The circadian night depression of photosynthesis analyzed in a herb, Pulmonaria vallarsae. Day/night quantitative relationships.

Although many photosynthesis related processes are known to be controlled by the circadian system, consequent changes in photosynthetic activities are poorly understood. Photosynthesis was investigated during the daily cycle by chlorophyll fluorescence using a PAM fluorometer in Pulmonaria vallarsae subsp. apennina, an understory herb. A standard test consists of a light induction pretreatment followed by light response curve (LRC). Comparison of the major diagnostic parameters collected during day and night showed a nocturnal drop of photosynthetic responses, more evident in water-limited plants and consisting of: (i) strong reduction of flash-induced fluorescence peaks (FIP), maximum linear electron transport rate (Jmax, ETREM) and effective PSII quantum yield (ΦPSII); (ii) strong enhancement of nonphotochemical quenching (NPQ) and (iii) little or no change in photochemical quenching qP, maximum quantum yield of linear electron transport (Φ), and shape of LRC (θ). A remarkable feature of day/night LRCs at moderate to high irradiance was their linear-parallel course in double-reciprocal plots. Photosynthesis was also monitored in plants subjected to 2-3 days of continuous darkness ("long night"). In such conditions, plants exhibited high but declining peaks of photosynthetic activity during subjective days and a low, constant value with elevated NPQ during subjective night tests. The photosynthetic parameters recorded in subjective days in artificial darkness resembled those under natural day conditions. On the basis of the evidence, we suggest a circadian component and a biochemical feedback inhibition to explain the night depression of photosynthesis in P. vallarsae.

Population genetics and biogeography of the lungwort lichen in North America support distinct Eastern and Western gene pools.

PREMISE: Populations of species with large spatial distributions are shaped by complex forces that differ throughout their ranges. To maintain the genetic diversity of species, genepool-based subsets of widespread species must be considered in conservation assessments. METHODS: The population genetics of the lichenized fungus Lobaria pulmonaria and its algal partner, Symbiochloris reticulata, were investigated using microsatellite markers to determine population structure, genetic diversity, and degree of congruency in eastern and western North America. Data loggers measuring temperature and humidity were deployed at selected populations in eastern North America to test for climatic adaptation. To better understand the role Pleistocene glaciations played in shaping population patterns, a North American, range-wide species distribution model was constructed and hindcast to 22,000 years before present and at 500-year time slices from then to the present. RESULTS: The presence of two gene pools with minimal admixture was supported, one in the U.S. Pacific Northwest and one in eastern North America. Western populations were significantly more genetically diverse than eastern populations. There was no evidence for climatic adaptation among eastern populations, though there was evidence for range-wide adaptation to evapotranspiration rates. Hindcast distribution models suggest that observed genetic diversity may be due to a drastic Pleistocene range restriction in eastern North America, whereas a substantial coastal refugial area is inferred in the west. CONCLUSIONS: Taken together the results show different, complex population histories of L. pulmonaria in eastern and western North America, and suggest that conservation planning for each gene pool should be considered separately.

Pulmonaria obscura and Pulmonaria officinalis Extracts as Mitigators of Peroxynitrite-Induced Oxidative Stress and Cyclooxygenase-2 Inhibitors-In Vitro and In Silico Studies.

The Pulmonaria species (lungwort) are edible plants and traditional remedies for different disorders of the respiratory system. Our work covers a comparative study on biological actions in human blood plasma and cyclooxygenase-2 (COX-2) -inhibitory properties of plant extracts (i.e., phenolic-rich fractions) originated from aerial parts of P. obscura Dumort. and P. officinalis L. Phytochemical profiling demonstrated the abundance of phenolic acids and their derivatives (over 80% of the isolated fractions). Danshensu conjugates with caffeic acid, i.e., rosmarinic, lithospermic, salvianolic, monardic, shimobashiric and yunnaneic acids were identified as predominant components. The examined extracts (1-100 µg/mL) partly prevented harmful effects of the peroxynitrite-induced oxidative stress in blood plasma (decreased oxidative damage to blood plasma components and improved its non-enzymatic antioxidant capacity). The cellular safety of the extracts was confirmed in experimental models of blood platelets and peripheral blood mononuclear cells. COX-2 inhibitor screening evidently suggested a stronger activity of P. officinalis (IC50 of 13.28 and 7.24 µg/mL, in reaction with synthetic chromogen and physiological substrate (arachidonic acid), respectively). In silico studies on interactions of main components of the Pulmonaria extracts with the COX-2 demonstrated the abilities of ten compounds to bind with the enzyme, including rosmarinic acid, menisdaurin, globoidnan A and salvianolic acid H.

