【作者】 王中铎；

【导师】 刘楚吾； 刘筠；

【作者基本信息】 湖南师范大学 ， 发育生物学， 2009， 博士

【摘要】 分子系统学利用分子生物学方法探究生物的分类、系统发育、进化规律,是现代生物多样性、系统进化和物种形成机制研究的重要依据。笛鲷属（Lutjanus）鱼类隶属鲈形目（Perciformes）,是南海区海洋捕捞和海水养殖的重要对象,本论文围绕该属的分子系统学进行研究,获取了以下结果:（1）使用cox1 DNA条码（barcode）快速可靠的进行物种分子鉴定和物种多样性确定运用一对特异性引物扩增并测定了南海硬骨鱼类40个物种（包括笛鲷属15种）89个样本的线粒体DNA的细胞色素c氧化酶Ⅰ（cox1）约660bp的部分序列,即cox1条码序列。综合比较了《中国鱼类系统检索》、海洋鱼类志、FishBase鱼类形态学分类库、ITIS综合分类学信息系统和生物条码系统（The Barcode of Life DataSystem,BOLD）的相应形态学与DNA序列资料,在揭示完善我国鱼类检索系统必要性基础上,探讨了cox1条码序列在硬骨鱼类辅助物种鉴别和适用性,并对运用该条码序列库完善我国鱼类检索系统的可能途径进行了初步摸索。结果表明:cox1条码序列获取便捷,广泛适用硬骨鱼类物种鉴别,能为笛鲷属的分子生物学辅助分类,资源有序利用和保护提供参考。（2）快速有效地测定笛鲷属4个物种和军曹鱼（Rachycentroncanadum,军曹鱼科,军曹鱼属）的线粒体DNA基因组数据,并加以深入分析和合理应用将Long-PCR和常规PCR技术整合,运用同34对通用引物测定了马拉巴笛鲷（L.malabaricus）、千年笛鲷（L.sebae）、孟加拉笛鲷（L.bengalensis）、四带笛鲷（L.kasmira）以及军曹鱼（R.canadum）等的线粒体基因组全序列,GenBank序列号分别为FJ171339,FJ416614,FJ824741,FJ824742和NC₀11219。实验结果表明本论文的全序列测定体系的通用性较强,操作简单。线粒体基因组的比对分析表明测定的mtDNA基因组的绝大部分区段与GenBank中现有的脊椎动物的序列有较高的同源性。聚类分析中各支与形态学分类阶元关系吻合,但黑带鳞鳍梅鲷（Caesioninae:Pterocaesio tile）与勒氏笛鲷聚为一支。考虑到其他研究者也发现类似问题,作者认为有必要对黑带鳞鳍梅鲷（P.tile）和梅鲷科（Caesionidae）的形态学分类地位进行更深入的评估。由于GenBank等数据库中尚缺乏鱼类同属多个物种的mtDNA基因组完整序列数据,笛鲷属多个物种的全序列测定为研究鱼类属内种间的mtDNA进化规律提供了重要的数据资源。（3）探讨线粒体DNA蛋白编码基因的适用性一方面,整理GenBank上已有的DNA序列数据,构建本地的鲈形目鱼类mtDNA基因组数据库;另一方面,整理本研究以及本研究室以往测定的DNA序列数据,构建笛鲷属的本地核酸序列库。通过单个基因和基因拼接序列比较,综合考虑邻位连接法构建系统进化树的置信度和序列的信息量,对13种蛋白质编码基因（细胞色素氧化酶1（cox1）,细胞色素氧化酶2（cox2）,细胞色素氧化酶3（cox3）,细胞色素b（Cyt b）,ATP合成酶亚分子6（ATP6）,ATP合成酶亚分子8（ATP8）,NADH脱氢酶复合物亚单位1（ND1）,ND2,ND3,ND4L,ND4,ND5,ND6）)的系统进化分析能力进行了评估。在参考以往研究者做法的基础上,依据本研究结果,作者按分类阶元不同将基因都分成不同的四等。在鲈形目的科间阶元,最好的为ND6和cox2;好的序列为ND5和ND4;ND4L、ND3和ATP8差;包括Cytb、cox1在内的其余6种基因为中等;在属内种间阶元,最好的为ATP6和cox2;好的序列为ND2和cox1;ND6、ND3和ATP8差;包括Cytb在内的其余6种基因为中等。另外,分析揭示序列长度的增加可以提高系统进化树的置信度,且属内物种间比较时序列长度的影响小于高级阶元。由于信息分布的不均一性,建议依分析对象的分类阶元言褡罴鸦蚝妥楹稀?（4）测定笛鲷属鱼类中性遗传标记进行系统进化分析利用DNA条码筛选出的各物种典型标本,综合mtDNA基因序列以及RAG1和RAG2等核基因序列,构建笛鲷属分子系统进化树,最大程度地还原了笛鲷属鱼类种系发生历程。本章首先对本论文测定的笛鲷属DNA条码和生物条码系统已有的共20种笛鲷鱼类的72条DNA条码进行聚类分析,筛选出采自南海的13种笛鲷鱼类的典型样本;然后,利用这13个典型样本的线粒体DNA的3个基因（cox1,cox2和Cyt b）全序列和核DNA的2个基因（RAG1,RAG2）的部分序列组合而成的5389 bp对这13个物种进行了系统进化关系的推测。数据表明分子进化关系与Allen的形态学分类系统所强调的体色、体侧带型特征相关性明显。典型活动水域为外海岩礁的蓝色笛鲷（L.kasmira,L.bengalensis和L.quinquelineatus）和近海水域的红色笛鲷（L.malabaricus,L.erythropterus和L.sebae）分别聚在一起。这提示在缺乏地理隔离的海洋岩礁鱼类,笛鲷的成种机制可能是通过体色或视觉系统的适应性进化驱动物种分化。（5）初步探讨笛鲷属鱼类种系发生的驱动力——分子进化与物种形成综合系统进化、形态学、生态学等数据结果,作者认为笛鲷属成种可能服从视觉驱动假设:视觉及其信号系统对于分布区域的不同光环境的适应导致了群体间的生殖隔离,并进一步引发了体色多样性和成种。本章研究测定了10种笛鲷属鱼类的长波段敏感性视蛋白基因（LWS）,序列组成分析揭示出LWS密码子第三位的信息位点含量为0.49,低于线粒体基因（约0.90）和RAG基因（0.74）。在高级阶元上,氨基酸序列构建的进化树与形态学分类一致,笛鲷属、鲤形目（斑马鱼、金鱼和盲眼鱼）和陆生动物（马、牛、猫和蜥）各自聚为一支。但笛鲷属一支的聚类关系与中性标记构建的系统进化树相比,画眉笛鲷和奥氏笛鲷的发生了明显的分离。以上证据暗示,LWS的选择性进化在笛鲷鱼类生态重叠区域的物种分化过程中可能扮演了重要角色。更多还原

