海洋生态与环境科学重点实验室知识库

海草是一种在全球温带和热带地区广泛分布的海洋被子植物，海草床与红树林、珊瑚礁生态系统并称为三大典型海洋生态系统。川蔓草(Ruppia spp.)是一种广泛分布于热带和温带滨海、瀉湖、盐沼地等浅水水域中的海草，承担着栖息地构建与水体-沉积物环境调节等重要生态功能。近年来，随着全球气候变化与人类活动影响加剧，包括川蔓草在内的众多海草在世界范围内正已惊人速度衰退。本研究调查了我国温带地区川蔓草属分布现状；探究了不同生活史类型中国川蔓草种群形成条件及其与底质环境的相互作用；比较了两类种群在干燥胁迫下的种群稳定性；对中国川蔓草种子萌发、建苗与保存条件进行系统研究；评估了中国川蔓草对重金属Cu与Cd污染的耐受能力及其毒性效应。

1. 我国温带地区川蔓草属分布现状调查

本研究调查了我国辽宁省至浙江省7个省(直辖市)沿海地区川蔓草属的分布现状，总结发现海水养殖池、咸水沟渠、盐场沟渠、湿地公园、瀉湖、潮间带是其主要的分布环境生境类型，其中海水养殖池是温带地区川蔓草属分布最常见生境类型。通过重访发现，部分文献中记录的川蔓草种群已消失，综合分析文献记录及实地调查信息后认为，人类活动干扰是影响川蔓草分布的主要原因。

2. 黄河三角洲中国川蔓草种群特征

对中国川蔓草而言，上覆水季节变化可导致种群表现为一年生或多年生生活史特征。本研究调查了黄河三角洲两处不同生活史类型的中国川蔓草种群的生态特征及其对沉积物性质的影响。通过调查其生长旺盛季节的生物量和各季节种子库规模，评估种群状态。通过测定中国川蔓草床沉积物营养元素组成及硫化物含量了解草床植被和沉积物之间的相互作用关系。研究结果表明，相邻地理种群，一年生中国川蔓草种群较多年生种群拥有更大的生物量与种子库规模；中国川蔓草植株对栖息地沉积物总氮含量与有机物组成特征有显著影响；一年生的中国川蔓草种群经历干燥、重新覆水后恢复力显著强于同种情况下的多年生种群。

本研究还调查了干燥对中国川蔓草床沉积物、种子库及其种群补充的影响。本次调查的中国川蔓草床表层沉积物会不定期遭遇长期干涸暴露影响。研究结果显示，经历2017年12月至2018年4月长达5个月的干燥后，该中国川蔓草床再次被水层覆盖，而后种子迅速萌发，2018年5月幼苗密度达到1360.86 ± 330.66苗/m²，规模与2017年3月只经历3个月干燥暴露时的情况相似。由此认为，干燥胁迫会显著地延迟中国川蔓草自然种群中种子的萌发与建苗。与大多数海草不同，中国川蔓草种子对栖息地干燥有相当强的耐受与适应能力，表层沉积物中的种子经历近5个月的干燥暴露后依然具有活力。本部分研究为干燥环境下中国川蔓草的抵抗力和稳定性提供了新的认知，并对川蔓草属种群特征进行补充。

3 环境因素对中国川蔓草种子萌发影响

本研究调查了温度与盐度对采自北方沿海地区的中国川蔓草种子萌发与建苗的影响。本研究共进行了0 – 50 psu范围内7个盐度与0 - 30℃范围内6个海水温度对中国川蔓草种子萌发影响的实验，确定了适宜中国川蔓草种子萌发和幼苗生长的环境条件。研究发现：(1)种子最佳萌发条件为盐度5 psu、温度30℃；(2)高盐度(40 - 50 psu)与低温度(0℃)会显著阻碍种子萌发，但当其转移至适宜萌发条件后，其萌发率升高；(3)在盐度适宜时(5 - 10 psu)，随着温度升高，中国川蔓草种子萌发率存在明显的双峰分布；(4)将高盐度或低温度条件下储存的种子转移至适宜萌发条件下后，其萌发率显著升高；(5)温度能够显著的影响中国川蔓草建苗过程，而盐度对建苗过程的影响不显著。本部分研究可为中国川蔓草种群保护恢复提供基础信息。

