Phylogeny of slug species of the genus Arion: evidence of monophyly of Iberian endemics and of the existence of relict species in Pyrenean refuges

The Iberian Peninsula contains the majority of the Paleartic land slug species of the genus Arion, which exhibits diverse taxonomic problems. The present study investigated Arion taxonomy on the basis of analyses of the mitochondrial ND1 gene and nuclear internal transcribed spacer 1 (ITS1) sequences. The Iberian endemic species were monophyletically clustered in two divergent sister clades. The topotype specimens of Arion lusitanicus and the closely related species Arion nobrei and Arion fuligineus, as well as Arion hispanicus and Arion flagellus, were grouped into an ‘Atlantic’ clade, whereas Arion baeticus, Arion gilvus, Arion anguloi, Arion wiktori and Arion paularensis were included in a ‘Continental–Mediterranean’ clade. Calibration of mutation rate in the ND1 gene suggested that the divergence of these two clades occurred around the Pliocene–Pleistocene boundary, with subsequent speciation events during the Pleistocene. A group of ancestral and divergent endemic species with distribution centred in the Pyrenean mountain range (Arion molinae, Arion lizarrusti, Arion antrhacius and Arion iratii) arose in the Pliocene and survived through the Pleistocene in geographically confined small populations. Arion lusitanicus showed up to be polyphyletic: specimens, sampled outside the geographic range of the topotype in the north‐western Iberian Peninsula, were included in a non‐monophyletic clade together with the widely distributed species Arion ater and Arion rufus. The divergent species with a wide European distribution (Arion subfuscus, Arion hortensis, Arion fagophilus and Arion intermedius) were located in basal positions in all topologies. The evolutionary history of these slug species (highly sensitive to climatic factors, with capacity for both outcrossing and selfing, and with low dispersal ability) appears to have been moulded by Pliocene–Pleistocene climate events and by the rugged topography of southern Europe, giving rise to repeated cycles of population isolation during periods of glaciation alternating with interglacial expansions limited by geographic barriers.     

The mapping of phytochrome genes and photomorphogenic mutants of tomato

The map positions of five previously described phytochrome genes have been determined in tomato (Lycopersicon esculentum Mill.) The position of the yg-2 gene on chromosome 12 has been confirmed and the classical map revised. The position of the phytochrome A (phy A)-deficient fri mutants has been refined by revising the classical map of chromosome 10. The position of the PhyA gene is indistinguishable from that of the fri locus. The putative phyB1-deficient tri mutants were mapped by classical and RFLP analysis to chromosome 1. The PhyB1 gene, as predicted, was located at the same position. Several mutants with the high pigment (hp) phenotype, which exaggerates phytochrome responses, have been reported. Allelism tests confirmed that the hp-2 mutant is not allelic to other previously described hp (proposed here to be called hp-1) mutants and a second stronger hp-2 allele (hp-2 ^j ) was identified. The hp-2 gene was mapped to the classical, as well as the RFLP, map of chromosome 1. Received: 24 May 1996 / Accepted: 14 June 1996     

Improvement of Aspergillus oryzae for hyperproduction of endoglucanase: expression cloning of cmc-1 gene of Aspergillus aculeatus

FI-Carboxymethylcellulase (cmc1; family 12) is one of the endoglucanases of Aspergillus aculeatus and consists of single polypeptide chain of 221 amino acids. The cmc1 gene was expressed in Aspergillus oryzae niaD300 (niaD⁻) under promoter 8142. The plasmid pCMG14 carrying the cmc1 gene at PstI site was used as a source of the gene (920 bp) and Aspergillus oryzae was successfully transformed by the plasmid pNAN-cmc1 (harboring cmc1 gene). The plasmid was integrated in Aspergillus oryzae niaD300 genome at niaD locus and the transformed fungus constitutively produced very high amounts of endoglucanases when grown on glucose, maltose, soluble starch and wheat bran.     

