地黄属和崖白菜属的分子系统学研究--兼论地黄属和崖白菜属的科级系统位置

地黄属（Rehmannia Libosch.ex Fisch.et Mey）和崖白菜属（Triaenophora Solereder）是广义玄参科（Scrophulariaceae sensu lato ）种类较少的两个属。地黄属包括六个种，崖白菜属包括三个种。地黄属植物的根可以入药，是中国传统的著名中药。但在传统的分类学上，地黄属的种间系统关系及地黄属与崖白菜属的系统关系一直没有进行深入的研究。这两个属在科级的系统位置也一直都不确定。针对以上问题，本文通过大量的标本查阅、野外考察、以及分子系统学研究，得出了一些初步的结果。 1. 形态学 通过大量的野外考察及标本观察，对地黄属和崖白菜属的形态性状及其变异式样进行了详细地比较分析，重新评价了各性状的分类学价值。认为地黄属和崖白菜属植物的叶型，小苞片的形状，花冠的折叠式样在属下种类区分和系统关系的分析方面均具有较高的系统学价值。过去种间处理所依据的性状（如植株高低，花冠的颜色等）并不可靠，这些性状不宜作为属下种间的划分。 2．分子系统学 利用叶绿体DNA 片段trnL-F 和rps16 及核基因ITS 片段，通过数据单独和联合的最大简约法和贝叶斯法的系统发育分析，结合地黄属所有六个种和崖白菜属的两个种的形态特征，探讨它们属间及属下种间的系统关系。分析结果支持地黄属和崖白菜属的姐妹群关系，同时也支持两个属各自的单系起源。在地黄属内，天目地黄位于地黄属的基部位置可能与它独特的花冠裂片形状及地理分布有关。茄叶地黄和地黄亲缘关系最近，细胞学和形态学的证据同样支持二者姐妹群关系。裂叶地黄和高地黄的近缘关系也得到分子和形态学证据的支持。 一系列的分子系统学的研究揭示广义玄参科至少包括五个主要单系的科，由于地黄属和崖白菜属在这些研究中缺乏取样，他们的科级系统位置仍然是不确定的。因此，在上述研究的基础上，我们在广义唇形目中对与地黄属和崖白菜属可能近缘的类群进行广泛取样，通过五个DNA 片段（ITS, trnL-F, rps16, rbcL 和rps2）进行最大简约法和贝叶斯法分析，来探讨地黄属和崖白菜属在科级的系统位置。我们的分析分为两步，一、我们对所有取样的种类进行ITS 数据单独分析和四个叶绿体DNA 合并分析。二、根据第一步的分析结果，选择合适的外类群，内类群聚焦于与地黄属和崖白菜属近缘的列当科，透骨草科，泡桐属来探讨地黄属和崖白菜属及其近缘类群的系统发育关系，以及它们在科级的系统位置。数据分析采用ITS 数据的单独分析，四个叶绿体DNA 数据合并分析及ITS 和叶绿体数据的联合分析。结果显示地黄属和崖白菜属组成的单系分支和列当科构成姐妹群关系，并具有较高的支持率。植物化学和形态学的证据也支持地黄属和崖白菜属与列当科的姐妹群关系。因此，地黄属和崖白菜属可能隶属于列当科，代表列当科第二个非寄生类群的分支与列当科的余下类群构成姐妹群关系，或应该将地黄属和崖白菜属组成的单系分支上升到一个科级的水平，与列当科互为姐妹群关系。

羊草遗传多样性分析和赖草属的分子系统发育

羊草（Leymus chinensis（Trin.）Tzvel.或Aneurolepidium chinense Trin.），隶属禾本科（Gramineae），大麦族（Hordeae），小麦亚族（Triticieae），赖草属（Leymus），兼具重要的生态价值和经济价值。面向国家重大需求，本论文选择羊草作为主要研究对象，开展了两方面的研究工作，即我国羊草种质资源的遗传多样性评价和赖草属物种的系统发育分析。 羊草是一种多年生根茎型禾草，根据叶色可以划分为黄绿型和灰绿型两种生态型。其分布范围横穿亚欧草原的东部，包括朝鲜和蒙古的西部，以及西伯利亚的西北部，集中分布于我国的东北部。羊草是我国典型草原植物群落中的优势种。它可以在多种土壤和气候条件下正常生长，如松嫩平原、内蒙古草原和黄土高原。从另一个角度讲，不同类型的生境也造就了羊草丰富的遗传多样性。 自上世纪90年代中期开始，我们陆续从我国东北部6个省市收集了293份羊草种质，包括205份灰绿型羊草和88份黄绿型羊草。经过连续3年的观测记录，通过37个重要农艺性状，对293份羊草的遗传多样性进行了评估。依据10个质量性状和27个数量性状，统计了羊草不同性状和不同地域羊草的Shannon遗传多样性指数，同时还使用了主成分分析和通径分析做了相关统计。结果显示：（1）与黄绿型羊草相比，灰绿型羊草具有更高的遗传变异（P<0.05）。结合这两个趋异型羊草的分布范围，可以得出两种类型羊草之间存在稳定的遗传差异;（2）通径分析显示，羊草营养生长性状和遗传多样性两者的组合效应可以解释羊草生殖特性中20.6％的遗传变异;（3）在124-128ºE这一区域，羊草的遗传多样性指数最高（H＝2.252），表明这一地区具有最丰富的羊草种质资源。 赖草属（Leymus Hochst.）是一个异源多倍体属，约有34个物种，该属未知基因组的起源一直争论不休。其倍性范围从四倍体（2n=4x=28）、八倍体（2n=8x=56）一直到十二倍体（2n=12x=84）。新麦草属（Psathyrostachys Nevski）只含有Ns一个基因组，约有9个物种。这两个属都是多年生牧草，具有抗旱、抗病和耐盐碱等生物学特性。 应用核糖体ITS（Internal Transcribed Spacer）序列和叶绿体trnL-F序列，我们对13个赖草属物种、小麦族18个属（40份二倍体材料）、以及Elymus californicus和Bromus catharticus，共计57份材料进行了系统发生分析。ITS序列分析表明，赖草属分别与新麦草属和小麦族中一个未知属在进化上具有紧密的关系。ITS谱系树表明赖草属内部存在大量的分化，以及赖草属物种具有多次起源的特征。trnL-F序列分析表明，赖草属物种的母本，部分来自Ns基因组，部分来自Xm基因组，这可能与赖草属物种的地理分布有关，分布于亚欧大陆的物种其母本是新麦草属，而分布于北美的大部分物种其母本是Xm基因组。trnL-F序列分析还表明，E. californicus和赖草属未知的基因组具有紧密的关系。以上研究结果表明：（1）从分子层面证明，赖草属的未知基因组并非来自薄冰草属，或是一个修正的新麦草基因组，其基因组组成应是NsNsXmXm;（2）赖草属物种的母本，部分来自Ns基因组，部分来自Xm基因组，这可能与赖草属物种的地理分布有关;（3）E. californicus的母本是Xm基因组，父本是Ns基因组，该物种应从披碱草属转移至赖草属。