Recent hybrid speciation at the origin of the narrow endemic Pulmonaria helvetica.

BACKGROUND AND AIMS: Hybridization is known to drive plant speciation through the establishment of homoploid or allopolyploid hybrid species. Here we investigate the origin of Pulmonaria helvetica, a narrow endemic species described across a restricted area of Switzerland that was entirely covered by ice during the last glacial maximum. This species presents an original number of chromosomes (2n = 24) and morphological traits suggestive of a hybrid origin. METHODS: We sequenced a plastid locus and 1077 double-digest restriction-site-associated DNA (ddRAD) loci in 67 individuals from across the distribution range of P. helvetica and candidate progenitor species growing in the same area. Assignment of genotypes to main genetic clusters within and among taxa using STRUCTURE tested whether P. helvetica represents a genetically differentiated lineage and addressed the hypothesis of its hybrid origin. Comparative ecological modelling further addressed possible niche differentiation among taxa. KEY RESULTS: Pulmonaria helvetica was highlighted as a genetically homogeneous species distinct from co-occurring taxa. Consistent with a scenario of hybrid speciation, it presented clear evidence of balanced admixture between Pulmonaria officinalis (2n = 16) and Pulmonaria mollis s.l. (2n = 18, 22), which was also highlighted as a maternal progenitor based on plastid sequences. Limited genetic structure within the maternal progenitor is consistent with an origin of P. helvetica through either homoploid hybridization with considerable karyotype changes or via complex scenarios of allopolyploidy involving a dysploid taxon of P. mollis s.l. Comparative niche modelling indicated non-significant ecological differences between P. helvetica and its progenitors, supporting intrinsic factors resulting from hybridization as main drivers of speciation. CONCLUSIONS: Hybridization appears as a major process having promoted the postglacial origin of the narrow endemic P. helvetica, suggesting hybrid speciation as an effective process that rapidly produces new species under climate changes.

The Pros and Cons of Cystic Fibrosis (CF) Patient Use of Herbal Supplements Containing Pulmonaria officinalis L. Extract: the Evidence from an In Vitro Study on Staphylococcus aureus CF Clinical Isolates.

The justification for the use of herbal supplements with Pulmonaria officinalis L. extract (POE) in the case of staphylococcal lung colonization/infections characteristic for cystic fibrosis (CF), was examined in vitro. The impact of POE phenolic-rich fraction on the virulence attributes of CF-associated Staphylococcus aureus (S. aureus) clinical strains has been assessed, including pathogen adhesion, biofilm formation on native and protein-conditioned surfaces (mucin, elastin), mature biofilm eradication, staphylococcal protein A expression, α-toxin release, and S. a. adhesion to A549 cells. Cytotoxicity of the extract to lung epithelial cells was also investigated. It was found that POE has bacteriostatic effects at MIC 1⁻2 mg/mL, recognized as of limited efficacy, but at MIC/subMICs it targeted virulence not viability. It usually decreased S. aureus adhesion and less frequently inhibited biofilm formation on native and protein-conditioned surfaces. Observed effect seems to be related to significant reduction by POE of sortase A activity. However, in some cases POE favored the creation of biofilm by staphylococci and S. aureus adhesion to the lung epithelium was not limited. On the other side POE caused significant decrease of S. a. α-toxin synthesis and slightly weakened the expression of SpA. When used at supraMICs POE eradicated mature biofilm, but in some cases with unsatisfying outcomes. Promisingly, POE has been recognized as a safe product, with no cytotoxicity up to 4 mg/mL. These results reflect the positive, negative or neutral anti-staphylococcal properties of POE. It seems that POE may be beneficial as a prophylactic, but not as a therapeutic or supportive agent in the area of CF-integrative medicine. However, introduction the official recommendations needs further in vivo studies.

Novel Phenolic Constituents of Pulmonaria officinalis L. LC-MS/MS Comparison of Spring and Autumn Metabolite Profiles.

