【作者】 张敏；

【导师】 马恩波；

【作者基本信息】 山西大学 ， 动物学， 2008， 博士

【摘要】 蛱蝶科Nymphalidae隶属鳞翅目Lepidoptera凤蝶总科Papilionoidea,为蝶类中较大的科,具有很高的观赏价值和经济价值,但其幼虫绝大多数专以植物体为食料,虫口密度大,是粮食作物、果树、蔬菜、纤维作物、林木、竹类、药用植物以及绿肥牧草等的主要害虫。对蛱蝶科物种的分类研究由来已久,早期主要以形态特征为依据进行分类,近来,有学者采用分子生物学的方法来重建蛱蝶科部分物种的系统发育关系。然而,由于蛱蝶科物种形态多变且生活史各不相同,该科物种的系统发育关系一直比较混乱,成为昆虫分类学家争论的焦点。此外,由于蛱蝶科内各亚科、族及属间的系统发育关系并未澄清,以致无法阐明诸多研究结果的进化意义。研究该科物种的遗传多样性和系统分类学关系不仅具有理论意义,也对其生物多样性保护和农业植保实践中的防治具有较为重要的应用价值。RAPD(随机扩增多态性DNA)技术是1990年由Willianms和Welsh同时提出的一项DNA分子水平的多态性检测技术,因其简捷、成本低且信息含量丰富,被广泛用于生物类群种属分类鉴定、遗传图谱构建、物种亲缘关系和种群遗传学等领域的研究。该方法很快被昆虫学家所认可,广泛用于种群遗传学和系统发育研究。线粒体DNA基因和核基因的序列分析是重建物种系统发育关系及进化研究的有效分子标记,早已被广泛用于研究种群结构、基因缺失、杂交后代、生物地理学和系统发生关系。本文采用RAPD技术探讨了蛱蝶科闪蛱蝶亚科大紫蛱蝶和粉蝶科菜粉蝶不同地理种群之间的遗传多样性;采用线粒体DNA细胞色素b(Cyt b)、细胞色素c氧化酶亚基Ⅰ(COI)和核延长因子(EF-1α)基因作为分子标记,对我国闪蛱蝶亚科、蛱蝶亚科、线蛱蝶亚科和蛱蝶科部分物种的系统发育关系进行初步的探讨;结合GenBank下载序列和自测序列(DataⅠ),采用NJ、ML和Bayesian方法分别构建蛱蝶科部分物种系统发育树,并将后翅臀角斑、眼状斑和后翅外缘突起这3个形态性状标记在ML系统发育树上,对这3个形态性状进行进化分析,为更好地开展蛱蝶科系统分类学研究、生物多样性保护和防治提供了分子生物学的实验依据。此外,基于自行测定的和GenBank收录的线粒体COI基因和核EF-1α基因序列(DataⅡ)蛱蝶科亚科间的系统发育关系,结合眼蝶亚科、蛱蝶亚科和Dynamine alexaen物种的化石资料,进一步计算出蛱蝶科所有12个亚科间的首次分歧时间。本文的研究内容主要包括以下几个方面:1.主要采用实验室保存的干标本,比较了酚-氯仿法和饱和NaCl法提取蝶类干标本胸部、腹部和中后足肌肉基因组DNA的效果及对RAPD-PCR和线粒体Cyt b、COI基因及核EF-1α基因部分序列扩增结果的影响。研究结果表明:两种DNA提取方法从干制标本中后足肌肉提取的基因组DNA带型整齐,无拖尾;两种方法得到的基因组DNA均适用于RAPD-PCR扩增,而酚-氯仿法得到的基因组DNA在随后的线粒体Cyt b、COI基因及核EF-1α基因部分序列扩增中效果更好。2.采用RAPD技术分析了大紫蛱蝶三个地理种群的遗传多样性,并以同属的黑紫蛱蝶指名亚种及近缘属种黑脉蛱蝶指名亚种为外群,探讨了它们之间的亲缘关系及RAPD技术在蛱蝶属种间的适用性。经筛选的10个随机引物对供试的58只蝶类标本共产生200条扩增谱带,这些谱带具有明显的属、种间多态性。根据Nei’s遗传距离,分别用UPGMA和NJ法对其进行聚类,构建了分子系统树。聚类结果表明:大紫蛱蝶三个地理种群亲缘关系的远近与地理距离存在一定的相关趋势,湖北种群与山西种群间存在一定的基因交流。聚类结果与Nei’s遗传距离均表明:大紫蛱蝶与黑紫蛱蝶的亲缘关系较近,而黑脉蛱蝶与上述两物种的亲缘关系较远。聚类图所显示物种间亲缘关系与传统的形态分类结果基本一致。3.采用12条10 bp的随机引物,对菜粉蝶不同种群共75个个体的基因组DNA进行扩增,共产生143条清晰、稳定的谱带。Nei’s遗传距离表明菜粉蝶5个种群间的遗传距离差异较小。用UPGMA和NJ两种聚类方法对菜粉蝶5个种群所有个体构建聚类图,除长治种群和太原种群的位置有所不同外,聚类结果基本一致。UPGMA结果显示:代县种群和夏县种群最先相聚,随后这两个种群与大同种群聚为一支,太原种群再与上述三个种群相聚,长治种群与其余四个种群亲缘关系最远。菜粉蝶种群间遗传距离与其地理距离之间不存在相关性。4.测定了重要林业害虫闪蛱蝶亚科11属16种蝶类的线粒体细胞色素氧化酶Ⅰ基因的部分序列,并结合由GenBank下载的该亚科4种蝶类的相应序列进行分析,探讨了闪蛱蝶亚科各属间的系统发育关系。以玉杵带蛱蝶,白斑眼蝶,忘忧尾蛱蝶和白带螯蛱蝶作为外群,采用PAUP~* v 4.0b4a软件构建了闪蛱蝶亚科的MP和NJ分子系统树。在所构建的系统树中,由分子数据得到的蛱蝶亚科系统发育关系与传统分类学的基本一致,其中迷蛱蝶属为单系群;累积蛱蝶为明窗蛱蝶的姐妹群且支持两物种亲缘关系近的自展置信度很高;支持将白斑迷蛱蝶,迷蛱蝶,夜迷蛱蝶,栗铠蛱蝶,黄带铠蛱蝶,铂铠蛱蝶以及银白蛱蝶等物种由闪蛱蝶属中移出的修订。5.参照周尧《中国蝶类志》中蛱蝶亚科的分类系统,通过测定蛱蝶亚科15属24种蝶类的线粒体细胞色素氧化酶Ⅰ基因部分序列,并结合由GenBank下载的该亚科8种蝶类的相应序列,首次基于分子数据研究了国内蛱蝶亚科部分物种的系统发育关系。采用NJ,ML和Bayesian三种方法分别构建了蛱蝶亚科的分子系统树,所产生的拓扑结构完全一致且置信度高。在所构建的分子系统树中,由分子数据得到的蛱蝶亚科系统发育关系与传统分类学观点存在一些分歧,其中发现蛱蝶族和斑蛱蝶族非单系类群;支持将眼蛱蝶属由蛱蝶族移置斑蛱蝶族(=眼蛱蝶族)中;盛蛱蝶属和蜘蛱蝶属与蛱蝶族内其余属的关系较远;而蛱蝶族中各属间的关系为((((蛱蝶属+琉璃蛱蝶属)+钩蛱蝶属)+麻蛱蝶属)+红蛱蝶属)+(盛蛱蝶属+蜘蛱蝶属),为我国蝶类分子系统发育研究提供了新的资料。6.为了探讨线蛱蝶亚科(参照Harvey的分类系统,1991)的分类地位,根据自行测定的和GenBank收录的线粒体COI基因和核EF-1α基因序列构建了线蛱蝶亚科、闪蛱蝶亚科、蛱蝶亚科、眼蝶亚科、斑蝶亚科、绢蛱蝶亚科、秀蛱蝶亚科、螯蛱蝶亚科、摩尔福蝶亚科和喙蝶亚科(参照Harvey的分类系统,1991)部分物种的NJ、ML和Bayesian系统树。系统树显示曾被Harvey归为苾蛱蝶族、线蛱蝶族、丝蛱蝶族和秀蛱蝶族的物种各自聚为相应的聚类簇,且线蛱蝶族的物种与其余3个族的亲缘关系很远,支持将苾蛱蝶族、线蛱蝶族、丝蛱蝶族和秀蛱蝶族分别变更为相应亚科的分类学观点。此外,根据重新界定的线蛱蝶亚科分类群,测定了该亚科9属21种蝶类的线粒体细胞色素氧化酶Ⅰ基因的部分序列,并结合由GenBank下载的该亚科17种蝶类的相应序列进行分析,首次探讨了线蛱蝶亚科各属的系统发育关系。结果与传统分类学的观点基本一致,支持线蛱蝶亚科的单系性,并对线蛱蝶亚科中各族间的系统学关系给出了合理的假设。7.