Morphology, phylogenetic position, and ecophysiology of Alexandrium ostenfeldii (Dinophyceae) from the Bohai Sea, China

Alexandrium ostenfeldii is a potentially toxic dinoflagellate that often occurs in coastal areas at high latitudes.Here we report the presence of A.ostenfeldii in the Bohai Sea,China,for the first time.The vegetative cells ofA.ostenfeldii are characterized by a narrow first apical plate and a large ventral pore located on the anterior right side.Partial large subunit sequence comparison revealed that the Chinese strain differs from the Finnish strains at only three positions,and from A.peruvianum of Spain at five positions.Maximum parsimony analysis revealed that A.ostenfeldii from China and Finland and A.peruvianum from Spain grouped together.They were the nearest sister group to a clade with A.ostenfeldii from New Zealand,Europe,and North America.In culture,growth did not occur at temperatures below 9 ℃ and occurred at salinities between 7 and 27 psu.It took 10-20 days for newly formed cysts to mature at 20 ℃.Lower temperature delayed germination,but the germination rate exceeded 90％ at temperatures from 12 to 24 ℃.No germination occurred below 9 ℃ after 1 month of incubation.The Chinese strain ofA.ostenfeldii produced neither spirolides nor paralytic shellfish poisoning toxins.     

Diversity of luciferase sequences and bioluminescence production in Baltic Sea Alexandrium ostenfeldii

The toxic dinoflagellate Alexandrium ostenfeldii is the only bioluminescent bloom-forming phytoplankton in coastal waters of the Baltic Sea. We analysed partial luciferase gene (lcf) sequences and bioluminescence production in Baltic A. ostenfeldii bloom populations to assess the distribution and consistency of the trait in the Baltic Sea, and to evaluate applications for early detection of toxic blooms. Lcf was consistently present in 61 Baltic Sea A. ostenfeldii strains isolated from six separate bloom sites. All Baltic Sea strains except one produced bioluminescence. In contrast, the presence of lcf and the ability to produce bioluminescence did vary among strains from other parts of Europe. In phylogenetic analyses, lcf sequences of Baltic Sea strains clustered separately from North Sea strains, but variation between Baltic Sea strains was not sufficient to distinguish between bloom populations. Clustering of the lcf marker was similar to internal transcribed spacer (ITS) sequences with differences being minor and limited to the lowest hierarchical clusters, indicating a similar rate of evolution of the two genes. In relation to monitoring, the consistent presence of lcf and close coupling of lcf with bioluminescence suggests that bioluminescence can be used to reliably monitor toxic bloom-forming A. ostenfeldii in the Baltic Sea.     

Morphology,toxicity, and phylogeny of Alexandrium (Dinophyceae) species along the coast of China

The toxigenic genus Alexandrium includes ∼30 species, but information about its biogeography at a regional scale is limited. In this study, we explored the diversity of Alexandrium along the coast of China by incubating resting cysts collected from 7 sites. A total of 231 strains of Alexandrium belonging to 7 morphospecies were found. Among them, Alexandrium andersonii, Alexandrium fraterculum, Alexandrium leei, Alexandrium pseudogonyaulax, and Alexandrium tamutum were recorded from the China Sea for the first time. Partial large subunit (LSU) and/or internal transcribed spacer region (ITS1, ITS2, and 5.8S rDNA) sequences revealed two ribotypes of Alexandrium andersonii, Alexandrium leei, and Alexandrium tamarense: Atama complex Group I and IV. Atama complex Group I was exclusively distributed in the Yellow Sea and the Bohai Sea, whereas Group IV was restricted to the East China Sea and South China Sea. Atama complex Group I produced mainly N-sulfocarbamoyl toxins (C1/C2, 61–79% of total toxins) and gonyautoxins (GTX1/4, 17–37%). Alexandrium ostenfeldii strain ASBH01 produced NEO and STX exclusively (65% and 35%, respectively). Our results support the premise that Atama complex Group I is endemic to the Asian Pacific and includes cold water species, whereas Atama complex Group IV tends to inhabit warmer waters.     

