用线粒体Cytb基因序列探讨冬虫夏草寄主蝠蛾的系统进化关系

为探讨冬虫夏草寄主蝠蛾种属间的系统进化关系,采用CTAB法分别从18个冬虫夏草居群的虫草虫体头部和2种寄主蝠蛾中提取基因组DNA,PCR扩增获得冬虫夏草寄主线粒体Cytb基因片段序列.结合GenBank中的蝙蝠蛾科24种冬虫夏草寄主的同源序列,构建NJ系统树.结果表明,双栉蝠蛾属(Bipectilus)先于蝠蛾属(Hepialus)和类蝠蛾属(Hepialiscus)分化;蝠蛾属的冬虫夏草寄主种类众多形态各异,且均有特定的地理分布,蝠蛾属的种间遗传分化也较明显,蝠蛾属可能是多系起源;Cytb基因序列在冬虫夏草寄主蝠蛾种属间存在较丰富的变异,在蝠蛾属中除草地蝠蛾和金沙蝠蛾的该段序列完全一致外,其他种类蝠蛾种间变异率为0.23%—9.24%,Cytb基因序列可用于冬虫夏草寄主昆虫种的鉴定,但有必要获取更多种类的寄主昆虫来进一步验证.通过对冬虫夏草虫体头部提取的DNA进行PCR扩增获得其寄主蝠蛾Cytb基因序列的方法准确有效,有助于进一步获取更多的基因序列对更多的冬虫夏草居群和寄主种类进行系统发育、居群遗传结构及菌虫相互关系等研究.     

拟步甲科部分种类Cytb基因序列比较及系统发育分析

测定了拟步甲科(Tenebrionidae)10种昆虫线粒体细胞色素b(Cytb)基因长度为579 bp的序列片段,结合Genbank的赤拟粉甲(Tribolium castaneum)的同源序列进行了分析.结果表明,11种拟步甲昆虫该片段中碱基T,C,A,G的平均含量分别为34.0%,22.5%,32.8%和10.7%, A T平均含量明显高于G C平均含量.密码子第3位点A T含量高达75.3%,而该位点G的平均含量仅为3.2%.碱基替换多发生于第3位点,转换略多于颠换,转换主要以C←→T为主,颠换以A←→T为主.以步甲科Blackburnia fossipennis为外群构建的系统发育树表明,土甲族、拟粉甲族和琵甲族有着很近的亲缘关系,三者聚为一支,漠王族和漠甲族聚为一支;而鳖甲族这一分支的系统关系与传统的分类存在一定的分歧,表现出与拟步甲亚科、漠甲亚科有着平行的进化关系,对产生这种分歧的原因进行了探讨.     

蜻科部分种类Cytb基因序列比较及系统发育分析(蜻蜒目:差翅亚目)

从分子生物学角度入手,以mtDNA的Cytb(Cytochrome b)基因作为分子遗传标记,用昆虫Cytb基因的特异性引物进行PCR扩增及DNA测序,共获得蜻蜓目差翅亚目蜻科7属8种(基斑蜻Libelluladepressa,黄蜻Pantala flavescens,庆褐蜻Trithemis festive,晓褐蜻Trithemis aurora,纹蓝小蜻Diplacodestrivialis,网脉蜻Neurothemis fulvia,六斑曲缘蜻Palpoleura sex-maculata,玉带蜻Pseudothemis zxmata)及2种外群(黄翅绿色螅Mnais auripennis,华艳色螅Neurobasischinensis)的Cytb基因部分序列(576 bp).这8种蜻蜓Cytb基因的部分核苷酸序列(长度为576 bp)中碱基T,C,A,G的平均含量分别为37.0%,18.3%,31.3%和13.4%,A T平均含量为68.3%,明显高于G C平均含量(31.7%).密码子第3位点的A T平均含量较高,为81.0%,碱基替换多发生在密码子第3位点.以束翅亚目的黄翅绿色螅和华艳色螅作为外群构建系统发育树,结果显示玉带蜻属与褐蜻属、脉蜻属与曲缘蜻属的亲缘关系较近.     

