Chemometric evaluation of the anti-cancer pro-drug podophyllotoxin and potential therapeutic analogues in Juniperus and Podophyllum species

Introduction – Podophyllotoxin, deoxypodophyllotoxin, demethylpodophyllotoxin and podophyllotoxone are four therapeutically potent secondary metabolites. There is a dearth of information on the holistic analysis of their distribution pattern in both phylogenetic and ecological contexts. Objectives – To analyse the continuum of the above metabolites in Juniperus and Podophyllum species collected from natural populations in Himalayan environments and the botanical gardens of Rombergpark and Haltern (Germany) using multi‐component LC‐ESI‐MS/MS, coupled with statistically relevant chemometric assessment. Methodology – We evaluated the individual and holistic metabolite profiles and chemometrically correlated the phytochemical loads between various species (infraspecific), organic and aqueous extracts, and populations of the same species from different locations, different species from same location, different species from different locations and infrageneric populations from same and different locations. Results – Multivariate analysis revealed Juniperus x‐media Pfitzeriana as a suitable alternative to Podophyllum hexandrum for commercial exploitation. A significant positive correlation of podophyllotoxone with both podophyllotoxin and demethylpodophyllotoxin, and a negative correlation of podophyllotoxin with both deoxypodophyllotoxin and demethylpodophyllotoxin (infraspecific among Podophyllum), were observed by Kruskal's multidimensional scaling and corroborated by principal component analysis, indicating probable similarity and/or difference between the biosynthetic pathways, and synergistic and/or antagonistic principles, respectively. Finally, linear discriminant analysis and hierarchical agglomerative cluster analysis revealed considerable infrageneric and infraspecific variability in secondary compound spectra and load of the different populations under study. Conclusion – Such holistic studies of plants and their therapeutic metabolites ought to assist in selecting plants, geographical areas and environmental conditions for bioprospecting and global‐scale phytochemical and phylogenetic diversity studies in the future. Copyright © 2010 John Wiley & Sons, Ltd.     

Ancestry and evolution of seasonal migration in the Parulidae

Seasonal migration in birds is known to be highly labile and subject to rapid change in response to selection, such that researchers have hypothesized that phylogenetic relationships should neither predict nor constrain the migratory behaviour of a species. Many theories on the evolution of bird migration assume a framework that extant migratory species have evolved repeatedly and relatively recently from sedentary tropical or subtropical ancestors. We performed ancestral state reconstructions of migratory behaviour using a comprehensive, well-supported phylogeny of the Parulidae (the 'wood-warblers'), a large family of Neotropical and Nearctic migratory and sedentary songbirds, and examined the rates of gain and loss of migration throughout the Parulidae. Counter to traditional hypotheses, our results suggest that the ancestral wood-warbler was migratory and that losses of migration have been at least as prevalent as gains throughout the history of Parulidae. Therefore, extant sedentary tropical radiations in the Parulidae represent losses of latitudinal migration and colonization of the tropics from temperate regions. We also tested for phylogenetic signal in migratory behaviour, and our results indicate that although migratory behaviour is variable within some wood-warbler species and clades, phylogeny significantly predicts the migratory distance of species in the Parulidae.     

The uropygial gland of the monk parakeet Myiopsitta monachus: Histology,morphogenesis, and evolution within Psittaciformes (Aves)

We describe the morphology, histology, and histochemical characteristics of the uropygial gland (UG) of the monk parakeet Myiopsitta monachus. The UG has a heart‐shape external appearance and adenomers extensively branched with a convoluted path, covered by a stratified epithelium formed by different cellular strata and divided into three zones (based on the epithelial height and lumen width), a cylindrical papilla with an internal structure of delicate type and two excretory pores surrounded by a feather tuft. Histochemical and lectin‐histochemical techniques performed showed positivity against PAS, AB pH 2.5, AB‐PAS, and some lectines, likely related to the granivorous feeding habits. Also, we describe the morphogenesis of the UG of the monk parakeet, which appears at embryological stage 34 as a pair of ectodermal invaginations. Heterochronic events in the onset development of the UG when compared with other birds could be recognized. Finally, to examine the phylogenetic occurrence of the UG within the Psittaciformes and infer its evolutionary history, we mapped its presence/absence over a molecular phylogeny. The reconstruction of the characters states at ancestral nodes revealed that the presence of the UG was the plesiomorphic feature for Psittaciformes and its loss evolved independently more than once.     

