Development of high-resolution chloroplast markers for intraspecific phylogeographic studies of Phaeocystis globosa

Phaeocystis globosa is an important harmful algal bloom causative species distributing widely in temperate and tropical coastal waters in the world.The morphological,physiological,and biochemical characte ristics are different amo ng geographic strains,which can not be distinguished with nuclear ribo somal DNA markers at present.Therefore,the genetic distance and phylogeographic relationships of nuclear 28 S rDNA D1-D2 and ITS regions,and three chloroplast intergenic spacers(petN-trnS1,trnM1-psbA,and rbcS-rpl27) were analyzed and compared among 13 strains of P.globosa isolated from the Pacific Ocean and Atlantic Ocean in this study.In addition,the nucleotide polymorphisms of 28 S rDNA D1-D2,ITS,and rbcS-rpl27 regions were evaluated in two P.globosa strains.The various levels of nucleotide polymorphism were in the nuclear 28 S rDNA D1-D2 region and ITS region,but no polymorphism was in the chloroplast rbcS-rpl27 intergenic spacer.A reasonable intraspecific phylogeographic relationship was presented by rbcS-rpl27 intergenic spacer,which had the strongest distinction to geographic strains compared to those of 28 S rDNA D1-D2 and ITS regions.In the phylogenetic tree of rbcS-rpl27 intergenic spacer,the two strains from the North Sea of the Atlantic Ocean were divided firstly from the species of P.globosa,and then formed an independent clade,while the other Atlantic strains and all of Pacific strains j oined up to build the other clade.It was implied that at least two genetically distant populations of P.globosa existed in the Atlantic coastal regions.This study provided a high-resolution chloroplast marker to analyze intraspecific phylogeographic populations of P.globosa,and preliminarily clarified the genetic relationships of the Pacific and Atlantic strains of P.globosa.     

Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

The wild Porphyra yezoensis collected from the Qingdao coast was used to prepare protoplasts by enzyme digestion. The pure line was constructed by cultivating the protoplasts. The 18S rDNA of the P. yezoensis pure line was cloned and sequenced. Sequence analysis was executed for this sequence and other 22 sequences retrieved from GenBank. A phylogenetic tree was constructed using the neighbor-joining method. The results revealed a high diversity of 18S rDNA sequences in genus Porphyra and the considerable variation of 18S rDNA sequences in different strains of the same species P. yezoensis and P. tenera. Significant difference of 18S rDNA sequence was observed between P. yezoensis from Qingdao, China, and the two strains of P. yezoensis from Japan, but the three strains of P. yezoensis formed a stable clade in the phylogenetic tree. These results indicate the possibility of interspecies and intraspecies discrimination of Porphyra using the 18S rDNA sequences.     

Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

1　Introduction　PorphyraisthemainobjectforalgafarmingandplaysaveryimportantroleinChinesemarineindus tries.Recently ,therehasbeenagreatlossinPor phyracultivationduetothedegenerationoftheculti var,sothereisanincreasingdemandforgoodPor phyracultivars .Theprerequisiteofthetraditionalbreed ingandbioengineeringresearchofPorphyraistheconstructionofpurelines .Traditionally ,theclassifi cationofPorphyrawasaccordingtotheirmorphologi calcharacteristics .However ,mostmorphologicalfea turesofPorphyraar…     

Characteristics of phytoplankton community structure during and after a bloom of the dinoflagellate Scrippsiella trochoidea by HPLC pigment analysis