蜻科部分种类Cytb基因序列比较及系统发育分析（蜻蜓目：差翅亚目）

从分子生物学角度入手，以mtDNA的Cytb（Cytochrome b）基因作为分子遗传标记，用昆虫Cytb基因的特异性引物进行PCR扩增及DNA测序，共获得蜻蜓目差翅亚目蜻科7属8种（基斑蜻Libellula depressa，黄蜻Pnntala flavescens，庆褐蜻阢thernis festive，晓褐蜻研themis aurora，纹蓝小蜻Diplacodes trivialis，网脉蜻Neurothemis fulvia，六斑曲缘蜻Palpoleura sex-maculata，玉带蜻Pseudothemis zonata）及2种外群（黄翅绿色螅Mnais auripennis，华艳色螅Neurobasis chinensis）的Cytb基因部分序列（576bp）．这8种蜻蜓Cytb基因的部分核苷酸序列（长度为576bp）中碱基T，C，A，G的平均含量分别为37．0%，18．3％，31．3％和13．4％，A＋T平均含量为68．3％，明显高于G＋C平均含量（31．7％）．密码子第3位点的A＋T平均含量较高，为81．0％，碱基替换多发生在密码子第3位点．以柬翅亚目的黄翅绿色螅和华艳色蟪作为外群构建系统发育树，结果显示玉带蜻属与褐蜻属、脉蜻属与曲缘蜻属的亲缘关系较近．     

基于Cyt b基因序列变异探讨鲱形目系统发育关系

鲱形目内科间分类关系一直以来存在较多争议,为探讨鲱形目系统发育关系,对黄鲫、凤鲚的线粒体Cyt b基因全序列进行扩增、克隆及测序,得到1 141 bp序列。结合Genbank中已发布的鲱形目鱼类Cyt b基因全长序列利用生物信息学软件进行比较分析后,共检测到553个变异位点,总变异为48.5%,其中含504个简约信息位点,未见序列插入或缺失。碱基的替换多在密码子第三位,序列中转换多于颠换,Ts/Tv为2.448,基因突变未达到饱和。基于K2P模型计算的遗传距离表明,宝刀鱼科同鲱科及鳀科的科间遗传距离(0.042~0.072)小于鲱科内属间遗传距离(0.065~0.106)。在以南美肺鱼为外群构建的NJ分子系统树中,齿头鲱科最先分化,是鲱形目中原始类群;鳓属、多齿鳓属独立聚为一支,且具较高的自举检验值,支持将其归入锯腹鳓科;宝刀鱼科并入到鲱科一支中去,与鲱科亲缘关系更近。     

基于线粒体蛋白编码基因探讨飞虱科Delphacidae13属系统发育关系

飞虱科昆虫隶属于半翅目蜡蝉总科,是世界上最重要的农业害虫之一。昆虫的线粒体基因是昆虫分子与进化研究中常用且有效的分子标记。本研究基于线粒体蛋白编码基因探讨了飞虱科13属18种的系统发育关系,分别采用距离法、最大似然法和贝叶斯法构建的系统发育树得到的结论是一致的,即在飞虱科13属中,Ugyops属较为原始,处于系统树的基部,其余12个属聚为2支,Bambusiphaga属、Epeurysa属、Tropidocephala属和Saccharosydne属聚为一支,Peregrinus属、Perkinsiella属、Changeondelphax属、Chloriona属、Ishiharodelphax属、Sogatella属、Laodelphax属和Nilaparvata属聚为另一支。18种的进化地位和分类归属与传统分类结果基本一致。     

从COI基因序列探讨叶甲科部分昆虫的系统关系

对叶甲科21种昆虫的COI基因（长度为716bp）的序列组成和变异情况进行了分析．结果表明,21种叶甲碱基T、C、A、G的平均含量分别为39．5％、13．9％、32．1％和14．5％,A＋T含量明显高于G＋C含量,密码子第三位点A＋T平均含量高达90．4％;该序列片段中共有315个碱基发生变异,变异率达到44％;核苷酸替换主要发生在C←→T和A←→T之间,第三位点的替换频率显著高于前两个位点．以中华萝摩叶甲（Chrysocus chinensis）为外群,采用MEGA4．0软件构建系统发育树．结果显示,21种叶甲聚为2大分支,叶甲亚科的种类形成一大分支,位于系统树的基部,较其他2个亚科分化得早;跳甲亚科与萤叶甲亚科共同聚为另一分支,二者的关系较之与叶甲亚科的关系更近．系统树所反映的三亚科间的关系与传统分类结果一致．     