5种东北红豆杉植物群丛及其物种多样性的比较

东北红豆杉(Taxus cuspidata)是我国数量极少的珍贵濒危树种, 了解其天然群落的组成和特征对东北红豆杉种群的保护利用和恢复有重要意义。本文对吉林省天然东北红豆杉群落进行调查, 根据物种组成进行系统聚类分析。将20块40 m × 40 m样地划分为5种群丛类型, 分别以优势种进行命名, 即: Ⅰ. 舞鹤草-五味子+狗枣猕猴桃-紫椴+臭冷杉群丛; II. 东北羊角芹-狗枣猕猴桃-臭冷杉群丛; III. 盾叶唐松草-狗枣猕猴桃-臭冷杉群丛; IV. 舞鹤草-软枣猕猴桃-红松+紫椴+臭冷杉群丛; V. 舞鹤草-软枣猕猴桃-紫椴+臭冷杉群丛。对群丛的物种组成、群落结构和群丛类型、物种多样性进行了分析。物种多样性选用Menhinick丰富度指数、Pielou均匀度指数、Simpson优势度指数以及Shannon-Wiener多样性指数, 对比分析不同群丛特征。结果显示: 东北红豆杉植物群落组成中蔷薇科的种和属数所占比例最大; 5个群丛的多样性指数顺序为群丛V > 群丛III > 群丛IV > 群丛II > 群丛Ⅰ; 群丛Ⅰ和II具有较低的多样性和较高的优势度, 群丛II和群丛III的乔木层的多样性指数差异不明显, 但其丰富度指数和优势度指数却呈现了相反的特征; 群丛II丰富度低而优势度高, 而群丛III丰富度高而优势度低; 群丛III中的草本层的多样性高于乔木层, 群落郁闭度较低; 群丛IV和群丛V均位于和龙市荒沟林场, 随着海拔上升, 其物种多样性随之下降。结果表明, 不同物种组成的东北红豆杉植物群丛的群落特征存在显著差异。     

Phylogeny and evolution of sexually selected tail ornamentation in widowbirds and bishops (Euplectes spp.)

Despite similar ecology, mating systems and female preferences for supernormal tails, the 17 species of African widowbirds and bishops (Euplectes spp.) show astonishing variation in male tail ornamentation. Whereas bishops retain their brown nonbreeding tails in nuptial plumage, widowbirds grow black nuptial tails, varying in length from a few centimetres in E. axillaris to the extreme half metre train of E. progne. Here, we phylogenetically reconstruct the evolution of the discrete trait, nuptial tail and the continuous trait, tail length, using a molecular phylogeny of 33 Euplectes subspecies. Unlike many recent findings of labile evolution of plumage ornaments, our results suggest that the nuptial tail of Euplectes is a derived and phylogenetically conserved ornamental trait that, once gained, shows directional evolution in its expression. Directionality is demonstrated in the trivial sense of a short‐tailed ancestor, and by contingency and randomization tests suggesting that branches with increasing tail length are overrepresented. This supports an early origin and strong retention of directional female mate choice in widowbirds and bishops, as previously indicated by empirical and experimental results, and provides a less labile, yet rapid scenario of sexually selected diversification.     

The evolution of male nuptial colour in a sexually dimorphic group of fishes (Percidae: Etheostomatinae)

To test hypotheses explaining variation in elaborate male colouration across closely related species groups, ancestral‐state reconstructions and tests of phylogenetic signal and correlated evolution were used to examine the evolution of male body and fin colouration in a group of sexually dichromatic stream fishes known as darters (Percidae: Etheostomatinae). The presence or absence of red–orange and blue–green male colour traits were scored across six body regions in 99 darter species using a recently estimated amplified fragment length polymorphism (AFLP) phylogeny for comparative analyses. Ancestral‐state reconstructions infer the most recent common ancestor of darters to lack red–orange colour and possess blue–green colour on different body regions, suggesting variation between species is due to independent gains of red–orange and losses of blue–green. Colour traits exhibit substantial phylogenetic signal and are highly correlated across body regions. Comparative analyses were repeated using an alternative phylogenetic hypothesis based on one mitochondrial and two nuclear genes, yielding similar results to analyses based on the AFLP phylogeny. Red–orange colouration in darters appears to be derived; whereas, blue–green appears to be ancestral, which suggests that different selection mechanisms may be acting on these two colour classes in darters.     

