinerator is low, long-term hazard due to Hg accumulation in the surrounding environment should be seriously taken into account.

Purpose: To assess the histopathologic changes in a postmortem sample derived from an eye donor with macular telangiectasia (MacTel) type 2 to gain further insight into the cause of the disease., Design: Clinicopathological case report., Participants: Postmortem tissue was collected from 5 different donors: 1 MacTel type 2 patient; 1 healthy control; 2 type 2 diabetic patients, 1 with retinopathy and 1 without retinopathy; and 1 patient with unilateral Coat's disease., Methods: Macular pigment distribution in the posterior part of freshly dissected eyes was documented by macrophotography. Paraffin sections from both the macular and peripheral regions were assessed using antigen retrieval and immunohistochemistry to study the distribution of cell-specific markers. Blood vessels were visualized with antibodies directed against collagen IV and claudin 5; glial cells with antibodies against glial fibrillary acidic protein (GFAP), vimentin, glutamine synthetase (GS), and retinaldehyde binding protein (RLBP1, also known as CRALBP); microglia with an antibody against allograft inflammatory factor 1 (also known as Iba1); and photoreceptors with antibodies against rhodopsin and opsin. Using anatomic landmarks, the sections then were matched with the macular pigment distribution and a fluorescein angiogram of the patient that was obtained before the patient's death., Main Outcome Measures: Presence and distribution of macular pigment and cell-specific markers., Results: Macular pigment was absent in the macula. Furthermore, abnormally dilated capillaries were identified in a macular region that correlated spatially with regions of fluorescein leakage in an angiogram that was obtained 12 years before death. These telangiectatic vessels displayed a marked reduction of the basement membrane component collagen IV, indicating vascular pathologic features. The presence of GFAP was limited to retinal astrocytes, and no reactive Müller cells were identified. Importantly, reduced immunoreactivity with Müller cell markers (vimentin, GS, and RLBP1) in the macula was observed. The area that lacked Müller cells corresponded with the region of depleted macular pigment., Conclusions: These findings suggest that macular Müller cell loss or dysfunction is a critical component of MacTel type 2, which may have implications for future treatment strategies., (Copyright © 2010 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

The hypothesis that CO(2) gas exchange and chlorophyll a fluorescence (ChlaF) of lichens vary according to the light regimes of their original habitat, as observed in vascular plants, was tested by analysing the photosynthetic performance of 12 populations of seven dorsoventral, foliose lichens collected from open, south-exposed rocks to densely shaded forests. Light response curves were induced at optimum thallus water content and ChlaF emission curves at the species-specific photon flux at which the quantum yield of CO(2) assimilation is the highest and is saturating the photosynthetic process. Photosynthetic pigments were quantified in crude extracts. The results confirm that the maximum rate of gross photosynthesis is correlated with the chlorophyll content of lichens, which is influenced by light as well as by nitrogen availability. Like leaves, shade tolerant lichens emit more ChlaF than sun-loving ones, whereas the photosynthetic quantum conversion is higher in the latter.

Sterile cultured isolates of lichen-forming aposymbionts have not yet been used to investigate lichen-rock interactions under controlled conditions. In this study mycobionts and photobiont of the endolithic lichens Bagliettoa baldensis and Bagliettoa marmorea were isolated and inoculated with coupons of one limestone and four marbles commonly employed in the Cultural Heritage framework. After one year of incubation, microscopic observations of polished cross-sections were performed to verify if the typical colonization patterns observed in the field may be reproduced in vitro and to evaluate the receptivity of the five lithotypes to endolithic lichens. The mycobionts of the two species developed both on the surface of and within all the lithotypes, showing different penetration pathways which depend on mineralogical and structural features and highlight different receptivity. By contrast, algae inoculated with the coupons did not penetrate them. Observations suggest that the hyphal penetration along intrinsic discontinuities of rocks is a relatively fast phenomenon when these organisms are generally considered as slow-growing. Samples from limestone outcrops and abandoned marble quarries, colonized by the same species or other representatives of Verrucariaceae, showed penetration pathways intriguingly similar to those reproduced in vitro and highlighted that lichen-driven erosion processes only increase the availability of hyphal passageways after a long-term colonization. These results show that in vitro incubation of sterile cultured lichen-forming ascomycetes with rock coupons is a practicable experimental system to investigate the lichen-rock interactions under controlled conditions and, together with analysis in situ, may support decisions on conservative treatments of historical and cultural significant stone substrata.

A revision based on the morphological and genetic analyses of 133 specimens of black-fruited, endolithic Caloplaca belonging to subgenus Pyrenodesmia is presented. The material was collected in 16 sites distributed along a transept from Gargano (Central Italy) to the southeastern Alps, from sea level to ca 1500m. The nuclear ITS was sequenced for all the mycobionts and selected representatives of photobionts. Except for the sorediate C. erodens, all species share the same algal lineage of Trebouxia as photobiont. The haplotype analysis of the mycobionts revealed an unexpected, high genetic heterogeneity. Three main morphotypic clusters were recognized among five species: C. albopruinosa (syn. C. agardhiana auct.), C. alociza, C. badioreagens, C. erodens, and C. variabilis. A phylogenetic analysis, including already available Caloplaca sequence data, revealed that these lichens form a monophyletic group within the genus. For each species, notes on ecology, distribution in Italy, and nomenclature are given.

Transplants of Flavoparmelia caperata (L.) Hale were used to test possible relationships between chlorophyll a fluorescence (CaF) and ambient atmospheric conditions (temperature, precipitation, SO2 and NOx levels). Portions of the same thalli collected in a pristine site (A) of the Trieste Karst were exposed at that site, as the control, and in four other sites (B-E) in NE Italy, near to pollution monitoring stations. These sites had been selected in order to provide similar two by two climatic conditions (sites B,C: more humid; D,E: drier) and air pollution load (sites B,D: low; C,E: high). Before exposure and after 43 and 90 days of exposure, CaF measurements were carried out in the laboratory under controlled conditions. A classification of meteorological and pollution parameters recorded during exposure substantially confirmed the differences between site couplets. After 90 days, samples from sites A (control) and B (with very low pollution load) showed only slightly reduced NPQ, qN, Fo, and Fm values. Samples from site D, with medium air pollution load, and sites C,E, with high air pollution loads, showed proportionally greater variation for most of the CaF parameters. A highly significant correlation was found between NPQ, qN, Fm, and NOx pollution but not with SO2 or O3. Effects of NOx on lichens and possible action mechanisms are discussed. The results strongly suggest that CaF measurements of lichen transplants can be a valid tool in biomonitoring studies.