An investigation of pectin methylesterification patterns by two independent methods: capillary electrophoresis and polysaccharide analysis using carbohydrate gel electrophoresis

The analysis of partially methylesterified oligogalacturonides plays a key role both in the elucidation of the fine structure of the polysaccharide pectin and in the study of pectin-acting enzymes. Experimental methods performing the separation, detection and quantification of oligogalacturonides are, therefore, of crucial importance in the drive to understand structure-function relationships in pectin containing systems, both in vitro and in vivo. In this work standard samples of unesterified and partially methylesterifed galacturonides, and enzymatic digests of several pectin samples possessing distinct intramolecular patterns of methylesterification were studied using capillary electrophoresis (CE) and polysaccharide analysis using carbohydrate gel electrophoresis (PACE). In addition to yielding interesting information regarding the fine structures of the different pectic substrates digested, the study has been used as a vehicle in order to compare the two recently reported methods.     

Influence of the degree of polymerization of oligogalacturonates and of esterification pattern of pectin on their recognition by monoclonal antibodies

The ability of galacturonic and oligogalacturonic acids with degrees of polymerization (DP) from 2 to 10 to inhibit the recognition of homopolygalacturonic acid by a monoclonal antibody specific for dimers of pectin (F Liners, J-J Letesson, C Didembourg, P Van Cutsem [1989] Plant Physiol 91: 1419-1424) has been tested by enzyme-linked immunosorbent assays. Oligomers of DP9 and above preincubated with the antibodies clearly inhibited the association between the antibodies and immobilized pectin. A minimum DP of nine consecutive galacturonic residues is thus necessary to be associated through calcium cations to form dimers. Randomly deesterified pectin was recognized by the antibody if its degree of methylesterification was <30%, whereas blockwise deesterified pectin was recognized up to 40% of methylesterification. The replacement of calcium ions by magnesium prevented the recognition of polygalacturonic acid by the antibody.     

Determining the degree of methylesterification of pectin by ATR/FT-IR: Methodology optimisation and comparison with theoretical calculations

The application of FT-IR to the study of the structure and interactions of the major plant-cell wall polysaccharide pectin has been reported for many decades. Nevertheless, here we show that the generally reported methodology for one of its most commonly utilised applications, the measurement of the degree of methylesterification (DM), requires careful interpretation and sample handling; including consideration of the moisture content and ionisation state. We propose instead a different methodology based on the assessment of the magnitude of C–H stretches in the methyl groups relative to those in the backbone and demonstrate experimentally the advantage of this method. In addition, we add a theoretical dimension to our work, performing full quantum chemical (DFT) calculations of monomeric-, dimeric-, and trimeric-pectic compounds, in various states of partial methylesterification. These extensive calculations not only confirm the identity of the proposed methyl-band and illustrate its scaling with DM; but also demonstrate the success of the theoretical approach. Thus, DFT calculations are expected to be a valuable tool in the interpretation of IR spectra obtained from more complex systems such as polysaccharide conjugates.     

Capillary electrophoresis of homogeneous pectin fractions

Capillary electrophoresis (CE) has been used to characterize pectin fractions obtained from mother samples of varying degrees (and intramolecular patterns) of methylesterification (DM). These samples have been carefully produced by fractionation, (a) from the original mother samples on the grounds of molar mass, and (b) subsequently from the daughter fractions so obtained, on the grounds of charge. The results confirm theoretical predictions regarding the dependence of electrophoretic mobility on molar mass and DM and give further evidence that CE is able to give realistic information regarding the intermolecular DM of pectins.     

Innovative enzymatic approach to resolve homogalacturonans based on their methylesterification pattern

Three series of model homogalacturonans (HGs) covering a large range of degree of methylesterification (DM) were prepared by chemical and/or enzymatic means. Randomly demethylesterified HGs, HGs containing a few long demethylesterified galacturonic acid stretches, and HGs with numerous but short demethylesterified blocks were recovered. The analysis of the degradation products generated by the action of a purified pectin lyase allowed the definition of two new parameters, the degree of blockiness, and the absolute degree of blockiness of the highly methylesterified stretches (DBMe and DB(abs)Me, respectively). By combining this information with that obtained by the more traditional endopolygalacturonase digestion, the total proportion of degradable zones for a given DM was measured and was shown to permit a clear differentiation of the three types of HG series over a large range of DM. This double enzymatic approach will be of interest to discriminate industrial pectin samples exhibiting different functionalities and to evaluate pectin fine structure dynamics in vivo in the plant cell wall, where pectin plays a key mechanical role.     

