【作者】 杨频；

【导师】 宋大祥； 陈立侨；

【作者基本信息】 华东师范大学 ， 水生生物学， 2007， 博士

【摘要】 沼虾属Macrobrachium是一类生存环境多样,起源与分化过程尚不甚明了,分类地位具争议性的动物类群。众多学者在分类方面已做了大量的研究,然而由于其鉴别特征的保守性及形态性状多态性丰富等特点,导致了沼虾属种类鉴定的困难,使得相关研究难以深入。本研究以中国境内分布的沼虾属种类为主要研究对象,利用DNA测序、PCR-RFLP技术和ISSR技术等分子生物学实验手段,运用支序系统学与分子进化生物学理论及分析方法,以地理群体为基本研究单元,展开从地理种群-物种-属的逐级水平的系统学研究。探讨长臂虾亚科各属、沼虾属各种及日本沼虾不同地理群体的分子遗传学特征,并重建系统发育树。其次,在重建系统发育关系树的基础上,诠释现存的种、属分类问题。此外,本研究对揭示沼虾属种间的系统发育关系、澄清其种属分类问题、对整个沼虾属的系统演化与分类格局关系的研究具有重要意义。还可揭示主要经济种类日本沼虾地理群体之间的遗传多样性信息,为有效保护和合理开发利用野生资源提供重要的理论基础。本研究主要从以下几个方面进行研究:1.应用支序系统学的原理和方法,选取长臂虾亚科9属60物种和Procaridae科2物种(外群)的形态和生态性状。并对其分类学、系统学、形态学及部分生物学等方面的文献资料进行综合分析,进行形态学观察和系统发育研究。构建的严格一致树显示,(1)两个外群聚为一支,与内类群的物种分开,位于支序图的基部。内类群长臂虾亚科的60个物种由3个主要特征(即尾节常具2对末端刺,有2或更多对中央刚毛,第三颚足常具2关节鳃)构成一支,形成一个单系群。(2)沼虾属也形成一个单系群,由头胸甲具肝刺,无鳃甲刺支持与其它几个属区分开来。(3)其他几个属间的亲缘关系并不明确。说明Holthuis(1950)和Chace(1992)所建立的分类系统虽然能够将一些种类区分开来,但是无法反映出这些类群的亲缘关系。2.利用线粒体16S rDNA部分序列对我国沼虾属的系统发育关系进行了探讨,使用PAUP软件构建NJ、MP、ML系统树,使用MrBayes3.0b4软件构建BI树。在忽略小于50%的支持率后,这四种系统树所显示的拓扑结构具有一定一致性,所有结果均支持沼虾属为单系群,但在几种沼虾的相互关系上有所区别。为了得到四种分析方法一致的沼虾属系统发育关系,我们构建了这四棵树的50%合一树,结果发现沼虾属可分为第二对步足左右不对称的类群和第二对步足左右对称的类群。另外,外形上极易混淆的三种沼虾(日本沼虾、胖掌沼虾和细螯沼虾)的分子数据显示其亲缘关系很近。3.使用分子鉴别方法对我国代表地区的日本沼虾、细螯沼虾和胖掌沼虾进行分类研究。共使用了3种分子标记,即线粒体16S rDNA、COⅠ和D-loopPCR-RFLP。线粒体16S rDNA和CO I基因序列分析结果显示这三种沼虾之间的差异小于沼虾属不同种间的遗传差异,并与沼虾属同种不同群体间的遗传差异有重叠现象。D-loop PCR-RFLP的结果亦显示这三种沼虾间的遗传距离比罗氏沼虾与这三种沼虾间的距离小得多。三种分子数据构建的系统发育树也显示不同采集地点的日本沼虾、细螯沼虾和胖掌沼虾之间的亲缘关系很近。因此结合三种分子标记的结果推测这三种沼虾间的差异处于种内水平,它们可能是同一物种。4.日本沼虾Macrobrachium nipponense是我国分布范围广、经济价值较大的一种重要淡水虾类。使用线粒体CO I基因片段与ISSR标记技术,对我国日本沼虾长江水系和澜沧江水系的10个野生群体的遗传结构进行分析,显示大致可分为长江上游及澜沧江族群、长江中游族群和长江下游族群。群体间存在遗传差异,且各群体间的遗传差异与其地理分布关系密切。两种分子标记的研究结果均显示湖北武汉群体的遗传多样性高,意味着该群体对外界环境变化的适应能力较强。遗传距离和聚类图显示浙江湖州(HZ)、江苏苏州(SZ)与上海(SH)群体的亲缘关系较近。从整个日本沼虾物种来说,存在较高的遗传多样性。线粒体COⅠ序列的研究结果相对于其他间接分子标记,能够直接揭示物种的遗传变异,但是相对耗时,花费较高。因此在无相关DNA序列信息的情况下,ISSR标记技术是一个非常有效的检测群体遗传变异的遗传标记。更多还原