实验测定蛱蝶科(参照周尧的《中国蝶类志》)9亚科51种蝶类的线粒体细胞色素b基因的部分序列,以凤蝶科金凤蝶作为外群,采用PAUP~* v 4.0b4a软件构建了蛱蝶科的NJ系统树。系统树显示各亚科均为非单系类群,各属种间的系统发育关系较为混乱,多数分支的自展支持率很低。可能由于本实验所测定线粒体DNA Cyt b基因的片段较短,加上线粒体基因进化速率相对较快,密码子第三位点可能存在碱基替换饱和的现象,导致信息含量偏低而不能正确反应所研究类群的系统发育关系。因此,我们基于线粒体DNA COI基因和核EF-1α基因部分序列的联合数据构建蛱蝶科部分物种的系统发育树,以期揭示蛱蝶科各亚科间更为真实可靠的系统发育关系。采用DataⅠ和DataⅡ数据集构建的系统发育树均表明蛱蝶科部分亚科关系为:((((蛱蝶亚科+闪蛱蝶亚科)+(丝蛱蝶亚科+苾蛱蝶亚科))+秀蛱蝶亚科)+(袖蛱蝶亚科+线蛱蝶亚科))。比较采用DataⅠ和DataⅡ数据集构建的系统发育树,结果显示应将摩尔福蝶亚科归入眼蝶亚科中,而闪蛱蝶亚科中的各物种科分为两个族:闪蛱蝶族和铠蛱蝶族。8.基于自行测定的和GenBank收录的线粒体COI基因和核EF-1α基因部分序列(DataⅠ数据集)构建系统发育树,并将后翅臀角斑、眼状斑和外缘突起这3种形态性状标记在基于DataⅠ数据集构建的分子系统发育树上。结果表明这3个性状在蛱蝶科内可能是多次起源的。9.基于自行测定的和GenBank收录的线粒体COI基因和核EF-1α基因部分序列(DataⅡ数据集),应用最大似然法、贝叶斯推断法及马尔可夫链蒙特卡罗方法构建世界蝶类最大科——蛱蝶科亚科间的系统发育树,并对该科分子系统发育树各分支间的进化速率进行了差异显著性检验。结合眼蝶亚科、蛱蝶亚科和Dynamine alexaen物种的化石资料,计算获得了蛱蝶科所有12个亚科间的首次分歧时间的平均估计值为44.2-87.1百万年前(MYA)。结果有助于人们深入了解该科的起源与进化以及估计蝶类和其它复杂类群的分歧时间。更多还原

【Abstract】 Nymphalidae（Lepidoptera:Papilionoidea） is a big family in Papilionodea.Butterflies in this family are beautiful and distribute in all continents of the world except Antarctica.Larvae of most species in this family represents important pest to foodcrops,fruit tree,vegetable,fibre crop, forest,bamboo,and so on.The systematics of Nymphalidae butterflies based on morphological characteristics has been studied by many authors for more than one century.In recent years,some researchers restructure the phylogenetic relationships among part of species from Nymphalidae through using methods and techniques of molecular biology.However,as the diverse shapes and life history of Nymphalidae butterflies,the phylogenetic relationships of the species in this family are very unclear all the time,which have been argued by taxonomist.Furthermore,it can not be illustrated that the evolution significance of many research results because the unclear phylogenetic relationships of subfamilies,tribes and genera in Nymphalidae. Therefore,research on the genetic diversity and phylogenetic relationship of this family not only has theory significance,but also possesses important practical value for protection of biology diversity as well as pest management.RAPD（Random Amplification Polymorphic DNA） method using for the identification of polymorphism and analyzing phylogenetic relationships among and within closely related species was first described by Willianms and Welsh in 1990.RAPD markers are based on the amplification of unknown DNA sequences using single,short,random oligonucleotide primers.The RAPD method is advantageous over RFLPs in technical ease, quickness,low cost and fewer amounts of samples required.It has been widely applies to the organisms including bacteria,plants and vertebrates for taxonomy and species identification,as well as construction of genetic mapping,genetic relationships among species and population,and locating pathological important genes.The RAPD method has also been soon adopted by entomologists and widely used to study taxonomy and phylogenetic relationships of insects.The sequence analyses of mtDNA and nuclear