Factors shaping the range-size frequency distribution of the endemic fish fauna of the Tropical Eastern Pacific

Aim To assess the effect of habitat fragmentation and isolation in determining the range‐size frequency distribution (RFD) of the shorefish fauna endemic to a discrete biogeographical region. Location The Tropical Eastern Pacific (TEP). Methods Habitat isolation represents the separation between oceanic islands and the continental shore of the TEP and habitat fragmentation the degree of spatial continuity of habitats (i.e. reefs, soft bottom, nearshore waters) along the continental coast of the TEP. The effects of habitat isolation and fragmentation were quantified by comparing the RFDs of (1) the species found on oceanic islands vs. the continental shore, and (2) species on the continental shore that use different habitat types. Results The RFD of the entire TEP fauna was bimodal, with peaks at both small‐ and large‐range ends of the spectrum. The small‐range peak was due almost entirely to island species and the large‐range peak due mainly to species found in both the continental shore and oceanic islands. RFDs varied among species using different habitats on the continental shore: reef‐fishes had a right‐skewed RFD, soft‐bottom species a flat RFD, and coastal‐pelagic fishes a left‐skewed RFD. Main conclusions Variation in dispersal capabilities associated with habitat isolation and fragmentation in the TEP appears to be the main mechanism contributing to differences among RFD structure, although variation in tolerances arising from the dynamic regional environment may contribute to some patterns. Because diversity patterns are strongly affected by RFD structure, it is now evident that the insular and continental components of a fauna should be treated separately when analysing such patterns. Furthermore, contrasts in RFD structure among species using different habitats demonstrate that a full understanding of the causes of diversity patterns requires analyses of complete regional faunas in relation to regional geography.     

Analysis of methylated and unmethylated polygalacturonic acid structure by polysaccharide analysis using carbohydrate gel electrophoresis

The electrophoretic migration in polyacrylamide gels of oligogalacturonic acids (OGAs) derivatized by a fluorophore (2-aminoacridone) was studied. We found conditions such that OGAs can be separated up to a degree of polymerization (DP) of 40. The migration was dependent on degree of methylation and DP, because the OGA mobility relies on the charge of the galacturonic acid residues. Since both methylated and unmethylated oligosaccharides can be resolved, polysaccharide analysis using carbohydrate gel electrophoresis (PACE) is a powerful method for studying the fingerprint of pectin hydrolysis. It can be used to characterize endopolygalacturonase (Endo-PG) tolerance of methylation. Furthermore, using an Endo-PG that can distinguish low and highly methylated pectin, PACE can be used to investigate the blockwise or nonblockwise distribution of methylation of polygalacturonic acid. We show that the method can be applied to crude cell wall preparations of Arabidopsis inflorescence stems. Using chemical deesterification before or after Endo-PG digestion, we show that in the Arabidopsis cell wall, the pectins have both nonesterified and highly esterified regions.     

宏生态尺度上景观破碎化对物种丰富度的影响

生物多样性的地理格局及其形成机制是宏生态学与生物地理学的研究热点。大量研究表明,景观尺度上的生境破碎化对物种多样性的分布格局具有重要作用,但目前尚不清楚这种作用是否足以在宏生态尺度上对生物多样性地理格局产生显著影响。利用中国大陆鸟类和哺乳动物的物种分布数据,在100 km×100 km网格的基础上生成了这两个类群生物的物种丰富度地理格局,进一步利用普通最小二乘法模型和空间自回归模型研究了物种丰富度与气候、生境异质性、景观破碎化的相关关系。结果表明,景观破碎化因子与鸟类和哺乳动物的物种丰富度都具有显著的关联关系,其方差贡献率可达约30%—50%(非空间模型)和60%—80%(空间模型),略低于或接近于气候和生境异质性因子。方差分解结果显示,景观破碎化因子与气候和生境异质性因子的方差贡献率的重叠部分达20%—40%。相对鸟类而言,景观破碎化对哺乳动物物种丰富度的地理格局具有更高的解释率。     