Purification of Cytochrome a of an L-Glutamate-producing Microorganism,Brevibacterium thiogenitalis

The respiratory chain system of Brev. thiogenitalis grown in the presence of copper ions contained cytochromes a, b and c. The cytochrome a was solubilized and purified from the cell-free extracts by means of Triton X-100 and cholate extraction, and DEAE-cellulose chromatography. It was purified about 130-fold from the cell-free extracts and was free from other cytochromes, The purified preparation contained 1.4 mμatom copper and 1.9 mμatom iron per mμmole heme a, respectively, and approximately 5 mμmoles heme a per mg protein.     

Comparison of the killer toxin of several yeasts and the purification of a toxin of type K2

A total of 13 killer toxin producing strains belonging to the genera Saccharomyces, Candida and Pichia were tested against each other and against a sensitive yeast strain. Based on the activity of the toxins 4 different toxins of Saccharomyces cerevisiae, 2 different toxins of Pichia and one toxin of Candida were recognized. The culture filtrate of Pichia and Candida showed a much smaller activity than the strains of Saccharomyces. Extracellular killer toxins of 3 types of Saccharomyces were concentrated and partially purified. The pH optimum and the isoelectric point were determined. The killer toxins of S. cerevisiae strain NCYC 738, strain 399 and strain 28 were glycoproteins and had a molecular weight of M_r=16,000. The amino acid composition of the toxin type K₂ of S. cerevisiae strain 399 was determined and compared with the composition of two other toxins.     

3种卫矛属植物叶片秋冬季转色期生理生化特征研究

以疏花卫矛(Euonymus laxiflorus)、丝棉木(E.maackii)和卫矛(E.alatus)3种卫矛属植物为试验材料,测定叶片转色期叶色参数和相关生理生化指标,探讨各项指标的变化规律和内在联系,为优良色叶植物的筛选提供依据。结果表明:(1)转色期,疏花卫矛的明亮度参数L~*和色素参数b~*(黄/蓝)呈上升趋势,色素参数a~*(红/绿)变化不大;丝棉木的L~*和b~*呈先上升后下降的单峰曲线,a~*呈上升趋势;卫矛的L~*和b~*变化不大,a~*呈上升趋势。(2)3种植物的叶绿素含量在转色期呈明显下降趋势;疏花卫矛的花色素苷相对含量、花色素苷/叶绿素的值较为平稳,类胡萝卜素/叶绿素的值呈上升趋势;丝棉木和卫矛的花色素苷相对含量、花色素苷/叶绿素的值均呈上升趋势,类胡萝卜素/叶绿素的值保持平稳。(3)3种植物苯丙氨酸解氨酶(PAL)活性呈下降趋势,查尔酮异构酶(CHI)活性呈先上升后下降的单峰曲线;卫矛的过氧化物酶(POD)和多酚氧化酶(PPO)活性保持平稳,疏花卫矛和丝棉木的POD活性呈上升趋势,疏花卫矛的PPO活性保持平稳,丝棉木的PPO活性呈下降趋势。(4)疏花卫矛和卫矛的可溶性糖和淀粉的质量分数呈先上升后下降的单峰曲线,丝棉木的可溶性糖和淀粉的质量分数呈上升趋势。(5)相关性分析显示,疏花卫矛呈现黄色主要是因为叶绿素的分解,丝棉木和卫矛呈现红色是因为花色素苷的合成,可溶性糖、淀粉、CHI对花色素苷的合成有一定作用,POD对叶片呈现红色有促进作用,PAL和PPO活性对花色素苷的合成无显著影响。     

