【作者】 刘刚；

【导师】 周立志；

【作者基本信息】 安徽大学 ， 生态学， 2014， 博士

【摘要】 近年来,动物线粒体DNA全序列的测定和系统发生关系的重建已成为分子系统学研究的热点之一,尤其是线粒体DNA全序列已经广泛应用于鸟类的系统学研究上,已成功用于解决某些鸟类类群的系统发生关系。雁形目(Anseriformes)鸟类是一个庞大的类群,其系统发育关系及部分物种的分类地位复杂且具有争议,也是鸟类系统学研究关注的焦点。由于雁形目种类繁多,目前不同的研究方法得到的雁形目鸟类的系统发生关系均存在较大的分歧,甚至各个阶元的系统发生关系等并不一致。长期以来,雁形目中一些重要物种的分类地位一直存在争议,如中华秋沙鸭(Mergus squamatus)、豆雁(Anser fabalis)、赤麻鸭(Tadorna ferruginea)和鸳鸯(Aix galericulatata)等物种的分类归属及亲缘关系是争论的焦点之一。此外,雁形目鸟类中的一些重要类群的分类地位及系统发生关系也存在争论,如鹊雁(Anseranas semipalmata)、针尾鸭(Stiff-tailed ducks)和树鸭(Whistling ducks)的分类阶元地位；鸭科(Anatidae)中在属的阶元上的系统发生关系也受到关注。传统的形态学研究对雁形目鸟类的系统发生关系存在较大的分歧,而这些争论仅仅从形态学上的证据难以得到解决。随着基因组学分析技术的发展和分子系统学研究的深入,线粒体DNA全序列作为重要的分子标记已经广泛用于研究物种之间的亲缘关系、推导物种的进化和系统发生关系。为了进一步澄清雁形目部分物种的分类地位和雁形目鸟类的系统发生关系,本研究选取了中华秋沙鸭、豆雁、赤麻鸭和鸳鸯的线粒体DNA全序列作为研究对象。根据已知的雁形目鸟类的线粒体DNA全序列设计引物进行PCR扩增,获得四种雁形目鸟类线粒体DNA全序列,分析其结构和特征,进一步研究雁形目鸟类线粒体基因的排列模式和结构,为雁形目鸟类线粒体谱系基因组学的研究提供新的数据资料。结合NCBI数据库上已有序列重建的16种雁形目鸟类的系统发育树,澄清四种雁形目鸟类的分类地位。同时,以部分线粒体基因序列(Cytb,ND2和COI)为联合标记基因,重建68种雁形目鸟类的系统发生树,探讨部分争议物种类群的分类地位,从分子系统学的角度为雁形目系统发育提供证据,以期为雁形目鸟类系统分类与进化研究提供新的信息。四种雁形目鸟类的线粒体DNA全序列均为典型的环状分子结构,其长度分别为16595bp、16688bp、16651bp和16639bp,包括37个基因：13个蛋白质编码基因(ND1-6、ND4L、ATP6、ATP8、COI-Ⅲ和Cyt b)、2个rRNA基因(16S rRNA和12S rRNA)、22个tRNA基因和1个非编码的控制区(D-Loop区)。四种雁形目鸟类的线粒体DNA各基因长度、位置与已知雁形目鸟类相似,其编码蛋白质区域和rRNA基因与其它雁形目鸟类具有很高的同源性,显示其线粒体DNA在进化上十分保守。蛋白质编码基因(PCGs)中,主要以ATN和GTG为起始密码子,终止密码子以典型的TAA, T-或TAG为主。非编码区包括少量的基因间隔区和控制区,控制区均位于tRNAGlu和tRNAPh。之间。预测了22个tRNA基因的二级结构,除tRNASer(AGN)和tRNAPhe缺少DHU臂外,其余均能折叠成典型的三叶草结构。2个rRNA基因(16S rRNA和12S rRNA)位于tRNAPhe和tRNALeu之间,并以tRNAVal间隔。以线粒体DNA全序列为分子标记构建的16种雁形目鸟类的系统发生树显示鸳鸯和赤麻鸭应同属于麻鸭亚科(Tadorninae);中华秋沙鸭隶属于鸭亚科(Anatinae)、海鸭族(Mergini)、与鸭族(Anatini)物种共同构成姐妹群；豆雁隶属于雁亚科(Anserinae)、雁族(Anserini);而花脸鸭(Anas formosa)在系统发生树中与其他鸭族物种相距较远,其分类地位还需要进一步研究。利用已知雁形目鸟类线粒体的三种联合标记基因(Cyt b, ND2和COI)构建的68种雁形目鸟类的系统发育树结果表明：雁形目鸟类可以划分为三个科,即鸭科(Anatidae)、鹊雁科(Anseranatidae)和叫鸭科(Anhimidae)、支持鹊雁作为一个单独的科；鸭科划分为五个亚科：鸭亚科(Anatinae)、麻鸭亚科(Tadorninae)、雁亚科(Anserinae)、针尾鸭亚科(Oxyurinae)和树鸭亚科(Dendrocygninae),支持麻鸭、针尾鸭和树鸭提升为亚科。鸭亚科包括海鸭族(Mergini)、绒鸭族(Somaterini)、鸭族(Anatini)和潜鸭族(Aythyini),支持绒鸭族从海鸭族中分离出来构建单独的一个族；麻鸭亚科包括Aix、Cairina、 Tadorna和Chloephaga;雁亚科包括雁族(Anserini)和天鹅族(Cygnini)两个族。更多还原