Lungwort (Pulmonaria officinalis L., Boraginaceae) is considered to possess therapeutic properties and it has been traditionally used as a remedy against various lung disorders in many countries. Nevertheless, very few data concerning its phytochemical composition are available. This research aims to provide a detailed description of specialized metabolites from the aerial parts of lungwort. Nine previously undescribed and 36 known phenolic compounds were detected in the 50% methanolic extract. Following multistep preparative procedures, structures of newly discovered compounds were determined using one- and two-dimensional techniques of NMR spectroscopy. Among the identified compounds were caffeic acid esters with aliphatic hydroxycarboxylic acids, conjugates of dicaffeic acid with rosmarinic acid, and previously unknown isomers of isosalvianolic acid A and yunnaneic acid E, as well as other lignans. Concentrations of all identified phenolic derivatives in the investigated herbal material were estimated using a method based on liquid chromatography with high-resolution mass spectrometry detection. Seasonal changes in the concentration of metabolites were also investigated using targeted and untargeted metabolomics techniques.

Taxonomy of the Golovinomyces cynoglossi Complex (Erysiphales, Ascomycota) Disentangled by Phylogenetic Analyses and Reassessments of Morphological Traits

The name Golovinomyces cynoglossi s. lat. is traditionally applied to a complex of morphologically similar powdery mildews on hosts of the plant family Boraginaceae. The current species-level taxonomy within this complex is ambiguous due to the lack of phylogenetic examinations. The present study applied phylogenetic methods to clarify the taxonomy of G. cynoglossi s. lat. Phylogenetic analysis of rDNA ITS sequences retrieved from Asian, European and North American specimens revealed that G. cynoglossi s. lat. collections from different hosts involved several species in five clearly separated lineages. Clade I consists primarily of Golovinomyces cynoglossi s. str. on Cynoglossum. Clade III consists of Golovinomyces sequences retrieved from the host genera Symphytum and Pulmonaria. The taxa within clade III are now assigned to G. asperifoliorum comb. nov. Clade V encompasses G. cynoglossi s. lat. on the host genera Bothriospermum, Buglossoides, Echium, Myosotis, and Trigonotis. The taxa within clade V are now assigned to G. asperifolii comb. nov. The species concerned in this study were lecto- and epitypified to stabilize their nomenclature.

Yunnaneic Acid B, a Component of Pulmonaria officinalis Extract, Prevents Peroxynitrite-Induced Oxidative Stress in Vitro.

Our work reveals that the aerial parts of Pulmonaria officinalis L. are a new source of yunnaneic acid B. We studied antioxidant activity and cytotoxicity of this compound (1-50 µg/mL) and its contents in various plant extracts. This is the first study confirming the presence of yunnaneic acid B in P. officinalis L. and Pulmonaria obscura Dumort and hence in the Boraginaceae family. Determination of 1,1-diphenyl-2-picrylhydrazyl radical reduction and peroxynitrite-scavenging efficacy in inorganic experimental systems provided EC50 values of 7.14 and 50.45 µg/mL, respectively. Then we examined the antioxidant action of yunnaneic acid B in blood plasma under peroxynitrite-induced oxidative stress in vitro. Yunnaneic acid B effectively diminished oxidative damage to blood plasma proteins and lipids. Furthermore, it was able to prevent the peroxynitrite-induced decrease in nonenzymatic antioxidant capacity of blood plasma. Additionally, cytotoxicity of yunnaneic acid B (at concentrations ≤50 µg/mL) toward peripheral blood mononuclear cells was excluded.

Symbiotic Interplay of Fungi, Algae, and Bacteria within the Lung Lichen Lobaria pulmonaria L. Hoffm. as Assessed by State-of-the-Art Metaproteomics.

Lichens are recognized by macroscopic structures formed by a heterotrophic fungus, the mycobiont, which hosts internal autotrophic photosynthetic algal and/or cyanobacterial partners, referred to as the photobiont. We analyzed the structure and functionality of the entire lung lichen Lobaria pulmonaria L. Hoffm. collected from two different sites by state-of-the-art metaproteomics. In addition to the green algae and the ascomycetous fungus, a lichenicolous fungus as well as a complex prokaryotic community (different from the cyanobacteria) was found, the latter dominated by methanotrophic Rhizobiales. Various partner-specific proteins could be assigned to the different lichen symbionts, for example, fungal proteins involved in vesicle transport, algal proteins functioning in photosynthesis, cyanobacterial nitrogenase and GOGAT involved in nitrogen fixation, and bacterial enzymes responsible for methanol/C1-compound metabolism as well as CO-detoxification. Structural and functional information on proteins expressed by the lichen community complemented and extended our recent symbiosis model depicting the functional multiplayer network of single holobiont partners.1 Our new metaproteome analysis strongly supports the hypothesis (i) that interactions within the self-supporting association are multifaceted and (ii) that the strategy of functional diversification within the single lichen partners may support the longevity of L. pulmonaria under certain ecological conditions.