基于磷酸丙糖异构酶基因序列的蓝氏贾第鞭毛虫分子系统发育的研究

[目的 ]探讨蓝氏贾第鞭毛虫种内系统发育及遗传多样性。 [方法 ]对不同来源虫株的磷酸丙糖异构酶(tim)基因进行 PCR扩增、序列测定后 ,用简约法和 NJ法构建分子系统树进行系统学分析。 [结果 ]在所测序列中共有 12 4个位点存在变异 (2 3% ) ,且大多数为发生在第三密码子的同义突变 ,两种构树方法所得两树的分枝结构相似 ,均将受试的 16株蓝氏贾第鞭毛虫分为明显的两组。 [结论 ]tim基因可作为研究蓝氏贾第鞭毛虫群体遗传结构一个有效的遗传标记

Phylogenetic relationships of Danio within the order cypriniformes: A framework for comparative and evolutionary studies of a model species

The evolutionary relationships of species of Danio and the monophyly and phylogenetic placement of the genus within the family Cyprinidae and subfamily Rasborinae provide fundamentally important phyloinformatics necessary for direct evaluations of an array of pertinent questions in modern comparative biology. Although the genus Danio is not one of the most diverse within the family, Danio rerio is one of the most important model species in biology. Many investigations have used this species or presumed close relatives to address specific questions that have lasting impact on the hypothesis and theory of development in vertebrates. Largely lacking from this approach has been a holistic picture of the exact phylogenetic or evolutionary relationships of this species and its close relatives. One thing that has been learned over the previous century is that many organismal attributes (e.g., developmental pathways, ecologies, behaviors, speciation) are historically constrained and their origins and functions are best explained via a phylogenetic approach. Herein, we provide a molecular evaluation of the phylogenetic placement of the model species Danio rerio within the genus Danio and among hypothesized closely related species and genera. Our analysis is derived from data using two nuclear genes (RAG1, rhodopsin) and five mitochondrial genes (ND4, ND4L, ND5, COI, cyt b) evaluated using parsimony, maximum likelihood, and Bayesian analyses. The family Cyprinidae is resolved as monophyletic but the subfamily Rasborinae (priority over Danioinae) is an unnatural assemblage. Danio is identified as a monophyletic group sister to a clade inclusive of the genera Chela, Microrasbora, Devario, and Inlecypris, not Devario nor Esomus as hypothesized in previous studies. Danio rerio is sister to D. kyathit among the species of Danio evaluated in this analysis. Microrasbora and Rasbora are non-monophyletic assemblages; however, Boraras is monophyletic.

Non-monophyly of fish in the Sinipercidae (Perciformes) as inferred from cytochrome b gene

The sinipercids represent a group of 12 species of freshwater percoid fish, including nine in Siniperca and three species in Coreoperca. Despite several classification attempts and a preliminary molecular phylogeny, the phylogenetic relationships and systematic position of sinipercids remained still unsolved. The complete cytochrome b gene sequences from nine sinipercid species four non-sinipercid fish species were cloned, and a total of 12 cyt b sequences from 10 species of sinipercids and 11 cyt b sequences from 10 species of non-sinipercid fish also in Perciformes were included in the phylogenetic analysis. As expected, the two genera Siniperca and Coreoperca within sinipercids are recovered as monophyletic. However, nine species representing Moronidae, Serranidae, Centropomidae, Acropomatidae, Emmelichtyidae, Siganidae and Centrarchidae included in the present study are all nested between Coreoperca and Siniperca, which provides marked evidence for a non-monophyly of sinipercid fishes. Coreoperca appears to be closest to Centrachus representing the family Centrarchidae. Coreoperca whiteheadi and C. herzi are sibling species, which together are closely related to C. kawamebari. In the Siniperca, the node between S. roulei and the remaining species is the most ancestral, followed by that of S. fortis. S. chuatsi and S. kneri are sibling species, sister to S. obscura. However, the sinipercids do not seem to have a very clear phylogenetic history, for different methods of phylogenetic reconstruction result in different tree topologies, and the only conclusive result in favor of a paraphyletic origin of the two sinipercid genera is the parametric bootstrap test. The paraphyly of Sinipercidae may suggest that the "synapomorphs" such as cycloid scales, upon which this family is based, were independently derived at least twice within sinipercid fishes, and further study should be carried out to include the other two Siniperca species and to incorporate other genes.