A bloom of the dinoflagellate Scrippsiella trochoidea was detected for the first time in inner Tolo Harbor, Hong Kong in 2 000. Water samples were collected at eight stations along a transect passing through a red tide patch for microscopic analysis of phytoplankton composition and high-performance liquid chromatography (HPLC) analysis of phytoplankton pigments. During the bloom, the density of dinoflagellates was 1.1×10⁶ cells L⁻¹ within the patch and 8.6×10⁵ cells L⁻¹ outside the patch where the phytoplankton community was dominated by diatoms. After the bloom the S. trochoidea began to decrease in density and was replaced by diatoms as the dominating bloom-causing organisms at all stations, and the density of dinoflagellates at most stations was less than 1.0 × 10⁶ cells L⁻¹. The status of S. trochoidea as the causative species of the bloom was indicated by the presence of peridinin, the marker pigment for dinoflagellates. The shift from dinoflagellates to diatoms was marked by the decline of peridinin and the prevalence of fucoxanthin. Phytoplankton pigment markers also revealed the presence of other minor phytoplankton assemblages such as cryptomonads and blue-green algal.     

Molecular identification and culture trials of Eutreptiella gymnastica (Eutreptiales, Euglenophyceae)

During a survey for marine microalgal resources, we isolated a rare marine euglenoid from the coastal waters of Qingdao, China in 2009, and established a pure culture. Electron microscopic and molecular phylogenetic (18S rDNA and 16S rDNA sequences) analyses revealed a close affinity with Eutreptiella gymnastica, a bloom-forming species. Different culture conditions were monitored to understand optimal E. gymnastica growth characteristics. The optimal growth conditions in a batch culture of this isolate were 20°C, 160 μmol photons/(m2×s) of white light, and a salinity of 10-31. Nutrient experiments demonstrated that growth increased dramatically with a phosphorus concentration greater than 72 μmol/L. Understanding the effect of culture conditions on E. gymnastica may help understanding the blooming mechanism of this alga in its natural environment.     

Effect of nitrogen and phosphorus on the growth of a red tide dinoflagellateScrippsiella trochoidea (Stein) Loeblich III

Scrippsiella trochoidea (Stein) Loeblich III was grown in a nitrogen-or phosphorus-limited batch culture system in laboratory. Growth rates and cellular Chl-a were measured as functions of nitrate and phosphate concentrations. Growth rates were hyperbolic with both nitrate and phosphate concentration and fit the Monod equation. The minimum cell quota of nitrogen and phosphorus and then the optimum N∶P ratio ofS. trochoidea were estimated in this study. Measurement of phosphate concentration in Jiaozhou Bay suggest that phosphorus is the limiting factor ofS. trochoidea growth. Contribution No. 3679 from the Institute of Oceanology, Chinese Academy of Sciences. Project 39790110 supported by NSFC, the study aslo supported by the National Climbing Project B (PDBG-7-2) and partly supported by an MF grant from Hong Kong University of Science and Technology.     

Effects of Fe and Mn on the growth of a red tide dinoflagellateScrippsiella trochoidea (Stein) Loeblch III

Batch culture experiments were conducted with a red tide dinoflagellateScrippsiella trochoidea (Stein) Loeblch III collected from Jiaozhou Bay, Shangdong, China. Growth rates and oellular Chl—a were measured in media with iron and manganese ion concentrations controlled at different levels using EdTA-trace metal buffer systems. Cell density increased 3.2 times to 6.5 times over the range of lowest (0 mol/L) to highest (10⁻⁵ mol/L) iron and manganese ion concentrations. The range of cell density response was much lower than the range of total available iron and manganese, which was >100—fold that of Fe. This nonlinear relationship indicates that the cells adapt to make more efficient use of iron and manganese under limiting conditions. The cellular Chl—a content maximized after 3 days incubation and then decreased gradually under either iron or manganese limitation conditions. It indicated that the algae gained higher photosynthesis ability when transferred to a new environment. Growth responses to iron and manganese limitation can be both modeled according to the equation of Monod. The half—saturation constant for growth,k, is 4.6×10⁻⁸ mol/L for Fe and 5.1×10⁻⁸ mol/L for Mn. Our results showed that the iron availability in Jiaozhou Bay does not limit the growth ofS. trochoidea. Contribution No. 2831 from the Institute of Oceanology, Chinese Academy of Sciences. Project 9389008 supported by NSFC; Study supported by PDB6.     

