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Background Sequencing of marker genes amplified from environmental samples, known as amplicon sequencing, allows us to resolve some of the hidden diversity and elucidate evolutionary relationships and ecological processes among complex microbial communities. The analysis of large numbers of samples at high sequencing depths generated by high throughput sequencing technologies requires efficient, flexible, and reproducible bioinformatics pipelines. Only a few existing workflows can be run in a user-friendly, scalable, and reproducible manner on different computing devices using an efficient workflow management system. Results We present Natrix, an open-source bioinformatics workflow for preprocessing raw amplicon sequencing data. The workflow contains all analysis steps from quality assessment, read assembly, dereplication, chimera detection, split-sample merging, sequence representative assignment (OTUs or ASVs) to the taxonomic assignment of sequence representatives. The workflow is written using Snakemake, a workflow management engine for developing data analysis workflows. In addition, Conda is used for version control. Thus, Snakemake ensures reproducibility and Conda offers version control of the utilized programs. The encapsulation of rules and their dependencies support hassle-free sharing of rules between workflows and easy adaptation and extension of existing workflows. Natrix is freely available on GitHub (https://github.com/MW55/Natrix) or as a Docker container on DockerHub (https://hub.docker.com/r/mw55/natrix). Conclusion Natrix is a user-friendly and highly extensible workflow for processing Illumina amplicon data.

Factors shaping community patterns of microorganisms are controversially discussed. Physical and chemical factors certainly limit the survival of individual taxa and maintenance of diversity. In recent years, a contribution of geographic distance and dispersal barriers to distribution patterns of protists and bacteria has been demonstrated. Organismic interactions such as competition, predation and mutualism further modify community structure and maintenance of distinct taxa. Here, we address the relative importance of these different factors in shaping protists and bacterial communities on a European scale using high‐throughput sequencing data obtained from lentic freshwater ecosystems. We show that community patterns of protists are similar to those of bacteria. Our results indicate that cross‐domain organismic factors are important variables with a higher influence on protists as compared with bacteria. Abiotic physical and chemical factors also contributed significantly to community patterns. The contribution of these latter factors was higher for bacteria, which may reflect a stronger biogeochemical coupling. The contribution of geographical distance was similar for both microbial groups.

Here, we analyzed patterns of taxon richness and endemism of freshwater protists in Europe. Even though the significance of physicochemical parameters but also of geographic constraints for protist distribution is documented, it remains unclear where regional areas of high protist diversity are located and whether areas of high taxon richness harbor a high proportion of endemics. Further, patterns may be universal for protists or deviate between taxonomic groups. Based on amplicon sequencing campaigns targeting the SSU and ITS region of the rDNA we address these patterns at two different levels of phylogenetic resolution. Our analyses demonstrate that protists have restricted geographical distribution areas. For many taxonomic groups the regions of high taxon richness deviate from those having a high proportion of putative endemics. In particular, the diversity of high mountain lakes as azonal habitats deviated from surrounding lowlands, i.e. many taxa were found exclusively in high mountain lakes and several putatively endemic taxa occurred in mountain regions like the Alps, the Pyrenees or the Massif Central. Beyond that, taxonomic groups showed a pronounced accumulation of putative endemics in distinct regions, e.g. Dinophyceae along the Baltic Sea coastline, and Chrysophyceae in Scandinavia. Many other groups did not have pronounced areas of increased endemism but geographically restricted taxa were found across Europe.

Small bacterivorous eukaryotes play a cardinal role in aquatic food webs and their taxonomic classification is currently a hot topic in aquatic microbial ecology. Despite increasing interest in their diversity, core questions regarding predator–prey specificity remain largely unanswered, e.g., which heterotrophic nanoflagellates (HNFs) are the main bacterivores in freshwaters and which prokaryotes support the growth of small HNFs. To answer these questions, we fed natural communities of HNFs from Římov reservoir (Czech Republic) with five different bacterial strains of the ubiquitous betaproteobacterial genera Polynucleobacter and Limnohabitans. We combined amplicon sequencing and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) targeting eukaryotic 18 S rRNA genes to track specific responses of the natural HNF community to prey amendments. While amplicon sequencing provided valuable qualitative data and a basis for designing specific probes, the number of reads was insufficient to accurately quantify certain eukaryotic groups. We also applied a double-hybridization technique that allows simultaneous phylogenetic identification of both predator and prey. Our results show that community composition of HNFs is strongly dependent upon prey type. Surprisingly, Cryptophyta were the most abundant bacterivores, although this phylum has been so far assumed to be mainly autotrophic. Moreover, the growth of a small lineage of Cryptophyta (CRY1 clade) was strongly stimulated by one Limnohabitans strain in our experiment. Thus, our study is the first report that colorless Cryptophyta are major bacterivores in summer plankton samples and can play a key role in the carbon transfer from prokaryotes to higher trophic levels.