【Abstract】 Molecular phylogenetics,also known as molecular systematics,is the use of the structure of molecules to gain information on an organism’s evolutionary relationships.The most common approach is the comparison of sequences for genes using sequence alignment techniques to identify similarity.Another application of molecular phylogeny is in DNA barcoding,where the species of an individual organism is identified using small sections of mitochondrial DNA.The result of a molecular phylogenetic analysis is expressed in a phylogenetic tree,which is the base of revealing the mechanism of speciation.Lutjanid Fishes (Perciformes:genus Lutjanus) are important fishery resource and have been cultured in South China Sea.Through studying their molecular phylogenetics,we have achieved the fellowing results:1.Forty species of teleosts fish,including fifteen lutjanid fishes,in South China Sea,were sequenced(barcoded) for a 660 bp region of the mitochondrial cytochrome oxidase subunit I gene(cox1).Most species were represented by multiple specimens,and 89 sequences were generated.Average within-species,genus,family,and order Kimura two parameter(K2P) distances were 0.0084,0.1567,0.1839 and 0.2319, respectively.All species could be differentiated by their cox1 sequence, although single individuals of each of two species had haplotypes characteristic of a congener.Although DNA barcoding aims to develop species identification systems,some phylogenetic signal was apparent in the data.In the neighbour-joining tree for all 89 sequences,species within genera invariably clustered and three major clusters were apparent: snappers,groupers and the other fishes.The two former clades revealed after bootstrapping generally corresponded well with expectations.We conclude that cox1 sequencing,or ’barcoding’,can be used to identify lutjanid fish species.2.The entire mitochondrial DNA sequence(mitogenome) of five species, Lutjanus bengalensis,L.Malabaricus,L.Sebae,L.Kasmira and cobia (Rachycentridae:Rachycentron canadum) were determined using long PCR and primer-walking methodology,and their GenBank Accession numbers are FJ171339,FJ416614,FJ824741,FJ824742 and NC 011219, respectively.The mitochondrial genomic sequence in snappers and cobia are similar to other vertebrate mitochondrial genomes with respect to gene order and genomic organization.Phylogenetic relationships derived from mtDNA sequences of L.russellii,L.rivulatus,L.bengalensis,L. Malabaricus,L.Sebae,L.Kasmira,cobia and Dark banded fusilier (Caesioninae:Pterocaesio tile) were incongruent with those derived from morphological taxonomic analyses.Beyond expected,high homogeneities were observed between some lutjanids and fusilier, belonging to Lutjaninae and Caesioninae.Further efforts may clarify the phylogenetic relationships among these snappers.3.Two data sets were constructed firstly.One comprised 32 complete mitochondrial DNA genomes of Perciformes downloaded from GenBank. The other was mainly based on the snappers’ mitochondrial DNA we determined in this study.Then we investigated the performance of all mitochondrial protein-coding genes to recover two phylogenies of Perciformes and genus Lutjanus.According to these tests,mitochondrial protein-coding genes can be roughly classified into four groups,(1) at the level of family,of very good(ND6 and cox2),good(ND4 and