从叶绿体DNA rbcL序列分析探讨五味子科的系统发育

以广玉兰Magnolia grandiflora，蝙蝠葛Menispermum dauricum，牡丹Paeonia suffruticosa和Illicium parviflorum为外类群，对五味子科的叶绿体rbcL基因进行序列分析，探讨其系统发育关系．结果表明，五味子科为一单系类群，与八角科关系最近，与木兰科关系较远．在严格一致性树中(Length=256，CI=0．8725，RI=0．8192)，五味子科被分为二支，一支由五味子属中的中华五味子亚属Subgen．Sinoschisandra，多蕊五味子亚属Subgen．Pleiostema和少蕊五味子亚属Subgen．Maximowixzia构成，支持率82％；另一支包括南五味子属和五味子属中的团蕊五味子亚属Subgen．Sphaerostema，重瓣五味子亚属Subgen．Plenischisandra．由此推断，五味子属和南五味子属关系密切，它们之间存在交叉和重叠，两属可能起源于共同的祖先．通过基因树分析，结合形态学、解剖学和化学证据认为，五味子S．chinensis和红花五味子S．rubriflora是五味子属中的原始类群．     

基于RLCKVⅡ基因序列的蜘蛛抱蛋属系统发育分析

蜘蛛抱蛋属(Aspidistra Ker-Gawl.)是单子叶植物中同一属内物种多样性最为丰富的类群之一.由于缺乏分子序列方面的研究资料,该属种间系统发育关系一直都不清楚.本研究对20种具有代表性的蜘蛛抱蛋属植物的RLCKVⅡ基因进行了测序分析.结果表明,采用分子系统学证据所构建的该属系统发育关系不支持先前基于形态证据所建立的分类系统;蜘蛛抱蛋属植物复杂的平行进化关系很可能是该属分化早期频繁种间杂交的结果.RLCKVⅡ基因适用于禾本科、棕榈科和天门冬科等类群,可作为研究单子叶植物系统发育关系的备选基因.     

中国双翅目花蝇科（Diptera：Anthomyiidae）各属间系统发育关系研究

本文分别运用PAUP4.0b和MacClade 4.0软件,选取中国花蝇科目前已知的34个属的模式种或代表种为内群,蝇科Muscidae家蝇属家蝇Musca domestica Linnaeus,1758和丽蝇科Calliphoridae丽蝇属反吐丽蝇Calliphora vomitoria（Linnaeus,1758）为外群,对总计36个分类单元进行了支序分析研究,初步分析并探讨了中国花蝇科各属间的形态系统发育关系。     

Phylogenetic relationships of host insects of Cordyceps sinensis inferred from mitochondrial Cytochrome b sequences

This study used the sequence of the mitochondrial Cytochrome b(Cytb)to estimate phylogenetic relationships among host Hepialidae insects of Cordyceps sinensis.Genome DNA of host insect was extracted from the dead larva head part of 18 cordyceps populations and 2 species of Hepialus,and the Cytb fragment of host insect was amplified with PCR technique.The nucleotide sequence alignments and their homologous sequences of 24 species host Hepialidae insects of Cordyceps sinensis were obtained from GenBank and were used to construct phylogenetic trees based on neighbor-joining method.The results showed that genus Bipectilus diverged earlier than genus Hepialus and Hepialiscus.Hepialus host insects of Cordyceps sinensis have multitudinous species with different morphological characteristics and geographical distributions.The interspecific genetic differentiations are obvious in Hepialus.Thus,the genus Hepialus might be considered as polyphyletic origin.Cytb sequences have abundant variations among the host insects of Cordyceps sinensis on spe- cific and generic level.The divergence rate of Cytb sequences among the species in Hepialus ranged from 0.23% to 9.24%,except that Hepialus pratensis and Hepialus jinshaensis have the same sequence.Cytb sequence can be used for species identification of host insects of Cordyceps sinensis,but further confirmation in more host insect species is needed.To obtain the Cytb sequence of host insect by ampli- fying DNA extracted from the head part of dead larva in cordyceps turns out to be an effective and accurate approach,which will be useful for studies on phylogeny and genetic structure of host insects of cordyceps populations,especially for analyzing relationships between C. sinensis and its host insects.     

