Development of methods for detection and Agrobacterium-mediated transformation of the sterile,endophytic fungus Muscodor albus

Symbiotic endophytes, unlike plant pathogens, do not usually induce visible host response. This may constraint the researcher's decision whether a plant has been successfully infected by the endophyte. In order to properly study the establishment, development and progress of an endophyte in the host plant and host-endophyte interactions, methods for the identification and localization of endophytic microorganisms are needed. Towards this aim, we focused at two levels: (A) We constructed M. albus-specific primers for polymerase chain reaction (PCR). In vitro, these primers specifically detected only M. albus strains and not isolates of related fungi (such as Daldinia sp. and a Xylariaceae sp.). (B) For direct visualization of the fungi, we inserted a reporter gene (gfp) into M. albus hyphae using Agrobacterium-mediated transformation. Since M. albus is a sterile fungus (i.e., without spores or fungal fruiting bodies), we used chopped fungal mycelium for the transformation procedure. We transformed three different isolates of M. albus using Agrobacterium-mediated transformation. Fifty-nine different transformants were collected with a transformation efficacy of 0.0004–0.0026%. Although PCR-based detection and direct visualization of the transformants in planta were unsuccessful, all tested transformants (with one exception) exhibited similar biological activity to their cognate wild type. This work provides a significant step forward in molecular research of the relationships between this endophytic genus and their hosts.     

基于花粉形态数量分类的核果类系统关系研究

运用扫描电子显微镜(SEM)观察了李属、杏属、樱桃属和桃属的花粉形态,观察结果表明:核果类果树的花粉均为近扁球形,等极,辐射对称,极面观为三角形或钝三角形,赤面观均为椭圆形,花粉粒大小在各属间差异明显。核果类果树花粉属N3P4C5类型,3条孔沟沿极轴方向在赤道面上等间距环状分布,内孔位于沟的中央,完全独立于外壁,孔盖覆不规则的拟网状或脑纹状纹饰。供试核果类果树花粉的表面纹饰由平行条纹或不规则条脊及散落于条脊间的穿孔组成,各属间条脊的排列方式、宽窄、脊洼深浅及穿孔有无等性状上存在明显差异。根据花粉大小、表面纹饰及覆盖层穿孔等的一般演化规律,推测核果类果树由低到高的演化顺序为李属→杏属→樱桃属→桃属,并在聚类分析的基础上对属间亲缘关系进行了探讨,旨在为核果类系统关系的研究提供孢粉证据。     

Genetic differentiation amongst fragrant orchids (Gymnadenia conopsea s.l.) in the British Isles

Genetic variation was examined in five microsatellite loci to seek evidence of genetic differentiation and restricted gene flow that would support the taxonomic division of Gymnadenia into three species ( G. borealis , G. conopsea , and G. densiflora ). A total of 107 alleles was detected in 17 populations from England, Scotland, and Ireland. The mean expected heterozygosities ranged from 0.48 to 0.81. The differentiation in allele frequencies amongst populations that had been assigned to each taxon on the basis of morphology was sufficiently large to support the taxa as distinct species. Phylogenetic trees based on microsatellite allele frequencies, as well as assignment tests, supported the existence of three distinct groups with at least partial restriction of gene flow between them. There was substantial homozygote excess, leading to high F _IS estimates, for most loci in most populations. This is unlikely to have been a result of widespread null alleles, and more probably reflects a high level of inbreeding in G. conopsea . This inference requires further investigation. The implications of the results of this and other taxonomic studies for the conservation of Gymnadenia in Britain are discussed.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 155 , 349–360.     

