|导师||刘进贤 ; 周毅|
|摘要||矮大叶藻（Zostera japonica Asch. & Graebn）是中国唯一可在温带和亚热带广泛分布的一种海草，可在潮间带或较浅的潮下带形成密集的草床，是重要的沿海生态系统。近年来，随着沿海人类扰动和污染的加剧，矮大叶藻的生境剧烈萎缩，历史记载的很多种群已经消失或退化殆尽。种群补充是种群持续的动力，种群遗传学特征是种群适应环境变化的内在机制，二者均是海草保护和修复工作的理论前提。但对矮大叶藻的种群补充机制（有性生殖和营养生殖），尤其有性生殖部分研究非常缺乏。同时在种群快速衰退的趋势下，中国矮大叶藻种群的现存状况及其遗传多样性和种群遗传结构也未进行全面的评估。本研究选取三个典型生境（潟湖、海湾、河口）的自然种群进行补充机制的调查研究，同时调查矮大叶藻的分布现状，对现存种群的遗传多样性和遗传结构进行评估。主要研究内容及结果如下：|
研究方法为生态调查（固定样方法和随机取样法）和微卫星分子验证相结合。2014年3月-2015年12月，对荣成天鹅湖和青岛汇泉湾两个种群进行为期近两周年的补充机制调查。天鹅湖种群为混合一年生，其生殖补充循环可以分为4个过程。各关键环节均已定量，简单来说，种苗补充的贡献率为41.16±24.49%，2014年最大花枝比例和潜在种子产量分别为26.11±17.00 % 和19668.3±8438.5 seeds﹒m-2，但因人为扰动增加，二者在2015年分别上升为37.32±6.90 % 和 47578.9±18435.7 seeds﹒m-2。单枝产种能力较高27.4±8.15 seeds﹒shoot-1。底质种子库为瞬时种子库（持续时间<1年），大小为1460.5±417.4 seeds﹒m-2。天鹅湖有性生殖和营养生殖的相对比例主要决定于冬季茎枝的残余量，而这又受冬季温度所控制。
汇泉湾种群的补充模式与天鹅湖明显不同。因为汇泉湾具有较高的冬季存活量（1674.1±912.3 vs 172.2±80.9 shoots﹒m-2）和较低的产种能力（12.0±2.7 seeds﹒shoot-1，7505.7±3375.2 seeds﹒m-2），尤其是极其稀疏短暂的种子库（9.6±6.3 seeds﹒m-2，持续时间< 2个月）和极为少见种苗补充（整个草床只观察到1例）。以上均说明克隆生殖在汇泉湾种群补充中的绝对地位，而有性生殖很可能是偶然事件。推测冬季较高的温度使得是汇泉湾种群以克隆生殖为主的主要原因。
种子萌发期较短，3月底开始，4月底结束。根据5月初数据显示，黄河口草床核心区域的种苗补充远高于过冬茎枝，种苗密度和种苗茎枝密度最高达 1844.4±882.5 seedlings﹒m-2和2311.1±1012.7shoots﹒m-2，对应种苗补充的贡献率高达90.59±20.65%。但草床边缘低洼处为较多的过冬茎枝提供了庇护所，种苗补充稀少。
8月各站位花枝比例存在显著差异，范围是36.57±9.46% - 57.43±12.85%。但站位间花枝密度和单枝种子产量无显著差异平均值分别为1699.9±583.8 shoots﹒m-2 和19.5±2.1 seeds﹒shoot-1。估算单位面积潜在种子产量是33955.4±9861.8seeds﹒m-2。12月种子库密度1581.9±526.4 seeds﹒m-2，至次年3月无显著下降。观察到较多的腐败变质情况，推测可能是底质以黏土为主易形成缺氧条件。
黄河口矮大叶藻克隆生长的周年动态在生物量上尤其显著，其生物量从5月（92.1±10.5 wet g﹒m-2）至8月（1198.0±129.4 wet g﹒m-2）增加了13.0±0.2倍，但密度只增加了1.53±0.57倍，由2343.2±305.2 shoots﹒m-2增至3471.8±831.5 shoots﹒m-2。草床主体部分冬季生物量极低，极少数具1-2个茎节的地下茎可以过冬进入下一年的补充。
种子萌发实验结果显示，各深度种子萌发率为32.25±5.38% - 40.25±19.22%，无显著差异，说明种子萌发率与深度无关。但不同深度萌发速度明显存在差异，1米萌发最快，种苗数5月初即达到最大值，比其它梯度提前3-4周，这可能主要与表层水温较高有关。
|其他摘要||Zostera japonica was a unique seagrass that can be widely distributed both in the temperate and subtropical zones in China. It often forms intensive meadows in the intertidal and shallow subtidal zones，which support important coastal ecosystems. In recent years, with the increasing human disturbances and pollutions along the coasts, the habitats of Z. japonica have degraded severely and many populations reported in former literatures have totally, if not, almost disappeared. Population recruitment, the motivation of a population to persist, and the population genetics, underlying the response and adaptation of a population to changing environments, are both among the theory basis for seagrass conservation and restoration. However, knowledge of population recruitment (sexual and vegetative reproduction), especially the sexual aspect, is still lacking for Z. japonica. And with so rapid declines, the status and population genetics of the extant populations of this species in China have not been investigated fully. This study investigated natural Z. japonica populations from 3 typical habitats (lagoon, bay, estuary) for their recruitment mechanisms, surveyed the extant populations along the coasts of China, and examined their genetic diversity and genetic structure. The main results were showed as follows.|
1 Comparision between the population recruitment in Swan Lake (SL), Rongcheng and Huiquan Bay (HQ), Qingdao
Ecological survey methods (permanent quadart survey and random sampling) verified by microsatellites were employed to clarify the recruitment mechanisms of Z. japonica population in SL and HQ.