Evolutionary trends in the distylous genus Pulmonaria (Boraginaceae): Evidence of ancient hybridization and current interspecific gene flow.

The distylous genus Pulmonaria contains approximately 18 species that are widely distributed across Eurasia. Previous studies have shown that species delimitation in the genus is problematic, but have not yet explored the evolutionary history of the genus. Premating reproductive barriers between European species appear to be weak, as several species have strongly overlapping distribution areas, flower at the same time and share the same pollinators, suggesting that hybridization may have contributed to the evolutionary history of Pulmonaria. To test this hypothesis, phylogenetic analyses of nuclear ITS and plastid data (rps16, trnH-psbA, rpl16) from 48 allopatric and four sympatric populations were performed to (1) provide a molecular phylogeny for nine of the most common Pulmonaria species in Europe, (2) detect current and ancient hybridization events, and (3) assess the contribution of hybridization versus incomplete lineage sorting to the inferred phylogenetic patterns. Our results showed that gene trees displayed widespread, strongly supported incongruence associated with the conflicting position of hybrid samples rather than incomplete lineage sorting. Evidence was found of different degrees of hybridization, ranging from current interspecific gene flow at secondary contact zones to introgression at the population level and at least one event of hybrid speciation. Overall, these results suggest that hybridization and introgression were - and could still be - important processes affecting speciation in the genus Pulmonaria.

Differences in fine-scale spatial genetic structure across the distribution range of the distylous forest herb Pulmonaria officinalis (Boraginaceae).

BACKGROUND: Geographical ranges of plants and their pollinators do not always entirely overlap and it has been suggested that the absence of specialized pollinators at range margins may induce changes in mating systems. Because a species' mating system is known to have a considerable effect on within-population pollen movement, the extent of fine-scale spatial genetic structure (SGS) can be expected to differ between populations located at different parts of their geographical range. To test this prediction, we compared the fine-scale SGS between two core and two disjunct populations of the distylous forest herb Pulmonaria officinalis. Because in disjunct populations of this species the heteromorphic self-incompatibility system showed relaxation in the long-styled morph, but not in the short-styled morph, we also hypothesized that the extent of fine-scale SGS and clustering differed between morphs. RESULTS: Spatial autocorrelation analyses showed a significant decrease in genetic relatedness with spatial distance for both core and disjunct populations with the weakest SGS found in one of the core populations (Sp = 0.0014). No evidence of stronger SGS in the long-styled morph was found in the center of the range whereas one disjunct population showed a significantly (P = 0.029) higher SGS in the long-styled morph (SpL = 0.0070) than in the short-styled morph (SpS = 0.0044). CONCLUSIONS: Consistent with previous analyses on distylous plant species, we found weak, but significant spatial genetic structure. However, the extent of SGS varied substantially between populations within regions, suggesting that population characteristics other than mating system (e.g. local pollinator assemblages, population history) may be as important in determining variation in SGS.

Among-population variation in microbial community structure in the floral nectar of the bee-pollinated forest herb Pulmonaria officinalis L.