ND5), medium(ND1,ND2,cox3,Cyt b,cox1 and ATPase 6),and poor(ND3, ND4,ND4L,and ATPase 8);and(2) at the level of genus,of very good (ATPase 6 and cox2),good(cox1 and ND2),medium(cox3,ND1,ND4, ND4L and ND5),and poor(ND3,ND6,and ATPase 8).Moreover,we inform that simple length differences and rate differences between these genes cannot account for their different phylogenetic performance.We advice the careful selected genes and their combinations should be helpful for high resolved phylogenetic analysis for different level taxon.4.The phylogenetic relationships of thirteen snapper species from the South China Sea(SCS) have been established based on the combined DNA sequences of three full-length mitochondrial genes(cox1,cox2 and Cyt b) and two partial nuclear genes(RAG1,RAG2).These thirteen species(genus Lutjanus) were selected after DNA barcoding of 72 individuals representing twenty species.5389 base pairs of combined DNA sequence were used to infer the phylogenetic relationship.Our data strongly suggests that the inter-specific relationships of all thirteen recognized species in SCS are consistent with Allen’s morphology-based classification.There is a strong correlation between the molecular and morphological characteristics,particularly body coloration and band patterns.Our finding is also consistent with the findings in other coral reef fish.The two distinct classes of stripes,blue(L.kasmira,L. bengalensis and L.quinquelineatus,mainly distributed in outer shelf reefs) and red(L.malabaricus,L.erythropterus and L.sebae,distributed in inshore and estuaries),may be evidence for speciation through sensory drive without geographical isolation.In addition,our study suggested that although DNA barcoding aims to develop species identification systems, this approach can also aid the construction of phylogenies by aiding the selection of taxa.The combined mitochondrial and nuclear gene data has advantage over the individual dataset due to its higher resolution power.5.Comparisons of functionally important changes at the molecular level in model systems have identified key adaptations driving isolation and speciation.To test the hypothesis that the evolution of the speciation of snappers is associated with opsin gene diversity,we sequenced long wavelength-sensitive(LWS) opsin genes in ten species of the genus lutjanus.Relationships among higher taxonomic groups were well resolved in the tree reconstructed from LWS opsin sequences.There is high(＞75%) bootstrap support for monophyly of lutjnaus,the family Cyprinidae(goldfish,carp,blind cavefish and zebrafish) and terrestrial animals(horse,cat,bovine and green anole).In contrast to mtDNA and RAG gene,the ratio of phylogenetically informative characters is very low in the third codon positions of LWS.And the signifiant diversity have been observed between LWS opsins of L.vitta and L.ophuysenii.These evidences imply that selective adaption of LWS may play a important role in the sympatric diversity of snappers.更多还原