From March 2014 to December 2015, ecological surveys were conducted in situ. The growth form of SL population was mixed annual and four processes were involved in its recruitment cycle, with key links being quantified. Briefly, the seedling recruitment contributed 41.16±24.49% to population recruitment in early June; the flowering shoot ratios and potential seed production were 26.11±17.00 % and 19668.3±8438.5 seeds﹒m-2 in 2014, respectively, while both of them increased to 37.32±6.90 % and 47578.9±18435.7 seed﹒m-2 owing to increasing human disturbances in 2015; the seed production capability was 27.4±8.2 seeds﹒shoot-1; the sediment seed bank was transient (<1 year) with a biggest density of 1460.5±417.4 seeds﹒m-2. The relative contributions of sexual to vegetative reproduction was assumed to be determined by the amount of overwintering shoots, which was controlled by winter temperatures.
The recruitment strategy of HQ population was different from SL population. Because HQ population showed a much higher survival (1674.1±912.3 shoots﹒m-2 vs 172.2±80.9 shoots﹒m-2) through winter, lower seed production capacity ( 12.0±2.7 seeds﹒shoot-1 and 7506±3375 seeds﹒m-2), extremely rare seed bank (9.6±6.3 seeds﹒m-2, transient < 2 months) and almost absent seedling recruitment (only one seedling was observed). All above suggested the absolute status of vegetative reproduction in recruitment while the sexual reproduction probably an accidental event. It was probably the warmer winter that have made the vegetative reproduction more fit for persistence of this population.
To verify the roles of sexual reproduction in the two populations, microsatellites analysis based on seven loci was conducted on two sets of samples collected in 2012 and 2015 from SL and HQ, respectively. The results of SL population coincided with observation in situ and indicated frequent sexual recruitment. Because the heterozygosity and number of alleles were relatively high and the clonal diversity (R=1) was especially high with no shared clones among all the samples from 2012 and 2015. While the results of HQ population only partly conformed as expected that the much larger (~30 m) and old (at least 3 years old) clones, the lower number of alleles and clonal diversity all indicated the contributions of vegetative reproduction for meadow persistence. However, the heterozygosity of HQ was not significant different from SL, though its numbers of alleles were lower. What’s more, four confident full-families were detected out of the pool samples of 2012 and 2015 from HQ. These both suggested the exsitence of sexual reproduction in HQ, which played a critical role in maintaining population genetic diversity though happened in a very low probability
2 An unusual large meadow of Z. japonica discovered in the Yellow river estuary and its population recruitment mechanism
A large Z. japonica distribution with an area of 800-1200 ha was found in Yellow river estuary of Shandong province. The population recruitment of Z. japonica was investigated seasonally from May 8, 2015 and March 18, 2016.
Seed germination period was short, from end of March to mid of April. The central parts of the meadows were mainly recruited by seedlings, and the largest seedling density and seedling shoot density were 1844.4±882.5 seedlings﹒m-2 and 2311.1±1012.7 shoots﹒m-2 respectively, which meant a contribution of 90.59±20.65 % by seeds to population recruitment. Meanwhile, seedlings were rare in the margins where low areas provided shadows for relatively more overwintering shoots.