Advances in phylogenetic studies of Nematoda

Nematoda is a metazoan group with extremely high diversity only next to Insecta. Caenorhabditis elegans is now a favorable experimental model animal in modern developmental biology, genetics and genomics; studies. However, the phylogeny of Nematoda and the phylogenetic position of the phylum within animal kingdom have long been in debate. Recent molecular phylogenetic studies gave great challenges to the traditional nematode classification. The new phylogenies not only placed the Nematoda in the Ecdysozoan and divided the phylum into five clades, but also provided new insights into animal molecular identification and phylogenetic biodiversity studies. The present paper reviews major progress and remaining problems in the current molecular phylogenetic studies of Nematoda, and prospects the developmental tendencies of this field.

猕猴属的分子系统学与遗传多样性研究

用六个线粒体基因片段序列数据，对称猴属的分子系统发育关系进行了分析。8种称猴：台湾猴，北平顶猴，藏酋猴，熊猴，红面猴，J厦河猴，狮尾猴和食蟹猴共40个样本用于本研究。结合从GenBank里下载的雯猴的相应序列，用拂拂作为外群构建了系统发育树。由于六个目的片段均为线粒体基因片段，缺乏重组且作为单一位点遗传，因此我们将六个数据集合并为一个数据集进行分析。我们获得了比前人置信度更高的线粒体系统树。除了红面猴，我们的结果支持Delson（1980）的分组。与前人研究结果一致，红面猴与食蟹猴组成员关系较近（尤其是食蟹猴）。恒河猴（相对于台湾猴），熊猴（相对于藏酋猴）和北平顶猴（相对于狮尾猴）都形成了并系。通过线粒体控制区序列和20个微卫星位点对14个恒河猴地理群体的群体结构进行了研究。微卫星数据和线粒体控制区数据均显示恒河猴有较高的遗传多样度。线粒体数据表明各地区的恒河猴群体间的差异显著大于微卫星数据显示的结果。也就是说，基于微卫星数据所得到的群体间基因流远大于基于线粒体数据所得到的群体间基因流。两种分子标记所得到的结果差异应归因为雌性恒河猴的归家冲动和雄性恒河猴的扩散。线粒体数据显示所有恒河猴群体近期没有发生过快速扩张。微卫星数据显示大部分恒河猴群体近期没有经历过瓶颈效应。恒河猴是分布较广的非人灵长类动物，但它的地理差异研究一直颇有争论且不足以划分其亚种。我们对采自包括印度、越南、缅甸和18个中国地区在内的21个地区共35个恒河猴样品的四个线粒体基因片段（COI，Cofl，COlll和12srRNA）进行了研究。食蟹猴、红面猴、台湾猴和史猴的相应序列作为外群。大约长为100bp的序列用于构建最大似然树和贝叶斯树。与前人结果一致（彭燕章等，1993；蒋学龙等，1995；Groves，2001），西部恒河猴（印度）．相当于M.m.mulatta。根据王应祥（2003）的分类，我们的东部恒河猴可分为四组：M．最早分离出来且一个来自缅甸的恒河猴位于该组基部；M.m.brachyurus和M.m．littoralis组成一个大枝，这个大枝与M.m，lasiotus构成姐妹群。