Red-Headed Woodpecker Nest-Habitat Thresholds in Restored Savannas

Abstract: The red-headed woodpecker (Melanerpes erythrocephalus) is a widespread, adaptable species that continues to decline across North America. We examined stand, nest-tree, and cavity characteristics of red-headed woodpeckers in restored savannas within the Necedah National Wildlife Refuge, Wisconsin, USA, during 2002 and 2003. Based on availability, red-headed woodpeckers selected snags and trees with greater dead limb length. Red-headed woodpeckers nested in areas with greater basal area, cavity density, snag density, limb-tree density, and total dead limb length. Red-headed woodpeckers exhibited a decadent-tree threshold that was most accurately measured by the number of trees with dead limbs around (0.04 ha) nests. We found that the probability of a red-headed woodpecker nest being present greatly increased above the decadent-tree threshold. Woodland managers throughout the red-headed woodpecker's extensive breeding range can use our results and recommendations to guide decadent-tree retention for this species.     

Data-Mining Discovery of Pattern and Process in Ecological Systems

ABSTRACT Most ecologists use statistical methods as their main analytical tools when analyzing data to identify relationships between a response and a set of predictors; thus, they treat all analyses as hypothesis tests or exercises in parameter estimation. However, little or no prior knowledge about a system can lead to creation of a statistical model or models that do not accurately describe major sources of variation in the response variable. We suggest that under such circumstances data mining is more appropriate for analysis. In this paper we 1) present the distinctions between data-mining (usually exploratory) analyses and parametric statistical (confirmatory) analyses, 2) illustrate 3 strengths of data-mining tools for generating hypotheses from data, and 3) suggest useful ways in which data mining and statistical analyses can be integrated into a thorough analysis of data to facilitate rapid creation of accurate models and to guide further research.     

Extreme long-distance dispersal of the lowland tropical rainforest tree Ceiba pentandra L. (Malvaceae) in Africa and the Neotropics

Many tropical tree species occupy continental expanses of rainforest and flank dispersal barriers such as oceans and mountains. The role of long-distance dispersal in establishing the range of such species is poorly understood. In this study, we test vicariance hypotheses for range disjunctions in the rainforest tree Ceiba pentandra, which is naturally widespread across equatorial Africa and the Neotropics. Approximate molecular clocks were applied to nuclear ribosomal [ITS (internal transcribed spacer)] and chloroplast (psbB-psbF) spacer DNA sampled from 12 Neotropical and five West African populations. The ITS (N=5) and psbB-psbF (N=2) haplotypes exhibited few nucleotide differences, and ITS and psbB-psbF haplotypes were shared by populations on both continents. The low levels of nucleotide divergence falsify vicariance explanations for transatlantic and cross-Andean range disjunctions. The study shows how extreme long-distance dispersal, via wind or marine currents, creates taxonomic similarities in the plant communities of Africa and the Neotropics.     

Free-air exposure systems to scale up ozone research to mature trees

Because seedlings and mature trees do not necessarily respond similarly to O(3) stress, it is critically important that exposure systems be developed that allow exposure of seedlings through to mature trees. Here we describe three different O(3) Free-Air Exposure Systems that have been used successfully for exposure at all growth stages. These systems of spatially uniform O(3) release have been shown to provide reliable O(3) exposure with minimal, if any, impact on the microclimate. This methodology offers a welcome alternative to chamber studies which had severe space constraints precluding stand or community-level studies and substantial chamber effects on the microclimate and, hence physiological tree performance.     

Antioxidative defence of old growth beech (Fagus sylvatica) under double ambient O3 concentrations in a free-air exposure system