Synthesis of stereoisomers of antithrombotic nipecotamides

The stereoisomers of α,α′-bis[3-(N,N-diethylcarbamoyl)-piperidino]-p-xylene ( 1 ) were synthesized. Rac ethyl nipecotate was resolved by diastereomeric (-)-D - and (+)-L-tartrate salt formation. The enantiomeric esters were hydrolyzed to the corresponding nipecotic acids, which were then converted into t-BOC derivatives. Treatment of the latter with diethylamine/isobutyl chloroformate and removal of the t-BOC protecting group afforded (R)- and (S)-N,N-diethylnipecotamides. Condensation of the latter with α,α′-dibromo-p-xylene gave (R,R)- and (S,S)- 1 . The meso-diastereomer was obtained by stereospecific synthesis in addition to our earlier procedure involving fractional crystallization of the diastereomeric mixture obtained by synthesis. The latter was resolved earlier into 1A , 1B , and 1C using chiral high-performance liquid chromatography (HPLC). Based on the stereospecific synthesis now achieved, 1A and 1B are assigned the configurations, (R,R) and (S,S) respectively, and 1C is assigned the meso configuration. The (R,S) structure of the latter is also confirmed by X-ray crystallography. © 1995 Wiley-Liss, Inc.     

Characterization of the fixABC region of Azorhizobium caulinodans ORS571 and identification of a new nitrogen fixation gene

Summary The fast growing strain, Azorhizobium caulinodans ORS571, isolated from stem nodules of the tropical legume Sesbania rostrata, can grow in the free-living state at the expense of molecular nitrogen. Five point mutants impaired in nitrogen fixation in the free-living state have been complemented by a plasmid containing the cloned fix-ABC region of strain ORS571. Genetic analysis of the mutants showed that one was impaired in fixC, one in fixA and the three others in a new gene, located upstream from fixA and designated nifO. Site-directed Tn5 mutagenesis was performed to obtain Tn5 insertions in fixB and fixC. The four genes are required for nitrogen fixation both in the free-living state and under symbiotic conditions. The nucleotide sequence of nifO was established. The gene is transcribed independently of fixA and does not correspond to fixX, recently identified in Rhizobium meliloti and R. leguminosarum. Biochemical analysis of the five point mutants showed that they synthesized normal amounts of nitrogenase components. It is unlikely that fixA, fixC and nifO are involved in electron transport to nitrogenase. FixC could be required for the formation of a functional nitrogenase component 2.     

Cytogeography of European perennial species of Cyanus (Asteraceae)

To reveal the general cytogeographical pattern of Cyanus section Protocyanus in Europe, DNA ploidy and/or chromosome numbers were newly examined for 160 populations by flow cytometry (450 plants) and/or chromosome counting (30 plants). Furthermore, previously published karyological data were revised (236 records). Our analyses confirmed chromosome counts of 2n = 22 for all newly investigated samples of the C. triumfetti group (the records for C. semidecurrens and C. ternopoliensis are new), C. diospolitanus and C. achtarovii; 2n = 44 for C. montanus and C. mollis; and 2n = 20 for C. lingulatus, C. napulifer, C. nissanus, C. orbelicus, C. thirkei, C. tuberosus and C. velenovskyi. The chromosome count of 2n = 20 is the first report for C. epirotus. The cytotype 2n = 40 was newly recorded for the Crimean endemic C. fuscomarginatus and Calabrian and Greek populations of C. graminifolius. The cytotypes 2n = 20 and 2n = 40 were confirmed for C. pindicola. For the first time triploidy (2n～3x～30) was found in C. nissanus, C. thirkei and in a newly discovered hybrid, C. epirotus × C. graminifolius. Two contrasting ecogeographical patterns emerged: cytotypes derived from the base chromosome number x = 11 (2n = 22, 44) are widespread in northern latitudes and ecologically diverse, whereas cytotypes with x = 10 (2n = 20, 30, 40) are confined to mountains in southern Europe. In general, tetraploids have smaller ranges than diploids. The new combinations Cyanus section Protocyanus (Dobrocz.) Ol?avská comb. nov. and Cyanus ternopoliensis (Dobrocz.) Ol?avská comb. nov. are provided. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 230–257.     

