【作者】 王金锋；

【导师】 秦松；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2010， 博士

【摘要】 基于核酸序列的分子系统发育分析在藻类生物地理分布、遗传多样性和进化研究中的作用突出。近年来,黄海海域连续发生大规模绿潮,对沿海生态造成巨大影响,有必要从分子水平对该海域的绿潮藻主要构成种类——石莼和浒苔的分类、分布,及其与世界其它地区特定物种的遗传多样性和亲缘关系进行探讨。本文基于ITS、rbcL和psbA等基因序列的分析,发现青岛漂浮绿藻样本之间ITS序列分歧度极低,仅为0.0%².5%;漂浮样本与定生样本之间的分歧度较高,从7.6%到24.4%。系统发育构建将15个定生样本分散到5个分枝中,漂浮样本形成独立的分枝,揭示了所有的漂浮绿藻为单一物种,青岛本地不存在漂浮绿藻的定生类群,2007青岛沿岸出现的漂浮绿藻为区域性外来物种。21个2008黄海漂浮样本ITS序列中的19个与2007青岛样本序列相同,其余2个与之相比分歧度也极低（0.2%）,表明2008与2007漂浮绿藻为同一物种;rbcL和psbA序列分析得到相似的结果。系统发育分析中,黄海漂浮浒苔聚类于浒苔linza-procera-prolifera复合群,形态上倾向于Enteromorpha prolifera,无论从种类还是形态上均与石莼相差较大;与欧洲和北美相比,黄海漂浮浒苔和日本E. prolifera具有更近的亲缘关系,充分反映出地理距离与该物种遗传多样性之间的联系。现有调查研究结果显示黄海两岸至少分布着8种浒苔和石莼属绿藻,但在本研究采集的该海域绿藻样本中暂未发现定生的漂浮浒苔E. prolifera。分子系统发育分析方法也被用于藻类进化研究方面。本文克隆了两种颜色的长心卡帕藻的藻胆蛋白基因,发现藻体颜色变化与藻胆蛋白基因碱基组成和排列无关。在PE-β分枝中,与高光适应型原绿球藻相比,红藻与低光适应型原绿球藻表现出更近的亲缘关系。卡帕藻与低光适应型原绿球藻PE-β相同且与高光适应型不同的位点达48个（而与高光适应型相同的位点仅为4个）,这些位点多处于α-螺旋区和一些功能已知区域,如色基结合、亚基和连接蛋白结合区域等,可见红藻的进化与对低光环境的适应有关,但同时也对高光环境有响应和调节机制。光系统II光合效率分析结果也显示卡帕藻半饱和光强低于115μmol m^-2 s^-1,与紫菜相似,同样证明红藻多数属于低光适应型物种。光合生理参数同样反映出红藻对高光的适应性。红藻的低光适应性,推测与上述48个位点中的一些关系密切;藻红蛋白中的4个位点可能与红藻的高光适应有关。藻蓝蛋白和别藻蓝蛋白氨基酸序列比对分析结果显示,相对于藻蓝蛋白而言,红藻、褐藻和蓝藻的别藻蓝蛋白保守性较高;总体上相比含有藻红蛋白种类的聚球藻,红藻、褐藻的藻蓝蛋白与藻胆体中不含有藻红蛋白的聚球藻RS9917和WH5701相似性更高,进一步验证了藻胆蛋白由蓝藻向红藻再到褐藻的进化过程中,别藻蓝蛋白同步进化,而藻蓝蛋白和藻红蛋白独立、分开进化,从而使藻类能够快速适应周围光环境变化的观点。更多还原

【Abstract】 Phylogenic analysis based on nucleotide sequences is excellent in the studies of algal biogeography and evolution.In recent years, large-scale green tides bloomed successively in the Yellow Sea, which caused in ecological and social problems. It is necessary to carry out molecular analysis on classification and distribution of mainly green-tide-forming algae, Ulva and Enteromorpha in this sea area, in addition to the genetic diversity and phylogenetic relationship between the green-tide-forming algae in the Yellow Sea and the reported species all over the world. In the present study, based on the analysis of nuclear rDNA ITS, chloroplast rbcL, and psbA sequences, it’s found that ITS sequence divergencies among all free-floating samples were very low （0.0%–2.5%）, while sequence divergency between free-floating samples and attached samples were much higher, ranging from 7.6% to 24.4%. According to the phylogenetic tree, all free-floating samples were grouped to one cluster; 15 attached samples were resolved into five other clades, which demonstrated that free-floating samples were unialgal, and the attached Ulvaceae species from Qingdao coasts in summer 2007 were not the biogeographic origin of the free-floating one. In the ITS sequence, 19 out of the 21 Yellow Sea samples of 2008 were identical to those of a sample collected from Qingdao in 2007. A low divergence （0.2%） was found in two remaining samples. Similar evidence was shown by pairwise distances of rbcL and psbA gene sequence data, implying the uniformity of the Yellow Sea blooms in 2007 and 2008. Molecular phylogenetic results grouped the free-floating alga into one clade with E. procera, E. linza and E. prolifera, while the morphological character of this alga is identical to E. prolifera. Both molecular and morphological analysis revealed the free-floating samples are not Ulva species. The haplotypes of the Yellow Sea free-floating E. prolifera were closely related to those from the Japanese coast but less to European and American algae, which reflect the relationship between biogeographic distance and genetic diversity. Recent investigation showed there are at least 8 species of Ulva and Enteromorpha distribute at both side of the Yellow Sea. However, none attached population of free-floating E. prolifera was found in this sea area.Molecular analysis was also carried out on evolution of algae. Phycobiliprotein genes of two color-differing K. alvarezii were cloned in the study, but there was not any difference which indicates that color variation was independent of nucleotide composition and arrangement of phycobiliprotein genes. Phycobiliprotein sequences of K. alvarezii, other representative red macroalgae （e.g. Porphyra, Gracilaria） together with those of cyanobacteria were used to illuminate the phylogenetic position and evolution of red algae. When comparing to high-light-adapted （HL-） Prochlorococcu, the red algae have a closer relationship with low-light-adapted （LL-） Prochlorococcu in this phylogenetic tree. Forty-eight amino acid sites of PE-βin red algae were identical to LL-Prochlorococcu but different from HL-Prochlorococcu, while 4 amino acid sites of PE-βin red algae were identical to HL-Prochlorococcu but divergent with LL-Prochlorococcu. These sites were generally located at domain ofα-helix, chromophore or linker interaction, which revealed that the evolution of red algae was not only associated with low light environment but also with high light adaptation. Similar to that of Porphyra, analysis of PSII photochemical efficiency of K. alvarezii displayed a low minimum saturating irradiance of 115μmol m^-2 s^-1, which also proved that these representative red macroalgae belong to low-light-adapted species. However, which is also reflected by PSII photochemical parameters, K. alvarezii can adapt the high light. The low-light-adapted adaptability of red algae might be correlated with the above mentioned forty-eight amino acid sites, while 4 amino acid sites in PE correspond to high-light-adapted adaptability. APC sequences of red algae, Cryptophytes and cycanobacteria were conservative when it was compared to PC. Generally, PC sequences of red algae and brown algae were more similar to those of PE deficient Synechococcus RS9917 and WH5701 compared to PE contained Synechococcus species, which also improved that APC has likely evolved together, while PC and PE have evolved independently during the evolution of phycobiliproteins from cycanobacteria to red algae and brown algae, allowing these algae rapid adaptation to a variety of light niches.更多还原