海洋生态与环境科学重点实验室知识库

作为一种独特的生活史策略，休眠孢囊在甲藻地理扩散和藻华爆发等生态学过程中具有极为重要的意义。共生藻科（Symbiodiniaceae）是甲藻中最为重要的类群之一，该科甲藻能够与珊瑚互利共生，使珊瑚礁生态系统具有极高的生产力水平。当前，珊瑚礁生态系统正在面临着全球气候变化及人类活动所带来的风险与挑战，具体表现为珊瑚因失去共生藻科甲藻而引发的白化现象。全球范围内日益增长的珊瑚白化事件不断激发对共生藻科甲藻生物生理学特征及应对环境变化潜力等方面开展广泛研究。然而在众多研究中，迄今为止，尚未有确凿的证据表明共生藻科中的任何物种能够产生休眠孢囊。本研究基于对海洋沉积物中孢囊的直接检测以及对实验室内纯培养的实验和观察，提供了一种隶属于共生藻科且营自由生活（free living）的甲藻—— Effrenium voratum 在自然水体和实验室内产生休眠孢囊的形态学、生活史过程、倍性检测和分子系统学证据。主要研究结果如下：

（1）通过对于海洋沉积物的一系列检测，获得 E. voratum 在自然水体中产生休眠孢囊的证据：首先，通过高通量测序在经由多钨酸钠（SPT）浓缩的中国近海沉积物孢囊样品中检测到 E. voratum 扩增子序列变异子（amplicon sequence variants, ASVs）；第二，利用基于特异性引物的PCR（polymerase chain reaction）技术进一步检测到沉积物样品中 E. voratum 的存在；第三，采用自主设计的特异性探针开展荧光原位杂交（fluorescence in situ hybridization, FISH）实验，检测到FISH阳性孢囊并对其进行光镜形态学观察；最后，利用单细胞PCR技术对FISH阳性孢囊进行测序鉴定，证实了海洋沉积物中存在 E. voratum 休眠孢囊。

（2）通过室内培养观察、倍性测定实验与萌发实验，获得 E. voratum 在实验室内产生休眠孢囊的证据：首先，在4°C 黑暗的环境条件下保存二周至6个月的 E. voratum 孢囊形态完好，当将其转移至常规培养条件后，高达76%（保存6个月）、84%（保存3个月）和92%（保存35天）的孢囊可以通过孢囊内部的发育过程在3-21天内成功萌发；第二，二株纯培养都可形成休眠孢囊，其呈球形，表面光滑无修饰物，每一孢囊内有一个大的红体或脂质体；第三，孢囊从被观察到孢囊壁内呈现似营养细胞形态的细胞（即幼殖体）到幼殖体从萌发孔（archeopyle）释出一般需时一天左右，幼殖体释出过程在五分钟之内完成。每一孢囊通过萌发孔释放出2或4个营养细胞，但未观察到单个细胞从萌发孔释出；第四，流式细胞术和荧光LM检测结果证明，实验室培养的 E. voratum 所产生的孢囊与营养细胞一样，绝大部分都是单倍体，且在实验采用的所有胁迫条件下（营养限制和温度、盐度胁迫）均未观察到配子体交配和融合，也没有观察到具有双纵鞭毛的二倍体运动合子（planozygote），表明孢囊是通过无性方式形成。但本研究不能完全排除 E. voratum 存在有性生殖的可能性，需要更详尽深入的研究做出确证。野外沉积物检测和实验室纯培养的生活史观察证据共同证明，E. voratum 能以无性方式形成休眠孢囊。

E. voratum 是甲藻共生藻科中首个被证实能够产生休眠孢囊的物种。迄今为止可能由于该科种类细胞体积微小、形态简单、丰度较低等共同特征，尚未在海洋沉积物中检测到该科其它种类的休眠孢囊。本研究填补了对于共生藻科甲藻是否可能产生休眠孢囊研究的空白，丰富了人们对共生藻科甲藻生活史过程的认识，也为该科其它物种休眠孢囊的检测与鉴定提供了一种可行的方法，对该种乃至该科甲藻的生物学和生态学具有重要意义。

As a unique life history strategy, resting cyst production is of great significance in the ecology process of many dinoflagellates, such as geographic expansion and harmful algal blooms. As one of the most important group of dinoflagellates, dinoflagellates belonging to the family Symbiodiniaceae have a mutually-beneficial relationship with corals, resulting in extremely high productivity levels of coral reef ecosystems. Coral reef ecosystems are currently facing risks and challenges from global climate change and human activities, which are manifested in coral bleaching due to the loss of Symbiodiniaceae species. The increasing number of coral bleaching events worldwide has stimulated researches of the physio-ecological characteristics of Symbiodiniaceae and their potential to cope with environmental change. However, in the numerous studies to date, there has been no confirmative evidence for the production of resting cyst in any Symbiodiniaceae species. In this study, based on the direct detection of cysts from the marine sediments and the morphological observations of cultures in the laboratory, here we provide both morphological and molecular evidences for the production of resting cysts in natural waters and in the laboratory by Effrenium voratum, the free-living, type species of the genus Effrenium in Symbiodiniaceae. The main research results are as follows:

(1) Evidences for the production of resting cysts in natural waters by E. voratum were obtained through a sequential detections in marine sediment samples: Firstly, E. voratum amplicon sequence variants (ASVs) were detected in the sediments concentrated with the sodium polytungstate (SPT) method from the China Seas by high-throughput sequencing; Secondly, the presence of E. voratum in the sediments were further detected by polymerase chain reaction (PCR) using the species-specific primers; Thirdly, the presence of cysts were confirmed via fluorescence in situ hybridization (FISH) using the species-specific probes and light microscopic (LM) observation of cyst morphology; Finally, the FISH-positive cysts were identified by a subsequent single-cyst PCR sequencing, which confirmed the existence of E. voratum resting cysts in marine sediments.