genes are effective molecule markers for reconstructing the phylogenetic relationships and evolution research,which has been widely used to investigate population structure,deletion of gene,hybridization, biogeography and phylogenetic relationships.In this study,we investigated the population-level genetic diversity of Sasakia charonda or Pieris rapae in China based on RAPD markers; preliminarily examined phylogenetic relationships of Apaturinae, Nymphalinae,Limenitidinae and Nymphalidae by analyzing sequences of the mtDNA Cyt b,COI and nuclear EF-1αgenes;reconstructed the phylogenetic trees of Nymphalidae based on the mtDNA COI and nuclear EF-1αgenes （Data I） by using NJ,ML as well as Bayesian methods,and then mapped 3 morphological characters on the ML tree for investigating the evolutionary patterns of these morphological characters.On the basis of the results of the phylogenetic relationships of Nymphalidae reconstructed based on the sequences determined from the 13 species in our laboratory and additional sequences of other 43 species found in GenBank（DataⅡ） and fossil information of Satyrinae,Nymphalinae and Dynamine alexaen,the average divergence times among the subfamilies are then estimated for the first time. The main contents and conclusions are as follows:1.Genome DNA of butterflies from the breast muscle,the abdomen muscle, and the midleg and hindleg muscle of dry samples was extracted using phenol-chloroform protocol and saturated NaCl methods.Genome DNA extracted by two methods from the midleg and hindleg muscle was quite good for RAPD analyses.However,the genome DNA extracted by phenol-chloroform protocol methods from the midleg and hindleg muscle suits to analyses of sequences of mtDNA and nuclear genes.2.RAPD markers were applied to analyze genetic divergence among three populations of Sasakia charonda,Sasakia funebris and Hestina assimilis. Genomic DNA of fifty-eight individuals was extracted from dissected leg muscle using saturated NaCl method and then amplified by 10 random primers（10 bp） which were previously selected,a total of 200 clear and reproducible bands were generated,the RAPD patterns showed polymorphic variations among and within different genera and species. The amplified fragments were analyzed by the Phyltools 6.0,the molecular phylogenetic tree based on Nei’s genetic distance of RAPD markers was constructed using UPGMA and Neighbor-Joining.The dendrogram indicated strong similarities within populations.Among three populations of Sasakia charonda,the individuals of Hubei population and Shanxi population closely clustered together;and then they were clustered with Liaoning population.These data suggested that several implications. Firstly,RAPD is very useful as an effective molecular marker to distinguish the divergence in Sasakia and its closed related genera. Secondly,it seems to be a tendency of correlations between the genetic and geographic distance among the 3 different populations of Sasakia charonda from the south to