【作者】 李涛；

【导师】 马恩波；

【作者基本信息】 山西大学 ， 动物学， 2011， 博士

【摘要】 稻蝗(Oxya)隶属于昆虫纲(Insecta),直翅目(Orthoptera),蝗总科(Acridoidea),斑腿蝗科(Oedipodidae),是重要的农业害虫之一。近年来,对稻蝗属物种的研究工作在形态、细胞、蛋白质标记和分子标记等方面均有报道,其中对中华稻蝗研究较为透彻,对于日本稻蝗和小稻蝗的研究仅局限于在中国境内的局部地区,采样点覆盖面较小,种群数量较少。本研究以日本稻蝗、小稻蝗和稻蝗属一待定种为研究对象,通过采用线粒体DNA等基因序列测定分析、AFLP分子标记和形态性状测定等方法,对稻蝗属物种分子遗传多样性、种群遗传结构、谱系分布进行研究,结合生物地理学信息,对该属物种分化及其生态适应规律进行探讨。本文的研究内容有如下几个方面：1)本研究采用AFLP分子标记对日本稻蝗进行种群遗传结构和遗传多样性研究,探讨不同生境中(栽培稻田和野生稻田)的日本稻蝗种群间是否存在遗传多样性差异和遗传分化。采用8对选择性引物组合对采自我国南方的7个日本稻蝗种群共计104个个体进行AFLP扩增,共得到564条清晰可辨的条带,其中563条为多态性条带。日本稻蝗种群间遗传多样性分化明显,基因多样性(HE)分布范围为0.1103-0.2035。野生稻田内的3个日本种群平均遗传多样性(HE=0.1635)高于栽培稻田内4个种群的平均遗传多样性(HE=0.1327)。种群分化系数FsT值(0.4172-0.7652)表明在七个种群之间存在明显的遗传分化现象。Mantel检验表明地理距离和遗传距离之间没有显著相关性(r=0.3541；p=0.0689)。生存在野生稻田的日本稻蝗种群与栽培稻田的日本稻蝗种群间出现明显的遗传分化,这可能是由于人类在栽培稻田的农业耕作行为(例如杀虫剂和农药的使用、施肥、灌溉等)对日本稻蝗种群遗传结构起到明显的影响。2)通过对100个日本稻蝗个体进行线粒体DNA COI基因部分序列测定分析,得到650bp的碱基序列,共计55个变异位点,并产生48个单倍型,其中27个单倍型为共享单倍型。共享单倍型仅分布于各个种群内部,种群间和组间并不存在共享单倍型。基于最大简约法构建的系统发育树表明：聚类树的拓扑结构与地理格局相吻合,单倍型首先以种群为单位相聚,大部分相邻的种群率先聚为一支并最终形成五个大的聚类簇。TCS分析形成了具有明显间隔的网络结构图,相邻的5个组间均有较多的突变步骤。分子方差分析结果表明,10个日本稻蝗种群具有很明显的地域分化,按照地域分隔形成的5个组间的遗传变异达到了总变异度的62.96%,遗传分化极其显著(P<0.001)。LAMARC结果表明,在同一地域的不同种群之间有频繁的基因交流,但是不同地域之间的种群基因交流不明显。FST值和网络结构图显示,5个组起源于共同的祖先种群,但是由于长时间的时空隔离,使不同组间的基因交流明显减少,最终形成遗传分化。Fu’s Fs检验结果显示所有检验结果均为负值,中国西南部组(TC)和菲律宾南部组(ILO和DAV)中性检验结果显著(表5)。5个组经过错配分布计算转化得到的扩张曲线显示,中国西南部组腾冲(TC)种群和菲律宾北方组(BAL和IRRI种群)呈现单峰倒钟形曲线,其余各组均为多峰曲线(图3),表明这两个组在近期经历了瓶颈效应和种群扩张。TC种群的扩张时间约为92820年前,菲律宾北部组的扩张时间约为70940年前。MDIV结果表明,TC种群与其他种群间的分歧时间为2.13百万年前(2.13 Mya),菲律宾北方组与菲律宾组南方组的分歧时间为0.74百万年前(0.74 Mya),马来半岛组合中国南部组之间的分歧时间为1.13百万年前(1.13Mya),4个菲律宾种群与大陆种群的分歧时间为1.55百万年前(1.55 Mya)。3)采用DNA序列测定和AFLP分子标记分别对采自东南亚四个国家的小稻蝗进行检测,以探讨地质事件和气候变迁对小稻蝗种群遗传结构和谱系地理学格局的影响。175个个体用于线粒体DNA COI序列片段测定,232个个体用于AFLP分子标记研究。结果表明,在不同的地理区域间,遗传多样性并未随着海拔或者经纬度的变化而发生明显的差异,聚类树形成三个深度分歧的拓扑分支结构,这与由于高山和海峡隔离形成的三个地理结构相吻合。TCS网络结构图显示出三个不相连的网络结构,AMOVA结果表明在不同区域间的种群遗传分化极显著,说明不同区域间存在长期的时空隔离。所有单倍型首先以种群为单位相聚,在同一区域的不同种群之间有频繁的基因交流,但是不同区域之间的种群基因交流不明显。过去的地质事件和冰期过程中的气候反复是形成小稻蝗现有种群遗传结构和谱系格局的最重要因素。同时,栖息地、植被以及人为因素的影响也对该物种的基因流和基因渗入产生一定影响。此外,适宜的温度、充足的雨水和大量禾本科植物是小稻蝗扩散的有利条件。较高的遗传多样性、明显的种群分化、负的Fu’s Fs检验值和单峰及多峰分布模型,均表明12个小稻蝗种群经历了复杂的扩张过程,这可能是由于第四季冰川时期环境的不断变化所造成的。4)本研究以我国和菲律宾的小稻蝗12个种群和日本稻蝗10个种群为研究对象,选取体长(LB)、前翅长(LEL)、前胸背板长(LP)、后足股节长(LF)、后足股节宽(WF)5个性状,使用电子游标卡尺进行长度测量并统计数据。采用SPSS 11.5统计软件进行虫体测量值比较、性状的单因素方差分析和各性状间及其与海拔的相关性分析。结果表明：小稻蝗和日本稻蝗标本所有雌虫体长均明显大于雄虫。小稻蝗形态测量结果表明,中国腾冲(TC)、西藏(XZ)和万宁(WN)种群与菲律宾种群相比较在各个性状均具有显著性差异,其余种群间差异不显著。日本稻蝗形态测量结果表明,中国种群在各个性状特征测量值上均大于菲律宾种群,且彼此差异显著。各稻蝗种群由于生活在不同的气候中,环境因素导致不同种群的稻蝗外部形态特征出现较明显的差异；地理屏障导致的种群隔离也是形成形态差异的主要原因。日本稻蝗和小稻蝗TC种群在大部分性状上均与其他种群差异明显,相关性分析结果也表明5个性状均与海拔高度呈现显著的正相关,说明体型较大、运动能力较强的稻蝗更有利于在高海拔环境中生存。5)对采自云南腾冲的一稻蝗属待定种进行形态学鉴定和系统发育分析。形态学结果显示,该物种具有稻蝗属物种的一般形态特征。主要差异在于,体长比日本稻蝗和中华稻蝗等物种为小,与小稻蝗长度相似,但体宽大于小稻蝗。前翅长与稻蝗属其它物种具有明显差异,长度仅达后足股节的一半。雄性肛上板形状与小稻蝗相似,但两侧无疣状突起,端部中央向后延伸,长大于宽,呈三角形。雌性下生殖板中央无凹槽,两侧无隆起的脊,末端无齿。对该物种进行线粒体DNA Cytb基因片段序列测定,并与从GenBank下载的9个稻蝗属物种Cytb序列进行比对,采用NJ、MP和ML方法构建系统进化树,系统进化树显示12个Oxya sp个体构成独立的支,证明该物种是稻蝗属一新种。6)基于稻蝗属物种及近缘属种的大量RAPD-PCR结果,筛选出稻蝗属物种特异性的RAPD条带,对特异条带进行克隆、测序,序列同源比对后设计特异引物,以稻蝗属不同物种和蝗总科其它物种基因组DNA为模板进行PCR扩增。以随机引物S823扩增得到约650 bp的RAPD条带,这些条带在稻蝗属不同物种中序列高度同源,G+C含量大于15%,并富含大量的A、T重复区。基于同源区域设计的特异引物对稻蝗属物种可以扩增出目的条带,而对稻蝗属外的其它物种均无扩增条带。因此认为该条带属于稻蝗属特异RAPD条带,基于序列设计的特异引物可用于稻蝗属物种的快速分子鉴定。更多还原

