中国科学院海洋研究所机构知识库

      自2007年起，我国黄海浒苔（Ulva prolifera）绿潮已成为一种常态化发生的海洋灾害，大量研究表明，苏北浅滩紫菜栽培筏架上的附生绿藻为绿潮提供了重要初始生物量。分子鉴定结果表明，筏架绿藻由多个物种组成，随时间有明显的物种演替，而浒苔最终占比可能与当年绿潮规模存在相关性。因此，及时、准确监控筏架附生绿藻的组成特征，对于绿潮预测预警具有重要意义。由于筏架区面积广大、绿藻组成具有空间差异、尤其绿潮早期阶段时间短暂，因此，亟待建立一种基于形态特征、适用现场大量样本的绿藻物种鉴定方法。另外，黄海绿潮由浒苔漂浮生态型主导，普遍认为其具有高度分枝的表型特征，可能赋予其显著的种间竞争优势，但始终缺乏形态学的量化表征，与定生浒苔相比，其分枝程度与分枝表型的可塑性是否存在明显差异尚缺乏依据。

    针对上述问题，本研究在苏北浅滩紫菜养殖筏架区设置了多个站位，通过多航次广泛采集了附生绿藻样本。基于分子鉴定结果，对常见优势种的关键形态学特征进行了观察、比较与统计分析。针对浒苔的分枝表型，通过广泛采集漂浮生态型与定生群体样本，利用量化表征的新指标，比较了二者在分枝程度及其可塑性方面的差异，取得以下主要研究结果：

    （1）对529个苏北浅滩紫菜筏架附生绿藻样本开展了分子鉴定，共检出五个优势种，分别是：缘管浒苔（U. linza）、浒苔（U. prolifera）、U. aragoënsis、扁浒苔（U. compressa）和盘苔（Blidingia sp.），符合历年基本规律，其中，鉴定U. aragoënsis为新记录种，对之前被广泛接受的物种鉴定结果——曲浒苔（U. flexuosa）进行了修正；

    （2）针对上述五个优势种，经形态学观察、测量与比较分析，提出了一个基于关键形态学特征进行物种鉴定的检索表：首先，用藻体最高分枝级数将物种分为高分枝（HB）组和低分枝（LB）组，在LB组中，可首先使用藻体的长宽比（length-to-width ratio，LWR）区分出Blidingia sp.，然后再用淀粉核数量分辨U. linza和U. aragoënsis；在HB组中，根据表面细胞排列方式和一级分枝与主枝连接处的形态特征区分U. prolifera和U. compressa。为了最大程度方便现场应用，联合使用藻体最高分枝级数和长宽比两个指标，开展了准确率检验，结果表明，平均准确率为81.3%，对浒苔的鉴定准确率达到89.1%，可以满足现场应用的要求；

    （3）针对浒苔分枝表型的量化表征，提出最高分枝级数和一级分枝密集度是两个可应用的指标，对比分析结果表明，定生浒苔普遍有一级分枝或无分枝，而漂浮生态型分枝级数最高可达五级，其一级分枝密集度是定生浒苔的5.8倍，说明漂浮生态型的分枝程度显著高于定生浒苔。另外，无论自然条件还是人工培养条件下，发现漂浮生态型分枝表型的可塑性也明显强于定生群体。提示高度分枝表型是漂浮生态型重要的漂浮适应性特征；而分枝表型的强可塑性，可能有助于其适应漂浮过程中多变的海洋环境。

    上述研究结果将为黄海绿潮的早期监测与规模预测提供重要的方法学基础，为后续绿潮生物学机制研究提供科学依据和必要的形态学评价指标。

Since 2007, the green tide of Ulva prolifera in the Yellow Sea has become a normalized marine disaster. Many studies have shown that green algae attached on Pyropia yezoensis aquaculture rafts in Subei shola provide an important initial biomass for the green tide. The molecular identification results show that the green algae attached on the rafts are composed of several species, which have species succession over time, and the final proportion of U. prolifera may be related to the scale of the green tide in that year. Therefore, timely and accurate monitoring of the composition characteristics of raft-associated green algae has important sense to prediction and early warning of green tide. Because of the large area of rafting area, the spatial diversity of species composition, especially the short time in the early stage of green tide, it is essential to establish a species identification method based on morphological characteristics and suitable for a large number of samples on site. In addition, the Yellow Sea green tide is dominated by the floating ecotype of U. prolifera, and it is generally believed that it has a highly branched phenotype, which may give it significant interspecific competitive advantage. However, since insufficiency of indexes to the characteristic of branch phenotypic, whether branching phenotype and its plasticity are a significant difference between attached U. prolifera and floating ecotype is still unknown.