Generalist Life Cycle Aids Persistence of Alexandrium ostenfeldii (Dinophyceae) in Seasonal Coastal Habitats of the Baltic Sea

In seasonal environments, strong gradients of environmental parameters can shape life cycles of phytoplankton. Depending on the rate of environmental fluctuation, specialist or generalist strategies may be favored, potentially affecting life cycle transitions. The present study examined life cycle transitions of the toxin producing Baltic dinoflagellate Alexandrium ostenfeldii and their regulation by environmental factors (temperature and nutrients). This investigation aimed to determine whether genetic recombination of different strains is required for resting cyst formation and whether newly formed cysts are dormant. Field data (temperature and salinity) and sediment surface samples were collected from a site with recurrent blooms and germination and encystment experiments were conducted under controlled laboratory conditions. Results indicate a lack of seasonal germination pattern, set by an endogenous rhythm, as commonly found with other dinoflagellates from the Baltic Sea. Germination of quiescent cysts was triggered by temperatures exceeding 10°C and combined nutrient limitation of nitrogen and phosphorus or a drop in temperature from 16 to 10°C triggered encystment most efficiently. Genetic recombination was not mandatory for the formation of resting cysts, but supported higher numbers of resistant cysts and enhanced germination capacity after a resting period. Findings from this study confirm that A. ostenfeldii follows a generalist germination and cyst formation strategy, driven by strong seasonality, which may support its persistence and possibly expansion in marginal environments in the future, if higher temperatures facilitate a longer growth season.     

Vertical distribution and germination ability of Alexandrium spp. cysts (Dinophyceae) in the sediments collected from Kure Bay of the Seto Inland Sea, Japan

The vertical distribution of Alexandrium tamarense/ catenella (hereinafter Alexandrium spp.) cysts was investigated with special attention to living cysts filled with fresh protoplasm and empty cysts. In addition, based on the incubation experiments of Alexandrium spp. cysts, the germination ability of the cysts was examined. A sediment core 63 cm in length, collected from Kure Bay of the Seto Inland Sea, West Japan, in September 2000, was provided for an analysis on the vertical distribution of Alexandrium spp. cysts. Samples from every 1 cm interval depth from the top down to 13 cm depth of the same core were taken to examine the germination ability of the cysts. Results show that Alexandrium spp. cysts were continuously observed from 59 to 60 cm depth to the top. The cyst densities in the upper parts of the core (from 9 to 10 cm depth to the top) were much more abundant those that in the lower parts (below 10–11 cm depth). The relationship between living and empty cysts in each depth did not reveal a positive correlation with the sediment depth. Based on the sedimentation rate of the core sediment (approximately 1.6 cm/year), Alexandrium spp. cysts have been produced since 1962, and a remarkable increase of these cysts was observed from ca 1993. Such a rapid increase of Alexandrium spp. cysts has probably been as a result of dense blooms of A. tamarense occurring since 1992 in Hiroshima Bay, including Kure Bay. The germination of Alexandrium spp. cysts was observed in samples collected from the top to 12–13 cm depth of the core. It suggests that the Alexandrium spp. cysts can keep the germination ability for more than 8 years.     

Characterization of spirolide producing Alexandrium ostenfeldii (Dinophyceae) from the western Arctic

Toxin producing dinoflagellates of the genus Alexandrium Halim represent a risk to Arctic environments and economies. This study provides the first record and a characterization of Alexandrium ostenfeldii in the western Arctic. During a cruise along the coasts of western and southern Greenland 36 isolates of the species were established in August 2012. Plankton samples taken at three different stations from the upper water layer at water temperatures of approx. 4–7 °C, contained low amounts of A. ostenfeldii. Sequencing of SSU and ITS-LSU rDNA and subsequent phylogenetic analyses identified all Greenland strains as members of a NW Atlantic spirolide producing phylogenetic clade. Molecular results were confirmed by morphological features typical for this group (=Group 5 of a recent ITS-LSU phylogeny of A. ostenfeldii). The Greenland isolates did not contain either Paralytic Shellfish Poisoning toxins or gymnodimines, but produced several spirolides. Altogether 12 different analogs were detected, of which only SPX-1, C, 20-meG and H have been described earlier. The remaining 8 spirolides have not been identified so far. Some of them were found to dominate the toxin profiles of a number of isolates. Among the 36 investigated strains spirolide composition varied considerably, particularly isolates from western Greenland (Station 516) exhibited a high diversity of analogs, with different profiles in nearly all 22 isolates. All of the 34 tested Greenland strains showed considerable lytic capacity when exposed to Rhodomonas salina.     

