中国沿海新报道的三种产毒拟菱形藻

为了明确我国海域拟菱形藻属(Pseudo-nitzschia)物种的产毒特征, 从中国沿海建立了15个拟菱形藻单克隆培养株系, 利用高效液相色谱-质谱联用法对其多莫酸特征进行检测, 在10个株系中检测到多莫酸。结合光学显微镜下的群体特征和透射电镜下的超微形态学特征, 以及基于核糖体转录间隔区的分子系统学数据, 确认上述10个产毒株系分别隶属于3个物种:尖细拟菱形藻P. cuspidata、伪柔弱拟菱形藻P. pseudodelicatissima、伪善拟菱形藻P. fraudulenta, 其中伪善拟菱形藻是我国的新记录种。建立尖细拟菱形藻的11个尖细拟菱形藻株系, 其中3个株系未检出多莫酸, 其余8个株系有检出, 单细胞产毒水平为0.4—5.5 fg。建立伪柔弱拟菱形藻株系2个, 1个未检出多莫酸, 另1个株系的单细胞产毒量为1 fg。建立伪善拟菱形藻株系2个, 纯种培养株系均未检出多莫酸。利用卤虫(Artemia salina)对部分藻株进行混培诱导, 其中尖细拟菱形藻(MC4049)和伪柔弱拟菱形藻(MC3015)的产毒水平略有下降, 单细胞产毒水平分别由2、1 fg降至0.2、0.4 fg, 而伪善拟菱形藻(MC4074)的产毒能力则有显著改变, 单细胞产毒水平由未检出上升至17.5 fg。研究丰富了我国产毒拟菱形藻的物种多样性, 明确了其物种信息和产毒水平, 可为后续深入研究提供基础数据。     

我国沿海拟菱形藻属的2新记录种及其产毒特征分析

为澄清我国沿海拟菱形藻属(Pseudo-nitzschia)的物种多样性,并确认中国海域拟菱形藻属是否具有产生多莫酸(Domoicacid)的能力,采用毛细管显微操作技术从我国沿海水体中分离、纯化拟菱形藻细胞,建立了单克隆培养株系,并基于核糖体转录间隔区ITS1-5.8S-ITS2 序列构建了分子系统树。结果表明,结合在光学显微镜和透射电镜下观察的形态学特征和分子系统发育分析数据,鉴定到我国拟菱形藻属的2新记录种:银河拟菱形藻(P. galaxiae Lundholm & Moestrup)和微孔拟菱形藻(P. micropora Priisholm, Moestrup & Lundholm),对其形态学特征进行了详细描述,并与相似种类进行了比较研究。利用高效液相色谱(HPLC)技术对多莫酸特征进行了检测,结果表明培养株系并不产生多莫酸。这些为我国拟菱形藻属物种多样性和产毒特征研究提供了基础数据。     

广东省水库微囊藻的产毒特征和ITS序列的遗传多样性分析

为了解广东省水库微囊藻的产毒特征和ITS 序列的遗传多样性,从广东省供水水库中分离得到28 株微囊藻(Microcystisspp.),对它们的产毒特征和15 株微囊藻的ITS 序列进行了分析.高效液相色谱(HPLC)和微囊藻毒素合成酶基因mcyE 的检测结果表明,广东省水库中的微囊藻以产毒藻株占优势,微囊藻毒素的主要类型为MC-RR.广东省15 株藻株的ITS 序列相似性大于93.2%,在用相邻法(NJ)构建的系统树上,不同形态的种和不同地理区域的藻株没有区分开,产毒和非产毒藻株没有形成独立分支.这说明微囊藻ITS 序列的遗传多样性较低,ITS 序列和mcyE 存在没有相关性,表型不能够反映藻株的进化关系.因此,有必要将藻类传统分类方法与分子方法结合起来对蓝藻进行重新分类.     