the north.Thirdly,the RAPD similarity levels follows the order of individuals > populations > species > genera.The results of dendrogram based on RAPD markers are consistent with the previous conclusions of morphologic classification.3.The genetic diversity and genetic relationship of five populations of the Pieris rapae in Shanxi province determined using random amplified polymorphic DNA（RAPD）.Nei’s genetic distances between populations were very low and minor different,showed that a high similarity among the five populations.The dandrogram based on Nei’s genetic distance of RAPD markers was constructed using UPGMA and Neighbor-Joining. This result indicated that Daixian population was closer with Xiaxian population,then the two populations clustered with Datong population, and finally,aider the Taiyuan population grouped together with them,the four populations clustered with the CZ population.The genetic relationship was not consistent with the geographic distribution.4.The phylogenetic relationships of genera in the subfamily Apaturinae were examined using mtDNA sequence data from 1471 bp of cytochrome oxidase subunit I（COI）.The mitochondrial COI gene from a total of 16 species in 11 genera were sequenced to obtain mtDNA data,along with those of 4 species obtained from GenBank,to construct the MP and the NJ trees using Athyma jina,Penthema adelma,Polyura nepenthes and Charaxes bernardus as outgroups.Within the Apaturinae,the clustering results are approximately identical to the classical morphological classification.The mtDNA data suggests the genus Mimathyma as a monophyletic group.Lelecella limenitoides and Dilipa fenestra have close relationship with very strong support in all phylogenetic trees.It also supports the taxonomic revision of removing several species from Apatura to other genera,namely Mimathyma schrenckii,M.chevana,M. nycteis,Chitoria subcaerulea,C.fasciola,C.pallas,and Helcyra subalba.5.The phylogenetic relationships of the subfamily Nymphalinae（sensu Chou,1994） was analyzed based on 1488 bp of mtDNA cytochrome oxidase subunit I（COI） gene sequence data obtained from 24 individuals, along with those of 8 species obtained from GenBank.The phylogenetic trees were reconstructed by NJ,ML and Bayesian methods through using Byblia anvatara as outgroup.Our data indicated that the tribes Nymphalini and Hypolimni（sensu Chou,1994） are not monophyletic groups,and the genus Junonia should be removed from Nymphalini to Hypolimni（= Junoniini）.Based on our data,the Symbrenthia and Araschnia had a relative distant relationship with the rest of Nymphalini. The relationships of species in the Nymphalini were confirmed via the NJ, ML and Bayesian methods,namely（（（（Nymphalis + Kaniska） + Polygonia） + Aglais） + Vanessa） +（Symbrenthia + Araschnia）.This investigation provides a little novel information for Chinese researches of butterflies.6.To investigate the phylogenetic position of the subfamily Limenitidinae （sensu Harvey’s,1991）,we reconstructed the phylogenetic