4 中国川蔓草种子长期保存方法

川蔓草属的种子是进行大规模的沉水植物修复的理想材料，在其繁殖季收集成熟的种子，并保存至人工种子库，可以提高新生的川蔓草种子利用效率，为后续川蔓草床修复提供充分的原料。一定的低温、高盐及干燥条件均能有效地抑制川蔓草种子萌发。该部分实验尝试利用不同的温度、盐度和干燥条件对中国川蔓草种子进行长期保存。实验结果表明：0℃/30 - 40 psu盐度的湿法保存是中国川蔓草种子长期保存的最佳保存方法。而5℃/相对湿度为33 ± 10 %的干燥保存法虽会一定程度降低中国川蔓草种子活力，但也是一种可行的种子长期保存方法。以上结果为建立中国川蔓草人工种子库提供相应基础知识。

5 中国川蔓草对重金属Cu和Cd污染耐受及毒性响应

川蔓草不仅能适应较宽范围盐度环境，也能耐受水环境中较高的重金属浓度，故认为其具有较高的生物修复潜力。然而，川蔓草在生活史早期阶段对重金属暴露的耐受能力还有待研究。本研究调查了中国川蔓草在三个生活史早期阶段对重金属Cu和Cd的吸收能力，并分别从个体水平、亚细胞水平与转录组水平观察相关毒性效应。研究表明，中国川蔓草的幼苗阶段最为脆弱；当Cu和Cd的胁迫浓度分别达到50 μM、500 μM时，中国川蔓草幼苗出现明显的毒性响应；两种重金属元素进入细胞后，迅速积累在细胞液泡和细胞质组织中，降低其对植物细胞的毒性作用。经4天Cu和Cd暴露后，中国川蔓草植株中与信号识别与蛋白质加工有关的基因表达显著下调。本研究初步探究了中国川蔓草生活史早期对重金属元素Cu和Cd的耐受能力、富集途径与毒性响应，评估了其作为咸水环境重金属污染修复工具物种的应用潜力。

Seagrass is a kind of marine angiosperms that are widely distributed in temperate and tropical regions of the world. Seagrass meadow is known as one of the three key marine ecosystems with mangrove and coral reefs. Ruppia is a kind of seagrass widely distributed in shallow waters such as tropical and temperate coasts, lagoons, salt marshes, etc. It is an important part of these water ecosystems, and undertakes the significant ecological functions such as habitats establishment and regulation of water-sediment environments. With the intensifying climate change and human impact, Ruppia has been declining at an alarming rate worldwide with numerous seagrasses. We investigated the current status of the distribution of Ruppia in temperate coastal regions of China, analyzed the influence of major environmental factors on the seed germination of R. sinensis and its favorable preservation conditions, explored the formation conditions of R. sinensis population in different life history types and their interactions with the substrate environment, and compared their stability under extreme dry inhibition. The tolerance of R. sinensis on Cu and Cd contamination and relative toxicity effects were investigated.

1 Investigation on the distributing status of Ruppia in China

This study investigated the distribution status of R. sinensis in the coastal areas from Liaoning to Zhejiang in China. It was concluded that marine aquaculture ponds, saltwater lagoons, ditches, wetland park, and intertidal area were the types of distribution environment in the temperate coastal regions of China, especially in aquaculture ponds. Several R. sinensis populations recorded in previous documents had disappeared in our revisiting investigation. After comprehensive analysis of the literature records and field survey information, it is believed that the disturbance of human activities is the main reason affecting the distribution of R. sinensis.

2 Population characteristics of R. sinensis in Yellow River Delta

In this study, we investigated the influence of desiccation on the sediment seed bank and population recruitment in a R. sinensis seagrass meadow where the surface sediment occasionally experiences long-term air exposure in the Yellow River Delta. Results showed that habitat desiccation had significantly impacted the R. sinensis population. However, a considerable number of sediment seeds survived the long-term desiccation of the surface sediment. After a 5-month period of desiccation (from December 2017 to April 2018) of the surface sediment, followed by re-submersion, the R. sinensis seedling density in May 2018 was 1360.86 ± 330.66 seedlings/m², which was similar to that in March 2017. Thus, desiccation appeared to have retarded seed germination and seedling establishment. These results indicated that sediment R. sinensis seeds can survive the long-term desiccation of their habitat. Unlike most seagrass species, we observed for the first time that R. sinensis seeds in sediment exhibited considerable tolerance and adaptability to habitat desiccation. Our results provide new insights into the resilience of R. sinensis populations affected by desiccation and supple the Ruppia populations characteristics.