数量性状水平上甘、青两省春小麦品种间的遗传多样性现状及演变趋势

在数量性状水平上,调查了43个春小麦品种的19个数量性状,利用主成分分析法计算品种间的欧氏平方遗传距离。并在此基础上用最小方差法做了聚类分析。发现43个品种间在数量性状水平上的遗传距离变异范围很大（0.926-67.942)。平均遗传距离为18.000,说明供试品种的19个考察性状上存在较大的表型变异,从聚类结果来看,地方品种基本上被聚在一起,说明地方品种同引进品种和育成品种至少在表型上存在较大差异。从20世纪40年代以来,在数量水平上甘,青两省春小麦地方品种间的遗传多样性水平最高（GD=31.389),其次是20世纪50年代引进品种（GD=26.308),而育成品种间的遗传多样性水平最低,总体上呈下降趋势,说明随着育种进程的深入,作为育种目标追求的经济性状趋于一致,其变异集中在一个狭小的范围之内。品种间的遗传多样性下降。     

The legacy of a vanished sea: a high level of diversification within a European freshwater amphipod species complex driven by 15 My of Paratethys regression

The formation of continental Europe in the Neogene was due to the regression of the Tethys Ocean and of the Paratethys Sea. The dynamic geology of the area and repetitious transitions between marine and freshwater conditions presented opportunities for the colonization of newly emerging hydrological networks and diversification of aquatic biota. Implementing mitochondrial and nuclear markers in conjunction with a large‐scale sampling strategy, we investigated the impact of this spatiotemporal framework on the evolutionary history of a freshwater crustacean morphospecies. The Gammarus balcanicus species complex is widely distributed in the area previously occupied by the Paratethys Sea. Our results revealed its high diversification and polyphyly in relation to a number of other morphospecies. The distribution of the studied amphipod is generally characterized by very high local endemism and divergence. The Bayesian time‐calibrated reconstruction of phylogeny and geographical distribution of ancestral nodes indicates that this species complex started to diversify in the Early Miocene in the central Balkans, partially in the shallow epicontinental sea. It is possible that there were several episodes of inland water colonization by local brackish water lineages. Subsequent diversification within clades and spread to new areas could have been induced by Alpine orogeny in the Miocene/Pliocene and, finally, by Pleistocene glaciations. The present distribution of clades, in many cases, still reflects Miocene palaeogeography of the area. Our results point out that investigations of the historical aspect of cryptic diversity in other taxa may help in a general understanding of the origins of freshwater invertebrate fauna of Europe.     

海南岛3个林区热带云雾林植物多样性变化

以分布在海南岛西部(霸王岭国家级自然保护区, 21个样方)、西南部(尖峰岭国家级自然保护区, 12个样方)和中部(黎母山省级自然保护区, 15个样方)的热带云雾林为研究对象, 研究α及β物种多样性、功能多样性、谱系多样性的变化, 为植物多样性的保护提供科学依据。结果表明: 尖峰岭群落树木个体多度、物种丰富度最大, 黎母山群落树木个体多度、物种丰富度最小; 黎母山群落间物种组成差异最大, 霸王岭群落间物种组成差异最小, 海南岛霸王岭(西部)、尖峰岭(西南部)和黎母山(中部) 3个林区热带云雾林物种多样性差异可能与空气温度和相对湿度有关。尖峰岭群落内功能丰富度、Rao’s二次熵最低, 功能均匀度最高, 群落间平均成对性状距离最小, 反映群落构建主要受环境筛影响; 霸王岭群落Rao’s二次熵最高, 功能均匀度最低, 群落间平均成对性状距离最大, 反映群落构建主要受限制相似性影响; 黎母山群落内功能丰富度最高, 群落间平均最近性状距离最大, 表明限制相似性在黎母山热带云雾林群落构建中的作用更重要。霸王岭群落内谱系多样性、物种间平均最近相邻谱系距离均较大, 反映物种间谱系关系趋于发散; 黎母山群落内谱系多样性、物种间平均成对谱系距离及群落间平均成对谱系距离均最小, 反映物种间谱系关系趋于聚集; 而尖峰岭群落内物种间平均成对谱系距离、群落间平均成对谱系距离最大, 但物种间平均最近相邻谱系距离最小, 反映物种间谱系关系既具有发散又具有聚集的共存格局。因此, 海南岛热带云雾林群落植物多样性变化格局与环境及物种间相互作用有关。     