Protists influence ecosystems by modulating microbial population size, diversity, metabolic outputs and gene flow. In this study we used eukaryotic ribosomal amplicon diversity from 218 European freshwater lakes sampled in August 2012 to assess the effect of mountain ranges as biogeographic barriers on spatial patterns and microbial community structure in European freshwaters. The diversity of microbial communities as reflected by amplicon clusters suggested that the eukaryotic microbial inventory of lakes was well-sampled at the European and at the local scale. Our pan-European diversity analysis indicated that biodiversity and richness of high mountain lakes differed from that of lowland lakes. Further, the taxon inventory of high-mountain lakes strongly contributed to beta-diversity despite a low taxon inventory. Even though ecological factors, in general, strongly affect protist community pattern, we show that geographic distance and geographic barriers significantly contribute to community composition particularly for high mountain regions which presumably act as biogeographic islands. However, community composition in lowland lakes was also affected by geographic distance but less pronounced as in high mountain regions. In consequence protist populations are locally structured into distinct biogeographic provinces and community analyses revealed biogeographic patterns also for lowland lakes whereby European mountain ranges act as dispersal barriers in particular for short to intermediate distances whereas the effect of mountain ranges levels off on larger scale. CA Boenigk

OA hybrid Inferring ecosystem functioning and ecosystem services through inspections of the species inventory is a major aspect of ecological field studies. Ecosystem functions are often stable despite considerable species turnover. Using metatranscriptome analyses, we analyse a thus‐far unparalleled freshwater data set which comprises 21 mainland European freshwater lakes from the Sierra Nevada (Spain) to the Carpathian Mountains (Romania) and from northern Germany to the Apennines (Italy) and covers an altitudinal range from 38 m above sea level (a.s.l) to 3110 m a.s.l. The dominant taxa were Chlorophyta and streptophytic algae, Ciliophora, Bacillariophyta and Chrysophyta. Metatranscriptomics provided insights into differences in community composition and into functional diversity via the relative share of taxa to the overall read abundance of distinct functional genes on the ecosystem level. The dominant metabolic pathways in terms of the fraction of expressed sequences in the cDNA libraries were affiliated with primary metabolism, specifically oxidative phosphorylation, photosynthesis and the TCA cycle. Our analyses indicate that community composition is a good first proxy for the analysis of ecosystem functions. However, differential gene regulation modifies the relative importance of taxa in distinct pathways. Whereas taxon composition varies considerably between lakes, the relative importance of distinct metabolic pathways is much more stable, indicating that ecosystem functioning is buffered against shifts in community composition through a functional redundancy of taxa.

Shifts in the nutritional mode between phototrophy, mixotrophy and heterotrophy are a widespread phenomenon in the evolution of eukaryotic diversity. The transition between nutritional modes is particularly pronounced in chrysophytes and occurred independently several times through parallel evolution. Thus, chrysophytes provide a unique opportunity for studying the molecular basis of nutritional diversification and of the accompanying pathway reduction and degradation of plastid structures. In order to analyze the succession in switching the nutritional mode from mixotrophy to heterotrophy, we compared the transcriptome of the mixotrophic Poterioochromonas malhamensis with the transcriptomes of three obligate heterotrophic species of Ochromonadales. We used the transcriptome of P. malhamensis as a reference for plastid reduction in the heterotrophic taxa. The analyzed heterotrophic taxa were in different stages of plastid reduction. We investigated the reduction of several photosynthesis related pathways e.g. the xanthophyll cycle, the mevalonate pathway, the shikimate pathway and the tryptophan biosynthesis as well as the reduction of plastid structures and postulate a presumable succession of pathway reduction and degradation of accompanying structures., Different pathway repertoirs in heterotrophic chrysophytes indicate the stepwise loss of photosynthesis related pathways in the course of evolution of heterotrophy.

