【作者】 黄华；

【导师】 郑荣泉；

【作者基本信息】 浙江师范大学 ， 生态学， 2012， 硕士

【摘要】 隐种,是指形态上几乎完全相同或尚未发现差异但遗传组成上存在显著分化的物种。隐种很难从形态学上加以区分,有时甚至是不可能区分,经常导致隐种被错误的归于一个物种。分子生物学技术的迅猛发展,为生物学家们提供了一个研究和探索隐种的新途径,并为揭示物种遗传结构、种内和种间多样性提供了有力工具。由于两栖动物迁移能力弱,种群分化程度高,因而较其他物种包含有更丰富的隐种多样性。棘胸蛙(Paa spinosa David)是棘蛙属(Paa)中在我国分布最广的一种蛙类。前期基于Cyt b较端片段序列研究表明,棘胸蛙遗传结构差异较大,暗示可能存在隐存物种。为了验证这一假设,本研究拟采用多基因结合的方法,探讨棘胸蛙的系统地理结构,以期揭示棘胸蛙的隐种多样性。具体结果如下：本研究测定了中国大陆棘胸蛙及其近缘种的线粒体16S rRNA和12S rRNA基因序列,这些样品包括：10个地理种群170个棘胸蛙(P. spinosa)样本、9个九龙棘蛙(P. jiulongensis)样本、6个棘腹蛙(P. boulengeri)样本、8个多疣棘蛙(P. verrucospinosa)样本、13个小棘蛙(P. exilispinosa)样本和23个棘侧蛙(P.shini)样本。共获得的基因序列总长度为1338bp,一共发现了118个变异位点分别定义了38个和65个单倍型。基于邻接法(neighbor joining、最大简约法(Maximum parsimongy、最大似然法(Maximum likelihood, ML)和最小进化法(Maximum evolution, ME)构建的单.倍型系统发生树结构基本一致,结果表明：来自10个地理种群的棘胸蛙种群基因组成上分为2个单系,即单系AI和AⅡ。单系AⅠ主要包括浙江丽水、浙江金华、安徽黄山、福建武夷山种群,对应于棘胸蛙东部分布区域；单系AⅡ所对应的地理种群主要是江西井冈山、湖南平江、广东阳山、江西庐山、广西永福、广西龙胜,在地理分布上为棘胸蛙中部种群。基于系统发生树计算棘胸蛙之间的遗传距离,16S rRNA结果显示单系AⅠ与小棘蛙的平均遗传距离为2.863%,单系AⅡ与小棘蛙的平均遗传距离为2.013%,而单系AI与All之间的平均遗传距离为2.675%,大于单系AⅡ与小棘蛙的遗传距离,达到了种间以上水平。基于12S rRNA的结果与16S rRNA保持一致,单系AⅠ与小棘蛙的平均遗传距离为2.845%,单系AⅡ与小棘蛙的平均遗传距离为2.392%,而单系AⅠ与AⅡ之间的平均遗传距离为2.738%,大于单系AⅡ与小棘蛙的遗传距离,达到了种间以上水平。结合以往的研究,广泛分布在中国南部的棘胸蛙可能至少包含2个隐种。今后,进一步对2个单系接触带的分子生物学及其形态、行为学等研究将有利于阐明棘胸蛙隐种形成的机制。更多还原

【Abstract】 Cryptic species are morphologically undetected yet genetically divergent species. It’s very difficult to distinguish in morphological and sometimes even impossible to distinguish so that cryptic species was incorrectly attributed to a single species, with the rapid development of molecular biology In recent years, biological diversity hidden within species are increasingly being discovered. There are many challenges on biodiversity estimation from the prevalence of cryptic species. Due to the weak of migration and strong philopatric, amphibians usually have a strong genetic differentiation and might be storehouses of tremendous cryptic diversity. The distribution of Giant spiny frogs (Paa spinosa) in China is the most extensive in Paa. Early genetic structure based on short fragment Cyt b of spinosa quite different, suggesting that there may be hidden to cryptic species. To test this hypothesis, this study intends to adopt multi-gene combination of methods to explore the phylogeographic structure of P. spinosa. We did research on genetic diversity and population genetic structure of the giant spiny frog population from different geographies by using mitochondrial DNA as genetic markers. The major results are as follows:Determination of this study in China’s mainland10geographic populations of170P. spinosa samples,9Paa jiulongensis samples,6Paa boulengeri,8Paa verrucospinosa,13Paa exilispinosa samples and23Paa shini samples (total number of samples to229) of the mitochondrial16S rRNA,12S rRNA gene sequence, its gene sequence length1338bp, found a total of118variable sites, defined38(16S rRNA) and65(12S rRNA) haplotypes. Based on method of the neighbor-joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and minimum evolution (Maximum evolution, ME), constructed haplotype phylogenetic tree structure, the results show that:the P. spinosa populations are divided into two monophyletic from10geographic populations, the Clade AI and Clade All. Clade AI including Lishui, Jinhua, Huangshan, Wuyishan population, corresponding to the eastern area of P. spinosa in distribution; monophyletic All including geographic populations of Jinggangshan, pingjiang, Yangshan, Lushan, Yongfu, Longsheng, corresponding to the central of geographical distribution of P. spinosa. Calculation the genetic distance of P. spinosa based on the phylogenetic tree, The results of16S rRNA gene showed that the average genetic distance is2.863percent between monophyletic AI and P. exilispinosa, the average genetic distance is2.013percent between monophyletic All and P. exilispinosa, the average genetic distance is2.675percent between monophyletic AI and All, greater than the genetic distance between monophyletic All and P. exilispinosa, reach the level above the species. the results of12S rRNA gene keep consistent with16S rRNA gene, The results of12S rRNA gene showed that the average genetic distance is2.845percent between monophyletic AI and P. exilispinosa, the average genetic distance is2.392percent between monophyletic All and P. exilispinosa, the average genetic distance is2.738percent between monophyletic AI and All, greater than the genetic distance between monophyletic All and P. exilispinosa, reach the level above the species. Combined with previous research, there are two cryptic species at least in p. spinosa in widely distributed in southern China. In the future, further study of two monophyletic contact with molecular biology and morphology, behavior and so on, will be beneficial to clarify the mechanism of formation of cryptic species in p. spinosa.更多还原