Structural Characterisation of Alkaloids in Leaves and Roots of Stephania kwangsiensis by LC‐QTOF‐MS

Introduction

The tuberous roots of Stephania kwangsiensis, which contain bioactive alkaloids, are used as a traditional Chinese medicine. Overexploitation of the roots has made the plant increasingly rare, and the abundant leaves of the same plant may offer a potential alternative. However, there is insufficient phytochemical information for a comparison of alkaloid compositions in the two parts.

Objective

To characterise and compare the alkaloids in the leaves and roots of S. kwangsiensis.

Methods

The alkaloids in S. kwangsiensis were characterised using high pressure liquid chromatography coupled with positive electrospray ionisation quadrupole time‐of‐flight tandem mass spectrometry (HPLC‐(+)ESI‐QTOF‐MS/MS). The alkaloid compositions in the leaves and roots were compared by visual inspection combined with principal component analysis (PCA) of the HPLC‐MS data.

Results

Seventy‐five alkaloids comprising aporphine‐, proaporphine‐, protoberberine‐, benzylisoquinoline‐, bisbenzylisoquinoline‐ and morphine‐type alkaloids were identified or tentatively identified in the roots and leaves of S. kwangsiensis. Sixty‐three of these alkaloids have not been previously reported in this species, and three have not been previously reported in the literature. The roots and leaves had similarities in alkaloid composition but differences in the peak intensities of most alkaloids. The PCA revealed that the samples were clustered into two distinct groups, which corresponded to leaves and roots.

Conclusion

This study further clarified the chemical constituents in the roots of S. kwangsiensis, and revealed that diverse alkaloids were also present in the leaves. The comparative chemical profiling of the two parts provides useful information on their potential medicinal use. Copyright © 2017 John Wiley & Sons, Ltd.     

太湖湿地昆虫群落结构及多样性

为了探讨太湖湿地昆虫群落的结构和变化规律,对太湖湿地各生境样点昆虫群落进行了调查研究.调查共获得昆虫标本24968号,隶属于13目88科301属318种;半翅目、鳞翅目、鞘翅目和直翅目等为优势类群.各样点昆虫群落多样性指数和均匀度指数特征值为湿地恢复示范区＞渔阳山＞三山岛＞三洋村＞灵湖村,优势集中性指数的特征值比较为:湿地恢复示范区＜渔阳山＜三洋村＜三山岛＜灵湖村.各样点昆虫群落的多样性指数与均匀度指数和丰富度指数表现一致.在时间序列上渔阳山样点昆虫群落的多样性指数波动最小.昆虫群落的极点排序表明渔阳山和湿地恢复示范区样点昆虫群落是最相近的.主分量分析的结果显示太湖湿地昆虫群落主要受捕食性和植食性昆虫群落的影响.湿地恢复示范区样点昆虫群落内天敌昆虫比例偏低,降低了昆虫群落对外界扰动的缓冲能力.综合昆虫群落的各项分析结果发现渔阳山样点昆虫群落稳定性最高,三洋村样点昆虫群落稳定性最低.     