隆肛蛙属Feirana种群的系统学与动物地理学研究

对隆肛蛙属的物种构成进行了订正，建立新属肛刺蛙属Yerana gen. nov.；订正后的隆肛蛙属现仅隶2种， 即隆肛蛙F. quadrana和太行隆肛蛙F. taihangnicus。运用形态学分析探讨了隆肛蛙属物种及种群的形态差异和分类关系，通过分子系统学研究探讨了隆肛蛙属物种及种群的分类和系统发育关系，运用动物地理学方法结合系统发育关系探讨了隆肛蛙属种群的地理分布格局成因与历史过程。主要结果和推论如下： 1．隆肛蛙属物种构成的订正及一新属建立 建立新属肛刺蛙属，将隆肛蛙属中的原叶氏隆肛蛙F. yei归隶新属肛刺蛙属并更名为叶氏肛刺蛙Y. yei，，新属建立的主要依据为：（1）雄性肛部隆起，肛孔下方有两个布满黑刺的大的白色球形隆起，具单咽下内声囊， 第一指具婚刺；（2）形态量度分析表明叶氏肛刺蛙与隆肛蛙和太行隆肛蛙的形态差异远大于后两者之间的差异；（3）叶氏肛刺蛙的分布区与隆肛蛙和太行隆肛蛙的分布区距离较远且呈隔离状态；（4）分子系统学研究资料（Jiang et al.,2005）证明叶氏肛刺蛙与隆肛蛙和太行隆肛蛙非单系发生；叶氏肛刺蛙在第二支中位于基部。因此，隆肛蛙属现仅隶2种，即隆肛蛙和太行隆肛蛙。 2．隆肛蛙属种群形态学研究 对隆肛蛙属中隆肛蛙和太行隆肛蛙的15个地理种群565只标本的28项形态性状进行了测量，运用典型判别分析法对其分析的结果表明：（1）太行隆肛蛙与隆肛蛙形态差异明显，支持其为不同的物种；（2）原隆肛蛙河南伏牛山种群和山西中条山种群应为太行隆肛蛙的地理种群；（3）隆肛蛙不同地理种群之间形态差异明显，其中四川安县种群、陕西周至种群和湖北利川种群与模式产地重庆巫山种群的差异可能达到了亚种或亚种以上分化水平。对隆肛蛙属量度分析的15个种群进行定性形态分析表明其分为三种形态型，对应隆肛蛙、过渡型和太行隆肛蛙，其变异特征主要为内跗褶、雄性肛部隆起及疣粒分布、第五趾外侧缘膜等，这与量度分析结果相似。 3．隆肛蛙属种群分子系统学研究 测定隆肛蛙属Feirana的2种19种群的线粒体12S rRNA和16S rRNA基因片段、ND2基因的DNA序列，比对后共计1953bps。（1）遗传多样性与距离分析：结果表明，隆肛蛙属种群具很高的遗传多样性，19个种群样品表现出19种单倍型（遗传多样性指数Hd＝1.0）； ND2基因的进化信息含量远高于12SrRNA和16SrRNA。隆肛蛙属2种群组内的种群间的遗传距离远小于两种群组间的距离，种群在不同基因上的遗传距离表现的关系与对应的系统树一致。（2）系统发育关系分析：结果表明，不同基因片断基于不同方法构建的隆肛蛙属种群系统发育树结构基本一致，基本表明隆肛蛙属种群为单系发生；它们在系统树中分为两大支，分别对应于隆肛蛙和太行隆肛蛙；支持中条山种群（沁水、历山和济源种群）和伏牛山种群（栾川和内乡种群）为太行隆肛蛙的地理种群，而原隆肛蛙秦岭中东段的部分种群（柞水、宁陕、长安大坝沟种群）也应为太行隆肛蛙的地理种群。（3）亚种分化分析：根据遗传距离分析和系统发育关系分析结果，并考虑形态上的差异情况以及地理分布信息，隆肛蛙所隶种群组可分为2亚种，即隆肛蛙指名亚种F. quadrana quadrana包括四川盆地东缘大巴山东段-巫山-武陵山北麓种群和秦岭中段（周至板房子和长安广货街）种群，他们在系统关系树上聚为一支；安县亚种F. quadrana anxianensis包括四川盆地西缘岷山东麓-龙门山-大巴山和秦岭西段的种群（安县、青川、文县、南江和凤县种群），他们在系统关系树上聚为一支。太行隆肛蛙所隶种群组也可分为2亚种，即太行隆肛蛙指名亚种F. taihangnicus taihangnicus包括中条山的种群（沁水、历山和济源种群）和中东秦岭的部分种群（柞水、长安大坝沟和宁陕种群），他们在系统关系树上聚为一支；太行隆肛蛙伏牛亚种F. taihangnicus funiuensis，为伏牛山地区的种群（栾川和内乡种群），他们在系统关系树上聚为一支。 4．隆肛蛙属种群动物地理学研究 隆肛蛙属19种群的分歧年代分析： 以长江巫山段和黄河三门峡段的形成历史时期为参考点，根据已测隆肛蛙属19种群及其外群包括N. pleski、P. yunnanesis、P. robertingeri、F. limnocharis的1953bps DNA序列构建分子钟，获得各支系的分歧年代。结果表明：①棘蛙族在70Ma左右开始其独立演化历程，这与Roelants et al.（2004）的分析结果～60±15Ma左右开始分化基本一致，后者印证了本文的分子钟。②隆肛蛙属的起始分化年代较早，隆肛蛙和太行隆肛蛙两种群组的最近祖先种群大概在46Ma～50Ma左右；隆肛蛙和太行隆肛蛙种群组内的种群分化年代相对两种群组间晚得多， 隆肛蛙种群组内两亚种分化起始年代约为10Ma左右，而太行隆肛蛙种群组内两亚种分化起始年代约为6Ma。 隆肛蛙属种群分布格局形成过程分析： ①隆肛蛙属的系统关系与地理分布格局密切相关，大部分系统分支分级与地理距离成正比；②隆肛蛙属最近祖先种群的分化中心可能位于秦岭中部地区， 隆肛蛙属的种群分布格局的形成表现为隔离分化与扩散相结合的机制，由隔离分化产生的隆肛蛙祖先种群主要从秦岭中部向西南方向扩散，后隔离分化为两亚种；太行隆肛蛙祖先种群向东北方向扩散也分化为两亚种。 隆肛蛙属种群分布区域地质历史的探讨：本文所建分子钟和种群分化方式印证了该区域的几次主要地质事件，包括岷山-龙门山-西秦岭等地区的快速差异隆起、第四纪冰期等。 The specific composition of the genus Feirana should be revised. A new genus Yerana gen. nov.（Ranidae：Dicroglossinae）was established based on morphological data-set and molecular phylogeny, as a result, only two species F. quadrana and F. taihangnicus are classified into Feirana now. Morphological differences and taxonomy of populations of Feirana were investigated based on morphological and morphometric data; phylogenetic relationships and taxonomy of populations of Feirana were elucidated using molecular data, and then the proceeding of the distribution pattern of populations of Feirana were discussed. The main results and conclusions and proposals were presented as following: 1. Revising of the specific composition of the genus Feirana and establishment of a new genus The new genus Yerana, only containing the type species Y. yei, was established based on the following evidences: (1) In adult male, distinct up-heaved circular vesicle presents around the anal, and under anal there are two white balls on which black spines exist, black horny spines scatter on the upper side of first finger, and internal single subgular vocal sac presents; (2) there is obvious morphometric differences between Yerana and Feirana; (3) Yerana is distributed far from Feirana; (4) evidences of molecular phylogeny（Jiang et al.,2005）suggested that Yerana take a special phylogenetic clade which is different from other genus included in the tribe Paini. As a result, there are only two species in Feirana, i.e., F. quadrana and F. taihangnicus. 2. Morphological research of populations of Feirana Twenty-eight characters of 565 individuals of 15 populations of the genus Feirana were measured, the results of Canonical Discriminant analysis of the morphometric data-set indicated that: (1) there are very prominent differences between the two species F. quadrana and F. taihangnicus. The validity of species F. taihangnicus was approved here; (2) Mt. Funiu population and Mt. Zhongtiao population should belong to the species F. taihangnicus; (3) Obvious differences exist among 12 populations of F. quadrana, the differentiation among Zhouzhi population, Anxian population, Lichuan population, and Wushan population together with the others probably reach sub-specific or specific level. Result of morphological comparison between 15 different populations show that 3 morphological types are recogenized in according with F. quadrana, F. taihangnicus and intergradation, this result conform to the result of morphometric analysis. 