Stream ecosystems are affected by multiple anthropogenic stressors worldwide. Even though effects of many single stressors are comparatively well studied, the effects of multiple stressors are difficult to predict. In particular bacteria and protists, which are responsible for the majority of ecosystem respiration and element flows, are infrequently studied with respect to multiple stressors responses. We conducted a stream mesocosm experiment to characterize the responses of single and multiple stressors on microbiota. Two functionally important stream habitats, leaf litter and benthic phototrophic rock biofilms, were exposed to three stressors in a full factorial design: fine sediment deposition, increased chloride concentration (salinization) and reduced flow velocity. We analyzed the microbial composition in the two habitat types of the mesocosms using an amplicon sequencing approach. Community analysis on different taxonomic levels as well as principle component analyses (PCoAs) based on realtive abundances of operational taxonomic units (OTUs) showed treatment specific shifts in the eukaryotic biofilm community. Analysis of variance (ANOVA) revealed that Bacillariophyta responded positively salinity and sediment increase, while the relative read abundance of chlorophyte taxa decreased. The combined effects of multiple stressors were mainly antagonistic. Therefore, the community composition in multiply stressed environments resembled the composition of the unstressed control community in terms of OTU occurrence and relative abundances.

Background Most protist lineages consist of members with diverging features e.g. different modes of nutrition and adaptations for life in different habitat types and climatic zones. The nutritional mode is particularly variable in chrysophytes and they are therefore an excellent model group to study the core genes and metabolic pathways of a functionally diverse lineage. The objective of our study is the identification of the joint genetic repertoire expressed in closely related chrysophytes as well as the extent of variation on species and strain level. Therefore, we investigated the transcriptomes of six strains belonging to four species of Ochromonadales. We performed analyses on metabolic pathway level as well as on sequence level. Results We could identify 1,574 core genes shared between all six investigated strains of Ochromonadales. Most of these core genes were affiliated with the primary metabolism. Phylogenetic analysis of 166 protein-coding core genes supported a close relation of Poteriospumella lacustris and Poterioochromonas malhamensis and resolved for more than 50% of investigated genes the relationship of strains affiliated with the species P. lacustris. Further, we found diverging phylogenetic patterns for genes interacting with the environment. Conclusions In Ochromonadales, a functionally diverse lineage, the core transcriptome represents only a minor part of the individual transcriptomes. But this small fraction of genes comprises the basal metabolism essential for life in several protist lineages. Phylogenetic analyses of these genes indicate a similar degree of conservation as observed for genes coding for ribosomal proteins.

Aquatic environments are heavily impacted by human activities including climate warming and the introduction of xenobiotics. Due to the application of silver nanoparticles as bactericidal agent the introduction of silver into the environment strongly has increased during the past years. Silver ions affect the primary metabolism of algae, in particular photosynthesis. Mixotrophic algae are an interesting test case as they do not exclusively rely on photosynthesis which may attenuate the harmful effect of silver. In order to study the effect of silver ions on mixotrophs, cultures of the chrysophyte Poterioochromonas malhamensis were treated in a replicate design in light and darkness with silver nitrate at a sub-lethal concentration. At five time points samples were taken for the identification and quantitation of proteins by mass spectrometry. In our analysis, relative quantitative protein mass spectrometry has shown to be a useful tool for functional analyses in conjunction with transcriptome reference sequences. A total of 3,952 proteins in 63 samples were identified and quantified, mapping to 4,829 transcripts of the sequenced and assembled transcriptome. Among them, 720 and 104 proteins performing various cellular functions were differentially expressed after eight days in light versus darkness and after three days of silver treatment, respectively. Specifically pathways of the energy and primary carbon metabolism were differentially affected by light and the utilization of expensive reactions hints to an energy surplus of P. malhamensis under light conditions. The excess energy is not invested in growth, but in the synthesis of storage metabolites. The effects of silver were less explicit, observable especially in the dark treatments where the light effect could not mask coinciding but weaker effects of silver. Photosynthesis, particularly the light harvesting complexes, and several sulphur containing enzymes were affected presumably due to a direct interference with the silver ions, mainly affecting energy supply. OA gold