trees of Limenitidinae,Apaturinae,Nymphalinae,Satyrinae,Danainae, Heliconiinae,Charaxinae,Morphinae and Libytheinae（sensu Harvey’s, 1991） based on COI and EF-1αsequences by using ML,NJ and Bayesian method.The results show that species classified into Biblidini,Cyrestini, Pseudergolini and Limenitidini by Harvey cluster together,respectively, and Limenitidini is farther with Biblidini,Cyrestini and Pseudergolini, supporting that Biblidini,Cyrestini,Pseudergolini and Limenitidini should be classified into subfamilies Biblidinae,Cyrestinae,Pseudergolinae and Limenitidinae,respectively.The phylogenetic analyses of the re-delimited Limenitidinae is presented based on 1471 bp of mtDNA cytochrome oxidase subunit I（COI） gene sequence data obtained from 21 individuals spanning 9 genera for the first time,along with those of 17 species obtained from GenBank.Our data support the monophyly of the subfamily Limenitidinae.The genus Limenitis can be split into two subclusters.Moreover,the results give one of the strongest hypotheses for the tribe relationships within Limenitidinae.7.The phylogenetic relationships of the Nymphalidae（sensu Chou,1994） was analyzed based on 432 bp of mtDNA Cyt b gene sequence data obtained from 51 individuals in 9 subfamilies.The phylogenetic trees were reconstructed by NJ method through using Papilio machaon as outgroup.The phylogenetic tree shows that each subfamily is not monophyletic and the phylogenetic relationships are disordered,resulting from short sequences of Cyt b and saturation on the 3^rd codon,which have led to reconstruct an untrue phylogenetic tree.Then,we reconstruct phylogenetic relationships of Nymphalidae based on the COI and EF-1αsequences（DataⅠand/or DataⅡ） to find the "true tree".The phylogenetic trees constructed based on DataⅠand DataⅡshow that phylogenetic relationships of partial subfamilies in Nymphalidae are as （（（（Nymphalinae + Apaturinae） +（Cyrestinae + Biblidinae）） + Pseudergolinae） +（Heliconiinae + Limenitidinae））.The results indicate that it seems reasonable to divide the subfamily into two Vibes:Apaturini and Chitorini and replaced the Morphinae into Satyrinae.8.We mapped the states of the three characteristics（i.e.,spot of anal angle, eyespots,and process from outer margin of hind wing） on the internal nodes of the combined phylogenetic tree,which was based on the COI and EF-1αsequences（DataⅠ） by using maximum likelihood method.The results suggested the 3 characteristics might have repeatedly evolved in the family Nymphalidae.9.We constructed the phylogenetic trees of Nymphalidae based on a combined dataset of the COI and EF-1αsequences（DataⅡ） using ML and Bayesian methods.On the basis of the results of the phylogenetic relationships among subfamilies in Nymphalidae and the relative-rate tests as well as fossil information,the average divergence times among the subfamilies are estimated as 44.2 - 87.1 million years ago.The results will help us for understanding the origin and evolution of this family.更多还原