BACKGROUND: Microbial communities in floral nectar have been shown to be characterized by low levels of species diversity, yet little is known about among-plant population variation in microbial community composition. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the microbial community structure (yeasts and bacteria) in floral nectar of ten fragmented populations of the bee-pollinated forest herb Pulmonaria officinalis. We also explored possible relationships between plant population size and microbial diversity in nectar, and related microbial community composition to the distance separating plant populations. Culturable bacteria and yeasts occurring in the floral nectar of a total of 100 plant individuals were isolated and identified by partially sequencing the 16S rRNA gene and D1/D2 domains of the 26S rRNA gene, respectively. A total of 9 and 11 yeast and 28 and 39 bacterial OTUs was found, taking into account a 3% (OTU0.03) and 1% sequence dissimilarity cut-off (OTU0.01). OTU richness at the plant population level (i.e. the number of OTUs per population) was low for yeasts (mean: 1.7, range: 0-4 OTUs0.01/0.03 per population), whereas on average 6.9 (range: 2-13) OTUs0.03 and 7.9 (range 2-16) OTUs0.01 per population were found for bacteria. Both for yeasts and bacteria, OTU richness was not significantly related to plant population size. Similarity in community composition among populations was low (average Jaccard index: 0.14), and did not decline with increasing distance between populations. CONCLUSIONS/SIGNIFICANCE: We found low similarity in microbial community structure among populations, suggesting that the assembly of nectar microbiota is to a large extent context-dependent. Although the precise factors that affect variation in microbial community structure in floral nectar require further study, our results indicate that both local and regional processes may contribute to among-population variation in microbial community structure in nectar.

Strong differences in genetic structure across disjunct, edge, and core populations of the distylous forest herb Pulmonaria officinalis (Boraginaceae).

PREMISE OF THE STUDY: Populations at the edge of a species' distribution area are often small and have low levels of gene flow resulting in lower genetic variation and higher differentiation compared to core populations. This study examined genetic variation among populations of the distylous temperate forest herb Pulmonaria officinalis located in the core, the edge, and outside the species' main distribution range. METHODS: We compared patterns of genetic variation for eight microsatellite loci between disjunct (Belgium), edge (western Germany), and core (eastern Germany) populations of P. officinalis. KEY RESULTS: Disjunct populations contained only a subset of alleles found in edge and core populations and had significantly lower within-population genetic variation. No significant differences, however, in within-population genetic variation were found between edge and core populations, except for allelic and genotypic richness. Genetic differentiation was highest among disjunct (F(ST) = 0.11) and lowest among core populations (F(ST) = 0.03). Significant (P < 0.01) isolation by distance was found for disjunct and edge populations (r(M) = 0.29 and 0.50, respectively), but not for core populations (r(M) = 0.18). CONCLUSIONS: The results are best interpreted from a "dynamic range" point of view in which the observed low levels of genetic diversity and high genetic differentiation in disjunct populations are best explained through historical processes, most likely the introduction of the species in medieval times. Lower levels of gene flow caused by the pronounced fragmentation of forests in Belgium may further have contributed to the genetic structure of P. officinalis in these disjunct populations.

Biased morph ratios and skewed mating success contribute to loss of genetic diversity in the distylous Pulmonaria officinalis.

BACKGROUND AND AIMS: In heterostylous plant species, skewed morph ratios are not uncommon and may arise from a range of factors. Despite the recognized importance of skewed morph ratios on overall reproductive success within populations, little is known about the impact of skewed morph ratios on population genetic diversity and differentiation in heterostylous species. This study specifically aimed to clarify the effect of population size and morph bias on population genetic diversity and differentiation in the temperate forest herb Pulmonaria officinalis. This species is characterized by a distylous breeding system and shows morph-specific differences in reproductive success. METHODS: Genetic diversity was determined for 27 P. officinalis populations in northern Belgium by using eight recently developed microsatellite markers. Multiple regressions were used to assess the relationship between genetic diversity, morph bias and population size, and F(ST)-values were calculated for short- and long-styled morphs separately to study genetic differentiation as a function of morph type. KEY RESULTS: For all genetic measures used, morph bias was more important in explaining patterns of genetic diversity than population size, and in all cases patterns of population genetic diversity followed a quadratic function, which showed a symmetrical decrease in genetic diversity with increasing morph bias. However, probably due to the reproductive advantage of L-morphs relative to S-morphs, maximum genetic diversity was found in populations showing an excess of L-morphs (60·7 % L-morph). On the other hand, no significant difference in pairwise genetic distances between populations was observed between L- (0·107) and S-morphs (0·106). CONCLUSIONS: Our results indicate that significant deviations from equal morph ratios not only affect plant reproductive success but also population genetic diversity of heterostylous plant species. Hence, when defining conservation measures for populations of heterostylous plant species, morph ratios should be considered as an important trait affecting their long-term population viability.

Histological study of some Echium vulgare, Pulmonaria officinalis and Symphytum officinale populations.