Phylogenetic relationships,morphological variation,and toxin patterns in the Alexandrium ostenfeldii (Dinophyceae) complex: implications for species boundaries and identities

Alexandrium ostenfeldii (Paulsen) Balech and Tangen and A. peruvianum (Balech and B.R. Mendiola) Balech and Tangen are morphologically closely related dinoflagellates known to produce potent neurotoxins. Together with Gonyaulax dimorpha Biecheler, they constitute the A. ostenfeldii species complex. Due to the subtle differences in the morphological characters used to differentiate these species, unambiguous species identification has proven problematic. To better understand the species boundaries within the A. ostenfeldii complex we compared rDNA data, morphometric characters and toxin profiles of multiple cultured isolates from different geographic regions. Phylogenetic analysis of rDNA sequences from cultures characterized as A. ostenfeldii or A. peruvianum formed a monophyletic clade consisting of six distinct groups. Each group examined contained strains morphologically identified as either A. ostenfeldii or A. peruvianum. Though key morphological characters were generally found to be highly variable and not consistently distributed, selected plate features and toxin profiles differed significantly among phylogenetic clusters. Additional sequence analyses revealed a lack of compensatory base changes in ITS2 rRNA structure, low to intermediate ITS/5.8S uncorrected genetic distances, and evidence of reticulation. Together these data (criteria currently used for species delineation in dinoflagellates) imply that the A. ostenfeldii complex should be regarded a single genetically structured species until more material and alternative criteria for species delimitation are available. Consequently, we propose that A. peruvianum is a heterotypic synonym of A. ostenfeldii and this taxon name should be discontinued.     

中国渤海海域甲藻孢囊的种类多样性和生态地理分布

在渤海湾24个站位采集0–10 cm表层沉积物144份样品, 进行甲藻孢囊的种类多样性和生态地理分布研究, 并分析渤海海域水体的富营养化趋势。在渤海海域共采集鉴定出6类32种甲藻孢囊, 包括原多甲藻类孢囊11种、裸甲藻类孢囊6种、膝沟藻类孢囊7种、钙质类孢囊5种、翼藻类孢囊2种及Tuberculodinioid类孢囊1种。其中, 自养型甲藻孢囊17种, 优势种为锥状斯氏藻(Scrippsiella trochoidea)孢囊, 丰富的孢囊库为锥状斯氏藻赤潮提供种源, 可作为该海域发生锥状斯氏藻赤潮的参考依据; 异养型甲藻孢囊15种, 优势种为无纹多沟藻(Polykrikos schwartzii)和锥形原多甲藻(Protoperidinium conicum)孢囊; 发现1种产麻痹性贝类毒素的孢囊--塔玛亚历山大藻(Alexandrium tamarense)孢囊, 但是仅在S6、S8和S14三个站位发现, 且孢囊数量较少。渤海甲藻孢囊组成与世界其它海域相似, 异养型甲藻孢囊种类丰富, 提示渤海海域富营养化程度较高。每个站位沉积物样品分为3层(每层约3 cm), 春夏两季表层、中层和底层之间孢囊组成差异不大, 平均种类数为5.58–7.29, 孢囊的平均密度范围在58.82–103.57 cysts·g^–1 DW之间; 孢囊种类丰富的站点, 孢囊密度也较高, 如S9、S14站位。对渤海海区表层沉积物中甲藻孢囊的生物多样性进行了统计分析, 发现表、中、底3层甲藻孢囊的平均种类多样性指数在春季(5月)分别为1.69、1.61和1.52; 略低于夏季(8月)的1.83、1.89和1.60。     

