蛙片虫的分类与系统发育研究

Opalinids are multinuclear, mouthless, osmotrophic protozoa, the majority of which live in the cloacae of anuran amphibians. Opalinids have long been controversial with respect to their systematic position among the protists, since the co-existence of both ciliate-like and flagellate-like features has confused their classification. They are also the representatives of last few higher eukaryotic taxa that were not studied with molecular phylogenetic methods because of lack of DNA sequence data. It is thus very significant for basic biology of opalinids and therapeutics of corresponding diseases to study the taxonomy and phylogeny of opalinids inhabitating anuran amphibians. In order to elucidate their origin and phylogeny, we will conduct a detailed investigation on Chinese local opalinids infecting anuran amphibians in different habitat types and study them from four aspects including morphology, phylogenetic analysis based on 18S rDNA, selection of DNA Barcoding candidate genes for opalinids, phylogenomic analysis based on transcriptome data. Modern taxonomic techniques and molecular phylogenetic methods will be used together to explore the validity of morphological species, to redescribe the typical genera, to reveal their phylogenetic relationships, and thus to infers their systematic evolution and origin. Moreover, the coevolution pattern of opalinids and their hosts will be analysed and their host specificity will also be evaluated. If completed according to the experimental plan, the controversy about origin and phylogeny of opalinids will be solved and a convincing taxonomical system of Opalinata will be provided. In addition, it will also provide referable foundation for the reasonable evaluation of the relationship between opalinids and their hosts.

蛙片虫是一类绝大多数种类共栖于无尾两栖动物泄殖腔内或肠道下端的原生动物；迄今为止其分类地位仍然存疑，分子系统学研究几为空白。本项目拟在对我国蛙片虫的本底资源与分布情况进行全面调查的基础上，从形态分类学、基于18S rDNA的分子系统学、DNA条形码基因的筛选、基于转录组数据的系统发育基因组学等四个方面开展研究。通过形态特征（显微、超微结构）与分子数据（标记基因、转录组）的相互补充与印证，阐明蛙片虫的系统分类地位，厘定本类群现存的分类学混乱和错误，重新刻画不同属的典型特征，探讨其系统发育模式，并最终修订其分类系统。本项目的完成将基本摸清我国蛙片虫的物种资源，构建完善的本类群形态学资料库和DNA条形码数据库，积累丰富的标记基因信息和典型物种的转录组学数据，极大地充实和填补国内外对本类群研究的欠缺和空白；同时为具有重要经济价值的无尾两栖动物体内蛙片虫的合理定性和病害评估提供基础资料。

蛙片虫是一类共栖于无尾两栖动物泄殖腔或肠道下端的原生动物。就整个类群而言，其基础研究非常匮乏，特别是基于现代分类学标准的形态学资料和分子数据的严重缺失，极大地限制了对蛙片虫系统分类地位的正确定位和内部阶元的合理界定及细化。本项目在广泛调查我国蛙片虫物种资源的基础上，通过形态特征和分子数据的联合探讨，研究了其分类和系统发育关系，阐明了蛙片虫的分类地位，填补了国际间对本类群系统发育研究的空白与欠缺；对不同蛙片虫属的典型特征进行了重新刻画；评估了核糖体rDNA不同片段作为蛙片虫类群条形码候选基因的可行性，积累了丰富的标记基因信息。（1）完成了3属14种蛙片虫的形态学刻画；成功获得了它们的rDNA全序列，填补了国际间本类群分子序列信息的空白（高AT含量，常超过70%）；（2）考量了蛙片虫的分类依据，认为虫体运动模式、缝线区特征、核器及附属结构、体表皮褶维管束与微丝系统的排布特点具有重要的分类学意义，其中核的数量和缝线的位置是重要的属级分类特征；（3）首次清晰阐释了蛙片虫的系统分类地位，认为蛙片虫类群与人原囊芽虫属Blastocystis亲缘关系最近，两者作为姊妹枝共同构成单系群；（4）分别对蛙片虫的核SSU、ITS1、ITS2 和 LSU rRNA 高变异区进行了遗传多样性研究，结果显示ITS是蛙片虫属、种间区分的有效分子标记，是蛙片虫类群最适合的DNA条形码基因；同时，ITS2的二级结构在属级阶元区分上具有重要参考价值；（5）选择长棍形蛙片虫Cepedea longa，开展了其单细胞转录组的测序、组装与分析工作；为后续深入探讨蛙片虫的寄生适应性分子机制打下了良好的基础。