Plants living in different ecological habitats can show significant variability in their histological and phytochemical characters. The main histological features of various populations of three medicinal plants from the Boraginaceae family were studied. Stems, petioles and leaves were investigated by light microscopy in vertical and transverse sections. The outline of the epidermal cells, as well as the shape and cell number of trichomes was studied in leaf surface casts. Differences were measured among the populations of Echium vulgare in the width and height of epidermis cells in the stem, petiole and leaf, as well as in the size of palisade cells in the leaves. Among the populations of Pulmonaria officinalis significant differences were found in the length of trichomes and in the slightly or strongly wavy outline of epidermal radial cell walls. Populations of Symphytum officinale showed variance in the height of epidermal cells in leaves and stems, length of palisade cells and number of intercellular spaces in leaves, and the size of the central cavity in the stem. Boraginaceae bristles were found to be longer in plants in windy/shady habitats as opposed to sunny habitats, both in the leaves and stems ofP. officinalis and S. officinale, which might be connected to varying levels of exposure to wind. Longer epidermal cells were detected in the leaves and stems of both E. vulgare and S. officinale plants living in shady habitats, compared with shorter cells in sunny habitats. Leaf mesophyll cells were shorter in shady habitats as opposed to longer cells in sunny habitats, both in E. vulgare and S. officinale. This combination of histological characters may contribute to the plant's adaptation to various amounts of sunshine. The reported data prove the polymorphism of the studied taxa, as well as their ability to adapt to various ecological circumstances.

Valores funcionales respiratorios pre y post- operatorios en pacientes sometidos a resección pulmonar / Preoperating and postoperating respiratory functional values in patients submissive of lung volume reduction surgery

Se realizó un estudio observacional, analítico, prospectivo, en el período 2006-2008, en el Hospital Neumológico Benéfico Jurídico, con el objetivo de comparar los valores predictivos espirométricos de los enfermos candidatos a resección pulmonar con los obtenidos después de la intervención quirúrgica. La muestra quedó formada por 28 pacientes, residentes en Ciudad de La Habana. Para comparar el valor predictivo del volumen espiratorio forzado en el primer segundo con el valor real, seis meses después de la resección pulmonar se calculó el cociente delta relativo que expresó en porcentaje el grado de coincidencia entre estas dos variables. Una vez recogida la información se sometió a un análisis exploratorio de datos, con pruebas de significación asociadas. Los resultados mostraron que la espirometría clínica tiene un espacio indiscutible en la evaluación de la función pulmonar preoperatoria de los candidatos a resección pulmonar. Los valores predictivos y reales después de la resección pulmonar del volumen espiratorio forzado en el primer segundo fueron semejantes. La lobectomía superior izquierda fue el tipo de intervención quirúrgica con mayor similitud entre estos valores, por el contrario la neumectomía izquierda resultó ser la técnica con mayores diferencias entre los mismos(AU)

We carried out an observacional, analytical, prospective study in the period 2006-2008, at Neumologico Hospital Benéfico Jurídico, with the objective to compare the spirometry predicted values of the patients candidates to pulmonary surgery with the obtained ones after the operation. The sample was formed by 28 patients, residents in Havana City. In order to compare the predictive value of the forced expiratory volume in the first second with the real value six months after the pulmonary surgery, we calculated the quotient relative delta that expressed in percentage the degree of coincidence between these two variables. The information was put under an exploratory analysis of data, with associate tests of meaning. The results showed that the spirometry has an unquestionable space in the evaluation of the preoperating pulmonary function of the candidates to pulmonary surgery. The predictive and real values after the pulmonary surgery of the forced expiratory volume in the first second were similar. The left upper lobectomy was the type of operation with greater similarity between these values, on the contrary the left neumectomy turned out to be the technique with greater differences between such(AU)

Floral display size and spatial distribution of potential mates affect pollen deposition and female reproductive success in distylous Pulmonaria officinalis (Boraginaceae).