Discordant patterns of genetic and phenotypic differentiation in five grasshopper species codistributed across a microreserve network

Conservation plans can be greatly improved when information on the evolutionary and demographic consequences of habitat fragmentation is available for several codistributed species. Here, we study spatial patterns of phenotypic and genetic variation among five grasshopper species that are codistributed across a network of microreserves but show remarkable differences in dispersal‐related morphology (body size and wing length), degree of habitat specialization and extent of fragmentation of their respective habitats in the study region. In particular, we tested the hypothesis that species with preferences for highly fragmented microhabitats show stronger genetic and phenotypic structure than codistributed generalist taxa inhabiting a continuous matrix of suitable habitat. We also hypothesized a higher resemblance of spatial patterns of genetic and phenotypic variability among species that have experienced a higher degree of habitat fragmentation due to their more similar responses to the parallel large‐scale destruction of their natural habitats. In partial agreement with our first hypothesis, we found that genetic structure, but not phenotypic differentiation, was higher in species linked to highly fragmented habitats. We did not find support for congruent patterns of phenotypic and genetic variability among any studied species, indicating that they show idiosyncratic evolutionary trajectories and distinctive demographic responses to habitat fragmentation across a common landscape. This suggests that conservation practices in networks of protected areas require detailed ecological and evolutionary information on target species to focus management efforts on those taxa that are more sensitive to the effects of habitat fragmentation.     

Morphology of complexes formed between β‐lactoglobulin nanofibrils and pectins is influenced by the pH and structural characteristics of the pectins

For the optimal use of β‐lactoglobulin nanofibrils as a raw material in biological composites an in‐depth knowledge of their interactions with other constituents is necessary. To understand the effect of electrostatic interactions on the morphology of resulting complexes, β‐lactoglobulin nanofibrils were allowed to interact with pectins in which the amount of available negative charge was controlled by selecting their degree of methylesterification. In this study, citrus pectins having different degrees of methylesterification (～48, 67, 86, and 97%) were selected and interacted with nanofibrils at pH 2 and pH 3, where they possess a net positive charge. Electrostatic complexes formed between β‐lactoglobulin nanofibrils and all pectin types, except for the sample having a degree of methylesterification of 97%. The morphology of these complexes, however, differed significantly with the degree of methylesterification of the pectin, its concentration, and the pH of the medium, revealing that distinct desired biological architectures can be attained relatively easily through manipulating the electrostatic interactions. Interestingly, the pectin with a degree of methylesterification of 86% was found to crosslink the β‐lactoglobulin nanofibrils into ordered ‘nanotapes’.     

Does plant diversity increase top–down control of herbivorous insects in tropical forest?

Higher trophic level interactions are key mediators of ecosystem functioning in tropical forests. A rich body of theory has been developed to predict the effects of plant diversity on communities at higher trophic levels and the mechanisms underlying such effects. The 'enemies hypothesis’ states that predators exert more effective top–down control of herbivorous insects with increasing plant diversity. Support for this hypothesis has been found in temperate forests and agroecosystems, but remains understudied in tropical forests. We compared incidence of attacks of different natural enemies using artificial caterpillars in a tropical forest landscape and investigated the role of plant community structure (i.e. species richness, composition and density), and the role of forest fragmentation (i.e. patch size, edge distance and canopy openness) on predation intensity. Plant community effects were tested with respect to three vegetation strata: trees, saplings and herbs. Observed predation was substantially due to ants. Predation rates increased with plant species richness for trees and herbs. Density of saplings, herb cover and herb species composition were important factors for predation. No significant patterns were found for fragmentation parameters, suggesting that forest fragmentation has not altered predation intensity. We conclude that in tropical forests, top–down control of herbivorous insects in the understory vegetation is affected by a combination of plant diversity, plant species composition and structural features of the plant community.     

