【作者】 叶维萍；

【导师】 黄原；

【作者基本信息】 陕西师范大学 ， 动物学， 2004， 硕士

【摘要】 斑翅蝗科(Oedipodidae)隶属于直翅目蝗亚目蝗总科(Orthoptera：Caelifera：Acrididae)。由于蝗虫对农牧业的影响，对蝗虫系统学以及蝗虫防治的研究一直受到广泛重视。分子生物学技术促进了昆虫分子系统学的兴起和发展，但目前国内外对斑翅蝗科昆虫的分子系统学研究远远落后于其它生物类群。本研究以12S rRNA基因为分子标记，探讨其分子进化机制并建立该科分子水平系统发育关系，为确定它们之间的关系提供分子生物学方面的证据。 12S rRNA基因由于其分布的普遍性、序列和结构的保守性以及进化速率镶嵌性而被广泛应用于不同分类阶元层次上的分子系统学研究。本研究采用PCR产物直接测序法测定了斑翅蝗科4个亚科10属19种昆虫12SrRNA基因546bp的片段，以从NCBI下载的一种蟋蟀Gryllus campestris的相应序列为外群序列，利用Clustal X进行序列比对，结合12S rRNA二级结构模型进行茎区(stem)和环区(loop)碱基的划分。MEGA 2.1进行序列组成统计。PAUP~*4.0b10(PPC)对数据集系统发育信号进行评估，并以NJ、MP，ML和贝叶斯系统发育推论法分别重建系统发生树。 最后得到的结论如下： 1．本研究得到19种斑翅蝗科昆虫长为546bp的12S rRNA基因片段，其序列组成分析显示典型的高AT含量(75.4％)和多变的距离依赖的转换／颠换(TS／TV)比。 2．12S rRNA基因546bp片段根据二级结构模型划分为茎区和环区，对全数据组、茎区数据组和环区数据组分别进行树长分布分析和PTP检验，两者均显示三个数据组包含较强的系统发育信号。对三个数据组分别重建的简约树显示，全数据组系统树分辨率最高，解决的分支最多，而茎区和环区数据组分别解决了部分分支的系统关系，说明茎区碱基和环区碱基各自包含不同的系统发育信息，在12S rRNA基因的系统发育应用中选用全序列比较合适。对茎区和环区碱基赋以不同权值得到的MP树与未加权结果相同，说明在该区段，茎区碱基的补偿性突变对本研究影响不大。 3．本研究采用MP，NJ，ML和BI法分别重建了斑翅蝗科19种昆虫的系统发生关系，并采用基于似然值的KH和SH检验比较了这四棵系统树，结果认为MP树的似然值(-Ln L)最小，但它们的似然值差异没有达到统计学显著性，所以认为这四种拓扑结构的系统树都很好的解释了该数据集。四种方法没有达到一致的结果，说明该数据集在解决这19个种的系统发生关系上提供的信息还是不够的。 4.四种方法合一树的结果中，包含清晰的两分支，即痴蝗支和飞蝗支。痴蝗支包括异痴蝗属、痴蝗属和皱膝蝗属，因为轮纹异痴蝗和两种皱膝蝗聚为一支，黄胫异痴蝗和两种痴蝗聚为一支，故本文认为异痴蝗亚科和痴蝗亚科应该合并为一个亚科。飞蝗支包含飞蝗属、小车蝗属、车蝗属、疵蝗属和绿纹蝗属5个属，建议合并为飞蝗亚科。这些类群的关系可以表示为:(((白边痴蝗，青海痴蝗)黄胫异痴蝗)((红翅皱膝蝗，鼓翅皱膝蝗)轮纹异痴蝗))(((亚洲飞蝗，东亚飞蝗，西藏飞蝗)(((黄胫小车蝗，红胫小车蝗)亚洲小车蝗)云斑车蝗)庆蝗)花胫绿纹蝗)。其它束颈蝗和蓝斑翅蝗的归属本研究没有得到一个定论。 5.125 rRNA基因部分片段没有完全解决斑翅蝗科19种的系统关系问题，关键问题在于所测序列长度太短，提供的系统发育信息不足以给出这19种昆虫全面正确的系统关系，测定基因全长或增加其它基因序列可能有助于该问题的解决。 本研究对斑翅蝗科19种125 rRNA基因序列的测定是科学上首次对中国斑翅蝗科昆虫进行的较大规模的线粒体基因序列分析，通过向GenBank提交所测蝗虫基因序列，与世界昆虫学界共享序列数据。通过后续分析，探讨斑翅蝗科内的系统发育关系，提供了分子数据方面的观点。更多还原