伪柔弱拟菱形藻复合群的形态分类学研究

于2007年夏季(8月)在广东省大亚湾海域观察到伪柔弱拟菱形藻复合群的6个种类:花形拟菱形藻(P.caciantha Lundholm,Moestrup Hasle)、靓纹拟菱形藻(P.calliantha Lundholm,Moestrup Hasle)、尖细拟菱形藻[P.cuspidata(Hasle)Lundholm,Moestrup Hasle]、曼氏拟菱形藻(P.mannii Amato Montresor)、伪柔弱拟菱形藻[P.pseudodelicatissima(Hasle)Lundholm,Hasle Moestrup]和中华拟菱形藻(P.sinica QiWang),其中花形拟菱形藻、靓纹拟菱形藻和曼氏拟菱形藻为我国新记录种类。对该复合群种类的形态学特征、生活习性和生态分布进行了观察和描述,并对其种类之间的形态学特征进行了比较研究。     

淡水藻种库(FACHB)库藏产2-MIB蓝藻的鉴定及其产嗅特征研究

为认识产二甲基异莰醇(2-MIB)蓝藻的形态和产嗅特征,从国家水生生物种质资源库淡水藻种库中筛选出24株可产2-MIB藻株,描述了这些藻株的形态特征和生境分布。结合形态和16S rRNA基因分析对藻株进行物种鉴定复核,修订了部分库藏藻株物种名称,例如发现库藏产2-MIB的浮丝藻属种类应当被重新鉴定为拉氏拟浮丝藻或索状气丝藻。基于mic基因系统发育树分析,显示蓝藻mic基因形成5个分支。通过2-MIB含量检测发现,不同藻株间单个细胞总2-MIB含量为6—2549 fg/cell,其含量通常为拉氏拟浮丝藻>索状气丝藻>灰假鱼腥藻。研究提供了产2-MIB蓝藻的形态、分子、生态和产嗅特性等的基础数据,首次报道气丝藻、沙丝藻、苏打丝藻种类可产2-MIB,并在国内首次报道了产2-MIB的微鞘藻,为进一步研究产2-MIB蓝藻生理生态特性提供重要的实验材料和科学依据。     

嵊山岛海洋药用生物资源状况调查

对嵊山岛礁生物资源以及邻近海域的微藻资源调查,共发现121种潮间带生物,其中有28种海洋药用生物,包括红条毛肤石鳖(Acanthochiton rubrolineatus)、马粪海胆(Hem icentiotus pulcherrimus)、海萝(Gloiopeltis furcata)等,其中多数是海洋中药或可制成中成药,嵊山岛丰富的海绵资源具有筛选天然活性物质的潜力。嵊山岛邻近海域的产毒藻类包括产麻痹性贝毒(PSP)的链状亚历山大藻(Alexandrium catenatum)、塔玛亚历山大藻(Alexandrium tamarens),产腹泻性贝毒(DSP)的倒卵形鳍藻(D inophysis forti)、具尾鳍藻(D inophysis caudata);产记忆缺失性贝毒(ASP)的尖刺拟菱形藻(Pseudo-Nitzschia pungens)、多列拟菱形藻(Pseudo-Nitzschia multiseries)和多纹拟菱形藻(Pseudo-Nitzschia multistriata);产神经性贝毒(NSP)的短凯伦藻(Kerina breve)以及红色裸甲藻(Gymnodinium sangium)、米氏凯伦藻(Kerina m ikim oto)、环状异甲藻(Heterocapsa circularisqua-ma)等能产生生理活性物质的藻类,其中嵊山岛邻近海域产石房蛤毒素和神经毒素的有毒藻类可能具有药用开发价值。     

南海局部海洋沉积物中真菌多样性及产酶活性

【目的】从11份南海海洋沉积物中分离耐盐真菌,并对其物种多样性及产酶活性进行研究。【方法】利用平板涂布法分离耐盐真菌,基于形态学和ITS序列的系统进化研究耐盐真菌多样性;利用6种筛选培养基对耐盐真菌进行产酶活性筛选。【结果】分离得到1689株耐盐真菌,共41个形态种。形态学和ITS序列分析表明,这些真菌归于15个属,其中曲霉属(Aspergillus)和青霉属(Penicillium)为优势菌群。对已测序的41株耐盐真菌的产酶活性研究表明,8株产纤维素酶,9株产淀粉酶,5株产复合酶,16株产蛋白酶,3株产脂肪酶,未发现产壳聚糖酶的菌株,其中Acrodontium sp.8m和Aspergillus sp.86b产复合酶的活性相对较高,而Penicillium sp.41m产蛋白酶的活性相对较高。【结论】南海局部海洋沉积物中耐盐真菌丰富,多数菌株具有产酶活性。     