Spatial analyses of intertidal assemblages on sheltered rocky shores

Abstract Understanding processes in complex assemblages depends on good understanding of spatial and temporal patterns of structure at various spatial scales. There has been little quantitative information about spatial patterns and natural temporal changes in intertidal assemblages on sheltered rocky shores in temperate Australia. Natural changes and responses to anthropogenic disturbances in these habitats cannot be accurately measured and assessed without quantitative data on patterns of natural variability in space and through time. This paper describes some suitable quantitative methods for examining spatial and temporal patterns of diversity and abundances of highshore, midshore and lowshore intertidal assemblages and the important component species for a number of shores in a bay that has not been severely altered by human disturbance. Despite a diverse flora and fauna on these shores, the midshore and lowshore assemblages on sheltered shores were characterized by a few species which were also the most important in discriminating among assemblages on a shore and, for each assemblage, among different shores. The same set of species was also important for measuring small-scale patchiness within each assemblage (i.e. between replicate sites on a shore). Therefore, these data provide a rationale for selecting species that are useful for measuring differences and changes in abundance among places and times at different scales and, hence, can be used in the more complex sampling designs necessary to detect environmental impacts. There was considerable spatial variability in all assemblages and all species (or taxa) examined at scales of metres, tens of metres and kilometres. There were no clear seasonal trends for most measures, with as much or more variability at intervals of 3 months as from year to year. Most interactions between spatial and temporal measures were at the smallest scale, with different sites on the same shore generally showing different changes from time to time. The cause(s) of this apparently idiosyncratic variability1 were not examined, but some potential causes are discussed. These data are appropriate for testing hypotheses about the applicability of these findings to other relatively undisturbed sheltered shores, about effects of different anthropogenic disturbances on sheltered intertidal assemblages and to test hypotheses about differences in intertidal assemblages on sheltered versus wave-exposed shores.     

景观历史对物种多样性的影响: 以内蒙古伊敏露天煤矿为例

矿产开采等人类活动极大地改变着生态环境和景观格局, 景观变化又是导致区域和全球物种多样性丧失的主要原因之一。然而, 物种多样性对周边景观变化响应的时间尺度问题往往被人们忽略。作者以内蒙古草原区伊敏露天煤矿为例, 从物种和功能群两个层次上, 探讨了不同的空间范围(1 km、2 km、3 km、4 km、6 km、8 km、10 km)内在不同时期(1975年、1990年、2000年、2010年)的景观格局(景观优势度指数、生境综合连接度指数和生境连接度概率指数)与生物多样性之间的关系。结果显示: 当前物种多样性与开矿前和开矿初期周边景观格局之间的相关性更高, 而且与4–8 km缓冲区范围内景观格局之间的关系更加密切。不同功能群物种丰富度与景观格局之间的关系不同, 其中, 多年生根茎禾草物种丰富度和当前小尺度(1–3 km)景观格局之间呈显著相关; 多年生杂类草和开矿前和开矿初期大尺度(4–10 km)景观格局之间相关显著; 多年生丛生禾草与景观格局的相关性并未达到显著水平, 但是随着空间尺度的增加出现单峰趋势, 在6 km范围上最高; 灌木、半灌木与景观格局的相关关系随着空间尺度的增加而增加; 一二年生草本与景观格局的相关性始终最低。为此, 本文得出如下结论: (1)物种多样性对周边景观格局变化的响应存在一定时间的滞后, 人类当前不合理的土地利用方式可能引起未来一段时间内该地区一些物种的消失; (2)区域种库决定小尺度物种多样性的大小, 研究区4–8 km范围内具有连通性的生境斑块是主要的种库资源; (3)植物的繁殖策略及种子传播方式是破碎化生境中物种多样性维持的重要机制。     

Comparison of phylogeographic structure and population history of two Phrynocephalus species in the Tarim Basin and adjacent areas