The Effect of Clp Proteins on DnaK-Dependent Refolding of Bacterial Luciferases

A study was made of the refolding of bacterial luciferases of Vibrio fischeri, V. harveyi, Photobacterium phosphoreum, and Photorhabdus luminescens. By reaction rate, luciferases were divided into two groups. The reaction rate constants of fast luciferases of V. fischeri and Ph. phosphoreum were about tenfold higher than those of slow luciferases of Ph. luminescens and V. harveyi. The order of increasing luciferase thermostability was Ph. phosphoreum, V. fischeri, V. harveyi, and Ph. luminescens. The refolding of thermoinactivated luciferases completely depended on the active DnaK–DnaJ–GrpE chaperone system. Thermolabile fast luciferases of V. fischeri and Ph. phosphoreum showed highly efficient rapid refolding. Slower and less efficient refolding was characteristic of thermostable slow luciferases of V. harveyi and Ph. luminescens. Chaperones of the Clp family were tested for effect on the efficiency of DnaK-dependent refolding of bacterial luciferases in Escherichia coli cells. The rate and extent of refolding were considerably lower in the clpB mutant than in wild-type cells. In E. coli cells with mutant clpA, clpP, of clpX showed a substantially lower luciferase refolding after heat shock.     

围封对天山高寒草原4种植物叶片和土壤化学计量学特征的影响

以中国科学院巴音布鲁克草原生态系统研究站长期围栏内外的羊茅(Festuca ovina)、天山赖草(Leymus tiansecalinus)、二裂委陵菜(Potentilla bifurca)和鹅绒委陵菜(Potentilla anserine)4种植物叶片和土壤为研究对象,分析了放牧与围封对植物叶片和土壤C、N、P的化学计量特征的影响。结果表明,围封样地土壤养分浓度整体高于放牧样地(P0.05),全氮(TN)浓度除外。围封显著增加羊茅叶片C、N浓度(P0.05),对P浓度影响不显著;围封显著增加鹅绒委陵菜叶片的C浓度,但是显著降低叶片的N和P浓度(P0.05),围封对天山赖草和二裂委陵菜养分含量影响不显著。围封显著增加鹅绒委陵菜C∶N和C∶P(P0.05);围封显著降低羊茅C∶N、C∶P和增加N∶P(P0.05);围封显著降低二裂委陵菜C∶N(P0.05),对天山赖草的化学计量特征影响不显著。不同植物对围封的响应不同,意味着长期围封可能会改变天山高寒草原生态系统的结构。围封降低优势种(羊茅)的固碳能力,增加退化期出现的代表性植物(鹅绒委陵菜)的固碳能力,表明在长期围封下植物凋落物中的杂类草(鹅绒委陵菜)可能更多的为土壤提供碳来源,也能促进优势禾本科物种的氮含量和碳含量的增加。     

Simple Measurement of Blood NADP and Blood Levels of NAD and NADP in Humans

The tryptophan synthase genes, trpA and trpB, of Bacillus stearothermophilus IFO13737 were cloned by transformation of tryptophan auxotrophic mutations of the trp genes into Escherichia coli. The genes are located in the order of trpB and trp A, according to their coding orientation, in a 2.5 kb EcoRy-Hindlll DNA fragment. The complete nucleotide sequence of this DNA was determined. The trp A and trpB genes consist of 810bp (269 amino acid residues) and 1215bp (404 amino acid residues), respectively. The 5′-proximal portion of the trpB gene was found to overlap 20 nucleotides of the upstream coding region of the trpA gene. The homology of the amino acid sequences of the trp gene products of trp A and trpB of B. stearothermophilus is 35 and 50 %, respectively, to those of E. coli, and 55 and 70 %, respectively, to those of B. subtilis.     

Cytogenetics of Brazilian species of Bromeliaceae

This paper presents the mitotic chromosome numbers of 18 species of Bromeliaceae. The diploid number 2n = 50 was observed in Aechmea comata, A. caudata, A. correia‐araujoi, A. recurvata, A. marauensis, A. bicolor, A. pineliana, Hohenbergia catingae, H. blanchetti, Alcantarea imperialis, Al. nahoumi, Neoregelia tenebrosa, Nidularium lyman‐smithii, N. scheremetiewii, N. innocentii var. innocentii, and N. innocentii × Neoregelia johannis hybrid, whereas 2n = 34 was observed in Cryptanthus maritimus and C. warren‐loosei. All of the determinations presented in this study are previously unpublished, except A. comata and H. catingae. These results confirm x = 25 as the basic number for the family and x = 17 as a secondary basic number probably generated by decreasing dysploidy. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158 , 189–193.     

