【作者】 胡自民；

【导师】 高天翔； 段德麟；

【作者基本信息】 中国海洋大学 ， 渔业资源， 2007， 博士

【摘要】 本论文利用核糖体18S rRNA、internal transcribed spacer(ITS)、线粒体cox2-3间区序列及inter simple sequence repeat(ISSR)标记方法对红藻角叉菜属的分子分类、系统发育和进化及居群遗传学进行了研究,揭示了角叉菜属种间的亲缘谱系关系、系统进化地位、北大西洋皱波角叉菜(Chondrus crispus)居群的分布机制和遗传多样性特征,为研究藻类的分子进化起源和过程打下了基础。研究结果如下:(1)采集了来自全球8个国家16个地点的角叉菜样品,采用PCR扩增和直接测序的方法分析了核糖体18S rRNA基因和内转录间隔区(ITS)序列,统计分析结果显示18S rRNA基因和ITS序列长度分别在1800 bp和710 bp左右。nrDNAITS数据表明采自中国大连的样本C.spl应该为C.nipponicus,尽管这与18SrRNA基因数据不一致(18S rRNA结果显示C.spl应为C.pinnulatus)。先前被命名为C.yendoi或Mazzaella japonica的样本C.sp2属于角叉菜属,但确切的分类地位还需要进一步的证实。初步推断在中国北方沿海至少存在3种角叉菜,其分别是C.ocellatus,C.nipponicus,C.armatus和C.pinnulatus。此外,分子数据显示采自爱尔兰Connemara的样本实际上不是C.crispus,而是Mastocarpusstellatus,尽管它在外部形态特征上与C.crispus非常相似。多种系统发育分析方法(NJ,MP,ML和ME)表明在角叉菜属内存在3个谱系,C.ocellatus,C.yendoi和C.nipponicus形成北太平洋的一个谱系,C.armatus和C.pinnulatus则形成北太平洋的另一个谱系,而所有C.crispus样本形成一个独立的北大西洋谱系。系统树的拓扑结构显示北大西洋的C.crispus可能起源于北太平洋。(2)以北太平洋的C.ocellatus,C.yendoi和C.pinnulatus作为外类群,利用nrDNA ITS序列初步研究了北大西洋C.crispus的分子地理进化过程。统计结果表明C.crispus的ITS序列变异比率在0.3～4.0%,NJ,MP和ME方法构建的系统进化树显示C.Crispus的不同地理样本形成两个主要的分支。我们认为北大西洋C.crispus形成现今的分布模式可能受到3个重要因素的影响:等温线阻隔、古气候和古海洋学。ITS数据不仅为分子支序发育,而且也为系统地理学研究提供了重要信息。(3)利用ISSR标记方法对C.crispus的7个野生居群(139个个体)进行了遗传多样性和种群结构分析,在进行PCR扩增条件优化和引物筛选后,18个ISSR引物共扩增出184个位点,其中174个为多态性。统计分析显示Nei’s基因多样性指数为0.1228,Shannon信息指数为0.2127。C.crispus居群水平上的基因多样性水平为0.2922,遗传分化指数为0.7463,这表明74.63%的变异发生在居群之间,25.37%的变异发生在居群内。这些数据证实北大西洋C.crispus居群内已出现了明显的遗传分化。(4)根据nrDNA ITS数据构建的系统进化树(ME和MP)显示:在进化水平上,真红藻纲的松节藻科(Rhodomelaceae)与红毛菜纲亲缘关系较近。在真红藻纲内,杉藻目的进化地位最高,其次是海膜科(Halymeniaceae)、石花菜科(Gelidiaeeae)、红叶藻科(Delesseriaceae)和粉枝藻科(Liagoraceae)等,而松节藻科进化地位最低。在杉藻目内,杉藻科(Gigartinaceae)和胶黏藻科(Dumontiaceae)关系密切,而外形上难以区分的角叉菜和红舌藻(Mazzaella)亲缘关系非常近。而mtDNA cox2-3数据分析表明,红藻门13个属的cox2-3基因间区域序列长度在134～400 bp之间,差异较大,A+T含量在49.1%至61.4%之间;红藻门属间平均核甘酸差异数达到34个,如不包括角叉菜属,则属内种间的核甘酸差异率达到了9.45～17.78%。NJ和MP系统树显示,在所研究的红藻门13个属间,石花菜目的沙菜属(Hypnea)和杉藻目的链藻属(Catenella)进化关系非常近,而仙菜目绒线藻科的绒线藻属(Dasya)和异管藻属(Heterosiphonia),及松节藻科的卷枝藻属(Bostrychia)和红叶藻科的鹧鸪菜属(Caloglossa)亲缘关系较近,柏按藻目的海门冬属(Asparagopsis)和串珠藻目的串珠藻属(Batrachospermum)亲缘关系也非常近。这些结果表明红藻线粒体cox2-3序列在进行红藻属间或属内种间亲缘关系分析和系统进化研究时,实用性较强,可用于重建红藻门内属间或属内的系统进化关系。更多还原

