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“褐潮”原因种抑食金球藻在中国海域的地 理分布与溯源及其生活史中存在休眠体的证明
马照鹏
Subtype博士
Thesis Advisor唐赢中
2019-11
Degree Grantor中国科学院大学
Place of Conferral中国科学院大学海洋研究所
Degree Name理学博士
Keyword褐潮 抑食金球藻 地理分布 沉积物测年 休眠体阶段
Abstract

抑食金球藻是一种非运动、球形、大小约2-3 μm的微微型浮游植物,隶属于海金藻纲。1985年以来,抑食金球藻在美国东海岸经常性地引发有害藻华褐潮1997年前后,在南非附近海域也爆发了几次抑食金球藻褐潮;2009年以来,在中国渤海沿岸海域频繁地发生抑食金球藻褐潮。褐潮的爆发对沿海生态系统有巨大的破坏作用,同时使水产养殖业遭受了重大损失,特别是造成了养殖贝类的大量死亡。有研究推测,抑食金球藻是近年来通过船舶压舱水运输的方式从美国传播到南非,从而扩大了其地理分布范围。那么作为中国海域的典型有害藻华藻种,抑食金球藻是近年来通过人工运输/自然转运的方式传播到中国海域的外来入侵物种,还是早就存在于中国海域的本地物种,是一个具有重要生态学意义的问题,并引起了广泛的关注。因此,对抑食金球藻在中国海域的地理分布以及历史溯源进行广泛深入的调查研究显然极为重要。此外,有害藻华藻种能否通过船舶压舱水运输的方式传播到其它海域的关键问题之一是该物种的生活史中是否存在休眠体阶段。因此,阐明抑食金球藻的生活史中是否存在休眠体阶段将有助于揭示中国海域抑食金球藻的来源、该物种在全球范围内进行地理扩散以及藻华年际复发的机制。

由于抑食金球藻个体微小、形态简单,在显微镜下很难与其它同样微小的藻类区分,使用物种特异性引物进行基于PCR方法的分子检测,是开展抑食金球藻广泛调查的最佳选择。此外,沉积物样品累积了所有季节和历史年代的物质沉积,可以提供更为准确和全面的生物群落信息,在本研究中被用来进行抑食金球藻的检测。通过对采集自中国四大海域(渤海、黄海、东海、南海)的201个表层沉积物样品进行18S28S rDNA片段的PCR扩增和克隆测序验证,发现其中160个样品为阳性,证明抑食金球藻广泛分布于中国四大海域:南北跨越纬度为29.841°(超过3000 km),东西跨越经度为15.674°(最宽处约1500 km),覆盖了近海(水深2.5 m)到开阔外海(水深3456 m),温带到热带的广阔水域。对采集自渤海海域的1个柱状沉积物样品(210Pb\137Cs结合AMS 14C测年方法完成年代测定)进行检测分析,发现抑食金球藻在渤海海域的存在历史至少可回溯到1500年前。这些结果将抑食金球藻在北半球的分布记录至少向南延伸了1700 km,同时明确地否证了中国海域的抑食金球藻是外来入侵物种的假说。本研究利用中国四大海域12个表层沉积物样品中得到的52993 bp大小的抑食金球藻28S rDNA序列(包括部分D2D3-D6区;GenBank accession Nos. MG782521-MG782572),和抑食金球藻CCMP1984藻株中得到的2028S rDNA序列(GenBank accession Nos. MG782499-MG782520)以及GenBank上提交的一条抑食金球藻28S rDNA序列(GenBank accession No. AF289042)共同构建了最大似然法系统进化树,发现中国海域的序列属于同一个种群,并且和美国东海岸的抑食金球藻种群没有发生明显的遗传分化。这说明,两者有一个共同祖先,而且地理分离的时间并不久远。