【Abstract】 The sequence research of mtDNA and reconstruction of the phylogenetic relationship of animals are becoming more and more popular at present. The complete mitochondrional genomes have been widely used as powerful molecular markers to resolve the controversial Aves species. Anseriformes is a highly differentiated group with worldwide distribution, which is regarded as one of the best-studied groups of birds. The phylogenetic relationship among Anseriformes is rather complex and controversial, which has been affected by rearrangements for several times. Aix galericulata, Tadorna ferruginea, Mergus squamatus and Anser fabalis are typically waterbirds in Anseriformes, and their phylogenetic sites are controversially for a long time. In addition, the relationship of some important groups in Anseriformes are controversial widely, such as the stiff-tailed ducks, Anseranas semipalmata and the whistling ducks. Traditionally, most available phylogenetic data came from morphological, anatomical and behavioral analyses. As the development of genetic analysis and molecular biology, the mitochondrial genome has become a highly useful molecular marker for the reconstruction of phylogenetic relationships at different animal taxonomic levels. In mis study, in order to have an insight into the phylogenetic relationship among Anseriformes, we used PCR-based method to determine the complete mitochondrial genome sequences of Mergus squamatus, Anser fabalis, Aix galericulata and Tadorna ferruginea, and analyzed the structure of some published Anseriformes species mitochondrial genome. In order to clarify the phylogenetic position of these four species, the phylogenetic trees were based on the complete mtDNA among16Anseriformes species. In order to have an insight into the phylogenetic relationship among Anseriformes, phylogenetic trees are reconstructed by multiple sequence alignments of three mitogonome (Cyt b, ND2and COI) sequences among68typical Anseriformes species.The complete mtDNA nucleotide sequences of the four birds are circular molecule, which are16595bp,16688bp,16651bp and16639bp in size. The organization of the sequence encodes13protein genes (ATP6, ATP8, COI-Ⅲ, ND1-6, ND4L, and Cyt b),22tRNA genes,2rRNA genes (12S rRNA and16S rRNA) and the non-coding control region (D-loop). The same gene order and arrangement illustrate extremely conservative and similar to other Anseriformes birds. The length among them is similar and very conservative in the PCGs in the four Anseriformes species, the ATG as start codon is generally observed and TAA is the most frequent stop codon. All tRNAs could be folded into canonical cloverleaf secondary structures except for tRNASer(AGY) and tRNALeu(CUN)、which are missing the dihydrouridine (DHU) arm. Like other Anseriformes,12S rRNA and16S rRNA genes are located between tRNAPhe and tRNALeu, separated by tRNAVal.The phylogenetic relationship among16Anseriformes species based on the complete mtDNA shows that Aix galericulata and Tadorna ferruginea are in the lineage, Tadorninae. Mergus squamatus belongs to Anatidae, and Anser fabalis belongs to tribes Anserini of Anserinae. Anas formosa has no close relatives among living ducks, so it should be put in a distinct genus other than Anas.Molecular phylogenetic analysis based on concatenated sequences (Cyt b, ND2and COI) support the studied Anseriformes birds being divided into three families: Anhimidae, Anatidae and Anseranatidae. Anseranas semipalmata does not belong to Anatidae, but represents an independent family, Anseranatidae. The results suggest Anatidae birds being divided into five subfamilies:Anatinae, Tadorninae, Anserinae, Oxyurinae and Dendrocygninae. Stiff-tailed ducks (Oxyurinae) and whistling ducks (Dendrocygninae) should not belong to Anserinae, but represents an independent subfamily respectively. The Anatinae, includes species of tribes Mergini, Somaterini, Anatini and Aythyini. The subfamily Anserinae, which comprises tribes Anserini and Cygnini. The phylogentic relationship supports that the genus Somateria is an early-diverging lineage in Mergini which is sometimes classified as a separate tribe, Somaterini.更多还原