古尔班通古特沙漠生物结皮微鞘藻分类学研究

实验研究了从古尔班通古特沙漠生物土壤结皮中分离纯化培养出的11株与微鞘藻(Microcoleus)形态接近的丝状蓝藻,通过形态特征、16S rRNA和ITS二级结构相结合的多相分析方法对其进行分类学研究。研究结果表明,实验藻株隶属于微鞘藻科(Microcoleaceae)的微鞘藻属(Microcoleus)和束脉藻属(Symplocastrum),其中包括2个中国新记录种:斯坦微鞘藻(Microcoleus steenstrupii)和细长束脉藻(Symplocastrum flechtnerii),另外还有具鞘微鞘藻(Microcoleus vaginatus)和类似斯坦微鞘藻的存疑物种。藻丝多少与排列方式、细胞大小与末端细胞形状,以及16S rRNA系统发育位置是确定微鞘藻(Microcoleus)与束脉藻(Symplocastrum)属于不同物种的关键依据, ITS二级结构是区分属内不同物种的重要参考。     

2005年夏季长江口水域浮游植物群集特征及其与环境因子的关系

根据2005年9月8—15日在长江口及其邻近水域(30.5°32.5°N,121.0°123.5°E)进行的多学科外业航次调查所获资料,对调查区浮游植物的群集特征及其与环境因子之间的关系进行了研究。结果表明:鉴定浮游植物98种(含变种和变型),硅藻是浮游植物中的主要类群;浮游植物生态类型多为温带近岸种,少数为暖水种和大洋种;优势种主要为多尼骨条藻、尖刺伪菱形藻、柔弱几内亚藻、翼鼻状藻细长变型和柔弱伪菱形藻;细胞丰度平均为256.4个·mL-1,高值出现在调查区的中部偏南及东北部海域;Margalef物种丰富度指数、Shannon-Wiener物种多样性指数、Pielou'均匀度指数的分布显示:调查区东南部海域浮游植物多样性程度较高,物种均一性较好。物种与环境的典范对应分析显示:甲藻与环境因子中溶解氧的关系较为密切;多尼骨条藻与海水中硅酸盐和硝酸盐的关系较为密切,与海水盐度有很大的负相关性;各物种在排序图中的位置反映了其对不同环境资源的生态需求。     

阿氏浮丝藻mcyT基因序列多样性研究

为研究我国浮丝藻（Planktothrix Anagnostidis et Komrek）的毒素相关基因,选取分离自我国不同省份水体的13株阿氏浮丝藻,通过PCR检测其微囊藻毒素合成酶基因mcyA、mcyE及mcyT研究其毒素基因特性。PCR结果表明除mcyT之外其他引物检测均无扩增产物,说明这13株浮丝藻不具备产微囊藻毒素的能力。通过克隆测序得到mcyT序列,并进行分子系统分析,构建了关于mcyT序列的Neighbor-Joining系统树,结果表明mcyT序列可以将产毒与不产毒浮丝藻分为两大独立的分支,两个分支之间的最低序列相似度分别为98.5%和99.1%。研究结果可为后续研究我国浮丝藻的微囊藻毒素合成相关基因的多样性以及分子监测提供参考。       

Pseudo-nitzschia simulans sp. nov. (Bacillariophyceae), the first domoic acid producer from Chinese waters

The genus Pseudo-nitzschia has attracted attention because of production of the toxin, domoic acid (DA), causing Amnesic Shellfish Poisoning (ASP). Pseudo-nitzschia blooms occur frequently in Chinese coastal waters, and DA has been detected in several marine organisms, but so far no Pseudo-nitzschia strains from Chinese waters have been shown to produce DA. In this study, monoclonal Pseudo-nitzschia strains were established from Chinese coastal waters and examined using light microscopy, electron microscopy and molecular markers. Five strains, sharing distinct morphological and molecular features differentiating them from other Pseudo-nitzschia species, represent a new species, Pseudo-nitzschia simulans sp. nov. Morphologically, the taxon belongs to the P. pseudodelicatissima group, cells possessing a central nodule and each stria comprising one row of poroids. The new species is characterized by the poroid structure, which typically comprises two sectors, each sector located near opposite margins of the poroid. The production of DA was examined by liquid chromatography tandem mass spectrometry (LC–MS/MS) analyses of cells in stationary growth phase. Domoic acid was detected in one of the five strains, with concentrations around 1.05–1.54 fg cell⁻¹. This is the first toxigenic diatom species reported from Chinese waters.     