Phylogenetic relationships of host insects of Cordyceps sinensis inferred from mitochondrial Cytochrome b sequences

This study used the sequence of the mitochondrial Cytochrome b (Cytb) to estimate phylogenetic relationships among host Hepialidae insects of Cordyceps sinensis. Genome DNA of host insect was extracted from the dead larva head part of 18 cordyceps populations and 2 species of Hepialus, and the Cytb fragment of host insect was amplified with PCR technique. The nucleotide sequence alignments and their homologous sequences of 24 species host Hepialidae insects of Cordyceps sinensis were obtained from GenBank and were used to construct phylogenetic trees based on neighbor-joining method. The results showed that genus Bipectilus diverged earlier than genus Hepialus and Hepialiscus. Hepialus host insects of Cordyceps sinensis have multitudinous species with different morphological characteristics and geographical distributions. The interspecific genetic differentiations are obvious in Hepialus. Thus, the genus Hepialus might be considered as polyphyletic origin. Cytb sequences have abundant variations among the host insects of Cordyceps sinensis on specific and generic level. The divergence rate of Cytb sequences among the species in Hepialus ranged from 0.23% to 9.24%, except that Hepialus pratensis and Hepialus jinshaensis have the same sequence. Cytb sequence can be used for species identification of host insects of Cordyceps sinensis, but further confirmation in more host insect species is needed. To obtain the Cytb sequence of host insect by amplifying DNA extracted from the head part of dead larva in cordyceps turns out to be an effective and accurate approach, which will be useful for studies on phylogeny and genetic structure of host insects of cordyceps populations, especially for analyzing relationships between C. sinensis and its host insects.     

Sequences of cytochrome b gene for primitive cyprinid fishes in East Asia and their phylogenetic concerning

1140 bp of cytochrome b gene were amplified and sequenced from 14 species of primitive cyprinid fishes in East Asia. Aligned with other ten cytochrome b gene sequences of cyprinid fish from Europe and North America retrieved from Gene bank, we obtained a matrix of 24 DNA sequences. A cladogram was generated by the method of Maximum likelihood for the primitive cyprinid fishes. The result indicated that subfamily Leuciscinae and Danioninae do not form a monophyletic group. In the subfamily Danioninae, Opsariichthys biden and Zacco platypus are very primitive and form a natural group and located at the root. But the genera in subfamily Danioninae are included in different groups and have not direct relationship. Among them, Aphyocypris chinensis and Yaoshanicus arcus form a monophyletic group. Tanichthys albonubes and Gobiocypris rarus have a close relation to Gobioninae. The genus Danio is far from other genera in Danioninae. In our cladogram, the genera in Leuciscinae were divided into two groups that have no direct relationship. The genera in Leuciscinae distributed in Europe, Sibera and North America, including Leuciscus, Rutilus, Phoxinus, N. crysole, Opsopoeodus emilae, form a monophyletic group. And the Leuciscinae in southern China including Ctenopharyngodon idettus, Mylopharyngodon piceus, Squalibarbus and Ochetobius elongatus have a common origination.     

黄河鮈Cytb基因克隆及其在鮈属中的系统发育

采用聚合酶链式反应克隆黄河鮈线粒体细胞色素b基因 (Cyt b),首次报道了该基因的全长序列(1140bp,FJ904648) ,并与鮈属中其他6个物种的Cyt b 基因进行同源性比较,分析了碱基组成和变异情况,以马口鱼属的马口鱼和鯝属的云南鯝作为外群构建了ML和NJ系统发育树.遗传距离和分子系统学分析表明,黄河鮈与犬首鮈具有较低的遗传距离(6.30%),在系统发育树上两者聚在一起,提示黄河鮈与犬首鮈存在较近的亲缘关系.     

Molecular phylogenetic status of Siberian roe deer （Capreolus pygargus） based on mitochondrial cytochrome b from Jeju Island in Korea

In order to elucidate the genetic diversity and taxonomic status of Siberian roe deer （Capreolus pygar- gus） from the Korean peninsula, partial sequences of the mitochondrial cytochrome b gene were analyzed. We col- lected 57 roe deer samples from Mainland and Jeju Island between July 2004 and June 2009. A total of 50 sequences （1,070 bp） were obtained, and seven haplotypes were defined. Five haplotypes were found from Mainland, and two were discovered from Jeju Island. The genetic diver- sity was lower in the Jeju Island population than in the Mainland population. In addition, both the consensus neighbor-joining tree and maximum parsimony tree showed an identical topology between the Mainland and Jeju Island clades. Based on the results, two subspecies of the Korean roe deer were confirmed. Siberian roe deer in Jeju Island showed a distinct difference in the cytochrome b gene compared with other Siberian roe deer, being aunique native species inhabiting only Jeju Island in Korea. Because Siberian roe deer in Jeju Island can be classified at subspecies level, the new subspecies in Jeju was named Capreolus pygargus jejuensis in this study. For this new subspecies, reasonable conservation and management effort is needed in the future.