3. Molecular phylogenetic study on populaions of Feirana Fragment of 12SrRNA and 16SrRNA genes, and ND2 gene of 19 populations of two species of Feirana were sequenced and aligned, from which 1953 bps were received. (1) analyses of genetic distance and hereditary diversity indicated that: genetic distance between populations in each group were less than distance between two groups of Feirana, 19 haplotypes were recognized from 19 samples of 19 populations, so the hereditary diversity of populations of Feirana was very high (Hd=1.0), phylogenetic information in ND2 gene is more than fragment sequence of 12SrRNA and 16SrRNA genes. (2) Result of molecular phylogeny indicate that the phylogenetic trees constructed using different methods based on different sequence data sets showed the revised genus Feirana is monophyletic since the 19 populations of Feirana were firstly clustered together as one large clade, which was further clustered into two major clades, corresponding to F. quadrana(GroupⅠ) and F. taihangnicus(GroupⅡ), respectively. So populations of Qinshui and Lishan in Mt. Zhongtiao, populations of Luanchuan and Neixiang in Mt. Funiu, and populations of Zhashui, Dabagou of Chang’an and Ningshan in eastern Mt. Qinling should belong to the species F. taihangnicus; (3) Subspecific differentiation. on the basis of genetic distance, phylogenetic trees and geographical distribution, F. quadrana should have two subspecies, i.e., F. quadrana qudadrana, consisting of the populations Guanghuojie of Chang’an and Zhouzhi in Mid-Mt. Qinling, populations in Wushan area and northern Mt. Wuling (Lichuan), and F. qudadrana anxianensis, consisting of the populations in eastern Mt. Ming shan-Mt. Longmen-western Mt. Daba-western Mt. Qinling (Anxian, Qingchuan, Wenxian, Nanjiang and Fengxian); F. taihangnicus should also has two subspecies, i.e., F. taihangnicus taihangnicus, consisting of the populations in Mt. Zhongtiao and eastern Mt. Qinling, and F. taihangnicus funiuensis, consisting of the populations in Mt. Funiu. 4. Zoogeography of populaions of Feirana Analysis for divergent time of 19 populations of Feirana: Using the dates of run-through of Wushan segment of Changjiang River as the time when the population of Lichuan started differentiated from the populations of Wushan and Shennongjia, and the dates of Sanmenxia segment of Yellow River as the time when the populations in Mt. Zhongtiao started differentiated from the population of Dabagou in Chang’an, molecular clock was established using sequences with 1953 bps of 19 populations of Feirana and outgroup including N. pleski, P. yunnanesis, P. robertingeri, F. limnocharis in order to estimate divergent time of all clades. Result of that indicated that: ① the tribe Paini started to evolve independently at about 70Ma when is in consistent with that estimated by Roelants et al.（2004）with result of about ～60±15Ma, they were corroborated by each other, this confirms the validity of this molecular clock; ② divergent time for speciation of Feriana is early, ancestral populations of F. quadrana and F. taihangnicus were found about 46Ma～50Ma; differentiation of populations within species is greatly late to the divergence of the two species, divergent time for F. quadrana is 10Ma and divergent time for F. taihangnicus is 6Ma. Proceeding of distribution pattern of Feirana. Phylogenetic relationships of populations of Feirana matched quite with distribution pattern of them, the relationships among clades showed in phylogenetic trees is direct ratio to geographical distance of them; the estimated date of speciation between two species of Feirana was as early as speciation of Paa yunnanesis and Nanara pleski; middle part of Mt. Qinling is the center of speciation of Feirana, combination of mult-events of dispersal and vicariance are probably the mechanism of speciation of Feirana, F. quadrana colonized the mid-Mt. Qinling and then differentiated into two subspecies in southwest direction, ancestral population of F. taihangnicus colonized the mid-Mt. Qinling and then differentiated into two subspecies in northeast direction. On geological history of the distribution of Feirana. According to molecular clock and speciation model of populations of Feirana, some geological events are confirmed, including special rise of Mt. Minshan- Mt. Longmen-western Mt. Qinling, glacial age.