戴云山国家级自然保护区黄山松群落类型与物种多样性分析

采用典型取样法对戴云山国家级自然保护区黄山松(Pinus taiwanensis Hayata)群落的类型和物种多样性进行了分析。以乔木层主要树种的重要值为参考指标,通过系统聚类分析,可将该黄山松群落分为8个群丛类型:黄山松+杉木-乌药-铁芒萁群丛(Assoc.P.taiwanensis+Cunninghamia lanceolata-Lindera aggregate-Dicranopterislinearis);黄山松+多脉青冈-细枝柃-里白群丛(Assoc.P.taiwanensis+Cyclobalanopsis multinervis-Eurya loquaiana-Hicriopteris glauca);黄山松+江南山柳-短尾越桔-铁芒萁群丛(Assoc.P.taiwanensis+Clethra cavaleriei-Vacciniumcarlesii-Dicranopteris linearis);黄山松+甜槠-肿节少穗竹-里白群丛(Assoc.P.taiwanensis+Castanopsis eyrei-Oligostachyum oedogonatum-Hicriopteris glauca);黄山松+吊钟花-短尾越桔-狗脊群丛(Assoc.P.taiwanensis+Enkianthus quinqueflorus-Vaccinium carlesii-Woodwardia japonica);黄山松+华丽杜鹃-短尾越桔-里白群丛(Assoc.P.taiwanensis+Rhododendron farrerae-Vaccinium carlesii-Hicriopteris glauca);黄山松+光叶水青冈-满山红-铁芒萁群丛(Assoc.P.taiwanensis+Fagus lucida-Rhododendron mariesii-Dicranopteris linearis);黄山松-满山红-平颖柳叶箬群丛(Assoc.P.taiwanensis-Rhododendron mariesii-Isachne truncata)。各群丛总体物种丰富度指数和多样性指数总体上呈随海拔升高而降低的趋势,总体均匀度指数Pielou指数和Alatalo指数波动不大。在群落垂直结构上,乔木层、灌木层和草本层物种丰富度指数、多样性指数和均匀度指数有差异;各群丛间乔木层和灌木层的物种丰富度指数、多样性指数和均匀度指数波动较大,草本层这3个指数波动较小且数值上小于前两者。分析结果显示:海拔是决定该黄山松群落多样性差异的主导因子。     

高原鼠兔干扰对高寒草甸植物物种和功能性状beta多样性的影响

采用野外空间多点同步取样,分析了高原鼠兔干扰对高寒草甸植物物种beta多样性和植物功能性状beta多样性的影响,确定了高原鼠兔干扰下高寒草甸植物物种和功能性状beta多样性的变化途径,分别提出了高原鼠兔干扰区域内,基于植物物种多样性和功能性状多样性的高寒草甸植物多样性维持策略。结果表明,高原鼠兔干扰使高寒草甸植物物种相似性显著降低了28.1%,植物功能相似性降低了28.7%。尽管高原鼠兔干扰没有改变高寒草甸植物物种和功能性状beta多样性的变化途径,且对植物物种和功能性状的嵌套组分不存在显著影响,但高原鼠兔干扰显著降低了植物物种和功能性状周转组分所占的比例,降幅分别为36.6%和34.3%。高原鼠兔干扰区域内,高寒草甸植物物种beta多样性的变化以周转为主导(周转占比81.4%;嵌套占比:18.6%),植物功能性状beta多样性的变化以嵌套为主导(嵌套占比64.9%;周转占比35.1%)。因此,针对划定的高原鼠兔干扰区,需要同时保护区域内所有高原鼠兔栖息地(多位点保护),以达到维持植物物种多样性的目的,而可以仅通过保护该区域内植物功能性状丰富的位点,即可维持较高的植物功能多样性。     

Variation of chemical compounds in wild Heliconiini reveals ecological factors involved in the evolution of chemical defenses in mimetic butterflies

Evolutionary convergence of color pattern in mimetic species is tightly linked with the evolution of chemical defenses. Yet, the evolutionary forces involved in natural variations of chemical defenses in aposematic species are still understudied. Herein, we focus on the evolution of chemical defenses in the butterfly tribe Heliconiini. These neotropical butterflies contain large concentrations of cyanogenic glucosides, cyanide‐releasing compounds acting as predator deterrent. These compounds are either de novo synthesized or sequestered from their Passiflora host plant, so that their concentrations may depend on host plant specialization and host plant availability. We sampled 375 wild Heliconiini butterflies across Central and South America, covering 43% species of this clade, and quantify individual variations in the different CGs using liquid chromatography coupled with tandem mass spectrometry. We detected new compounds and important variations in chemical defenses both within and among species. Based on the most recent and well‐studied phylogeny of Heliconiini, we show that ecological factors such as mimetic interactions and host plant specialization have a significant association with chemical profiles, but these effects are largely explained by phylogenetic relationships. Our results therefore suggest that shared ancestries largely contribute to chemical defense variation, pointing out at the interaction between historical and ecological factors in the evolution of Müllerian mimicry.     