Ecological gradients are one of the main reasons for changes in biodiversity. However, whether what is known to be true for plants and animals also applies to protists in the same way is largely unknown. Along an alpine elevation gradient, including 29 freshwater lakes, we investigated ecological drivers of protistan diversity and community structure, as well as the explanatory value of the species-area relationship for protistan diversity using a deep-sequencing approach. We found that species richness does not decrease with elevation, nor is elevation a major factor explaining the observed shifts in community composition. The observed protistan communities differ depending on many factors, with pH and nutrient concentrations being most important. Considering distinct subgroups, Chrysophyceae accurately reflect the pattern of the overall protistan community. Within the investigated elevation gradient, species richness was correlated with the area of individual lakes irrespective of their elevation. However, we found no support for decreasing species richness with elevation, as known for plants and animals. OA hybrid

Summary 1. Plants express multiple defensive traits, but little is known about the genetic stability and phenotypic plasticity of these traits in nature. To investigate sources of variation and their potential ecological consequences for herbivores, we combined field observations of cyanogenic lima bean with laboratory experiments. 2. Field studies in South Mexico revealed a distinct variability of cyanogenic traits within and among wild lima bean populations. To differentiate among genetic variation and the impact of ambient conditions on plant phenotypes, we used seed-grown plants as well as clones propagated from high- (HC) and low-cyanogenic (LC) wild type plants. 3. In growth chamber experiments, we cultivated plants under three intensities each of drought and salt stress, nutrient supply, and light availability. We consecutively quantified cyanogenesis and total phenolics as chemical defences, soluble proteins as measure of leaf quality, and leaf mass per area and tissue hydration as physical defence-associated traits. 4. Plant traits were genetically stable, as clones propagated from the HC- and LC-mother plants maintained their cyanogenic features and also showed constancy of other leaf parameters tested. 5. In contrast to genetically heterogeneous seed-grown plants, cyanogenesis and soluble protein concentration in clonal plants showed significant variation in response to reduced water supply, increased soil salinity, and fertilization. The other leaf traits, however, showed no significant phenotypic plasticity depending on treatment. 6. Among all traits analysed, genetic and phenotypic variation in cyanogenesis proved the best predictor of herbivore behaviour and development, as LC-plants were preferred by adult Mexican bean beetles and allowed for faster larval development. 7. Synthesis. We demonstrate that (i) functional analyses of plant responses to abiotic factors require methodical separation of genotypic variability and phenotypic plasticity, (ii) different abiotic parameters quantitatively affect the plants’ chemical phenotype and that (iii) changes of plant phenotype can have strong impacts on natural herbivores. Our results suggest that genetic variability and phenotypic plasticity of plant anti-herbivore defences allow plant populations to rapidly respond to changing environmental conditions.

Ontogenetic variability in chemical plant defenses against herbivores is a common phenomenon, but the effects of this variability on herbivore-plant interactions are little understood. In a previous study on lima bean (Phaseolus lunatus), we found a trade-off between cyanogenesis, a direct defense, and the release of herbivore-induced volatile organic compounds (VOCs; mainly functioning as an indirect defense). Moreover, the expression of these two defenses could change during plant ontogeny. The present study aimed at elucidating whether such ontogenetic changes in plant defense can affect herbivore-plant interactions. We quantified feeding rates of a natural insect herbivore, the Mexican bean beetle (Epilachna varivestis), on primary and secondary leaves of individual lima bean plants. These insects strongly preferred low cyanogenic primary leaves over high cyanogenic secondary leaves. Although weakly defended by cyanogenesis, lima beans' primary leaves showed protein concentrations and photosynthetic activities that did not differ significantly from secondary leaves at the time of analysis. Based on our findings, we suggest that lima beans' long-lived primary leaves function as efficient source organs, even beyond the stage of seedlings. This hypothesis may explain why primary leaves express a strong indirect defense by the release of herbivore induced VOCs.

Protists are perhaps the most lineage-rich of microbial lifeforms, but remain largely unknown. High-throughput sequencing technologies provide opportunities to screen whole habitats in depth and enable detailed comparisons of different habitats to measure, compare and map protistan diversity. Such comparisons are often limited by low sample numbers within single studies and a lack of standardisation between studies. Here, we analysed 232 samples from 10 sampling campaigns using a standardised PCR protocol and bioinformatics pipeline. We show that protistan community patterns are highly consistent within habitat types and geographic regions, provided that sample processing is standardised. Community profiles are only weakly affected by fluctuations of the abundances of the most abundant taxa and, therefore, provide a sound basis for habitat comparison beyond random short-term fluctuations in the community composition. Further, we provide evidence that distribution patterns are not solely resulting from random processes. Distinct habitat types and distinct taxonomic groups are dominated by taxa with distinct distribution patterns that reflect their ecology with respect to dispersal and habitat colonisation. However, there is no systematic shift of the distribution pattern with taxon abundance.