小鲵属分子系统发育研究

小鲵属为亚洲特有的有尾两栖类，是小鲵科之模式属。现记载小鲵属动物有29种，占全科物种数一半以上（Frost, 2007），为小鲵科第一大属。该属分布跨越古北界和东洋界，分布于中国、朝鲜、韩国、日本等地区，其系统学研究一直以来颇为中外学者所关注。澄清该属的物种分类问题，阐明其种间的系统发育关系对整个小鲵科的系统演化与分布格局关系的研究具有关键性意义。 本论文以中国及周边地区的小鲵属物种为主要对象，主要利用分子生物学实验与生物信息学途径相结合的手段，运用支序系统学与分子进化生物学理论及分析方法，展开系统发育的研究。在此基础上诠释现存的分类问题，并探讨该属系统发育关系。 研究材料上，本研究采用野外采集与网络下载数据相结合的方法，获取了较为全面的小鲵属物种DNA序列资料。技术手段上，选取了线粒体DNA的Cytb、12S、16S、NADH 2、COI等多个基因部分片段序列，对小鲵属开展了较为全面系统的研究。分析方法上，针对小鲵属物种各类群的具体情况，运用了处于领域前沿的多种分析方法。应用PAUP、MrBayes、Modeltest、Mega等软件，采用了最大简约法（MP）、邻接法（NJ）、贝叶斯推断（BI）及K2P遗传距离分析等方法。 本研究对小鲵属进行了较为全面的系统发育研究，弥补了有关小鲵属系统发育研究的不足，并得出了以下结果： （1）关于豫南小鲵Hynobius yunanicus的有效性，基于细胞色素b序列的系统发育关系联合形态和染色体组型等证据证明了豫南小鲵是商城肥鲵的同物异名。 （2）获得了较为全面的小鲵属物种系统发育树，并以此解释了北海道滞育小鲵、东北小鲵、中国小鲵与义乌小鲵等存在的分类问题。 （3）本研究利用DNA条形码技术对小鲵属及小鲵科物种进行了鉴定，再次证明豫南小鲵为商城肥鲵的同物异名；并认为猫儿山小鲵与挂榜山小鲵为同物异名。 综上，本研究较为完整地勾勒了小鲵属的系统发育关系全貌，并对小鲵属物种的起源进行了推测。 Hynobius, the type genus of the Family Hynobiidae, is the only exclusively Asian salamander genus. This genus which contains 29 species (beyond half of total Family), is the key group in Hynobiidae. The genus distributed across Palaearctic and Oriental Realm, and was found in China, Korea, and Japan. Systematics of genus Hynobius draws attention of researchers all the times. Resolving the taxonomic and phynogenetic questions of Hynobius is very important to the evolutionary research of Family Hynobiidae. Firstly, studies on systematics of genus Hynobius based on morphology, karyotype and molecular phylogeny of Hynobius are reviewed along with existing questions of this genus. The sequential reaserch project of phylogenetics is perspectively outlined. Using molecular data, we compared Hynobius yunanicus with a sympatric species Pachyhynobius shangchengensis. Our cytb sequences associating with karyotypic and morphological data supportted that H. yunanicus is not a valid species, but a synonym of P. shangchengensis. Because of phenotypic plasticity, some morphological characters are not even suitable for identifying hynobiids. The taxonomy of hynobiids is still controversial to a certain extent (Zhao et al. 1993; Fei, 1999; Chen et al. 2001; Zeng et al. 2006) and needs to be resolved by a new method. Here we examined the utility of COI barcoding for the discrimination of hynobiids. Meantime, the taxonomy of this Family was looked-over again. Our result show that the DNA Barcoding based on COI is easier and more rapidly than classic methods. And the DNA Barcodes data supported the actual taxonomy of Hynobiidae. Based on the achievements of our research, the phylogeny of Hynobius was reconstructed including some new species (H. maoershanensis, H. guabangshanensis, etc). Besides the phylogenetics of Hynobius was outlined, some questions and the hypothesis about the origin of genus Hynobius was put out.