In August, the flowering shoot ratios among plots significantly varied from 36.57±9.46% to 57.43±12.85%；however the flowering shoot density and seed production per shoot among plots were not significantly different with a mean of 1699.9±583.8 shoots﹒m-2 and 19.5±2.1 seeds﹒shoot-1 respectively. The potential seed production per unit area was 33955.4±9861.8 seeds﹒m-2.
The sizes of sediment seed bank in meadow center were 1581.9±526.4 seeds﹒m-2 in December and did not change significantly until March the next year. Considerable decayed seeds were observed and it may be resulted from the anoxia conditions due to the extremely high proportions of silt and organic matter in sediment.
The annual clonal growth dynamics was more revealed by changes of biomass, because the biomass increased 13.0±0.2 times from May（92.1±10.5 wet g﹒m-2） to August ( 1197.9±129.4 wet g﹒m-2)，while the density only increased 1.53±0.57 times from 2343.2±305.2 shoots﹒m-2 (May) to 3471.8±831.5 shoots﹒m-2 (August), which suggested more energy was allocated to the increase of shoot size more than shoot density. The overwintering biomass was extremely low in the main part of the meadow, and generally entered the next recruitment cycle through rhizomes with one or two small nodes.
3 The effects of water depth on the sexual and vegetative reproduction
From May 2014 to November 2015, twice shoot (with rhizomes) transplantations and once seed germination experiment were conducted with cylindrical PVC box hanging on the scallop culture rafts in Ailian Bay. The aim is to clarify the effects of water depth on clonal growth and seedling recruitment of Z. japonica.
The results of shoot transplantations showed that the clonal growth (shoot density, height, persistence) was positively related to the light density along the depth gradient. The highest density, fast growth rate, and longest persisit time were observed in the depth of 1 m. The shoots in 2 m showed a lower density and height but persistent more steadily because of the more stable hydrodynamic conditions. The shoots planted in 6 m cannot survived more than 3 months.
The results of seed germination experiment showed that germination rate of different depths were not significantly different with a range of 32.25±5.38% - 40.25±19.22%, which suggested no effects from water depths. But the germination speed varied along the depths, seeds in 1 m germinated more rapid with the highest seedling density appearing in early May, which is 3-4 weeks earlier than other depths. This may be mainly related to the higher temperature in water surface.
4 The status of the extant Z. japonica in China and the population genetics
Based on our field surveys, the habitats of Z. japonica along the coasts of China have declined severely. The status of South China Sea was much more alarming and this species was likely to have been extinct in coasts of Fujian province. A total of 9 distributed sites were found during surveys, within which 5 (Haxian Island, Dlian; Xingcheng, Huludao; Yellow river estuary, Dongying; Swan Lake, Weihai; Huiquan Bay, Qindao) located in the north the Yellow sea and Bohai Sea and 4 (Laichiwo, Hongkong; Shangchuan Island, Guangdong; Beigang village, Haikou; Fangchenggang, Guangxi) in the South China Sea. The largest meadow was in Yellow River estuary. The minimum were Huiquan Bay, Qingdao and Shangchuan Island, Guandong, but the former has colonized new habitats while the latter degraded rapidly to an area < 200 m2. The main cause for the declines was the human disturbances, among which the aquaculture and coast constructions occupying the habitats of seagrasses, repeatedly digging and fishing activities, and increasing pollutions were the most prominent.
A total of 10 microsatellite loci were newly developed to examine the genetic structure of extant Z. japonica populations. All analysis supported the divergence between the north and south populations. Meantime, all the populations within the north group significantly differentiated from each other and no exchanges were detected, and the south as well. The Haxian Island and Hong Kong populations had the largest genetic distances from other populations within group, and the Huiquan Bay and Shangchuan Island populations took the second places respectively. The limited dispersal of propagules and habitat fragmentation were the main reasons for the genetic distances among populations. The history geographic events, such as the isolations between the marginal seas of China during the last glacial maximum, probably be responsible for the divergence between north and south. In addition, the genetic diversity of all the populations were generally high, which also revealed the roles of sexual reproduction. Coincided with their population sizes, the genetic diversity of Huiquan Bay and Shangchuan Island were the lowest within groups. In-situ conservations for each population was recommended. What’s most urgent is some actions should be taken immediately to conserve to Shangchuan Island, such as isolating the habitat from access of human and appropriate restorations.
This study filled the gap of population recruitment, especially the sexual contribution, of Z. japonica. We examined the main factors controlling sexual and vegetative reproduction, and clarified the population genetic diversity and population structure of the extant Z. japonica along the coasts of China. These results would provide scientific and useful information for later conservation and restoration projects.
|张晓梅. 矮大叶藻种群补充机制与种群遗传学研究[D]. 北京. 中国科学院大学,2016.|