Gas exchange is generally regarded to occur between the leaf interior and ambient air, i.e. in vertical (anticlinal) directions of leaf blades. However, inside homobaric leaves, gas movement occurs also in lateral directions. The aim of the present study was to ascertain whether lateral CO 2 diffusion affects leaf photosynthesis when illuminated leaves are partially shaded. • Measurements using gas exchange and chlorophyll fluorescence imaging techniques were performed on homobaric leaves of Vicia faba and Nicotiana tabacum or on heterobaric leaves of Glycine max and Phaseolus vulgaris . • For homobaric leaves, gas exchange inside a clamp-on leaf chamber was affected by shading the leaf outside the chamber. The quantum yield of photosystem II ( Φ PSII ) was highest directly adjacent to a light/shade border (LSB). Φ PSII decreased in the illuminated leaf parts with distance from the LSB, while the opposite was observed for nonphotochemical quenching. These effects became most pronounced at low stomatal conductance. They were not observed in heterobaric leaves. • The results suggest that plants with homobaric leaves can benefit from lateral CO 2 flux, in particular when stomata are closed (e.g. under drought stress). This may enhance photosynthetic, instead of nonphotochemical, processes near LSBs in such leaves and reduce the photoinhibitory effects of excess light.

The effects of decreasing water potential (Ψ) on O2 evolution and fluorescence yield at room temperature and at 77 K were investigated using the lichen Lobaria pulmonaria. Changes in Ψ were created either by atmospheric desiccation or by osmotic dehydration, with either sucrose, sorbitol or NaCl as osmoticum. Independent of the method used to establish Ψ, similar inactivation patterns were obtained and were reversible after reincubation in pure water for 10 min. Our data indicate that exposure to increasing water stress acts at two levels. In the first phase, at ‘mild’ stress, i.e. at Ψ greater than −13, −16 and −20 MPa for drying, NaCl and sucrose treatments, respectively, a progressive decline in O2 production and the fluorescence yield (ΔF/Fm′ and Fv/Fm) was correlated with increases in non-photochemical quenching (qN). At the same time the photochemical quenching (qp) changed only sligthly, indicating the absence of overreduction. The Fo level remained relatively constant in this first stage of water loss. A ΔpH mediated down regulation and a donor side limitation of photosystem (PS) II are discussed. When the water stress was severe, a further decrease in the fluorescence yield was observed and correlated with a considerable decrease in Fo (second phase). Kinetic analysis of the 77 K emission showed that osmotic stress and atmospheric desiccation possibly lead to an increased spillover from PS II to PS I. In addition, a strong negative effect of NaF on the recovery from dehydration was found. This may indicate a state transition mediated by the displacement/recoupling of light harvesting complex (LHC) II from/to PS II. The photoprotective role of spatial rearrangements of antenna complexes during desiccation is discussed.