An aridification of the Tarim Basin and adjacent areas since middle Pleistocene has produced significant genetic structuring of the local fauna. We examined the phylogeographic patterns, population structure and history of Phrynocephalus axillaris and Phrynocephalus forsythii using a mitochondrial fragment ND4-tRNA(LEU). Phylogenetic hypotheses were constructed using maximum parsimony and Bayesian inference, and the divergence times of major lineages were estimated by BEAST. Population structure and history were inferred by nested clade analysis, neutrality tests, mismatch distribution, and isolation by distance analysis. The two species might have experienced different evolutionary history throughout their current distribution. For P. forsythii, a vicariant event, as a consequence of geological isolation and desert expansion, might have produced the significant divergence between the Tarim and the Yanqi populations. For P. axillaris, populations of the Yanqi, Turpan and Hami Basins might have been established through dispersal during demographic expansion. Climatic fluctuations caused alternate expansion and shrinkage of rivers and oases several times, which likely led to habitat fragmentation for both species. Interaction between vicariance, dispersal and habitat fragmentation produced the current distribution and genetic diversity. The observed difference between the two species may be due partially to their different reproductive modes (ovoviviparous vs. oviparous).     

Sol-gel-based solid-phase microextraction and gas chromatography-mass spectrometry determination of dextromethorphan and dextrorphan in human plasma

A novel solid-phase microextraction (SPME) method was developed for isolation of dextromethorphan (DM) and its main metabolite dextrorphan (DP) from human plasma followed by GC-MS determination. Three different polymers, poly(dimethylsiloxane) (PDMS), poly(ethylenepropyleneglycol) monobutyl ether (Ucon) and polyethylene glycol (PEG) were synthesized as coated fibers using sol-gel methodologies. DP was converted to its acetyl-derivative prior to extraction and subsequent determination. The porosity of coated fibers was examined by SEM technique. Effects of different parameters such as fiber coating type, extraction mode, agitation method, sample volume, extraction time, and desorption condition, were investigated and optimized. The method is rapid, simple, easy and inexpensive and offers high sensitivity and reproducibility. The limits of detection are 0.010 and 0.015 ng/ml for DM and DP, respectively. The precisions for both analytes are below 5% (n=5). The correlation coefficient was satisfactory (r(2)>0.99) for both DM and DP. Linear ranges were obtained from 0.03 ng/ml to 2 microg/ml for DM and from 0.05 ng/ml to 2 microg/ml for DP.     

荒漠草原“土岛”生境群落物种共存机制

经过长期破碎化, 荒漠草原原生硬质灰钙土斑块散布在广大沙化土地中, 形成类似“土岛”的土被结构。为揭示土岛生境的群落物种共存机制, 2016年在宁夏盐池县皖记沟村选取大(200-300 m²)、中(约100 m²)、小(约50 m²)土岛各3个开展调查, 采用Jaccard相异系数、物种生态位宽度和生态位重叠度、零模型、Meta分析, 综合计算和分析土岛内部与外部植物群落相似性、物种生态位宽度和生态位重叠、物种共存格局及其影响因子。研究发现, (1)随着破碎化加剧, 土岛内部植物多样性整体呈现下降趋势, 群落优势种从短花针茅(Stipa breviflora)转变为猪毛蒿(Artemisia scoparia)和短花针茅共优种, 土岛内外群落相似性增加。(2)土岛内外绝大多数物种生态位重叠较小, 生态位重叠在土岛内呈集中分布, 而土岛外则呈均匀发散分布。(3)环境过滤为主的生态过程决定了土岛生境群落物种的共存格局, 随着土岛面积减小, 环境因子对群落物种共存的调控强度降低, 关键性环境因子由土壤细砂粒和黏粒转变为粗砂粒, 显著性竞争物种共存格局在小岛出现。综上所述, 土岛生境对于维持草原物种具有重要作用, 环境过滤主导了荒漠草原物种共存格局。随着生境破碎化加剧或土岛面积减小, 物种共存格局及其调控因子发生转变。保护面积在200 m²以上的大土岛对于恢复荒漠草原区草原成分种和其物种多样性机制都十分必要。     

Dispersal, habitat differences, and comparative phylogeography of Southeast Asian seahorses (Syngnathidae: Hippocampus)