【Abstract】 Oxya Serville, which is in Oedipodidae, Orthoptera, Insecta, is a worldwide agricultural pest. In recent years, most researches of Oxya genus are focus on Oxya chinensis using different methods, such as morphology, cytology, biochemistry and molecular biology. The study of other Oxya species is still limited.In this study, genetic diversity, genetic structure and phylogeography of three Oxya species were examined using DNA sequences, AFLP technology and morphological marker. The results were analyzed combined with informations of biogeography to discuss the genetic differnciation and ecological adaption. The main contents and conclusions are as follows:1) In this study, we used AFLP marker to investigate the genetic diversity and population structure of rice grasshoppers collected from south China, with emphasis on testing the hypothesis that there was significant genetic differentiation among grasshopper populations associated with different hosts (i.e. wild vs. cultivated rice). Seven populations consisting of 104 individuals were sampled from Hainan Island and the mainland of south China. Eight primer combinations produced 564 reliable bands, of which 563 were polymorphic. Oxya japonica showed considerable genetic variation at population level, with gene diversity (HE) ranging from 0.1103 to 0.2035. Genetic diversity were studied on seven populations, and generally three populations from wild rice had higher levels of genetic diversity (HE=0.1635) than the other four populations feeding on cultivated rice (HE=0.1327). We observed high population differentiation, with Fst ranging from 0.4172 to 0.7652 among the seven populations. However, Mantel test detected no significant correlation between genetic distance and geographical distance (r=0.3541; p=0.0689). By contrast, we found significant genetic differentiation between groups collected from different hosts. These data suggested that the anthropogenic activity in cultivated rice fields (i.e. pesticides, fertilization and cultivation) could have played an important role in shaping the genetic structure of O. japonica.2) 100 individuals of Oxya japonica were analyzed by using mitochondrial cytochrome oxidase subunit one (COI) DNA sequences collected in four Southeast Asian countries, to examine whether phylogeographic pattern and population genetic is related to past geological events and/or climatic oscillations. Four populations of Philippines have higher haplotype diversity and nucleotide diversity than populations from mainland. Five geographic groups were identified which congruent with the geographically separated. It is notable that networks presented deep genetic divergence pattern.Several unobserved haplotypes were existed between ach two adjacent geographical groups. AMOVA and FST value showed that high genetic variance among groups were found which indicating a relatively strong geographic structure for this species. LAMARC