Molecular and morphological diversity ofTrebouxiamicroalgae in sphaerothallioidCircinariaspp. lichens1

Three vagrant (Circinaria hispida, Circinaria gyrosa, and Circinaria sp. 'paramerae') and one crustose (semi-vagrant, Circinaria sp. 'oromediterranea') lichens growing in very continental areas in the Iberian Peninsula were selected to study the phycobiont diversity. Mycobiont identification was checked using nrITS DNA barcoding: Circinaria sp. 'oromediterranea' and Circinaria sp. 'paramerae' formed a new clade. Phycobiont diversity was analyzed in 50 thalli of Circinaria spp. using nrITS DNA and LSU rDNA, with microalgae coexistence being found in all the species analyzed by Sanger sequencing. The survey of phycobiont diversity showed up to four different Trebouxia spp. as the primary phyc…

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

Lichens are adapted to desiccation/rehydration and accumulate heavy metals, which induce ROS especially from the photobiont photosynthetic pigments. Although their mechanisms of abiotic stress tolerance are still to be unravelled, they seem related to symbionts' reciprocal upregulation of antioxidant systems. With the aim to study the effect of Pb on oxidative status during rehydration, the kinetics of intracellular ROS, lipid peroxidation and chlorophyll autofluorescence of whole Ramalina farinacea thalli and its isolated microalgae (Trebouxia TR1 and T. TR9) was recorded. A genetic characterization of the microalgae present in the thalli used was also carried out in order to assess possib…

Untangling the hidden intrathalline microalgal diversity inParmotrema pseudotinctorum:Trebouxia crespoanasp. nov.

AbstractIntrathalline phycobiont diversity was investigated in a rosette-forming lichen,Parmotrema pseudotinctorum, using a combination of Sanger sequencing, 454-pyrosequencing, conventional light and confocal microscopy, and transmission electron microscopy. A total of 39 thalli sampled in five Canary Island populations were investigated. Three novel lineages of lichen phycobionts were detected, all being inferred within theTrebouxiaclade G. The most abundant phycobiont lineage, occurring in all lichen populations investigated, is described here asTrebouxia crespoanasp. nov. This species produces spherical to pyriform cells possessing a crenulate chloroplast with lobes elongated at their e…

Balanced Gene Losses, Duplications and Intensive Rearrangements Led to an Unusual Regularly Sized Genome in Arbutus unedo Chloroplasts

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 geno…

Preliminary assessment of terrestrial microalgae isolated from lichens as testing species for environmental monitoring: Lichen phycobionts present high sensitivity to environmental micropollutants

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 phyco…

A new method to isolate lichen algae by using percoll® gradient centrifugation

AbstractA 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.

Changes in net photosynthesis, chlorophyll fluorescence and xanthophyll cycle interconversions during freeze-thaw cycles in the Mediterranean moss Leucodon sciuroides

The tolerance to freezing and thawing of Leucodon sciuroides, a moss growing in mountainous areas of the Mediterranean (south-east Spain), was investigated by means of CO2 gas exchange, modulated chlorophyll (Chl) a fluorescence and pigment analysis by high-performance liquid chromatography. Evidence is presented for freezing-induced decreases in CO2 fixation that enhance non-radiative dissipation of absorbed light energy, a process which protects the photosynthetic apparatus. The photosynthetic apparatus of L. sciuroides remained fully recuperable after freezing, as indicated by the recovery of photosynthetic CO2 fixation and Chl fluorescence parameters to pre-freezing values during thawin…

Global Soil Biodiversity Atlas

SPE EA Pôle BIOME The Atlas is divided in 8 chapters covering all the aspects of soil biodiversity: - Chapter I: The soil habitat - Chapter II: Diversity of soil organisms - Chapter III: Geographical and temporal distribution - Chapter IV: Ecosystem functions and services - Chapter V: Threats - Chapter VI: Interventions - Chapter VII: Policy, education and outreach - Chapter VIII: Conclusions Soil biodiversity experts from all over the world are involved in the project aiming at the creation of a reference publication not only for soil biodiversity researchers but also policy makers and general public.; International audience; The Global Soil Biodiversity Initiative (GSBI) and the Joint Res…

Response of Spinach Leaves (Spinacia oleracea L.) to Ozone Measured by Gas Exchange, Chlorophyll a Fluorescence, Antioxidant Systems, and Lipid Peroxidation

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 c…

The impact of dehydration rate on the production and cellular location of reactive oxygen species in an aquatic moss.

† 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 produc…

LOOKING AT LICHENS

Effects of SO2 fumigations on photosynthetic CO2 gas exchange, chlorophyll a fluorescence emission and antioxidant enzymes in the lichens Evernia prunastri and Ramalina farinacea

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 activi…

Effects of long-term ozone exposure on citrus: Chlorophyll a fluorescence and gas exchange

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 phot…

Acclimation Potential to High Irradiance of Two Cultivars of Watermelon

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. A…

A single primer pair gives a specific ortholog amplicon in a wide range of Cyanobacteria and plastid-bearing organisms: applicability in inventory of reference material from collections and phylogenetic analysis.

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 phylogen…

Chlorophyll A Fluorescence and Chlorophyll Content in Parmelia Quercina Thalli from a Polluted Region of Northern Castellon (Spain)

AbstractMeasurements 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 …

The organic air pollutant cumene hydroperoxide interferes with NOantioxidant role in rehydrating lichen

Organic pollutants effects on lichens have not been addressed. Rehydration is critical for lichens, a burst of free radicals involving NO occurs. Repeated dehydrations with organic pollutants could increase oxidative damage. Our aim is to learn the effects of cumene hydroperoxide (CP) during lichen rehydration using Ramalina farinacea (L.) Ach., its photobiont Trebouxia spp. and Asterochloris erici. Confocal imaging shows intracellular ROS and NO production within myco and phycobionts, being the chloroplast the main source of free radicals. CP increases ROS, NO and lipid peroxidation and reduces chlorophyll autofluorescence, although photosynthesis remains unaffected. Concomitant NO inhibit…

Suitability of chloroplast LSU rDNA and its diverse group I introns for species recognition and phylogenetic analyses of lichen-forming Trebouxia algae

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 Trebouxi…

An automatic system for stepwise treatment of solid samples and application to pollution evaluation by measuring ion lixiviation rates in lichens

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…

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

The lichens Parmelia quercina, Parmelia sulcata, Evernia prunastri, Hypogymnia physodes, and Anaptychia ciliaris were exposed to ozone (O3) in controlled environment cuvettes designed to maintain the lichens at optimal physiological activity during exposure. Measurements of gas exchange, modulated chlorophyll (Chl) fluorescence, and pigment analysis were conducted before and after exposure to 300 mm3 (O3) m−3, 4 h per d for 14 d. No changes in the efficiency of photosystem 2 (PS2) photochemistry, the reduction state of 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 assimila…

Biomonitoring with cryptogams near the power station of la Robla (Leon, Spain)

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 …

Interactions between nitrogen fertilization and ozone in watermelon cultivar Reina de Corazones in open-top chambers. Effects on chlorophyll alpha fluorescence, lipid peroxidation, and yield

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…

Releasing rates of inorganic ions in lichens monitored by capillary zone electrophoresis as indicators of atmospheric pollution

Abstract 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 locat…