基于线粒体全基因组的角蟾科物种系统发育与进化研究

As a member of Mesobatrachia, thought as the transition animal group between the Archaeobatrachia and Neobatrachia, Megophryidae has a very important position on the evolution of the tailless amphibians. This family is endemic in Asia,mainly in the oriental realm，and distributes from Pakistan and western China east to the Philippines and the Greater Sunda Islands. According to Frost (2013), there are totally 10 genera and 171 species in this family, and in them there are 70% of genera and 88 species occur in China, So China always be thought as　a hotspot for the study and conservation for the megophryids. Owing to this animal group have the significantly phylogenetic position, complication on its' classification, unique sexual characters, reversal sexual size dimorphism, and significantly morphological variation of their tadpoles, the studies on the systematics and phylogeny of Megophryidae is keeping hot in the whole world, but previous studies had either limited number of taxa or limited number of informative characters, as well as some analytical shortcomings, the preferred phylogenetic relationship in the family is hard to get consistence. Based on the new generation DNA sequencing technic, this project will analyze the complete mitochondrion genomes of 28 species,represented 10 different genera in the　family respectively, both from the aspect of systematics and bioinformatics. Comparing with the present results of other anuran species, we will try to find the features of the Mt DNA genomic both from the structure and evolution among the different subfamily and genera. We will reconstruct the phylogenetic relationship of Megophryidae, and clarified it's evolution position in Anura. we will clarify the origin，speciation, and phylogenetic hypotheses for the family more completely and comprehensively. Moreover, by using molecular clock-independent approaches for inferring dating information from molecular phylogenies, a time scale for events in Megophryidae evolution will be given. The results of the project will present an important reference for the studies on the amphibians in oriental realm, and provide new evidences for the geographic history study in this region.

角蟾科被认为是始蛙亚目与新蛙亚目的重要过渡类群之一，在两栖动物的系统发育关系中占有极其关键的演化地位。该类群主要分布在东洋界，根据Frost（2013）最新统计，该类群现有10属171种，中国因具有近70%的属和88个物种,成为该类群保护的关键地区。由于该科分类地位独特、适应辐射广泛、属种分化复杂、第二性征独特，蝌蚪形态变化多样，其分类和系统适应与进化研究一直成为国内外研究的热点。但因为研究类群和选用分子标记以及分析技术和方法的局限性，关于该类群的系统发育关系仍存在较多分歧。本项目拟运用DNA测序技术，从系统学和生物信息学两个层面，对全科的不同类群和不同生态型的10属28种进行线粒体全基因组分析，结合其它近缘脊椎动物的比较基因组学研究，以期阐明不同亚科、属的线粒体基因组的结构和适应进化特点，为进一步厘清该科在两栖动物无尾类中的分类地位，科内的系统发育关系以及物种分化成因提供更加系统的证据。

线粒体基因组广泛地运用到分子系统和分子进化研究。角蟾科作为过渡类群在进化上有重要科学意义。本研究用PCR和LA PCR技术，研究了其线粒体基因组特点及相关系统发育关系，揭示角蟾科的进化特点。.一、.共测定了角蟾科3亚科9属27个物种的线粒体基因组序列。.二、.角蟾科物种线粒体基因组长度在14816~20267bp之间，最短的为大角蟾，最长的为胸腺猫眼蟾。使用频率最高的起始密码子为ATG；使用频率最高的终止密码子为T++。重叠区最短为1bp，最长的14bp；间隔区最短为1bp，最长的为1105bp。12SrRNA和16SrRNA长度浮动不大，分别为924~937bp和1583~1600bp。tRNA基因个数在各物种之间有差异(20~23)，最少的为20个。.三、 角蟾亚科的线粒体基因组呈现典型的脊椎动物排布方式；拟髭蟾亚科的线粒体基因组发生了排列的改变。齿蟾属、髭蟾属和拟髭蟾属的排列方式相同：均出现串联tRNA-Met，且tRNA-Trp(W)移位到tRNA-Pro和 D-Loop 之间。（2）而齿突蟾属的物种中，在ND1和ND2之间发现了新的一个D-loop区（D-loop1），且tRNA-Trp基因移位到了ND1下游；tRNA-Ile和tRNA-Gln 发生缺失。而齿突蟾属基于以上的排列，tRNA-Met也发生了串联重复。掌突蟾亚科2个物种均出现了tRNA-Met的复制。高山掌突蟾除tRNA-Met重复外，tRNA-Val移位到了tRNA-Met1的下游，tRNA-Pro移位到了tRNA-Met2的上游；tRNA-Trp发生移位并功能消失。姆鲁山小臂蟾2个tRNA-Met被从12S rRNA下游移位而来的tRNA-Val所隔开，tRNA-Trp移位到tRNA-Pro和 D-Loop之间，tRNA-Ala，tRNA-Thr和tRNA-Pro 发生缺失。研究共发现5种线粒体基因组新的基因排列方式。.四、线粒体基因组分析强烈支持锄足蟾超科为一单系；支持以锄足蟾超科为代表的中蛙亚目为始蛙亚目和新蛙亚目之间的过渡类群；支持角蟾科三个亚科的划分，支持角蟾亚科角蟾属为复系群的观点。.五、中蛙亚目作为过渡类群，既具有始蛙亚目所具有的脊椎动物线粒体基因组典型排列特点，又具有新蛙亚目所含有的一些线粒体基因组基因排列特点。