Four distinct phylogeographical patterns across Southeast Asia were observed for four species of seahorse (genus Hippocampus) with differing ecologies. For all species, genetic differentiation (based on cytochrome b sequence comparisons) was significantly associated with sample site (Phi(ST) = 0.190-0.810, P < 0.0001) and with geographical distance (Mantel's r = 0.37-0.59, P < 0.019). Geographic locations of genetic breaks were inconsistent across species in 7/10 comparisons, although some similarities across species were also observed. The two shallow-water species (Hippocampus barbouri and Hippocampus kuda) have colonized the Sunda Shelf to a lesser degree than the two deeper-water species (Hippocampus spinosissimus and Hippocampus trimaculatus). In all species the presence of geographically restricted haplotypes in the Philippines could indicate past population fragmentation and/or long-distance colonization. A nested clade analysis (NCA) revealed that long-distance colonization and/or fragmentation were likely the dominant forces that structure populations of the two shallow-water species, whereas range expansion and restricted dispersal with isolation by distance were proportionally more important in the history of the two deeper-water species. H. trimaculatus has the most widespread haplotypes [average clade distance (D(c)) of nonsingleton haplotypes = 1169 km], indicating potentially high dispersal capabilities, whereas H. barbouri has the least widespread haplotypes (average D(c) = 67 km) indicating potentially lower dispersal capabilities. Pleistocene separation of marine basins and postglacial flooding of the Sunda Shelf are extrinsic factors likely to have contributed to the phylogeographical structure observed, whereas differences among the species appear to reflect their individual ecologies.     

The elevational gradient of Andean plant endemism: varying influences of taxon-specific traits and topography at different taxonomic levels

Aim Little is known about the elevational gradient of plant endemism. It is mostly assumed that patterns are determined by topographical factors such as area of elevational belts and degree of habitat fragmentation, but comparative studies of different plant taxa along the same elevational gradient are lacking. The aim was to compare the elevational patterns of plant endemism of the entire flora and selected families and genera in a search for commonalities. Methods The elevational patterns of endemism for the entire Ecuadorean vascular plant flora, for twenty‐seven selected families, and for twenty‐four selected genera based on the Catalogue of Vascular Plants of Ecuador were analysed. Results Elevational patterns of endemism were non‐random at all taxonomic levels but there was no common elevational pattern. Rather, the study groups showed a wide variety of independent patterns at all taxonomic levels. Most groups had hump‐shaped patterns with maxima at different elevations and mostly at the same or at higher elevations than the maxima of species richness. The overall flora showed highest endemism in the narrowest and most fragmented elevational belts, presumably because of the consequent fragmentation of species populations. Main conclusions Patterns of endemism appear to be influenced both by taxon‐specific ecological traits (e.g. life form, reproduction, dispersal, demography, spatial population structure, competitive ability) in their specific interaction with historical processes and by environmental factors such as topographical fragmentation. The degree to which these influences become visible along the elevational gradient are determined by which combination of species is analysed: for a given genus or family, taxon‐specific traits dominate the patterns, for the entire flora taxon‐specific patterns are blurred by averaging and the signal of topography emerges. Beyond the elevational gradient as such, this study shows that the frequently stated assumption that given biogeographical settings lead to similar patterns of endemism among different taxa is wrong.     

Historic DNA reveals contemporary population structure results from anthropogenic effects, not pre-fragmentation patterns

Contemporary patterns of genetic structure among fragmented populations can either result from historic patterns or arise from human-induced fragmentation. Use of historic samples collected prior to fragmentation allows for the origin of genetic structure to be established and appropriate management steps to be determined. In this study, we compare historic and contemporary levels of genetic diversity and structure of an endangered passerine, the New Zealand mohua or yellowhead (Mohoua ochrocephala), using nuclear microsatellites. We found that a significant amount of allelic richness has been lost over the last 100 years. Close to half of this was due to extinction of birds from entire regions, but almost as much was due to loss of genetic diversity within extant populations. We found a pattern of isolation by distance among contemporary populations, which could have resulted from historic structure due to limited gene flow along a latitudinal cline. However, we found that minimal genetic structure existed historically. The pattern of increased structure over time was confirmed by factorial correspondence analysis. We conclude that the genetic structure apparent today resulted from anthropogenic effects of recent fragmentation and isolation. We emphasize the importance of assessing genetic structure of populations prior to their fragmentation, when determining the significance of contemporary patterns. This study highlights the growing importance of museum specimens as a tool in the conservation of threatened and endangered species.