In view of the above problems, several stations were set up in P. yezoensis aquaculture rafts in Subei shola to collect samples with multiple voyages in this study. Based on the results of molecular identification, key morphological characteristics of common dominant raft- associated species were observed, compared and statistically analyzed. According to the branching phenotype of U. prolifera, the differences of branching degree and plasticity between attached U. prolifera and floating ecotype were compared by widely collecting samples and using new quantitative indexes. The following important results are obtained:

(1) The molecular identification of 529 samples of raft- associated green algae in Subei shola was carried out, and five dominant species were detected: U. linza, U. prolifera, U. aragoënsis, U. compressa and Blidingia sp., which were consistent with previous studies. Importantly, U. aragoënsis was firstly identified as a new record in China, which has been mis-identified as U. flexuosa for long time.

(2) Through morphological observation, measurement and comparative analysis, a key based on morphological characteristics to above-mentioned five species identification is proposed: first, these five species were divided into high-branching (HB) group and low-branching (LB) group by the top branching order. In LB group, Blidingia sp. was distinguished by length-to-width ratio (LWR), U. linza and U. aragoënsis were distinguished by the number of pyrenoid. In HB group, U. prolifera and U. compressa were identified by cell arrangement and the shape of junction between primary branch and main axis. In order to maximize convenience of on-site application, a practical accuracy test was designed to evaluate the application of the top branching order plus LWR of the algae to distinguish the green seaweeds. The results showed that average accuracy was 81.3%, and identification accuracy of U. prolifera was 89.1%, which could meet the requirements of field application.

(3) Two quantitative indicators, i.e. the top branching order and the intensity of primary branches, were used to characterize the branching phenotypes of U. prolifera. The comparative analysis results showed that attached U. prolifera generally has primary branch or no branching, while the top branching order might reach five in the floating ecotype. The intensity of primary branches in the floating ecotype was 5.8 times higher than that of attached populations, indicating a significant difference in the branching phenotypes between floating ecotypes and attached U. prolifera. In addition, the plasticity of branching phenotypes was also proved much stronger in the floating ecotype than attached populations under both natural and artificial cultivation conditions. These results suggest that, the highly branched phenotype is an important adaptive feature of the floating ecotypes, and the strong plasticity of the branched phenotype may help them adapt to the changeable marine environment.

The above results will provide an important methodological basis for the early monitoring and scale prediction of green tide in the Yellow Sea, and provide scientific basis and necessary morphological evaluation indexes for further study of branching mechanism in U. prolifera.

第1章 引言... 1

1.1 绿潮与绿潮藻概述... 1

1.1.1 绿潮概述... 1

1.1.2 石莼属绿藻概述... 2

1.2 黄海绿潮——常态化的海洋灾害... 2

1.2.1 黄海绿潮概述... 2

1.2.2 黄海绿潮致因种的鉴定... 4

1.2.3 黄海绿潮地理源头的研究... 5

1.2.4 苏北浅滩筏架附生绿藻的物种组成及其变化规律... 5

1.2.5 苏北浅滩筏架附生U. prolifera的生物量占比与绿潮规模变化... 6

1.3 漂浮浒苔高度分枝的表型... 6

1.3.1 漂浮浒苔的生理适应性研究... 7

1.3.2 漂浮浒苔的表型适应性研究... 7

1.3.3 漂浮浒苔高度分枝表型的研究... 8

1.4 本论文的研究内容、目的及意义... 9

第2章 苏北浅滩筏架附生绿藻的形态学区分方法... 10

2.1 引言... 10

2.2 材料与方法... 11

2.2.1 样本采集... 11

2.2.2 DNA提取和分子鉴定... 12

2.2.3 形态学观察... 16

2.2.4 准确率检验... 18

2.3 结果... 18

2.3.1 筏架附生绿藻的物种组成... 18

2.3.2 形态观察与描述... 20

2.3.3 准确率检验... 26

2.4 讨论... 28

2.4.1 苏北浅滩筏架附生绿藻的物种组成... 28

2.4.2 苏北浅滩筏架附生U. aragoënsis的分类地位... 29

2.4.3 筏架附生物种的关键形态学特征... 30

2.4.4 苏北浅滩筏架附生绿藻形态鉴定方法的适用性... 31

2.5 小结与展望... 32

第3章 浒苔漂浮生态型的分枝表型及其可塑性... 33

3.1 引言... 33

3.2 材料与方法... 33

3.2.1 调查航次与样本采集... 33

3.2.2 样本DNA提取与分子鉴定... 35

3.2.3 浒苔分枝表型的量化表征... 36

3.2.4 浒苔的诱导生殖与培养... 36

3.3 结果... 37

3.3.1 分子鉴定... 37

3.3.2 浒苔分枝表型的量化表征... 40

3.3.3 漂浮生态型自然条件下分枝表型的可塑性... 42

3.3.4 漂浮生态型培养条件下分枝表型的可塑性... 43

3.3.5 漂浮生态型分枝表型在绿潮过程中的变化... 45

3.4 讨论... 46

3.4.1 漂浮生态型的高度分枝表型... 46

3.4.2 漂浮生态型的分枝表型可塑性高于定生浒苔... 47

3.4.3 漂浮生态型的分枝表型在绿潮过程中的变化... 48

3.5 小结与展望... 49

参考文献... 50

致 谢... 59

作者简历及攻读学位期间发表的学术论文与研究成果... 61