基于ITS 序列和matK 基因序列的中国药用石斛及其混伪品的分子鉴定

为了从分子水平对中国药用石斛及其混伪品进行鉴定，本文选取了核rDNA ITS 序列和叶绿体DNA 的matK 基因序列进行研究。采用改良的CTAB 法提取石斛的基因组DNA，PCR 产物直接测序法对17 种（共32 份）药用石斛的核糖体内转录间隔区ITS 全序列进行测定，克隆测序法对12 种（共22 份）药用石斛的叶绿体的matK 基因序列进行测定，运用BioEd it，MEGA4.0 等生物软件分析了石斛属植物的rDNA ITS 序列及叶绿体的matK 基因序列的特征，比较了石斛属间、种间、种内不同居群（品种）间的序列碱基差异及遗传距离，应用邻接法构建分子系统树。主要研究结果如下： （1）建立了17 种（共32 份）药用石斛rDNA ITS 区碱基全序列数据库，其中，ITS1 的长度为228~234 bp，GC 含量为45.7%~53.0%，变异位点167 个，占总位点67.34%，信息位点106 个，占总位点42.74%，ITS2 长度为241~247 bp，GC含量为44.8%~55.7%，变异位点165 个，占总位点66.27%，信息位点115 个，占总位点46.18%。 （2）建立了12 种（共22 份）药用石斛的叶绿体matK 基因全序列数据库，叶绿体matK 基因长1410 bp，变异位点51 个，信息位点11 个。除了存在碱基替换的遗传变异外，还存在碱基的插入和缺失。 （3）通过ITS 序列比较分析了各材料间的遗传距离和碱基差异，属间的遗传距离为0.295，石斛种间的平均遗传距离为0.142，碱基相差2~156 个，种内各居群间的平均遗传距离为0.002，碱基相差1~2 个。属间的遗传距离大于种间的遗传距离，种间的遗传距离大于种内不同居群（品种）间的遗传距离。 （4）根据分析石斛叶绿体的matK 基因序列得到，外类群（密花石豆兰）与石斛属间最小遗传距离为0.027，石斛种间的平均遗传距离为0.008，种间最大的遗传距离0.014， 最小的遗传距离为0.003，碱基相差8~20 个。种内不同居群（品种）遗传距离为0.001，相差1~5 个碱基。 （5）利用17 种石斛的全序列数据库及遗传分析软件，通过对待检种rDNA I TS区进行序列测定，成功地对10 个待检种进行了鉴定，并且在原植物开花后得到了验证。 （6）运用12 种石斛的matK 基因全序列数据库及遗传分析软件，成功地对4个待检种进行了鉴定，同样在原植物开花后得到了验证。 （7）本文利用石斛的核糖体内转录间隔区ITS 序列和叶绿体的matK 基因序列数据库分别构建了NJ 树，外类群与石斛属间石斛种间以及种内不同居群（品种）间均能在NJ 树中明显分化开来，二者构建的分子系统树一致，为石斛的分子鉴定提供了依据。 In order to identify Chinese Herba Dendrobii and its adulterant species on molecular level, we studied the sequences of rDNA ITS and chloroplast matK gene. Genomic DNA of Dendrobium was extracted using the modified cetyltrimethyl ammonium bromide (CTAB) method. The PCR products of the rDNA ITS sequences of Dendrobium (32 materia ls) were purified and then sequenced. The PCR products of chloroplast matK gene of Dendrobium (22 materia ls) were purified, cloned and then sequenced. The characteristic of the sequences and the genetic dista nce were compared between Bulbophyllum odoratissimum and Dendrobium, Dendrobium interspecies, and different populations. Phylogenetic trees were constructed using the NJ method by the biology softwares including BioEd it, MEGA4.0 etc. The ma in results as follows: (1) It was built up that the database of rDNA ITS sequences of 17 species of Herba Dendrobii (32 materia ls). The ITS1 was 228~234 bp, the GC content accounting for 45.7%~53.0%. Its variable sites were 167, accounting for 67.34%. The Parsim-Informative positions were 106, accounting for 42.74%. The ITS2 was 241~247 bp, the GC accounting for 44.8%~55.7%. The variable sites were 165, accounting for 66.27%. The Parsim-Informative positions were 115, accounting for 46.18%. (2) The database of the chloroplast matK gene sequences was built up, which contained 12 species of Herba Dendrobii (22 materia ls). The matK gene sequences were about 1410bp in length. There were 51 variable sites and 11 Parsim-Informative sites. And there were nucleotides insertions and deletions in some species , in addition to the nucleotides substitutions. (3) The rDNA ITS sequences were compared and analyzed by the biology softwares. The genetic dista nce between Bulbophyllum odoratissimum and Dendrobium was 0.295. The avera ge genetic dista nce was 0.142 between Dendrobium species, and there were 2~156 variable nucleotides. The avera ge genetic dista nce between different populations was 0.002, and there were 2~156 variable nucleotides. The genetic dista nce between Bulbophyllum odoratissimum and Dendrobium was greater tha n that of Denrobium interspecies. Meanwhile, the genetic dista nce between Denrobium species was also greater tha n that of different populations (variaties). (4) The characteristics of the chloroplast matK gene sequences were obtained after analyzing by the biology softwares. The minima l genetic dista nce was 0.027 between Bulbophyllum odoratissimum and Dendrobium . The ma xima l genetic dista nce was 0.014 between Dendrobium species, and there were 20 variable nucleotides. The minima l genetic dista nce between populations was 0.003, and there were 8 variable nucleotides.The genetic dista nce between populations was 0.001, and there were 1~5 variable nucleotides. (5) The molecular Phylogeny tree was constructed on the database of rDNA ITS the sequences of 17 species of Herba Dendrobii using the biology softwares. Then we authenticated 10 materia ls on molecular level. What’s more, they had been proved when these pla nts flowered. (6) The molecular Phylogeny tree was built up on the database of chloroplast matK gene sequences of 12 species of Herba Dendrobii with the biology softwares.Then 4 materia ls were authenticated on molecular level. Moreover, they had also been proved when these pla nts were in flower. (7) The Phylogenetic trees were separately constructed on the sequences of rDNA ITS and chloroplast matK gene B. odoratissimum and Dendrobium all could be distinguished on the Phylogenetic trees. Meanwhile, the Phylogenetic trees based on two groups of sequences were coincident. rDNA ITS and matK gene sequence could be used as molecular markers for authentication of Herba Dendrobii.