Development of a high-resolution molecular marker for tracking Pseudo-nitzschia pungens genetic diversity through comparative analysis of mitochondrial genomes

The harmful algal bloom (HAB) species Pseudo-nitzschia pungens is widely distributed in almost all continents. Accumulating evidence suggests that P. pungens has high genetic diversity and many strains can produce the toxin domoic acid (DA) that harms animals and humans. Nevertheless, different P. pungens strains cannot be distinguished using morphological features or using common molecular markers including 18S rDNA, 28S rDNA, ITS, cox1, and rbcL. As such, high-resolution molecular markers need to be developed to resolve P. pungens genetic diversity, facilitating accurate tracking of toxic P. pungens strains. We hypothesized that molecular markers with high resolution and high specificity can be designed through identifying regions with high genomic variations in the mitochondrial genome. Here, we describe the development of a new molecular marker Pseudo-nitzschia pungens mitochondrial 1 (ppmt1) with high resolution and high specificity through comparative analysis of mitochondrial genomes of nine P. pungens strains isolated from coastal regions of China. In conclusion, we have developed ppmt1 as a high-resolution and high-specificity molecular marker for tracking strains and genetic diversity of the HAB species P. pungens.

     

CRYPTIC AND PSEUDO‐CRYPTIC DIVERSITY IN DIATOMS—WITH DESCRIPTIONS OF PSEUDO‐NITZSCHIA HASLEANA SP. NOV. AND P. FRYXELLIANA SP. NOV.1

A high degree of pseudo‐cryptic diversity was reported in the well‐studied diatom genus Pseudo‐nitzschia. Studies off the coast of Washington State revealed the presence of hitherto undescribed diversity of Pseudo‐nitzschia. Forty‐one clonal strains, representing six different taxa of the P. pseudodelicatissima complex, were studied morphologically using LM and EM, and genetically using genes from three different cellular compartments: the nucleus (D1–D3 of the LSU of rDNA and internal transcribed spacers [ITSs] of rDNA), the mitochondria (cytochrome c oxidase 1), and the plastids (LSU of RUBISCO). Strains in culture at the same time were used in mating studies to study reproductive isolation of species, and selected strains were examined for the production of the neurotoxin domoic acid (DA). Two new species, P. hasleana sp. nov. and P. fryxelliana sp. nov., are described based on morphological and molecular data. In all phylogenetic analyses, P. hasleana appeared as sister taxa to a clade comprising P. calliantha and P. mannii, whereas the position of P. fryxelliana was more uncertain. In the phylogenies of ITS, P. fryxelliana appeared to be most closely related to P. cf. turgidula. Morphologically, P. hasleana differed from most other species of the complex because of a lower density of fibulae, whereas P. fryxelliana had fewer sectors in the poroids and a higher poroid density than most of the other species. P. hasleana did not produce detectable levels of DA; P. fryxelliana was unfortunately not tested. In P. cuspidata, production of DA in offspring cultures varied from higher than the parent cultures to undetectable.     

MORPHOLOGY OF THE MARINE DIATOM NITZSCHIA NAVIS-VARINGICA, SP. NOV. (BACILLARIOPHYCEAE), ANOTHER PRODUCER OF THE NEUROTOXIN DOMOIC ACID