Plant-soil interactions influence vegetation status and sediment characteristics. Annual and perennial life cycles are common in the same submerged aquatic angiosperm species, yet relationships between the life cycle strategies and sediment characteristics are poorly understood. This study aimed to investigate the interactions between different life cycles of the seagrass species R. sinensis and sediment characteristics including sediment nutrients. We surveyed two R. sinensis populations in the Yellow River Delta with different life cycles. The biomass of their peak growing seasons and their seasonal seed bank size were applied to evaluate their population status and potential recruitment capacity. Nine parameters were measured to assess sediment nutrient composition and variability. We found relatively higher biomass and more intensive seed bank sizes in annual life cycle R. sinensis populations. The appearance of overlying water impacted the surfer sediment moisture content and then R. sinensis life cycle strategies. Moreover, the appearance of R. sinensis shoots significantly impacted total nitrogen and organic matter in their habitat. Our results indicate that different life cycle strategies have different resilience capacities after exposure to desiccation of the habitat, while the appearance of R. sinensis impacted sediment nitrogen and organic matter features.

3 Effects of environmental factors on seed germination of R. sinensis

In this study, we investigated the effects of temperature and salinity on seed germination and seedling establishment of R. sinensis seeds collected from northern China. The effects of seven salinities (0 – 50 psu) and six water temperatures (0 - 30 °C) on seed germination were investigated to identify the environmental conditions that could potentially limit survival and growth. We found that: (1) optimum seed germination was salinity 5 psu at 30°C; (2) high salinity (salinity 40 – 50 psu) and low temperature (0°C) significantly inhibited seed germination, but which significantly increase after transferring to optimum conditions; (3) seed germination with increasing temperature showed a bimodal pattern at suitable salinities (5 - 10 psu); 4) storing seeds at high salinities (40 - 50 psu) or low temperature (0°C) promoted germination after transferal to optimal germination conditions. (5) Temperature had the significant influence on the germination of R. sinensis, however, salinity didn’t. These findings may serve as useful information for R. sinensis habitat establishment and restoration programs.

4 The long-term preservation methods of R. sinensis seeds

The seeds of Ruppia are ideal materials for its population restoration. Collecting mature Ruppia seeds during its breeding season and save them to the artificial seed bank, which can make full use of them for subsequent restoration. The low temperature, high salinity and desiccation all effectively inhibited the germination of R. sinensis seeds. Different temperature, salinity, and desiccation conditions were adopted to reserve R. sinensis seeds in experiments of this section. From the results of the previous part, we can know that certain low temperature, high salt and dry conditions can inhibit the seed germination of R. sinensis without reducing their activity, so the experiment of this part attempts to use different high salinity and low temperature conditions for its long-term preservation in water. Moreover, R. sinensis seeds were preserved at different temperatures in dry air. By comparing the viability of seeds after 9 months of storage with two preservation methods and their internal energy substances, it is considered that the wet preservation method of 0 °C and 30 - 40 psu salinity conditions is fit for the long-term preservation of R. sinensis seeds, and the best environmental condition of dry preservation is 5℃ and relative humidity of 33 ± 10%. Although the method of dry preservation will reduce the seed vigor to a certain extent, it is also a feasible method for long-term preservation of R. sinensis seeds. The above results provide corresponding basic knowledge for establishing the artificial seed bank of R. sinensis.

5 The tolerance of R. sinensis on Cu and Cd pollution and relative toxicity effects

Ruppia is tolerant not only of a wide salinity range, but also of high concentrations of trace metals. However, the tolerance of its early life stages to such trace metal exposure is unclear. Thus, the current study investigated the trace metal-absorbing capacity of three different life history stages of R. sinensis, a species that is widely distributed in China, observing toxic symptoms at the individual, subcellular, and transcription levels. The seedling period was the most vulnerable, with visible toxic effects at individual level in response to Cu 50 μM and Cd 500 μM after 4 days of exposure. The highest concentrations of trace metals occurred in the vacuoles and cytoplasmic structures of aboveground tissues. Genes related to signal identification and cellular components were significantly downregulated after 4 days of exposure to Cd. These results provided information relating to the strategies evolved by R. sinensis to absorb and isolate trace elements, and evaluated the phytoremediation potential of this species.