Phylogenetic relationships of five species of Dorippinae (Crustacea, Decapoda) revealed by 16S rDNA sequence analysis

A molecular phylogeny is presented for the subfamily Dorippinae (including 9 individuals, representing 5 species and 4 genera), based on the sequence data from 16S rRNA gene. Two-cluster test between lineages in these phylogenetic trees has been performed. On the basis of rate constancy, the rate of nucleotide substitutions of 16S rDNA sequence data is estimated as 0.27% per million years. The analysis strongly supports the recognition of the Dorippinae as a monophyletic subfamily. Phylogenetic tree indicates that the subfamily Dorippinae is divided into two main clades, and genus Dorippe appears basal in the subfamily, diverging from other species 36.6 Ma ago. It is also clear that the Heikea is closely related to the genus Neodorippe. The divergence time between them is 15.8 Ma.

Chromosomal rearrangement in Pectinidae revealed by rRNA loci and implications for bivalve evolution

Karyotype and chromosomal localization of major (18-5.8-28S) and minor (5S) ribosomal RNA genes were studied in two species of Pectinidae, zhikong (Chlamys farreri) and bay (Argopecten irradians irradians) scallops. using fluorescence in situ hybridization (FISH). C. farreri had a haploid number of 19 with a karyotype of 3m + 4sm + 7sm-st + 4st + 1st-t, and A. i. irradians had a haploid number of 16 with a karyotype of 5st + 11t. In C. farreri, the major and minor rRNA genes had one locus each and were mapped to the same chromosome-Chromosome 5. In A. i. irradians, the major rRNA genes had two loci, located on Chromosomes 4 and 8, and the 5S rRNA gene was found at a third chromosome-Chromosome 10. Results of this and other studies indicate that karyotype of A. i. irradians (n = 16, 21 arms) is secondary and derived from an ancestral karyotype similar to that of C. farreri (n = 19, 38 arms) through considerable chromosomal loss and rearrangements. The ability to tolerate significant chromosomal loss suggests that the modal karyotype of Pectinidae and possibly other bivalves with a haploid number of 19 is likely tetraploid; i.e., at least one genome duplication has occurred during the evolution of Bivalvia.

Origin of the Qinghai-Tibetan Plateau endemic Milula (Liliaceae): further insights from karyological comparisons with Allium

Milula, a monotypic genus endemic to the Qinghai-Tibetan Plateau, was found to be nested deeply within Allium by the molecular phylogeny despite the aberrant morphology. It remains unknown what had contributed to the rapid evolution of morphology and origin of this exceptional species. In contrast to a previous report of its karyotypes with 2n = 16 = 8M+8SM (2SAT), similar to most species of Allium, a rather different karyotype, 2n = 20 = 4M +10SM+6T (2SAT), was found in examined 31 individuals from 6 populations of M. spicata distributed in the central Tibet. Karyotypes of 7 Allium species occurring in the Qinghai-Tibetan Plateau were further reported. The basic number x = 8 was confirmed for all of them and their karyotypes consist mainly of metacentric and submetacentric chromosomes with rare subterminal and terminal chromosomes. The karyotype of M. spicata is distinctly different from that of most Allium species occurring in the plateau through a complete comparison of all available species in this region and adjacent areas. However, the same chromosome number and similar karyotypic structure were found in A. fasciculatum of Sect. Bromatorrhiza, indicating a possible close relationship between them. But this similarity is contradictory to the preliminary molecular phylogenetic analysis that Milula was closely related to A. cyathophorum of Sect. Bromatorrhiza with x=8, but the other species with x=10 and 11 in this section were clearly placed in the other clade. We therefore suggested that the paralleling evolution from x=8 to x=9, 10 and 11 with increasing asymmetry of karyotype possibly due to the chromosomal Robertsonian translocation might occur separately in the two recognized phylogenetic lineages of Allium. In addition to aneuploidy and following change of the chromosomal structures, the habitat isolation due to the recent uplift of the Qinghai-Tibetan Plateau and the Quaternary climatic oscillation, plays a greater role in origin of Milula and other endemic species (genera) with aberrant morphology from their progenitors.

Phylogeny and biogeography of the Petaurista philippensis complex (Rodentia : Sciuridae), inter- and intraspecific relationships inferred from molecular and morphometric analysis

With modified DNA extraction and Purification protocols, the complete cytochrome b gene sequences (1140 bp) were determined from degraded museum specimens. Molecular analysis and morphological examination of cranial characteristics of the giant flying squirrels of Petaurista philippensis complex (P. grandis, P. hainana, and P. yunanensis) and other Petaurista species yielded new insights into long-standing controversies in the Petaurista systematics. Patterns of genetic variations and morphological differences observed in this study indicate that P. hainana, P. albiventer, and P. yunanensis can be recognized as distinct species, and P. grandis and P. petaurista are conspecific populations. Phylogenetic relationships reconstructed by using parsimony, likelihood, and Bayesian methods reveal that, with P. leucogenys as the basal branch, all Petaurista groups formed two distinct clades. Petaurista philippensis, P. hainana, P. yunanensis, and P. albiventer are clustered in the same clade, while P. grandis shows a close relationship to P. petaurista. Deduced divergence times based on Bayesian analysis and the transversional substitution at the third codon suggest that the retreating of glaciers and upheavals or movements of tectonic plates in the Pliocene-Pleistocene were the major factors responsible for the present geographical distributions of Petaurista groups. (c) 2005 Elsevier Inc. All rights reserved.