为了检测沉积物中是否存在抑食金球藻的休眠体,本研究于201458日,在连续多年爆发抑食金球藻褐潮的秦皇岛沿岸渤海海域(站位BS10639.602°N, 119.913°E, 水深25 m)采集了表层沉积物样品,并将该样品置于黑暗4℃环境下保存了一年多时间,以保证沉积物样品中存活的细胞一定是休眠体而不是正常营养细胞。利用RT-PCR的方法对该沉积物样品进行检测后,通过开展萌发实验,结合异硫氰酸荧光素标记的单克隆抗体(FITC-MAb)检测和物种特异性的PCR方法,对萌发生长出来的细胞进行了鉴定,证实了沉积物样品中存在抑食金球藻的休眠体。同时,通过对实验室纯培养的抑食金球藻CCMP1984藻株进行不断观察,发现了其休眠体,并对休眠体的形态特征、生理特性、萌发的时序过程以及长时间黑暗低温(4℃)处理后转变为营养细胞的能力都开展了相关研究。这些研究结果充分证明了抑食金球藻的生活史中存在休眠体阶段,而且其休眠体能够在黑暗低温的海洋沉积物中长期存活(超过一年),一旦置于适宜的环境条件下,可以快速地萌发(几个星期内)并恢复营养生长。产生休眠体为藻华的年际复发以及通过人工运输(如船舶压舱水)或自然转运进行地理扩散的过程至少提供了一种重要的机制。因此,抑食金球藻生活史中休眠体阶段的发现,对这种海金藻纲的典型有害藻华藻种的基础生物学以及生态学,都具有重要的理论意义。

鉴于上述研究中的重要发现:抑食金球藻1)广泛分布于近海和远洋的水域中;2)在中国海域至少存在了1500年;3)生活史中存在休眠体阶段,自身可以进行持续跨洋运输,我们认为它很可能是一种全球性分布的物种。通过挖掘已发表论文(de Vargas et al., 2015)的补充材料,发现在太平洋、南大西洋,地中海,印度洋的众多水样中都检测到了抑食金球藻,从而明确地证实了抑食金球藻是一种全球各大洋广泛分布的物种,而产生休眠体可能是造成该物种全球分布和褐潮年际复发的重要原因。本研究得到的结果不仅为中国海域抑食金球藻的种源和其全球地理分布格局提供了决定性认识,也为进一步研究褐潮爆发成因提供了至关重要的科学问题导向,即褐潮爆发成因应当从本地环境条件的变迁中探索。此外,本研究采用的研究思路和方法也为研究其它近几十年来有明显地理扩散趋势的有害藻华藻种提供了一个范例。

Other Abstract

The nonmotile, spherical, picoplanktonic (2-3 μm) pelagophyte Aureococcus anophagefferens has caused numerous harmful algal blooms (HABs) ("brown tides") along the east coast of the USA since 1985, a few times in South Africa around 1997, and frequently in the coastal waters of the Bohai Sea, China since 2009. As a consequence, the harmful blooms have caused massive losses in aquaculture and coastal ecosystems, particularly mortalities in cultured shellfish. It was hypothesized that A. anophagefferens was introduced to South Africa recently and expanded its distribution via ships’ ballast water transport. Therefore, as a relatively novel HAB-forming species in China, whether A. anophagefferens was recently introduced to China via natural/artificial transport or has been an indigenous species has become a question of profound ecological significance and broad interest, which necessitated an extensive investigation on the geographic and historical presence of this species in the seas of China. Moreover, one of the key questions regarding whether a harmful algal species is able to be introduced from another site via ships’ ballast water is whether or not there is a resting stage in the life history of the species. Therefore, answering whether A. anophagefferens has a resting stage may help to explain the origin of A. anophagefferens found in China, the apparent geographic expansion of the species around the world, and the mechanisms of annual recurrances of blooms.