Impact of salinity stress on soil-borne fungi of sugarbeet

The sugarbeet cultivar Kaumera was found to be highly susceptible to infection by the root-rot pathogens Rhizoctonia solani and Sclerotium rolfsii in the absence of salinity stress. Under this environmental condition, R. solani was more efficient than S. rolfsii in producing cell wall-degrading enzymes in infected hypocotyls. Xylanase and galactanase were most effective. The rate of cell wall degradation by R. solani was nearly 2.5 times that of S. rolfsii when cells walls of healthy hypocotyls were used as sole carbon substrate for the in vitro produced crude enzymes.Under salinity stress the pathogenicity and the performance of cell wall-degrading enzymes of R. solani and S. rolfsii varied profoundly. Pathogenicity studies showed that R. solani appeared to be more tolerant than S. rolfsii of the salinity stresses applied, and relatively more virulent to cv Kaumera. The activities of cell wall enzymes of R. solani decreased and those of S. rolfsii increased with increased salt concentration when cell wall material was used as a sole carbon source. The metabolic products produced under salinity stress by R. solani and R. solani in the cell wall amended culture media shifted the initial pH towards neutrality or slight alkalinity for R. solani and to high acidity for S. rolfsii.When model substrates were used, xyland and galactan were the most responsive substrates for degradation by the cell wall enzymes of the two fungi studied. The rate of degradation was higher for S. rolfsii than for R. solani. The excessive acidity in salt stressed S. rolfsii culture media suggested reduced activities of the enzymes involved in cell wall degradation in vivo. This may explain the decreased virulence potentialities.     

The influence of different aphid prey species on the biology and life table parameters of Propylaea japonica

The influence of three aphid prey species – Aphis craccivora Koch, Megoura viciae Buckton and Rhopalosiphum maidis (Fitch) – on the biology of Propylaea japonica (Thunberg) was studied under laboratory conditions. The development, survivorship, longevity, reproduction and life table parameters of P. japonica differed significantly among different treatments. The shortest developmental period of P. japonica (from first-instar larvae to adult) was observed on A. craccivora, whereas the longest was observed on M. viciae. The highest survivorship was observed on A. craccivora, and the lowest survivorship was observed on M. viciae. The highest sex ratio, fecundity and the shortest pre-oviposition period were observed when A. craccivora was used as prey. The longevities of P. japonica females and males did not differ significantly when reared on different aphid species. The highest values of net reproductive rate, intrinsic rate of increase and finite rate of increase were observed on A. craccivora. The results suggest that A. craccivora is a suitable prey for P. japonica among the three aphid species tested and can serve as a diet for the mass rearing of P. japonica under laboratory conditions for possible use in integrated pest management.     

The ultrastructure of Cerebropollenites from the Jurassic and Cretaceous of Asia

The dispersed pollen taxon Cerebropollenites recovered from the Jurassic of Afghanistan and Cretaceous of China was investigated ultrastructurally. Cerebropollenites lacks the differentiation of the proximal and distal hemispheres, a faint proximal triradiate mark, and an equatorial fringe, all features found in extant Tsuga, however, the gross pollen morphology, and the wall ultrastructure of Cerebropollenites suggests an affinity with the extant genus Tsuga (Section Micropeuce). The differences observed between Cerebropollenites and Tsuga are no greater than the differences observed between the pollen of the two Sections of Tsuga, Hesperopeuce and Micropeuce. The occurrence of Cerebropollenites in the Jurassic and Cretaceous is, thus far, the only fossil evidence that the genus Tsuga may have been present in the Mesozoic, and suggests that the Pinaceae, which is unequivocally recognized in the Cretaceous, may have had an earlier origin.     