analysis showed that there had been frequent historical gene flow of O. japonica among local populations within region, while low gene flow existed among regions. Networks and high FST values all suggested that all clades diverged in allopatry from a common ancestor, and deep divergences were happened related to specific geologic events. Significantly negative values of Fu’s Fs tests and unimodal mismatch distributions of the frequencies of pairwise differences were presented in TC and north Philippines groups, which all indicated these populations had passed through recent demographic expansions after the bottleneck. The expansion times of TC and north Philippines groups were 92820 years ago and 70940 years ago respectively. The divergence time between north Philippines group and south Philippines group and Malaya group and south China group was 0.74 and 1.13 Mya respectively. The time between the Philippines populations and mainland populations was 1.55 Mya while the time between TC and other populations was dating back to 2.13 Mya during the late Miocene to early Pliocene.3) Population genetic diversity and structure of this Oxya species was examined using both DNA sequences and AFLP technology. The samples of 12 populations were collected from four Southeast Asian countries, among which 175 individuals were analysed using mitochondrial DNA cytochrome c oxidase subunit I (COI) sequences, and 232 individuals were examined using amplified fragment length polymorphisms (AFLP) to test whether the phylogeographical pattern and population genetics of this species are related to past geological events and/or climatic oscillations. No obvious trend of genetic diversity was found along a latitude/longitude gradient among different geographical groups. Phylogenetic analysis indicated three deep monophyletic clades that approximately correspond to three geographical regions separated by high mountains and a deep strait, and TCS analysis also revealed three disconnected networks, suggesting that spatial and temporal separations by vicariance, which were also supported by AMOVA as a source of the molecular variance presented among groups. Gene flow analysis showed that there had been frequent historical gene flow among local populations in different regions, but the networks exhibited no shared haplotype among populations. In conclusion, the past geological events and climatic fluctuations are the most important factor on the phylogeographical structure and genetic patterns of O. hyla intricata in Southeast Asia. Habitat, vegetation, and anthropogenic effect may also contribute to gene flow and introgression of this species. Moreover, temperature, abundant rainfall and a diversity of graminaceous species are beneficial for the migration of O. hyla intricata. High haplotype diversity, deep phylogenetic division, negative Fu’s Fs values and unimodal and multimodal distribution shapes all suggest a complicated demographic expansion pattern of these O. hyla intricata populations, which might have been caused by climatic oscillations during