(2) Evidences for the production of resting cysts in laboratory by E. voratum were obtained through observations of laboratory-reared clonal cultures, ploidy determination and germination experiments: Firstly, cysts could maintain intact for two weeks to six months when stored at 4°C in darkness, and up to 92% of them successfully germinated through an internal development processes within a time period of 3-21 days after being transferred back to the normal culturing conditions; Secondly, both strains formed resting cysts, which were spherical with smooth surfaces and no modifications, and each one contained a large red body or liposome; Thirdly, it generally took about one day from the observation of cells with morphological resemblance to vegetative cells in the cyst wall (i.e., germlings) to the release of germlings from the archeopyle, and the release of germlings was completed within five minutes. Two or four germlings were released from each cyst through the cryptopylic archeopyle, but no single cell was observed to be released from the archeopyle; Finally, the results of flow cytometry and epifluorescence LM measurements indicated that most of the cysts produced by laboratory cultures were similarly haploid to the vegetative cells, and gametes mating and fusion or planozygote (cells with two longitudinal flagella) were not observed under all the stress conditions used in the experiment (nutrient limitation, temperature and salinity stress), suggesting that these cysts were formed asexually. However, the possibility of sexual reproduction in E. voratum cannot be completely excluded in this study, and further detailed and in-depth studies are needed to confirm this possibility. Evidences from field sediment detection and life history observations of cultures in the laboratory suggest that E. voratum is capable of forming resting cysts asexually.

E. voratum is now the first case in Symbiodiniaceae species that has been proven to produce resting cysts. Up to date, no evidence for resting cysts production from marine sediments has been obtained for any other species of the family, probably due to their common features to E. voratum such as small sizes, simple morphology, and extremely low abundance. Our study filled the gaps in the knowledge about whether Symbiodiniaceae species are capable of producing resting cysts, and thus enriched our understanding of the life history of this group of dinoflagellates. Our work not only provides a feasible approach for the detection and identification of resting cysts of other species in this family, but also is of vital importance in the exploration of the biology and ecology of Symbiodiniaceae species.

第1章 绪论..................................................................................................................... 1

1.1 甲藻与休眠孢囊...................................................................................................... 1

1.1.1 甲藻及其在生态系统中的重要作用.................................................................. 1

1.1.2 甲藻生活史及休眠孢囊....................................................................................... 2

1.1.3 甲藻孢囊的种类................................................................................................... 4

1.1.4 甲藻休眠孢囊的重要生态学作用和应用...........................................................5

1.2 珊瑚礁生态系统与共生藻科（Symbiodiniaceae）甲藻...................................... 6

1.2.1 珊瑚礁生态系统的重要意义............................................................................... 6

1.2.2 共生藻科甲藻与珊瑚礁的关系........................................................................... 7

1.2.3 共生藻科甲藻分类研究进展............................................................................... 8

1.2.4 共生藻科甲藻生活史研究进展........................................................................... 9

1.3 共生藻科甲藻Effrenium voratum............................................................................ 11

1.3.1 E. voratum介绍...................................................................................................... 11

1.3.2 E. voratum研究现状.............................................................................................. 12

1.4 本论文的科学问题、研究目的与意义................................................................. 13

1.4.1 科学问题............................................................................................................... 13

1.4.2 研究目的与意义................................................................................................... 14

第2章 野外沉积物中存在E. voratum休眠孢囊的证据............................................... 15

2.1 材料与方法............................................................................................................... 15

2.1.1 沉积物样品采集.................................................................................................... 15

2.1.2 应用PCR技术检测海洋沉积物中E. voratum孢囊核酸信号.............................. 16

2.1.3 应用FISH检测E. voratum孢囊及其光镜形态观察............................................. 16

2.1.4 应用单细胞巢式PCR鉴定FISH阳性孢囊........................................................... 17

2.2 实验结果................................................................................................................... 18

2.2.1 海洋沉积物中E. voratum孢囊核酸信号PCR检测.............................................. 18

2.2.2 海洋沉积物中E. voratum孢囊FISH检测............................................................. 25

2.2.3 单细胞PCR及测序鉴定........................................................................................ 27

2.3 讨论........................................................................................................................... 28

2.4 小结........................................................................................................................... 29

第3章 实验室内E. voratum产休眠孢囊的证据........................................................... 31

3.1 实验材料与方法....................................................................................................... 31

3.1.1 实验藻株及培养条件............................................................................................ 31

3.1.2 E. voratum孢囊的观察及倍性检测...................................................................... 31

3.1.3 E. voratum孢囊的萌发.......................................................................................... 32

3.2 实验结果................................................................................................................... 32

3.2.1 E. voratum孢囊的形态及倍性.............................................................................. 32

3.2.2 E. voratum孢囊萌发过程的时间序列.................................................................. 34

3.3 讨论........................................................................................................................... 42

3.3.1 E. voratum可以无性方式形成休眠孢囊............................................................. 42

3.3.2 关于共生藻科甲藻生活史策略的探讨.............................................................. 43

3.4 小结........................................................................................................................... 44

第4章 结论、创新点与展望......................................................................................... 45

4.1 主要结论................................................................................................................... 45

4.2 创新点....................................................................................................................... 45

4.3 展望........................................................................................................................... 46

参考文献......................................................................................................................... 49

附录................................................................................................................................. 65

致谢................................................................................................................................. 73

作者简历及攻读学位期间发表的学术论文与其他相关学术成果.......................... 75