In this study the influence of chronic free-air ozone exposure and of different meteorological conditions in the very dry year 2003 and the more humid year 2004 on the antioxidative system in sun and shade leaves of adult FAGUS SYLVATICA trees were investigated. Contents of ascorbate, glutathione, and alpha-tocopherol, as well as chloroplast pigments were determined under ambient (1 x O(3)) and double ambient (2 x O(3)) ozone concentrations. Ozone affected the antioxidative system in June and July, causing lower ascorbate contents in the apoplastic space, a more oxidized redox state of ascorbate and glutathione and an increase in pigment contents predominantly in the shade crown. For all measured parameters significant differences between the years were observed. In 2004 the redox state of ascorbate and glutathione was in a more reduced state and leaf contents of alpha-tocopherol, pigments of the xanthophyll cycle, beta-carotene, lutein, neoxanthin, and alpha-carotene were lower compared to 2003. Contents of total glutathione and chlorophyll a + b were increased in the second year. These results indicate a strong influence of the drought conditions in 2003 on the antioxidative system of beech overruling the ozone effects. Shade leaves showed lower contents of ascorbate in both years and the redox states of ascorbate and glutathione were more oxidized compared to sun leaves. Contents of photoprotective and accessory pigments generally were enhanced and the de-epoxidation state of the xanthophyll cycle was lower in the shade compared to the sun crown. Exhibiting less antioxidants shade leaves seem to be more sensitive against ozone than sun leaves.     

Synopsis of the CASIROZ case study: carbon sink strength of Fagus sylvatica L. in a changing environment--experimental risk assessment of mitigation by chronic ozone impact

Databases are needed for the ozone (O(3)) risk assessment on adult forest trees under stand conditions, as mostly juvenile trees have been studied in chamber experiments. A synopsis is presented here from an integrated case study which was conducted on adult FAGUS SYLVATICA trees at a Central-European forest site. Employed was a novel free-air canopy O(3) fumigation methodology which ensured a whole-plant assessment of O(3) sensitivity of the about 30 m tall and 60 years old trees, comparing responses to an experimental 2 x ambient O(3) regime (2 x O(3), max. 150 nl O(3) l (-1)) with those to the unchanged 1 x ambient O(3) regime (1 x O(3)=control) prevailing at the site. Additional experimentation on individual branches and juvenile beech trees exposed within the forest canopy allowed for evaluating the representativeness of young-tree and branch-bag approaches relative to the O(3) sensitivity of the adult trees. The 2 x O(3) regime did not substantially weaken the carbon sink strength of the adult beech trees, given the absence of a statistically significant decline in annual stem growth; a 3 % reduction across five years was demonstrated, however, through modelling upon parameterization with the elaborated database. 2 x O(3) did induce a number of statistically significant tree responses at the cell and leaf level, although the O(3) responsiveness varied between years. Shade leaves displayed an O(3) sensitivity similar to that of sun leaves, while indirect belowground O(3) effects, apparently mediated through hormonal relationships, were reflected by stimulated fine-root and ectomycorrhizal development. Juvenile trees were not reliable surrogates of adult ones in view of O(3) risk assessment. Branch sections enclosed in (climatized) cuvettes, however, turned out to represent the O(3) sensitivity of entire tree crowns. Drought-induced stomatal closure decoupled O(3) intake from O(3) exposure, as in addition, also the "physiologically effective O(3) dose" was subject to change. No evidence emerged for a need to lower the "Critical Level for Ozone" in risk assessment of forest trees, although sensitive tree parameters did not necessarily reflect a linear relationship to O(3) stress. Exposure-based concepts tended to overestimate O(3) risk under drought, which is in support of current efforts to establish flux-related concepts of O(3) intake in risk assessment.     

Could the tree diversity pattern in Europe be generated by postglacial dispersal limitation?

The relative importance of contemporary climate and history as controls of geographical diversity patterns is intensely debated. A key example is the controversy over the extent to which temperate tree distributions and diversity patterns reflect postglacial dispersal limitation. Here, we focus on Central and Northern Europe, and show that recent estimates of tree migration rates < 100 m year⁻¹ imply that many species have probably not reached equilibrium with climate in this region. We then demonstrate that geographical accessibility from glacial refuges explains 78% of the geographical variation in the region's tree diversity and is a much stronger diversity predictor than climate. Finally, we show that realistic estimates of migration rates can be derived from the observed tree diversity pattern by assuming it to be purely dispersal driven. In conclusion, the tree diversity pattern in Central and Northern Europe could, to a large extent, be a result of postglacial dispersal limitation.