In animal-pollinated plants, both the spatial distribution of flowering individuals and the number of flowers that an individual displays affect pollen deposition rates and female reproductive success. Heterostylous species are likely to be particularly sensitive to the contingencies of spatial distribution, as they are reproductively subdivided into distinct mating groups, which usually exhibit self- and intra-morph incompatibility and differ in floral morphology. In this paper, we explore the joint effects of both spatial distribution of potential mates and floral display size on morph-specific pollen deposition rates and seed set patterns in two natural populations of Pulmonaria officinalis, a distylous species with a weak self-incompatibility system. Both total stigmatic pollen load and the proportion of legitimate pollen decreased with increasing spatial isolation. Legitimate (intermorph) pollen transfer was, however, asymmetric and decreased more rapidly with decreasing proximity to a compatible legitimate mating partner in the S-morph than in the L-morph. Total stigmatic pollen loads per flower increased with increasing floral display size, indicating that large plants are disproportionately more visited than smaller individuals. However, because legitimate pollen deposition decreased with increasing floral display size, these results also suggest that larger numbers of flowers increase the degree of geitonogamous pollination. In both the L- and S-morph, seed set significantly decreased with increasing isolation from a legitimate mating partner, but in the L-morph seed set was less dependent on the spatial distribution of the S-morph. In addition, seed set significantly increased with floral display size in the L-morph, but not in the S-morph. These findings indicate that the spatial distribution of potential mates and variation in floral display size may cause morph-specific differences in pollen deposition rates and female reproductive success.

Unravelling the roles of desiccation-induced xanthophyll cycle activity in darkness: a case study in Lobaria pulmonaria.

Desiccation-tolerance ability in photosynthetic organisms is largely based on a battery of photoprotective mechanisms. Xanthophyll cycle operation induced by desiccation in the absence of light has been previously proven in the desiccation-tolerant fern Ceterach officinarum. To understand the physiological function of xanthophyll cycle induction in darkness and its implication in the desiccation tolerance in more detail, we studied its triggering factors and its photochemical effects in the lichen Lobaria pulmonaria. We found that both the drying rate and the degree of desiccation play a crucial role in the violaxanthin de-epoxidase activation. De-epoxidation of violaxanthin to zeaxanthin (Z) occurs when the tissue has lost most of its water and only after slow dehydration, suggesting that a minimum period of time is required for the enzyme activity induction. Fluorescence analysis showed that Z, synthesised during tissue dehydration in the absence of light, prevents photoinhibition when rewatered tissues are illuminated. This is probably due to Z implication in both non-photochemical quenching and/or antioxidative responses.

Analytical characterisation of homoeopathic mother tinctures.

Quality of homoeopathic mother tinctures is assured by the definition of the starting material, the manufacturing process and the analytical characteristics described in the monograph. Traditionally analytical characterisation of the mother tincture comprises appearance, odour, identity, density and dry residue. According to annex I of directive 2001/83/EC an assay is only performed in case of a health hazard due to toxic compounds. The concept of marker substances as usually used in phytotherapy cannot be transferred to mother tinctures without research effort. For example the marker substances echinacoside, apigenin-7-glucoside and rosmarinic acid found in dried underground parts of Echinacea pallida Nutt., dried flower heads of Matricaria recutita L. and dried herb of Pulmonaria officinalis L. cannot be found in homoeopathic mother tinctures prepared from fresh material thereof.

Photoprotective implications of leaf variegation in E. dens-canis L. and P. officinalis L.

Variegated leaves occur rarely in nature, but there are some species, primarily in the forest understory, that possess this characteristic. We recently studied two variegated plants: Erytronium dens-canis L., which is characterised by a pattern of red patches and Pulmonaria officinalis L., with light green spots. These non-green areas could attenuate light reaching mesophyll cells with respect to green sections. The aim of the study was to verify whether such red and light green parts are more photoprotected than green ones and if this trait could be of adaptive value. Red patches in E. dens-canis were due to a single layer of red cells in the upper parenchyma, which accumulated anthocyanins. Light green spots in P. officinalis were caused by the presence of loosely arranged cells instead of a well-established layer of packed cells in the palisade parenchyma. Chlorophyll fluorescence imaging was performed under light treatment, showing a greater decrease of photochemical efficiency in red and light green patches than in green sections. Differences in the extent of photochemical efficiency among patches were not attributable to different activation of the xanthophyll cycle. These observations failed to confirm our initial hypothesis, but they questioned the physiological reason for this higher sensitivity in red and light green patches of photosynthetic tissues. Chlorophyll fluorescence imaging was therefore performed in the field. The same pattern of photochemical efficiency was maintained only in E. dens-canis. The current results demonstrate that in both species the benefits of variegation, if any, are different from enhanced photosynthetic performance.