Dinoflagellate cyst records in recent sediments from Daya Bay, South China Sea

Nine sediment cores of 8–26 cm in length were collected from two basins of Daya Bay, the South China Sea, by Tokyo University Fisheries Oceanography Laboratory core sampler in August 2001 to investigate the distribution of dinoflagellate resting cysts. In the present study, 51 different cyst morphotypes representing 22 genera were identified from 65 sediment samples. Among them, there were 21 autotrophic species and 30 heterotrophic ones. Cyst species richness in each sample varied from 12 to 29, while the values of Shannon‐Weaver diversity index (H′) were between 0.15 and 4.13. There were an obvious increase in both species richness and values of H′in 2–6 cm sediments. Cyst concentrations varied from 154 to 113 483 cysts per gram dry weight sediment, and were much higher in upper sediments. Scrippsiella trochoidea was the most dominant cyst type, which took up over 90% of cyst assemblages in the upper sediments. The abrupt increase of S. trochoidea cysts in the surface sediments reflected the bloom of this species in Daya Bay in 2000. The results from cyst assemblages showed some trend of changes in water quality in this area, and indicated a typical type of pollution caused by cultural eutrophication, which started in the 1980s and greatly accelerated in the middle of 1990s. Cysts of Alexandrium, mainly those of Alexandrium catenella and Alexandrium tamarense complex, occurred frequently and abundantly in this area, with the highest concentration and relative frequency of 503 cysts per gram dry weight sediment and 22.3%, respectively. The high abundance of Alexandrium cysts provided rich ‘seed bed’ for Alexandrium blooms and was also an important source of paralytic shellfish poisoning toxins, especially in winter.     

Comparative study of the life cycles of Alexandrium tamutum and Alexandrium minutum (Gonyaulacales,Dinophyceae) in culture1

The microalgal genus Alexandrium includes species known to produce paralytic shellfish poisoning (PSP). Due to the importance of discriminating between HAB‐forming species, we compared the undescribed life‐cycle pattern of Alexandrium tamutum Montresor, Beran et U. John and of its toxic relative Alexandrium minutum Halim. Sexual stages, asexual and sexual division, mating type, and nuclear morphology were studied in both species. Sexual cysts are known to be morphologically identical. However, the relative size of the U‐shaped nucleus may be used to differentiate between the cysts of these species since DNA packaging in the resting cysts was lower in A. tamutum than in A. minutum, species in which the planozygote nucleus was reduced to half its volume prior to encystment. The dormancy period of the cysts was <20 d for A. tamutum, but longer than 1 month for A. minutum. In both species, cyst appearance needed to be explained by the existence of more than two sexual types (+/–), which indicates a complex heterothallic mating type. However, planozygotes of both species may divide instead of encysting. This characteristic was used for nutritional and heritage studies. Isolated planozygotes of both species encysted in larger percentages in medium deficient in both nitrates and phosphates (L/15) than in medium without phosphates added (L‐P), a medium in which most planozygotes neither divide nor encyst. Parental strains of A. minutum with and without the ventral pore formed planozygotes and, later, offspring with the ventral pore, although apparently smaller than usual. A synchronization–flow cytometry method for discriminating diploids formed by sexual fusion (planozygotes) from cells with 2C DNA content resulting from self‐duplication of DNA (dividing cells) was described. The results indicated that the maximum percentage of A. minutum planozygotes (20%) was achieved only 3 to 5 d after crossing the parental strains, and that light might not be needed for the sexual fusion and formation of planozygotes.     

Diversity, distribution, and new phylogenetic information of calcareous dinoflagellates from the China Sea

Calcareous dinoflagellates have been recorded from subarctic to tropical areas, inhabiting both neritic and oceanic regions. In the present study we analyse 12 sediment samples from the China Sea to establish the existence of calcareous dinoflagellates in this area. A total of 11 calcareous dinoflagellate species were recorded, among them, three genera (Calciodinellum, Leonella, Posoniella) and seven species (Calciodinellum albatrosianum, C. levantinum, C. operosum, Leonella granifera, Posoniella tricarinel...     