The infrageneric taxonomy of Chaerophyllum (Apiaceae) revisited: new evidence from nuclear ribosomal DNA ITS sequences and fruit anatomy

Evolutionary relationships among 65 Chaerophyllum spp. were inferred from nuclear ribosomal DNA internal transcribed spacer (ITS) sequence variation. Thirty‐one species, represented by 158 mericarp samples, were analysed for fruit anatomical character variation, employing phylogenetic and phenetic methods to explore their congruence with infrageneric divisions based on molecular data. Phylogenetic trees inferred from molecular data using maximum likelihood (ML) and Bayesian inference (BI) methods corroborated the division of the genus into four sections: Chaerophyllum, Dasypetalon, Physocaulis and Chrysocarpum. From among the newly sequenced species, the Greek endemic C. heldreichii was grouped with section Chaerophyllum, whereas the highly variable Asian C. reflexum–C. villosum complex formed an early‐branching paraphyletic assemblage in section Chrysocarpum. The recently described C. karsianum has an identical ITS sequence to C. bulbosum, whereas C. aksekiense was clearly separated from the morphologically similar C. macrospermum. Our study confirmed the postulated synonymy of several species on the basis of morphology, but also demonstrated distant relationships between some morphologically similar species. With the exception of the monotypic section Physocaulis, we were unable to find carpological traits matching sectional divisions. We hypothesize that fruit characters evolved rapidly as a result of diversification of members of the genus in different habitats. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 298–313.     

Multivariate analyses of salt stress and metabolite sensing in auto- and heterotroph Chenopodium cell suspensions

Global warming increases plant salt stress via evaporation after irrigation, but how plant cells sense salt stress remains unknown. Here, we searched for correlation-based targets of salt stress sensing in Chenopodium rubrum cell suspension cultures. We proposed a linkage between the sensing of salt stress and the sensing of distinct metabolites. Consequently, we analysed various extracellular pH signals in autotroph and heterotroph cell suspensions. Our search included signals after 52 treatments: salt and osmotic stress, ion channel inhibitors (amiloride, quinidine), salt-sensing modulators (proline), amino acids, carboxylic acids and regulators (salicylic acid, 2,4-dichlorphenoxyacetic acid). Multivariate analyses revealed hirarchical clusters of signals and five principal components of extracellular proton flux. The principal component correlated with salt stress was an antagonism of γ-aminobutyric and salicylic acid, confirming involvement of acid-sensing ion channels (ASICs) in salt stress sensing. Proline, short non-substituted mono-carboxylic acids (C2-C6), lactic acid and amiloride characterised the four uncorrelated principal components of proton flux. The proline-associated principal component included an antagonism of 2,4-dichlorphenoxyacetic acid and a set of amino acids (hydrophobic, polar, acidic, basic). The five principal components captured 100% of variance of extracellular proton flux. Thus, a bias-free, functional high-throughput screening was established to extract new clusters of response elements and potential signalling pathways, and to serve as a core for quantitative meta-analysis in plant biology. The eigenvectors reorient research, associating proline with development instead of salt stress, and the proof of existence of multiple components of proton flux can help to resolve controversy about the acid growth theory.     