 第1章  文献综述... 1

1.1  海草生态系统... 1

1.1.1  海草简介... 1

1.1.2  川蔓草简介... 1

1.2  海草床退化原因... 2

1.2.1  自然胁迫... 3

1.2.2  人为影响... 3

1.3 海草种群特征... 3

1.3.1  种群与生境特征... 3

1.3.2  海草生活史策略... 4

1.4  海草床生态修复研究... 5

1.4.1  海草种群补充机制... 5

1.4.2  海草种子特性... 6

1.4.3  自然种子库... 7

1.4.4  人工种子库... 8

1.5 海草在重金属污染生物修复中的应用前景... 8

1.5.1重金属污染生物监测... 9

1.5.2 重金属污染植物修复... 9

1.6  本研究的目的、意义以及思路... 10

1.6.1  研究目的及意义... 10

1.6.2  科学问题... 10

1.6.3  内容与技术路线... 10

1.6.4  预期成果... 11

1.7  本章小结... 11

第2章  我国温带地区川蔓草属分布现状调查... 13

2.1  前言... 13

2.2  材料与方法... 13

2.2.1  资料收集... 13

2.2.2  实地调查... 13

2.2.3  数据分析... 13

2.3  实验结果... 14

2.3.1  文献调查结果... 14

2.3.2  实地调查结果... 15

2.4  讨论... 18

2.5  本章小结... 23

第3章  黄河三角洲地区中国川蔓草种群特征... 25

3.1  前言... 25

3.2  材料与方法... 26

3.2.1  调查地点... 26

3.2.2中国川蔓草种群生长环境调查... 27

3.2.3中国川蔓草种群特征调查... 29

3.2.4  数据分析... 30

3.3  实验结果... 30

3.3.1  黄河三角洲地区中国川蔓草的生长环境... 30

3.3.2  黄河三角洲地区中国川蔓草的生长情况... 37

3.3.3  极端干燥环境对中国川蔓草种群影响... 47

3.4  讨论... 55

3.5  本章小结... 58

第4章  环境因素对中国川蔓草种子萌发及建苗过程的影响... 59

4.1  前言... 59

4.2  材料与方法... 60

4.2.1  研究地点... 60

4.2.2  种子收集... 60

4.2.3  中国川蔓草种子形态... 60

4.2.4  温度、盐度对中国川蔓草种子萌发影响... 62

4.2.5  低温、干燥对中国川蔓草种子萌发影响... 63

4.2.6  数据分析... 64

4.3  实验结果... 64

4.3.1  中国川蔓草种子形态及萌发过程... 64

4.3.2  温度、盐度对中国川蔓草种子萌发的影响... 68

4.3.3  温度、盐度对中国川蔓草幼苗建苗的影响... 75

4.3.4  低温、干燥对中国川蔓草种子萌发的影响... 79

4.4  讨论... 82

4.5  本章小结... 84

第5章  中国川蔓草种子保存方法研究... 85

5.1  前言... 85

5.2  材料与方法... 85

5.2.1  种子收集... 85

5.2.2  种子湿法保存... 86

5.2.3  种子干燥保存... 87

5.2.4  数据分析... 87

5.3  实验结果... 87

5.3.1  种子湿法保存... 87

5.3.2  种子干燥保存... 90

5.4  讨论... 93

5.5  本章小结... 93

第6章  中国川蔓草对重金属Cu和Cd胁迫的响应... 95

6.1  前言... 95

6.2  材料与方法... 96

6.2.1 样品收集... 96

6.2.2 Cu和Cd对种子萌发过程影响... 97

6.2.3 Cu和Cd对休眠种子活力影响... 97

6.2.4 Cu和Cd对幼苗影响... 98

6.2.5 Cu和Cd在幼苗内积累转移过程研究... 99

6.2.6 幼苗转录组对Cu和Cd短期暴露响应... 100

6.2.7 数据分析... 101

6.3  实验结果... 101

6.3.1 自然条件下中国川蔓草植株和生长环境中Cu和Cd浓度... 101

6.3.2 Cu和Cd暴露后个体水平响应... 102

6.3.3 Cu和Cd在幼苗中的动态积累过程... 105

6.3.4 Cu和Cd胁迫下幼苗亚细胞结构响应... 106

6.3.5 Cu和Cd暴露后的转录水平响应... 108

6.4  讨论... 115

6.1  总结... 118

第7章  总结与展望... 121

7.1  总结... 121

7.2  创新性... 121

7.3  存在问题... 121

7.4  研究展望... 122

参考文献... 123

致  谢... 141

作者简历及攻读学位期间发表的学术论文与研究成果    143