During our investigations of the microflora in NRW (Duisburg, Düsseldorf and Essen incl. the greenhouse of the Botanical Garden) in 2007 and 2008, we were able to collect and identify about 55 species on trees, bushes and ornamental plants as parasites and saprophytes. Some of these species are new for Germany or have been only rarely found until now. Most of the species belong the Ascomycotina, Basidiomycotina and Deuteromycotina for example Arthrocladiella mougeotii (Lév.) Vassilkov. on Lycium barbarum L., Caudospora taleola (Fr.) Starb on Quercus robur L., Colletotrichum coffeanum F. Noak on Coffea arabica L. (new for Germany) Colletotrichum trichellum (Fr.) Duke on Hedera helix L., Erysiphe buhrii U. Braun on Lychnis cf. coronaria (L.) Desr. (Anamorph. Oidium dianthi Jacz.), Erysiphe spec. on Acer opalus Mill (new host), Erysiphe flexuosa (Peck) U. BraunS. Takam. on Aesculus spec. (new for Europe)), Erysiphe heraclei DC. on Tinguarra montana (Webb ex Christ ) A.HansenG.Kunkel, Erysiphe necator Schwein. = Uncinula necator (Schwein.) Burrill on Cissus cf. rhombifolia Vahl. (new for NRW), Erysphe trifolii Grev. on Trigonella caerulea (L.) Ser., Golovinomyces cichoracearum (DC.) V.P.Gelyuta (Oidium spec.) on Argyranthemum pinnatifidum (L.f.) R.T. Lowe (new host), Lobatopedis foliicola P.M. Kirk on Quercus robur L. (new for NRW), Lophodermium juniperinum (Fr.) de Not. on Juniperus communis L., Mamiania coryli De Not. on Corylus avellana L., Marssonina juglandis (Lib.) Magnus on Juglans regia L., Oidium hortensia Jørst on Philadelphus coronarius L., Oidium spec. on Dahlia variabilis (Willd.) Desf. (new for Germany), Oidium longipes NoordeloosLoerak on Petunia hybrida Vilm., Oidium pedilanthi M. Yen on Pedilanthus titymaloides (L.) Poit, Oidium pedaliacearum H.D. Shin sp. nov. (= Oidium sesami H.D. Shin) on Ibicella lutea (Lindl.) van Eselt. (= Martynia lutea Lindl.), Passalora pastinacae (Sacc.) U. Braun = Pseudocercosporella pastinacae (P. Karst.) U. Braun (new for Germany) on Pastinaca sativa L., Podosphaera tridactyla (WalIr.) de Bary on Prunus laurocerasus L., Septoria cornicola Desm. on Cornus sanguinea L., Stigmina tinea (Sacc.) M.B.Ellis on Viburnum opulus L., Torula herbarum (Pers.) Link on Potentilla argentea L., etc. All species are located in the herbarium Mycotheca parva collection G.B. Feige and N. Ale-Agha.

Non-destructive measurement of photosynthetic activity as an indication of the health and potential growth of individual lichen thalli and of lichen populations.

Chlorophyll fluorescence (CF) is the absorption of blue or red photons (= excitation) by chlorophyll molecules and the emission of far red photons (the energy difference is lost as heat). In contrast to chloroform solutions of chlorophyll or to fluorescent dyes, the emission from active photosynthetic systems does not remain constant, but changes in a complicated way after a constant excitation light is switched on. These changes (= variable fluorescence) are due to the onset of photosynthetic reactions that consume absorbed quanta. Accordingly, several derived fluorescence parameters (see below) provide diagnostic information about the state of the photosynthetic apparatus.

Inactivation of photosynthesis during atmospheric and osmotic (highly concentrated NaCl or sucrose solutions) dehydration was monitored by measurement of chlorophyll fluorescence induction (OIP-phase, Kautsky-curves) in three lichen species. The induction curves were changed in a very similar way by all three treatments. All dehydration effects were rapidly reversible after rehydration. At relatively mild water stress, the rise time to the transient peak Fp was prolonged, and the variable part of fluorescence was diminished. In addition, at severe water stress, a considerable decline of the F0 value was observed. For NaCl treatment this effect started at water potentials

Liquid water can restore the photosynthesis of most desiccated lichens within minutes. In contrast to lichens with green algal photobionts, cyanobacterial photobionts cannot uptake enough water from water vapor, i.e. they need liquid water to regain their photosynthetic activity (1). When exposed to wet air for several days, not even variable chlorophyll fluorescence can be detected in cyano-lichens (2). Obviously, their photosystem II reaction centres remain inactive. Moreover, the phycobilisomes remain functionally disconnected from photosystem II during this limited water uptake (3). In phycobilin (PB) fluorescence images (green excitation light), the fluorescence intensity becomes very low during desiccation (4), but until now there have been no data about the PB fluorescence intensity in the air-wetted state. Therefore we were very interested to know whether or not the PB fluorescence reappears in this phase. For this purpose we recorded PB fluorescence images in this and related states and compared the relative fluorescence intensity with chlorophyll fluorescence data obtained from an apparatus (MiniPAM) equipped with a small light guide (1.5 mm diameter). On the basis of 2-dimensional data, we discuss the surprising correlation between PB fluorescence intensity and the reappearance of variable chlorophyll fluorescence in some thallus regions. In addition, the role of the cortex + tomentum transparency and of the extent of pre-desiccation is assessed.

Mannitol-1-phosphate dehydrogenase [EC 1.1.1.17] activity was demonstrated in extracts of Pseudevernia furfuracea and Hypogymnia physodes. The reaction was found to be highly substrate specific for fructose-6-phosphate/NADH or mannitol-1-phosphate/NAD+. The pH optimum for fructose-6-phosphate reduction was 7.1, and apparent Km values were 1.2 mM for fructose-6-phosphate and 20 μM for NADH. The reaction did not require Mg++ or Ca++. For conversion of mannitol-1-phosphate into mannitol, the occurrence of mannitol-1-phosphatase in Pseudevernia furfuracea is postulated.