【Abstract】 Freshwater prawns of the genus Macorbrachium (Crustacea: Decapoda: Caridea: Palaemonidae) constitute one of the most diverse, abundant and widespread cruatacean genera. They inhabit a wide variety of environments from mountain steams and lowland rivers to estuaries and coastal lagoons. The palaemonids have historically been a taxonomically difficult group because they appear to be highly conservative in morphology. The problems have been attributed to difficulties in the interpretation of the significance of morphological characteristics used to classify palaemonid shrimp and have hindered the development of stable classification systems. Until recently, all major systematic treatments of palaemonids have been based on morphological characteristics alone. A few allozyme-based and DNA-based studies have been undertaken. However, phylogenetic relationships within Macrobrachium species remain poorly studied. In this study, collected all available specimen of Chinese Macrobrachium species, using morphological characters, mitochrondrial DNA sequencing and Inter-simple sequence repeats marker, employing MP, ML, BI and NJ to generate phylogenetic hypothesis, we intent to interpret the phylogenetic relationships, classification of the genus and study the genetic diversity of Macrobrachium nipponense populations in order to provide preliminary genetic information for the development of management strategies for this species.This study focuses on four subjects using morphological and molecular characters:1. A cladistic analysis of morphological and ecological characters was preformed in which 36 characters exhibited by 60 species of 9 genera of Palaemoninae. Two species of Procaridae were selected as outgroup for the character analysis. The cladogram shown that (1) the Palaemoninae species formed a monophyletic clade by three main characters (telson usually with 2 pairs of dorsal spines, 2 or more feathered setae on telson, third maxilliped with two orthrobranchia). P. ascensionis and V. chaceorum cluster together as related taxa at the base of the tree; (2) the Macrobrachium species forming a monophyletic clade. They are charaterised by the presence of hepatic spine on carapace and absence of branchiostergal spine; (3) the relationships among the rest genera remaining unclear. The results suggested that Holthuis (1950) and Chace (1992) classification provided a frame of reference to sort out species and some natural groups but failed to clarify the relationships of the group.2. Based on mitochondrial DNA fragement of the large subunit (16S) rRNA gene, the monophyletic phylogeny of the genus Macrobrachium, including freshwater and estuarine species, was supported, although there are some difference on the topology. The 50% consensus tree of MP, NJ, ML and BI trees showed two distinct clades: one clade includes the species with equal second pereiopod, the other clade separately clustered with the species with unequal second pereiopod. Furthermore, we found that three easy-confused species, M. nipponense, M. inflatum and M. superbum, have closer relationship.3. In order to identify the relationship of M. nipponense, M. inflatum and M. superbum which have the similar morphological characters and share similar distribution ranges, we chosed to use three markers proposed as most informative in delimiting species relationship with DNA barcodes, namely cytochrome c oxidase I (CO I), 16S ribosomal DNA and D-loop PCR-RFLP. The sequence analysis results of CO I and 16S rRNA gene showed that the much lower divergent level found among the ingroup samples in this study is more typical of what is seen among populations within species. The result of RFLP reinforced the above conclusion. And the trees produced by NJ analyses were identical. Haplotypes representing M. nipponense, M. inflatum and M. superbum collected from different locations across inland China form a tight monophyletic group, with very short branch lengths compared with the much longer branch lengths observed in other Macrobrachium species. Therefore, M. nipponense, M. inflatum and M. superbum may be a single species.4. The oriental river prawn M. nipponense is one of the most important freshwater prawn s for aquaculture in China, especially in southern regions. In present work we investigate the genetic diversity among the 10 populations from the Yangtze and Lancang River by CO I gene region and ISSR marker. The dendrogram produced resulted in the formation of three major clusters (Lancang River and upriver reaches of Yangtze River, middle reaches of Yangtze River and lower reaches of Yangtze River). The genetic differentiations among most of the populations corresponded to the geographic distances among the populations. The results of the two molecular markers also showed Wuhan population exhibited the high level of variability, which means this population can effectively adapt to environmental changes. Genetic distance and dendrograms showed that compared with other populations, Huzhou (HZ), Suzhou (SZ) population and Shanghai (SH) population are closer. At the species level, M. nipponense showed relatively high level of polymorphism. Compared to other indirect methods, the results of DNA sequencing can directly reveal the genetic variation of the species, but much expensive. Therefore, if the species do not have prior genome sequence information, ISSR is a powerful marker of choice for the assessment of genetic variation among populations.更多还原