Diversity and bioactivity of actinomycetes from marine sediments of the Yellow Sea

Among the 116 actinomycetes collected from marine sediments of the Yellow Sea,56 grew slowly and appeared after 2?3 weeks of incubation.Among the 56 strains,only 3 required seawater(SW) for growth,and 21 grew well in the medium prepared with SW rather than distilled water(DW),while the remaining 32 grew well either with SW or with DW.Six representatives with different morphological characteristics,including 1 SW-requiring strain and 5 well-growing with SW strains,were selected for phy-logenetic analysis based on 16S rRNA gene.Two strains belong to Micrococcaceae and Nocardiopsaceae respectively.The other 4 strains belong to the family of Streptomycetaceae.In the analyzed 6 strains,one was related to Nocardiopsis spp.and the other three were related to Streptomyces spp.,representing new taxa.Bioactivity testing of fermentation products from 3 SW-requiring strains and 21 well-growing with SW strains revealed that 17 strains possessed remarkable activities against gram-positive pathogen or/and tumor cells,suggesting that they were prolific resources for natural drug discovery.     

Isolation of novel psychrophilic bacteria from Arctic sea ice

The phylogenetic diversity of cuhurable psychrophilic bacteria associated with sea ice from the high latitude regions of Canadian Basin and Chukchi Sea, Arctic, was investigated. A total of 34 psychropilic strains were isolated using three methods of （ Ⅰ ） dilution plating （ at 4 ℃ ）, （ Ⅱ ） bath culturing （ at - 1 ℃ ） and dilution plating, and （ Ⅲ） cold shock （ -20 ℃ for 24 h）, bath culturing and dilution plating under aerobic conditions. Sea-ice samples were exposed to -20 ℃ for 24 h that might reduce the number of common microorganisms and encourage outgrowth of psyehrophilic strains. This process might be able to be introduced to isolation psychrophilic bacteria from other environmental samples in future study. 16S rDNA nearly full-length sequence analysis revealed that psychrophilic strains felled in two phylo- genetic divisions, γ-proteobacteria （in the genera ColweUia,Marinobaeter、Shewanella,Glaciecola,Marinomortas and Pseudoalteromortas ） and Cytophaga-Flexibacter-Baeteroides （ Flavobacterlum and Psyehroflexus）. Fifteen of bacterial isolates quite likely represented novel species （16S rDNA sequence similarity below 98% ）. One of strains （BSi20002） from Canadian Basin showed 100% sequence similarity to that of Marinobacter sp. ANT8277 isolated from the Antarctic Weddell sea ice, suggesting bacteria may have a bipolar distribution at the species level.     

Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum

The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.     

Decadal cimatic variations indicated by dulan tree-ring and comparison with other proxy data in China of the last 2000 years

Based on high-resolution tree-ring data from Dulan area of Qinghai Province, five spells have been divided: the warm period before 230’s A. D., the cold period between 240’s A. D. and 800’s A. D., the significantly warm period between 810’s A. D. and 1070’s, i. e. “Medieval Warm Period”, the cold period including the “Little Ice Age” 1420’ – 1870’s and the warming period since 1880’s. All the eleven coldest or warmest decades and several great abrupt changes took place before the Middle Ages, indicating that climatic system operated in great instability during the period 150’s – 1100’s A. D., Comparison of the tree-ring data with other temperature proxy data from East China, Guliya ice core as well as the south of Qinghai-Xizang Plateau shows that such great climatic events as Eastern Han warm period between the beginning of the 1st century and the previous fifty years of the third century, the cold period covering the span of the Wei, the Jin, and the Southern and Northern dynasties, the well-known “Medieval Warm Period” as well as the “Little Ice Age” appeared in these series such as East China and Dulan area. Only the first two climatic events were recorded conspicuously in Guliya ice core while the “Medieval Warm Period” and “Little Ice Age” is far weaker. Also, the well-defined “Medieval Warm Period” didn’t occurred in the south of Qinghai-Xizang Plateau. The warming since the 20th century is the warmest in the last 2000 years Guliya ice core, the second in Dulan area and East China, but it scarcely seems pronounced in the eastern part of Qinghai-Xizang Plateau. Foundation item: Under the auspices of the projects of the Chinese Academy of Sciences (KZ951-A1-204-02 and KZ951-A1 402-03). Biography: YANG Bao(1971–), male, a native of Yanggao County, Shanxi Province, Ph. D. His research interestinclude global change.     