Effects of temperature and salinity on the growth of Alexandrium (Dinophyceae) isolates from the Salish Sea

Toxin‐producing blooms of dinoflagellates in the genus Alexandrium have plagued the inhabitants of the Salish Sea for centuries. Yet the environmental conditions that promote accelerated growth of this organism, a producer of paralytic shellfish toxins, is lacking. This study quantitatively determined the growth response of two Alexandrium isolates to a range of temperatures and salinities, factors that will strongly respond to future climate change scenarios. An empirical equation, derived from observed growth rates describing the temperature and salinity dependence of growth, was used to hindcast bloom risk. Hindcasting was achieved by comparing predicted growth rates, calculated from in situ temperature and salinity data from Quartermaster Harbor, with corresponding Alexandrium cell counts and shellfish toxin data. The greatest bloom risk, defined at μ >0.25 d^?1, generally occurred from April through November annually; however, growth rates rarely fell below 0.10 d^?1. Except for a few occasions, Alexandrium cells were only observed during the periods of highest bloom risk and paralytic shellfish toxins above the regulatory limit always fell within the periods of predicted bloom occurrence. While acknowledging that Alexandrium growth rates are affected by other abiotic and biotic factors, such as grazing pressure and nutrient availability, the use of this empirical growth function to predict higher risk time frames for blooms and toxic shellfish within the Salish Sea provides the groundwork for a more comprehensive biological model of Alexandrium bloom dynamics in the region and will enhance our ability to forecast blooms in the Salish Sea under future climate change scenarios.     

渤海中、南部大型底栖动物的群落结构

在对渤海中、南部大部分海区 3个航次的大型底栖动物现存量、生产力和物种多样性研究的基础上 ,对群落结构及其与环境因子的关系进行了分析。结果表明 ,种群的相对重要性存在着年份和季节的差异 ,与大型底栖动物群落关系最为密切的环境因子为水深和底层水中的硝酸盐浓度。底层水中的 NO3-N和 PO4-P含量、N∶ P值及大型底栖动物的类群组成显示 ,渤海已成为典型的富营养化海域 ,有些群落已有受到污染或富营养化扰动的趋势 ,但总体上污染尚未给研究海域的大型底栖动物群落结构带来明显影响。与 2 0世纪 80年代初相比 ,渤海大型底栖动物群落组成发生了一些变化 ,高速的富营养化进程、渔业活动的加剧以及底栖动物捕食者的改变 ,可能是造成变化的原因     

大亚湾亚历山大藻种群周年变动与环境因子的关系

于1997年7月至1998年6月,在大亚湾澳头海域设立6个监测站位,连续监测亚历山大藻(Alexandrium sp.)种群周年变动规律及其与环境因子的关系。大亚湾亚历山大藻种群的密度高峰出现在春季(4月至5月),最高密度达到20.4cells·mL^-1,其它季节则密度较低。亚历山大藻密集出现的温度为22.8～30.0℃、盐度范围为25～30。在亚历山大藻种群密度高峰期里,N/P都较高,DIN含量维持较高水平,有利于亚力山藻的生长,DIP含量却很低,使得此藻比其他藻更具竞争优势。可溶性Fe含量可能成为亚历山大藻增殖的限制性因子。     

我国东南沿海亚历山大藻休眠孢囊的分布和萌发研究

对4个海域的塔玛亚历山大藻(Alerandrium tamarense)和链状亚历山大藻(A．catenella)休眠孢囊的分布及萌发进行了研究．结果表明,厦门港仅在X1和X2站位有分布,且密度很小(0．4个·g-1);广西只在G2站位有发现,密度较少(2．5个·g-1泥样)．闽江口有3个站位有分布,M4站位的4～6cm层密度最大,达到6个·g-1泥样;长江口的孢囊分布广、密度大,DG-26站位的8～10cm层孢囊密度达到了23．2个·g-1泥样．孢囊的分布与沉积物底质类型、沉积速率、海流都有一定的关系．光照对孢囊萌发没有影响,温度升高导致萌发率和存活率均增大,而萌发时间缩短;在低氧条件下(0．01mgO2L-1),孢囊萌发率为0．亚历山大藻孢囊在合适的环境条件下终年都会萌发     

Positive effects of continuous low nitrate levels on growth and photosynthesis of Alexandrium tamarense (Gonyaulacales, Dinophyceae)