八仙山不同类型松栎林群落主要特征分析

为探究八仙山保护区不同类型森林群落的更新潜力、多样性程度以及稳定性水平,阐明三者间的关系,该文以保护区内油松林、蒙古栎林和油松-栓皮栎混交林(松栎混交林) 3种不同类型天然次生林为对象,调查建群种径级结构和更新潜力,计算不同层次群落多样性,测定M. Godron稳定性,并采用主成分法建立评价模型。结果表明:(1)油松种群径级结构近似正态分布,处于成熟期,但幼苗较少;栓皮栎、蒙古栎以及阔叶杂木的幼苗、幼树较多,更新潜力较大。(2)松栎混交林的乔木层多样性较高,而灌木层多样性最低,油松林的草本层多样性最低;松栎混交林群落总体物种丰富度最低而均匀度最高。(3) M. Godron稳定性表明,蒙古栎林距离稳定点较近,而油松林偏离较远。(4) PCA双序图表明,M. Godron稳定性与种群更新潜力、Alatalo均匀度呈较强正相关,综合排序依次为松栎混交林、蒙古栎林和油松林。综上结果表明,建群种更新潜力和物种均匀度对群落稳定性影响较大,林地管理应注重对幼苗幼树的保护。     

Influences of transformation of cover values on classification and ordination of lake vegetation

Summary Numerical ordination (Program ORDINA) and classification (Program TABORD) have been applied to vegetation data from a lake in south Sweden.Full percentage values and different transformations of the estimated percented cover scale have been tested on relevés with nymphaeids (+ elodeids) and on relevés containing obly helophytes. The used scales have been: percentage (P), Hult-Sernander-Du Rietz (HSD), Log (%+1) (LP), 1+ log% (L), 100+ % (HP) and Presence/Absence (P/A). The main aim has been to find if there is an optimal transformation regarding both quantitative and qualitative information in vegetation data.In the classification of helophytes a dataset with 87 relevés and 20 species have been used applying P-and L-scales and P/A. The similarity between the different scales has been compared in relation to common relevés between every two corresponding clusters. L and P/A give very similar results, more similar than L and P or PA and P. It is also interesting that the result with P-scale is more similar to L than to P/A. The P-scale neglects with the species with a low cover value, whilst the L-scale takes notice of both quantity and quality.In the classification of 90 nymphaeid relevés containing only 6 species P, HSD, L, HP and P/A were applied. The L-scale (and partly PH-scale) gave a result similar to the most quantitative P-and HSD-scales. P/A-values gave a very deviating result. In the ordination of helophytes only 38 relevés with 18 species, taken along a transcect, were used and P, P/A and 4 different transformations were applied. The similarity between order of relevés in the graphs and real order of relevés along the transect was used as an objective test of the effectiveness of the scale applied. The P-scale gave a bad result, whilst P/A gave the best result. The intermediate scales L and HP gave acceptable results.In the ordination of nymphaeids (+ elodeids) 90 relevés, where only pattern was studied, the L-scale gave the best result most similar to P-and HSD, but applying the two extremes, P-scale and P/A, resulted in large reduction of information.In the submitted ordinations and classifications the applied L-scale (and HP-scale) always gave ecologically interpretable results, but the extreme transformation were less reliable.This work has been supported by grants from the University of Lund, for which I am duty grateful.Nomenclature follows Lid (1974).I am grateful to Professor Nils Malmer, head of the Department of Plant Ecology, Lund, Sweden, Dr. Eddy van der Maarel, University of Nijmegen, Holland, and my colleague Fil. Kand. Stefan Persson for valuable advice and discussions. I also thank my wife Fil. Kand. Eva Waldemarsson Jensén, Institute of Ecological Botany, Uppsala, for valuable discussions, Mr. Tommy Olsson for valuable technical advice, Dr. R.S. Clymo for advice with DIVINF and PCA and correction of the language, Mrs. Mimmi Varga for drawing the figures and Mrs. Brita Billstein for typewriting the text and the tables.