glacial periods in the Quaternary.4) Morphology of 12 Oxya hyla intricata populations and 10 Oxya japonica populations from China and Philippines was analyzed. Length of body, length of elytra, length of pronotum, length of femur, width of femur were chosen, measured by electronic vernier caliper and analyzed by using SPSS 11.5 software. Morphological difference between male and female individuals, morphological difference of different populations and the correlations among body measurement indices and between body measurements and elevation were analyzed in order to demonstrate the relation between morphology and environment. Female individuals were obviously larger than male individuals. The results of O. hyla intricata indicated that three populations (TC, XZ, WN) from China have have significant difference with all populations from Philippines,. The results of O. japonica indicated significant difference was exist between Chinese and Philippines populations. Based on the above results, we proposed that (i) Geographical barrier was the major reason for significant differentiation of morphological characters between Chinese and Philippines populations and (ii) these two Oxya species with large body and high mobile ability were benefit for survival in the high elevation area.5) Morphological description and phylogenetic analysis were conducted to indentify a species of the genus Oxya collected form Tengchong, Yunnan province. This species has most characters of genus Oxya, but differns in:1). the length of elytra was much smaller than other Oxya specie; 2). the epiproct was similar with O. hyla intricata but without wart at the edge.3). Female subgenital plate has no central fovea and edge teeth. Mitochondrial cytochrome b gene fragment sequence was analyzed and compared with other Cytb gene sequence of download from GenBank, and three phylogenetic trees were conducted base on NJ, MP and ML method. Phylogenetic tree indicated 12 individuals of Oxya sp. form a monophyletic group and suggested that this species belongs to genus Oxya.6) Oxya-specific RAPD band was screened out from lots of RAPD-PCR results, the specific band was cloned and sequenced, a pair of primer was designed based on homologue region after sequence alignment. PCR was performed by using designed specific primers, genomic DNA from different Oxya species and other Acridoidea species were used as template. A clear and reproductive band about 650 bp amplified from random primer S823 was present in different Oxya species, sequence analysis showed high nucleotide homology (92.3%-96.6%), each sequence determined consisted of 651-667 nucleotides, with G+C content >15% and repetitive A or T regions. Sequence characterized amplified region (SCAR) primers for specific PCR were developed, a expected specific band (550 bp) can be amplified using SCAR primers in seven species of Oxya examined but absent in Pseudoxya diminuta and other Acridoidea species. Oxya-specific SCAR molecular marker was identified, SCAR primers can be used for rapid identification of Oxya species.更多还原