The growth and photosynthesis of Alexandrium tamarense (Lebour) Balech in different nutrient conditions were investigated. Low nitrate level (0.0882 mmol/L) resulted in the highest average growth rate from day 0 to day 10 (4.58 × 10² cells mL^?1 d^?1), but the lowest cell yield (5420 cells mL^?1) in three nitrate level cultures. High nitrate‐grown cells showed lower levels of chlorophyll a‐specific and cell‐specific light‐saturated photosynthetic rate (P_m^{chl a} and P_m^cell), dark respiration rate (R_d^chla and R_d^cell) and chlorophyll a‐specific apparent photosynthetic efficiency (α^chla) than was seen for low nitrate‐grown cells; whereas the cells became light saturated at higher irradiance at low nitrate condition. When cultures at low nitrate were supplemented with nitrate at 0.7938 mmol/L in late exponential growth phase, or with nitrate at 0.7938 mmol/L and phosphate at 0.072 mmol/L in stationary growth phase, the cell yield was drastically enhanced, a 7–9 times increase compared with non‐supplemented control culture, achieving 43 540 cells mL^?1 and 52 300 cells mL^?1, respectively; however, supplementation with nitrate in the stationary growth phase or with nitrate and phosphate in the late exponential growth phase increased the cell yield by no more than 2 times. The results suggested that continuous low level of nitrate with sufficient supply of phosphate may facilitate the growth of A. tamarense.     

Development of microsatellite markers in the toxic dinoflagellate Alexandrium minutum (Dinophyceae)

Outbreaks of paralytic shellfish poisoning caused by the toxic dinoflagellate Alexandrium minutum (Dinophyceae) are a worldwide concern from both the economic and human health points of view. For population genetic studies of A. minutum distribution and dispersal, highly polymorphic genetic markers are of great value. We isolated 12 polymorphic microsatellites from this cosmopolitan, toxic dinoflagellate species. These loci provide one class of highly variable genetic markers, as the number of alleles ranged from four to 12, and the estimate of gene diversity was from 0.560 to 0.862 across the 12 microsatellites; these loci have the potential to reveal genetic structure and gene flow among A. minutum populations.     

Species boundaries and global biogeography of the Alexandrium tamarense complex (Dinophyceae)1

Alexandrium catenella (Whedon et Kof.) Balech, A. tamarense (M. Lebour) Balech, and A. fundyense Balech comprise the A. tamarense complex, dinoflagellates responsible for paralytic shellfish poisoning worldwide. The relationships among these morphologically defined species are poorly understood, as are the reasons for increases in range and bloom occurrence observed over several decades. This study combines existing data with new ribosomal DNA sequences from strains originating from the six temperate continents to reconstruct the biogeography of the complex and explore the origins of new populations. The morphospecies are examined under the criteria of phylogenetic, biological, and morphological species concepts and do not to satisfy the requirements of any definition. It is recommended that use of the morphospecies appellations within this complex be discontinued as they imply erroneous relationships among morphological variants. Instead, five groups (probably cryptic species) are identified within the complex that are supported on the basis of large genetic distances, 100% bootstrap values, toxicity, and mating compatibility. Every isolate of three of the groups that has been tested is nontoxic, whereas every isolate of the remaining two groups is toxic. These phylogenetic groups were previously identified within the A. tamarense complex and given geographic designations that reflected the origins of known isolates. For at least two groups, the geographically based names are not indicative of the range occupied by members of each group. Therefore, we recommend a simple group‐numbering scheme for use until the taxonomy of this group is reevaluated and new species are proposed.     

Development of microsatellite markers in the toxic dinoflagellate Alexandrium catenella (Dinophyceae)

The outbreak of paralytic shellfish poisoning caused by the toxic dinoflagellate Alexandrium catenella (Dinophyceae) is a worldwide concern from the economic and food hygiene point of view. To assess the dispersal mechanism, a population genetic study using highly polymorphic genetic markers is one of the crucial approaches. We isolated 12 polymorphic microsatellites from this species. These loci provide one class of highly variable genetic marker as the number of alleles ranged from three to 12 and the estimate of gene diversity was from 0.248 to 0.897 across the 12 microsatellites; we consider that these loci have a potential to detail the genetic structure and gene flow among A. catenella populations.