【Abstract】 Nuclear ribosomal 18S rRNA gene,internal transcribed spacer（ITS）,mitochondrial cox2-3 spacer sequences,and inter simple sequence repeat（ISSR） marker technique were applied to study the molecular taxonomy,phylogeny and evolution,population genetics of red algae Chondrus Stackhouse,aiming to understand the origin and mechanism of molecular evolution of this red algae.（1） The nuclear-encoded small-subunit ribosomal RNA gene（18S rDNA） and internal transcripbed spacer（ITS） of 16 isolates of Chondrus from 8 countries were sequenced.A total of 1796 nucleotides and 710 nucleotide were obtained and aligned with phylogenetic analysis conducted for 18S rRNA and nrDNA ITS,respectively. NrDNA ITS data indicate that the entity from Dalian,China,regarded as C.spl is C. nipponicus,although which is not consistent with the result from 18S rRNA gene analysis（C.sp1 may be C.pinnulatus）.The C.sp2 previously depicted as C.yendoi or Mazzaella japonica may belong to genus Chondrus.So,at least three Chondrus species,C.ocellatus,C.nipponicus,C.armatus and C.pinnulatus are distributed in China.However,the entity from Connemara,Ireland,named C.crispus,is not a Chondrus species but that ofMastocarpus stellatus,although it is similar to C.crispus morphologically.Phylogenetic analysis based on completel8S rDNA sequence data show that genus Chondrus includes 3 main lineages:the Northem Pacific lineage containing C.ocellatus,C.yendoi,and C.nipponicus;C.armatus and C.pinnulatus formed the sub- North Pacific lineage;and Northern Atlantic Ocean lineage comprises samples of C.crispus from Canada,Portugal,Ireland,Germany and France.The phylogenetic relationships indicate that genus Chondrus might have a North Pacific ancestral origin,and radiated to North Atlantic area and formed species C crispus.（2） Eighteen isolates of the-red algae Chondrus crispus were collected from Northern Atlantic sites,togethor with C.ocellatus,C.yendoi and C.pinnulatus from the North Pacific.The nuclear rDNA internal transcribed spacer（ITS） was sequenced and compared,spanning both the ITS regions and the 5.8S rRNA gene.Percentage of nucleotide variation for C.crispus ranged from 0.3%to 4.0%.Phylogenetic analyses were performed using maximum parsimony,neighbor-joining and minimum evolution methods.They showed that two main clades existed within the C.crispus samples examined and that suggested C.crispus had a single Atlantic origin.The clustering however did not follow the geographic origin.We hypothesized that the current distribution of C.crispus populations might be a result of three main factors: temperature boundaries,paleoclimate and paleoceanography.ITS data exhibited abundant molecular information not only for phylogeographical investigation but also for systematics studies.（3） Genetic diversity and population structure analysis were conducted on 7 wild populations（139 individuals） of C.crispus with ISSR markers.Based on the results of PCR amplifying optimization and primers screening,eighteen primers were totally yielded 184 loci,of which 174 were polymorphic.Statistic analysis showed that Ne’s gene diversity（He） and Shannon’s information index（I） were 0.1228 and 0.2127, respectively.Gene diversity（H_t） among 7 populations of C.crispus was 0.2922,and gene differentiation index(G_st) was 0.7463,which indicated that 74.63 percent of differentiation occurred among populations,the rest（25.37%） was in populations.All these data suggested that extinct genetic differentiation existed in Northern Atlantic C. crispus populations.（4） Phylogenetic analyses using minimum -evolution and maximum-parsimony methods from ITS data show that Rhodomelaceae in Florideophyceae is closely related to Bangiophyceae evolutionally.In Florideophyceae,Gigartinales has the highest evolutional status,then Halymeniaceae,Gelidiaceae,Delesseriaceae and Liagoraceae,and Rhodomelaceae the lowest.In Gigartinales,Gigartinaceae and Dumontiaceae have close relationship,while Chondrus and Mazzaella,which are difficult to discriminate from morphology,formed a fairly compact clade.The low divergence among Chondrus species is justified considering its recent origin,and suggests that ITS sequence would provide a valid method for molecular phylogenetic analysis,and to reconstruct the phylogeny within the Rhodophyta on order and family levels.The intergenic region cox2-3 ranged from 134 to 400 bp in length,with great variation in the 13 genus of Rhodophyta,and A+T content varied from 49.1%to 61.4%.The average intergeneric variant nucleotides number is 34,and if genus Chondrus excluded,the interspecific nucleotide variant rate is about from 9.45%to 17.78%.The NJ and MP phylogenetie trees show that in the 13 red algal genus,genus Hypnea in Gelidiales is close to genus Catenella in Gigartinales in evolutionary relationships.In order Ceramiales,genus Dasya and genus Heterosiphonia in family Dasyaceae have close relationships to genus Bostrychia in family Rhodomelaceae and genus Caloglossa in family Delesseriaceae,and genus Asparagopsis in order Bonnemaisoniales is close to genus Batrachospermum in family Batrachospermaceae. Our results suggest that mitochondrial intergenic region（cox2-3） sequence has great practicality in algal phylogenetic and systematic studies,especially to Rhodophyta at intergeneric or interspecific level,it could be used for reconstructing the red algal intergeneric or intrageneric phylogenetic relationships.更多还原