Isolation and identification of bacteria associated with the surfaces of several algal species

We conducted this study to assess the diversity of bacteria associated with the surfaces of algae based on 16S rDNA sequence analyses.Twelve strains of bacteria were obtained from the surfaces of the following four species of algae:Gracilaria textorii,Ulva pertusa,Laminaria japonica,and Polysiphonia urceolata.The isolated strains of bacteria can be divided into two groups:Halomonas and Vibrio,in physiology,biochemical characteristics and 16S rDNA sequence analyses.The phylogenetic tree constructed based on ...     

Sequence analysis of the ITS region and 5.8S rDNA of <Emphasis Type="Italic">Porphyra haitanensis</Emphasis>

The sequences of the ITS(internal transcribed spacer) and 5.8S rDNA of three cultivated strains of Porphyra haitanensis thalli(NB,PT and ST) were amplified,sequenced and analyzed.In addition,the phylogenic relationships of the sequences identified in this study with those of other Porphyra retrieved from GenBank were evaluated.The results are as follows:the sequences of the ITS and 5.8S rDNA were essentially identical among the three strains.The sequences of ITS1 were 331 bp to 334 bp,while those of the 5.8...     

Total lipid and fatty acid composition of eight strains of marine diatoms

Fatty acid composition and total lipid content of 8 strains of marine diatoms (Nitzschia frustrula, Nitzschia closterium, Nitzschia incerta, Navicula pelliculosa, Phaeodactylum tricornutum, Synedra fragilaroides) were examined. The microalgae were grown under defined conditions and harvested at the late exponential phase. The major fatty acids in most strains were 14∶0 (1.0%–6.3%), 16∶0 (13.5–26.4%), 16∶1n−7 (21.1%–46.3%) and 20∶5n−3 (6.5%–19.5%). The polyunsaturated fatty acids 16∶2n−4, 16∶3n−4, 16∶4n−1 and 20∶4n−6 also comprised a significant proportion of the total fatty acids in some strains. The characteristic fatty acid composition of diatoms is readily distinguishable from those of other microalgal groups. Significant concentration of the polyunsaturated fatty acid 20∶5n−3 (eicosapentaenoic acid) was present in each strain, with the highest proportion in B222 (19.5%). Project supported by the Hi-Tech “863” Programs of the China Ministry of Science and Technology (863-819-02-01).     

Polyphasic examination on Merismopedia tenuissima CHAB 7021 from Ganjiang River,China revealed the polyphyly of the genus Merismopedia(Cyanobacteria)

Species in the cyanobacterial genus Merismopedia are present in freshwaters at different trophic levels, with some species even as the components of cyanobacterial blooms. However, species diversity in this genus was not fully verified by molecular investigation and polyphasic taxonomic studies. In this study, Merismopedia-like strain tenuissima CHAB 7021 was isolated from Ganjiang River in Jiangxi Province, China, and polyphasic characterization of this strain was performed by morphological observation, ultrastructural examination, chemical detection of pigments and phylogenetic analysis based on 16 S rRNA gene sequences. Morphological identification of the strain was supported by the ultrastructural features, as the tiny species Merismopedia tenuissima Lemmermann. The phylogeny based on 16 S rRNA gene sequences revealed at least three clades formed by the strains of Merismopedia. The three M. tenuissima strains including M. tenuissima CHAB 7021 was gathered in clade III with distant relationship to the clade I formed by the six Merismopedia strains including the type species M. punctata, and such a genetic distance may propose Merismopedia tenuissima to separate from Merismopedia genus. However intermixture relationship in between strains of M. punctate and M. glauca in the phylogenetic tree still complicated the taxonomic status in the genus Merismopedia. The process for taxonomic revision in the Merismopedia genus still await for examination and further information on more strains of type species M. punctata.     