【Abstract】 Oedipodidae is a large family in Acridoidea (Orthopera: Caelifera). In China, it includes 37 genera 124 species. Researches on controlling over plague of locusts were of great significance on agriculture. Molecular biological techniques were applied to insect systematics and much important progress has been made. However, studies on molecular phylogeny of Chinese Oedipodidae are rather few, and a comprehensive phylogeny is lacking. Here we report a phylogenetic analysis of Chinese band-winged grasshoppers based on DNA sequence of mitochondrial 12S rRNA gene. The goals of our study were to better resolve the phylogenetic relationships among Chinese band-winged grasshoppers and investigate the effectiveness of 12S rRNA gene sequences for reconstructing the phylogeny of Oedipodidae.Mitochondrial small subunit ribosomal RNA (12S rRNA) gene has been used for phylogenetic analyses of a wide range of species and divergence levels for their universal occurrence, sequence and structure conservation and abundance. We adopted PCR products direct sequencing technique to sequence fragments of 12S rRNA gene of 19 Oedipodidae species in China (representing 4 subfamilies and 10 of 37 genera). An outgroup, Gryllus campestris ’s corresponding fragment from NCBI, and all the sequenced fragments were aligned using Clustal X and the alignment length was 546bp. All nucleotide sites were grouped to stem set and loop set according to the secondary structure model of 12S rRNA. Statistics of sequence compositions and nucleotide substitutions were obtained using MEGA2.1. PAUP*4.0blO (PPC) was used to evaluate the phylogenetic information content of the data and reconstruct NJ, MP and ML trees. Bayesian analyses were also performed with MrBayes V3.0. Sequences and phylogenetic analyses have been arrived at conclusions as follows:1. Fragments of 12S rRNA gene of 19 Oedipodidae taxa showed patterns, typical of many insects, such as high A+T content and variable distance-dependent transition/transversion (TS/TV).2. Analyses of tree length distribution and PTP test of combined data set, loop set and stem set showed strong phylogenetic signals and a strong correction of characteristics beyond that expected by random. MPT reconstructed from three data setssuggested that combined data set produced a more constructive topology while loop set and stem set separately resolved parts of the topology, which showed stem and loop of 12S rRNA gene contained different phylogenetic information and combined data was better for reconstruction. The weighted MP and unweighted MP analyses got the identical topology, which showed that stem bases’ compensatory substitution didn’t influence the phylogenetic analysis. Reflected from wMP analysis and corrected distance reconstruction, potential substitution saturation showed by saturation analyses also didn’t matter much in reconstruction.3. Likelihood-based KH and SH tests of four topologies from MP, NJ, ML and Bayesian inference (BI) showed that although the MP tree had the lowest likelihood score, the KH and SH tests showed no significant difference in the -Ln likelihood between four trees so that MP tree was not significantly better than other trees. Therefore we can’t reject NJ, ML and BI trees in favor of MP tree. Four trees were all considered good interpretations of the data provided. We concluded that the data couldn’t provide enough phylogenetic information to fully resolve the relationship of the 19 species in Oedipodidae.4. The 50% consensus tree of MP, NJ, ML, BI trees showed two distinct clades: One clade includes all species of Bryodeminae and Bryodemellinae sampled, and the two Bryodemella taxa separately clustered with Angaracris and Bryodema taxa, so we considered it was appropriate to merge them as one subfamily; the other clade includes Bryodemella, Oedaleus, Gastrimarygus, Trilophidia and Aiolopus. The relationship of the species in these two clades was: (((Bryodema luctuosum luctuosum, Bryodema miramae miramae)更多还原