Metabolic flux analysis in Escherichia coli by integrating isotopic dynamic and isotopic stationary 13C labeling data

The novel concept of isotopic dynamic 13C metabolic flux analysis (ID-13C MFA) enables integrated analysis of isotopomer data from isotopic transient and/or isotopic stationary phase of a 13C labeling experiment, short-time experiments, and an extended range of applications of 13C MFA. In the presented work, an experimental and computational framework consisting of short-time 13C labeling, an integrated rapid sampling procedure, a LC-MS analytical method, numerical integration of the system of isotopomer differential equations, and estimation of metabolic fluxes was developed and applied to determine intracellular fluxes in glycolysis, pentose phosphate pathway (PPP), and citric acid cycle (TCA) in Escherichia coli grown in aerobic, glucose-limited chemostat culture at a dilution rate of D = 0.10 h(-1). Intracellular steady state concentrations were quantified for 12 metabolic intermediates. A total of 90 LC-MS mass isotopomers were quantified at sampling times t = 0, 91, 226, 346, 589 s and at isotopic stationary conditions. Isotopic stationarity was reached within 10 min in glycolytic and PPP metabolites. Consistent flux solutions were obtained by ID-13C MFA using isotopic dynamic and isotopic stationary 13C labeling data and by isotopic stationary 13C MFA (IS-13C MFA) using solely isotopic stationary data. It is demonstrated that integration of dynamic 13C labeling data increases the sensitivity of flux estimation, particularly at the glucose-6-phosphate branch point. The identified split ratio between glycolysis and PPP was 55%:44%. These results were confirmed by IS-13C MFA additionally using labeling data in proteinogenic amino acids (GC-MS) obtained after 5 h from sampled biomass.     

In and out of refugia: historical patterns of diversity and demography in the North American Caesar's mushroom species complex

Some of the effects of past climate dynamics on plant and animal diversity make‐up have been relatively well studied, but to less extent in fungi. Pleistocene refugia are thought to harbour high biological diversity (i.e. phylogenetic lineages and genetic diversity), mainly as a product of increased reproductive isolation and allele conservation. In addition, high extinction rates and genetic erosion are expected in previously glaciated regions. Some of the consequences of past climate dynamics might involve changes in range and population size that can result in divergence and incipient or cryptic speciation. Many of these dynamic processes and patterns can be inferred through phylogenetic and coalescent methods. In this study, we first delimit species within a group of closely related edible ectomycorrhizal Amanita from North America (the American Caesar's mushrooms species complex) using multilocus coalescent‐based approaches; and then address questions related to effects of Pleistocene climate change on the diversity and genetics of the group. Our study includes extensive geographical sampling throughout the distribution range, and DNA sequences from three nuclear protein‐coding genes. Results reveal cryptic diversity and high speciation rates in refugia. Population sizes and expansions seem to be larger at midrange latitudes (Mexican highlands and SE USA). Range shifts are proportional to population size expansions, which were overall more common during the Pleistocene. This study documents responses to past climate change in fungi and also highlights the applicability of the multispecies coalescent in comparative phylogeographical analyses and diversity assessments that include ancestral species.     

HPLC fingerprints combined with principal component analysis,hierarchical cluster analysis and linear discriminant analysis for the classification and differentiation of Peganum sp. indigenous to China

Introduction – Seeds of wild Peganum harmala Linn., P. multisectum (Maxim) Bobr., P. nigellastrum Bunge and a probable indeterminate species, herein referred to as P. variety, are commonly used in Chinese medicine. These seeds cannot be differentiated based on morphology. Objective – Seeds of P. harmala Linn., P. multisectum (Maxim) Bobr., P. nigellastrum Bunge and P. variety were collected in different provinces in China and their HPLC profiles were recorded for statistical analysis and pattern recognition. Methodology – HPLC chromatograms of seed extracts were recorded under the same conditions. Individual HPLC chromatograms for each species were evaluated against the mean chromatogram for the same species generated using a similarity evaluation computer program. Data from chromatographic fingerprints were also processed using principal component analysis (PCA), hierarchical cluster analysis (HCA) and linear discriminant analysis (LDA). Results – The Peganum sp. seed extracts had similar HPLC fingerprints but with some inter‐specific differences. The chromatographic fingerprints combined with PCA, HCA and LDA could distinguish the seeds of the different species of Peganum investigated. Conclusion – HPLC fingerprints can be used to authenticate and differentiate the seeds of three different species of genus Peganum indigenous to China. The results indicated that the unidentified P. variety might indeed be a new species or variety. Copyright © 2009 John Wiley & Sons, Ltd.