中国蚌科DNA条形码序列筛选及其分子系统学研究

Accurate identification of the species is the basis of understanding biological diversity and sustainable using the biological resources. Recently, the DNA barcoding technique taking a standardized DNA region as a tag has been proved to be a new accurate approach in species identification and phylogenetic analysis of creatures. Moreover, both the accurate species identification and the phylogenetic analysis are critical to effective biodiversity conservation and management, so the DNA barcoding technique provides the theoretical basis for biodiversity conservation. As the largest family of freshwater mussels, Unionidae plays a key role in freshwater ecosystems. Now, Unionids have declined in both the diversity of species and abundance due to excessive commercial exploitation, pollution and loss of habitat, etc. They have been considered one of the most endangered group of creatures in the world. Currently, the species identification of Unionidae has been based mainly on morphological traits. Because of the pronounced phenotypic plasticity in shell morphology and other traits, it is disputable to classify the Unionidae and comfirm the phylogenetic relationships for Chinese Unionidae only based on morphological traits. In this project, more than 60 species of Chinese Unionidae will be sampled to construct the standardized DNA barcoding system based on mitochondrial DNA sequences and nuclear DNA sequences, such as COⅠ, COⅡ, 16SrRNA, ND1, ND5, Cytb and ITS, etc. Researches on the DNA barcoding and phylogenetic relationship of these Chinese Unionidae will not only have theory significance，but also possess important practical value for correcting taxonomy system, protecting biology diversity and providing conservation strategies. Furthermore, it is very important to combine the morphological traits with molecular data in constructing accurate species identification systems. We aim to develop some efficient and valid DNA barcodes for species identification and phylogenetic analysis for Chinese Unionidae. This research will provide us with a theoretical basis of resources conversation and development of DNA taxonomy for freshwater mussels in China and in the world.

物种的准确鉴定是认识生物多样性、持续利用生物资源的基础。DNA条形码技术能准确、快速地鉴定物种，对物种分类和生物资源保护有着重要意义。作为真瓣鳃目最大的一个科，蚌是淡水生态系统中的关键物种。由于过度捕捞、淡水资源污染和栖息地破坏等原因，蚌的数量大大减少，资源日益衰竭，很多物种受危严重甚至濒临灭绝，急需进行保护。同时，蚌科物种的鉴定目前主要是依据传统的形态学方法，由于表型的可塑性及蚌的趋同现象，物种的有效性及种间的系统发育关系还存在很大争议。在这种背景下，本项目拟收集全国60多种蚌，选取COⅠ、COⅡ、16SrRNA、ND1、ND5、Cytb、ITS等线粒体基因和核基因，筛选出适用于蚌科的DNA条形码标准基因，建立我国蚌科DNA条形码系统，结合传统的形态学手段，准确鉴定物种，识别疑难物种和发现隐存种，确定种间系统发育关系，为保护我国淡水蚌提供科学依据,为建立淡水贝类DNA条形码系统奠定基础。

以DNA序列标记技术为基础，与系统发育和分类学研究有一定关联的DNA条形码技术被广泛应用于物种分类鉴定、生物多样性评估。本项目完成了多个线粒体基因和核基因序列作为蚌科DNA条形码候选基因的筛选和可行性分析，并利用这些基因序列信息，对蚌科各物种间的系统发育关系进行了分析。本项目获得的主要研究结果如下：1. 确定了线粒体基因COI、ND1序列可作为DNA条形码对蚌科物种进行有效的鉴定。并利用COI和ND1基因信息，进行物种系统发育关系分析。 蚌科物种平均的种间遗传距离都为平均种内遗传距离的10倍以上。采用邻接法，分别对COI和ND1基因序列构建系统发育树，结果显示，两个基因的系统发育树均形成三个明显的分支，且每个物种都各自聚为一支，支持率均在90%以上。2.分析了核基因转录间隔区ITS1、ITS2基因序列作为蚌科DNA条形码鉴定物种的可行性。结果显示，这两个基因序列均可以作为蚌科DNA条形码候选基因，但在物种系统发育关系分析中显示，在分析属间亲缘关系的远近上两个基因序列存在着差异。ITS1、ITS2均适合作为蚌科DNA条形码鉴定物种。3.对线粒体蛋白质编码基因Cyt b部分序列分析表明，蚌科物种平均种内遗传距离和种间遗传距离相差不足10倍，不宜作为DNA条形码鉴定物种与系统发育分析。4.选取核基因28S rDNA的部分序列作为蚌科DNA条形码的候选基因进行分析，结果表明蚌科的28S rDNA保守性比较强，进化速率比较慢，适用于高阶元的物种分类。5. 对蚌科28S rDNA和COI 的序列信息联合分析显示，两个基因的联合数据集没有明显的异质性，联合两个数据集构建了蚌科贝叶斯系统发育树和最大似然树，确定了蚌科各属的单系性与复系性，并对中国蚌科属分类阶元和亚科分类阶元的系统发育关系进行了探讨。