Since A. anophagefferens cells are too small in size and too simple in morphology to be distinguished microscopically from other small algae, PCR-based molecular detection using species-specific primers became the best option for the extensive survey. In addition, sediment samples, which may provide a much more accurate and comprehensive record because of the nature of sediments in accumulating all seasonal and historical deposits, were used for the detection. In testing sediment samples with PCR amplification and sequencing for partial 18S and 28S rDNA, we discovered that 160 of 201 surface sediment samples collected from all four seas of China (Bohai Sea, Yellow Sea, East China Sea and South China Sea) were positive for the presence of A. anophagefferens, a geographic distribution covering a latitude range of 29.841° (i.e. >3000 km), a longitude range of 15.674°(~1500 km at the widest, and a water depth range from 2.5 to 3456 m (temperate to tropical and coastal to open oceans). The positive detection in one sediment core (dated via a combined approach of 210Pb and 137Cs measurements and AMS 14C dating) proved a presence of the species for >1500 years at least in Bohai Sea. These detections extended its southernmost tropical range of presence recorded in the northern hemisphere by 1700 km and disproved it to be an alien species recently-introduced into China. Fifty-two 28S rDNA sequences of A. anophagefferens with a length of 993 bp (covering partial D2 and D3-D6 domains; GenBank accession Nos. MG782521-MG782572) were obtained from 12 sediment samples covering all four China Seas. The Maximum Likelihood phylogenetic analysis based on these sequences and 20 sequences of the clonal culture of A. anophagefferens strain CCMP1984 (GenBank accession Nos. MG782499-MG782520) and a sequence of A. anophagefferens deposited in GenBank (GenBank accession No. AF289042) demonstrated that the sequences from China belong to one population and are not phylogenetically distant from that the population from the east coast of the USA, suggesting they share a common ancestor and have not been geographically separated for a long age.

In order to test whether A. anophagefferens presents in the sediment as resting stage cells, germination experiments were conducted for the sediment sample collected on 8 May, 2014 from the station BS106 in the coast of Qinhuangdao, Bohai Sea (39.602°N, 119.913°E, water depth 25 m), where brown tides have occurred since 2009. Before being used in the germination experiments, the sediment sample of BS106 had been stored in darkness at 4°C for more than one year to make sure that live cells existing in the sediment sample must be cells at resting stage rather than normal vegetative cells. At last, existence of a resting stage in A. anophagefferens was proved through germination experiments, combination of a specific monoclonal antibody conjugated to fluorescein isothiocyanate (FITC-MAb) staining and species-specific PCR identification for germlings, and RT-PCR detection of rRNA in sediment samples. In addition, an intensive study using clonal culture of A. anophagefferens strain CCMP1984 discovered the resting stage cells of A. anophagefferens. Characterization of the cellular morphology and physiological status of the resting stage cells, a time series observation for the germination processes of the resting stage cells and the ability of resting stage cells transforming into vegetative cells after prolonged treatment in the dark (4°C) were conducted. With all evidences presented in this study, we are now confident that, by definition, a resting stage does exist in the life history of A. anophagefferens which can survive stressful conditions such as near freezing temperature and darkness for a fairly long period of time (> 1 year) in the sediment and recover (or "germinate") to normal vegetative growth within a reasonable time (a few weeks) when they are placed back to a favorable environment. The resting stage cells, therefore, at least provide a facility for to seed annual blooms and expand its distribution via either artificial (e.g. ships' ballast water) or natural pathways. Discovery of a resting stage of A. anophagefferens is of vital significance not only in the basic biology but also in the ecology of this notorious HABs-causing pelagophyte.

Considering the above-described findings of this work that A. anophagefferens widely distributes from coastal to pelagic waters, a presence in the sediment for >1500 years, and the existence of resting stage, A. anophagefferens may perform transoceanic transportation by itself continuously and thus has achieved a possibly global distribution. After searched the supplementary data set of de Vargas et al. (2015), we found that A. anophagefferens was detected in water samples from many stations of the Atlantic Ocean, Indian Ocean, Mediterranean Sea, and the south Pacific Ocean, which clearly confirmed that this species has in fact a global, cosmopolitan distribution. Production of resting stage cells may be an important reason for the global distribution and annual recurrence of the brown tide. The results obtained in this study not only provide a decisive understanding of the origin of A. anophagefferens found in China and the global distribution pattern of this species, but also provide a crucial scientific guidance for further studies on the causes of brown tidesi.e. the causes of the outbreak of brown tide must be explored from the local environmental settings. We believe the approach adopted in this study also provides an exemplary case applicable to other harmful bloom-forming species which have appeared to expand their geographic distribution during the last decades.

MOST Discipline Catalogue理学
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/162423
Collection海洋生态与环境科学重点实验室
Recommended Citation
GB/T 7714
马照鹏. “褐潮”原因种抑食金球藻在中国海域的地 理分布与溯源及其生活史中存在休眠体的证明[D]. 中国科学院大学海洋研究所. 中国科学院大学,2019.
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