The sensitivity of Hypogymnia physodes, Lobaria pulmonaria and Peltigera aphthosa to six photosystem II herbicides and to DBMIB was tested in the laboratory by chlorophyll fluorescence and oxygen-exchange measurements. In addition, experiments with freshly isolated photobiont cells from H. physodes and L. pulmonaria were performed. Generally, the lichens were most sensitive to the urea herbicides diuron and isoproturon, whereas the triazines atrazine, terbuthylazine, and simazine and the triazinone metamitron were less inhibitory. Among the three lichen species investigated, H. physodes was the most sensitive to the urea herbicides. For the other agents, no significant differences between lichen species could be found. The highest pI50 values obtained from dose response curves were around 6.5 for isolated photobionts, but most values for lichen thalli were in the range 5–6. Thus, there is no particular sensitivity of green algal lichen photobionts to photosystem II herbicides as compared to other algae, higher plant chloroplasts or protoplasts. In nature, we observed recovery from (damaging) treatment with 10−5 mol diuron 1−1 for H. physodes within weeks. Therefore, damage to lichens from the use of photosystem-II herbicides in agriculture is probably only of very local occurrence.

Electron spin resonance (ESR) spectra of stearic acid spin labels incorporated into spinach thylakoids can be used to monitor membrane changes during freezing. Changes in the ESR parameters can be directly correlated to the extent of functional freeze damage. Freeze-induced changes in the ESR parameters strongly depend on the osmotic conditions of the incubation medium. Similar changes as on freezing can be observed by transferring thylakoids from an isotonic to a hypotonic medium, i.e., by swelling osmotically flattened thylakoids. This and computer simulations of spin label ESR spectra, which allow for variation of vesicle shape, lead to the conclusion that freeze-induced ESR spectral changes are due to swelling of the thylakoids. Indeed, van't Hoff plots of thylakoid packed volume indicate a freeze-induced increase in the apparent number of osmotically active molecules within the intrathylakoid lumen. During freezing, salt and/or sugar leak into the lumen. Simultaneously, proton channels are irreversibly opened. As the structural alterations obtained upon freezing are not accompanied by a change in bulk fluidity, these data are interpreted in terms of a local action of cryotoxic agents on critical microstructures, possibly at the rims of the thylakoid membranes.

In order to study the effect of freeze damage to chloroplast membranes microviscosity of spinach thylakoids was probed by stearic acid spin labels. Changes in ESR parameters have been determined either as a function of temperature or during freezing at −15 °C as a function of time. An empirical parameter h + h 0 (ratio of height of a low field line component h+ over height of the central line h0) proved to be very sensitive to minute changes in membrane structure. In cryoprotected chloroplast membranes Arrhenius plot breaks indicative of phase changes are observed at +15 and −10 °C. Breaks in the Arrhenius plots were not observed in vesicles prepared from chloroplast lipids by sonication. Instead, a melting zone was indicated below −30 °C. Freeze damage of thylakoids during storage at −15 °C is reflected in an increase of h + h 0 and a decrease in central line width W0. At +20 °C, differences between the ESR parameters of active as compared to freeze-damaged membranes could be detected, if the osmolarity of the suspending medium exceeded 200 mosm. The observed changes in line shapes are interpreted as an increase in mobility and/or orientation of the lipids following the swelling of thylakoids. They do not indicate a disorganization of the lipid phase. Sedimentation experiments indicated that the freeze-damaged swollen membranes still exhibited osmotic responses. It is suggested that freezing which is known to dissociate proteins from the membranes altered the charge distribution of the membranes leading first to membrane swelling and finally, by the opening of hydrophilic channels, to membrane collapse.

The distribution of sorediate lichens in different ecological scenarios is depicted on the basis of data obtained from ITALIC, an on-line database on the lichens of Italy. Sorediate species are ca. 15% of the Italian flora. They are most frequent under humid-shaded conditions, and – limited to certain types of substrata – under moderate to high levels of eutrophication. Most of the sorediate species of Italy are relatively rare, only a few are abundant in anthropized habitats. It is suggested that the presence of sorediate species is highest under the same conditions which favour the proliferation of free-living algae.