A new marine diatom, Nitzschia navis-varingica , sp. nov., isolated from Vietnamese waters, is described by light, transmission, and scanning electron microscopy, including thin sectioning. The new species has been found to produce the neurotoxin domoic acid (DA), better known from several species of Pseudo-nitzschia Peragallo and one species of Amphora Ehrenberg. Production of DA is therefore more widespread among diatoms than previously thought. Taxonomically, the genus Nitzschia Hassall is exceptionally difficult, with about 900 described taxa. Grunow (in Cleve and Grunow 1880 divided the genus into 24 sections, and this system is still used with modifications. Nitzschia navis-varingica , sp. nov. fits best into a group of sections that includes Dubiae, Bilobatae , most of the Lanceolatae , and Lineares , all sensu Grunow, as the cell is slightly indented in the middle in girdle view and has a moderately eccentric raphe and a weak longitudinal fold on the valve. Many species within these sections have features similar to N. navis-varingica , but no species seems to be identical. Because both Pseudo-nitzschia and Nitzschia belong to the family Bacillariaceae, it seems reasonable to look for further producers of DA in this family, including freshwater species, which mainly comprise species within the sections Dubiae, Bilobatae, Lanceolatae , and Lineares.     

Genetic diversity of Pseudo-nitzschia brasiliana (Bacillariophyceae) from Malaysia

To clarify the genetic diversity of a potentially toxic pennate diatom, Pseudo-nitzschia brasiliana found in Malaysian waters, 30 strains of P. brasiliana were established into clonal culture since May 2008. The ultrastructure of these strains was examined for confirmation of species identification. The genetic marker, internal transcribed spacer (ITS) of nuclear-encoded ribosomal DNA was used to examine the genetic diversity of P. brasiliana isolated from different geographical localities. The ITS sequences of P. brasiliana were highly conserved in their secondary structures, with five helices in the first internal transcribe spacer (ITS1) and four universal helices in the second internal transcribe spacer (ITS2) with a pseudo-helix. No compensatory base change was observed among the strains examined. Genetic divergences among the Malaysian strains ranged from 0.07 to 0.54%. The present study revealed a high genetic homogeneity of Malaysian P. brasiliana strains.     

我国沿海双唇形突海链藻的形态学研究

唇形突是海链藻中常见的一类突起, 其形态学特征和排列方式是重要的分类学依据。绝大多数海链藻只有一个唇形突, 两个唇形突的现象仅报道于极少数海链藻种类。我国关于双唇形突海链藻的报道较少。研究利用扫描电镜(SEM)技术, 对采自我国沿海海域的自然水样, 以及分离获得的单克隆培养藻株进行了形态学观察, 针对其中的海链藻属Thalassiosira Cleve种类开展了形态学的专题研究。报道了4个具有双唇形突的海链藻种类, 分别是有翼海链藻T. bipartita (Rattray) Hallegraeff、脆弱海链藻T. fragilis Fryxell、亨氏海链藻T. hendeyi Hasle &; Fryxell和维斯吉思海链藻T. visurgis Hustedt, 其中有3个为我国新记录种类: 脆弱海链藻、亨氏海链藻和维斯吉思海链藻。对每个种类的形态学特征、生活习性和生态分布进行了描述, 提供了扫描电镜照片, 对相似种类的形态学特征进行了比较研究, 分析了唇形突的变化类型及其分类学价值。     

基于染色体计数和ITS序列初步探讨横断山区柴胡属植物(伞形科)的系统发育

横断山区是中国柴胡属Bupleurum植物的分布中心。本文对横断山区6个种2变种进行了染色体记数报道,其中4个种2变种是首次报道。对横断山区的10个种4个变种、中国北方（河北和黑龙江）的3个种的nrDNA ITS进行测序,同时从GenBank里面下载同属的来自非洲和地中海西部的16个nrDNA ITS序列数据,结合染色体数目变化结果,初步探讨了横断山区柴胡属植物的系统发育。结果表明横断山区可能是现代柴胡属植物的频度中心和多样分布中心之一。它们的祖先种可能是非洲北部的木本柴胡属植物B.fruticosum,或者是地中海西部的柴胡属植物,推测是通过中东和高加索扩散而形成的,其中与非洲南部特有种B.mundtii的亲缘关系也较近;染色体基数演化趋势是：8是较原始基数,6和7是次生基数,其染色体异基数变异和多倍化可能是物种形成、进化以及向外扩散的主要方式;在ITS系统发育树中,中国柴胡属植物染色体基数为8的种类聚为一支,染色体基数为6和7的种类聚为了一支,不支持舒璞等（1998）关于中国柴胡属的属下分类系统。结合已有的形态学、细胞学、孢粉学证据和ITS系统发育树,建议窄竹叶柴胡B.marginatum var.stenophyllum独立成种。