Production of DMS and DMSP in different physiological stages and salinity conditions in two marine algae

Dimethylsulfide(DMS) and dimethylsulfoniopropionate(DMSP) production by Scrippsiella trochoidea and Prorocentrum minimum was investigated to characterize the effects of physiological stage and salinity on DMS and DMSP pools of these two marine phytoplankton species.Axenic laboratory cultures of the two marine algae were tested for DMSP production and its conversion into DMS.The results demonstrated that both algal species could produce DMS,but the average concentration of DMS per cell in S.trochoidea(12.63 fmol/L) was about six times that in P.minimum(2.01 fmol/L).DMS and DMSP concentrations in algal cultures varied significantly at different growth stages,with high release during the late stationary growth phase and the senescent phase.DMS production induced by three salinities(22,28,34) showed that the DMS concentrations per cell in the two algal cultures increased with increasing salinity,which might result from intra-cellular DMSP up-regulation with the change of osmotic stress.Our study specifies the distinctive contributions of different physiological stages of marine phytoplankton on DMSP and DMS production,and clarifies the influence of salinity conditions on the release of DMS and DMSP.As S.trochoidea and P.minimum are harmful algal bloom species with high DMS production,they might play an additional significant role in the sulfur cycle when a red tide occurs.     

IS CHINA LEARNING FROM ITS NEIGHBORS? A COMPARISON OF OVERSEAS INVESTMENT BETWEEN CHINA AND OTHER EAST ASIAN ECONOMIES

1INTRODUCTIONAfter2decades'opendoorpractice,Chinahasnotonlybeenabigplayerasanimportanthostnationtoforeigndirectinvestment(FDI),butalsothelargestFDIhomenationamongthedevelopingcountries(SUN,1998;ZHANG,1995;WorldInvestmentReport,1998).Infact,sincethelate1980s,theChinesegovernmenthasnotsimplyputitseffortintoexportingmade-in-China"productstoearnforeigncurrencyorintoencouragingforeigninwardcapital.Ithasactivelyencourageditsmanufacturerstoinvestoverseas,andhasdeliberatelyandstrategical…     

A new type of isotherm for zinc liquid-solid partitioning on δ−MnO2, γ−MnOOH and manganite in seawater

This article deals with the interaction of zinc with δ−MnO₂, γ−MnOOH and manganite existing in natural water systems. The mechanism of the reaction has been studied in detail. From the fact that the “ratio of ion exchange(%)-pH” graph is an “S shaped” curve, it is possible to deduce that the chemical reaction is of the nature of cation exchange. And since the pH range of ion exchange=4, it is possible to further deduce that the reaction can be explained by the mechanism of monovalence cation exchange. The main result of this article is the discovery of a new type of isotherm which has not been mentioned in previous literature here and abroad. This isotherm cannot be represented by any presently available adsorption isotherm equations in marine chemistry. The characteristics of this new type of isotherm are as follows: the isotherm has two “knees” and three “plateaus”, the heights of these three “plateaus” are in the ratio 1:2:3. In order to explain theoretically our new isotherm, this article suggests the application of the principle of interfacial stepwise ion exchange for liquid-solid distribution of minor elements on suspended particulate matter. The corresponding isotherm equation was derived from this theory. The results obtained in this article will be of theoretical guiding significance in the study of the marine geochemistry of zinc. Projects supported by the Science Fund of the Chinese Academy of Sciences.