基于ITS2和16S rRNA的西施舌群体遗传差异分析

目前,我国西施舌群体分子遗传差异研究结果存在争议。分析我国南北沿海（9个群体）、与广西北海毗邻的越南（1个群体）西施舌核DNA的内转录间隔区2（ITS2）和线粒体DNA的16S rRNA基因（16S）片段核苷酸序列及其二级结构,为解决争议问题提供分子生物学资料。扩增获得西施舌ITS2片段和16S序列,其长度分别为389-402 bp和306 bp,加之下载序列共147条;序列分析显示,74个ITS2序列共有17种基因型,73个16S序列有15种单倍型,其中,长乐（CL）群体独享9种ITS2基因型和5种16S单倍型,非长乐群体（nCL）多数为群体间交叉共享1种或几种基因（单倍）型;基因（单倍）型核苷酸变异位点占5.7%（ITS2）和11.8%（16S）;基于ITS2和16S的CL群体和nCL群体间的遗传距离与群体内遗传距离之比分别为2.42和11.08,nCL群体间的平均遗传距离均为0.007;二级结构显示CL群体ITS2的9种基因型和16S的5种单倍型均区别于nCL群体,nCL的ITS2和16S二级结构分别相似;ITS2和16S基因的系统发育分析显示,CL西施舌形成支持率很高（98,96）的单系支,而nCL群体则交叉聚为另一支（98,96）。研究结果揭示,福建西施舌是腔蛤蜊属（Coelomactra）的一个新种。     

基于ITS2序列的獐牙菜属六种药材的分子鉴定

本文以獐牙菜属的六种药材为研究对象,采用MEGA 6.0软件确定ITS2序列的GC含量,基于K2P模型计算遗传距离,构建NJ树,ITS2 database在线分析二级结构等方法,研究ITS2条形码序列对六种药材的鉴别效果。结果表明,六种药材的ITS2序列长度范围为230~232 bp,平均GC含量为61.2%~64.0%。六种药材的种内变异均较小,其种间平均遗传距离为0.03~0.09。ITS2二级结构的茎环结构和四个臂均不同,NJ树结果显示,六种药材可分别聚为一支,呈现出明显的单系性。因此,ITS2序列可准确、有效地鉴别本研究的獐牙菜属六种药材,为这些药材的后续相关深入研究奠定了基础,也为獐牙菜属其他物种鉴定提供了参考。     

基于部分18S rDNA, 28S rDNA和COI基因序列的索科线虫亲缘关系

通过PCR扩增获得我国常见昆虫病原索科线虫6属10种18S rDNA、28S rDNA(D3区)和COI基因序列,结合来自GenBank中6属10种索科线虫的18S rDNA同源序列,用邻接法和最大简约法构建系统进化树。结果显示:12属索科线虫分为三大类群,第一大类群是三种罗索属线虫(Romanomermis)先聚在一起,再与两索属(Amphimermis)和蛛索属(Aranimermis)线虫聚为一支;在第二大类群中,六索属(Hexamermis)、卵索属线虫(Ovomermis)和多索属(Agamermis)亲缘关系最近,先聚在一起,再与八腱索属(Octomyomermis)和Thaumamermis线虫聚为一支。第三大类群由索属(Mermis)和异索属(Allomermis)线虫以显著水平的置信度先聚在一起,再与蠓索属(Heleidomermis)和施特克尔霍夫索属(Strelkovimermis)线虫聚为一支。从遗传距离看,基于3个基因的数据集均显示索科线虫属内种间差异明显小于属间差异,武昌罗索线虫(R.wuchangensis)和食蚊罗索线虫(R.culicivorax)同属蚊幼寄生罗索属线虫,其种间的遗传距离最小。     

中国库蚊属四十个种(亚种)的分子系统发育关系研究(双翅目:蚊科)

【目的】对在中国有分布的库蚊属Culex种(亚种)的COⅠ序列和ITS2序列进行测序,构建和讨论这些种(亚种)的分子系统发育关系。【方法】测定了库蚊属20个种(亚种)的COⅠ和ITS2序列,并从NCBI数据库中下载了库蚊属另外20个种(亚种)的COⅠ和3个种(亚种)的ITS2序列。对库蚊属40个种(亚种)的COⅠ序列和其中23个种(亚种)的ITS2序列进行碱基构成、种间遗传距离和饱和度分析,并对COⅠ+ITS2序列进行ILD(incongruence length difference)检验。分别使用2种分子数据集(COⅠ和ITS2)的核苷酸序列,用最大似然法(ML)、贝叶斯法(BI)、邻接法(NJ)和最大简约法(MP)推断这些种的系统发育关系。通过Kishino-Hasegawa(KH)和ShimodairaHasegawa(SH)检验评估这4种系统树间的差异,确定最为合理的系统发育树。【结果】本研究新测序获得20种(亚种)的COⅠ和ITS2序列的长度范围分别为625~685 bp和300~559 bp。COⅠ和ITS2序列在库蚊属成对蚊种间的遗传距离分别是0.002~0.198和0.006~1.807。库蚊属23个种(亚种)的COⅠ+ITS2序列的ILD检验结果显示,数据集具有不相容性,因此COⅠ+ITS2序列不适用于这些种(亚种)的系统发育研究。经KH和SH检验显示,4种系统发育树中,基于COⅠ序列构建的BI树最为合理,而基于ITS2序列构建的MP树最为合理。基于COⅠ核苷酸序列所构建的BI树显示,除幼小库蚊Cx.infantulus和短须库蚊Cx.brevipalpis外,各亚属间成员聚类结果与传统的形态学归类结果吻合;路蚊亚属Lutiza和包蚊亚属Barraudius都归入库蚊亚属Culex内;梅蚊亚属Maillotia和新库蚊亚属Nexoculex聚为一支;库状蚊亚属Culiciomyia、真黑蚊亚属Eumelanomyia和簇角蚊亚属Lophoceramyia均显示为单系。基于ITS2序列所构建的MP树显示,各亚属间和种(亚种)间关系混乱。【结论】重建的分子系统发育树表明库蚊亚属不是一个单系群。在重建库蚊属系统发育关系时,相较于ITS2和COⅠ+ITS2,COⅠ是更为理想的分子标记。本研究构建的分子系统发育关系为中国库蚊属中各亚属和各种(亚种)之间的亲缘关系的研究奠定了基础。     

基于18S rDNA遗传距离与GC含量对游走类纤毛虫的分子系统学研究

为了揭示游走类纤毛虫的系统发生,对寄生于淡水鱼类的车轮虫科中的6种车轮虫进行了18S rDNA的测序并获得了9个序列。采用了最大似然法(ML)与贝叶斯法(BI)对GenBank中所有游走类纤毛虫的18S rDNA序列进行了系统树的构建,并首次将SPSS与18S rDNA遗传距离结合分析了游走类纤毛虫的系统发生。研究结果进一步证实了车轮虫属(Trichodina)的非单系发生与小车轮虫属 (Trichodinella) 的有效性。此外,研究结合18S rDNA 的GC含量与遗传距离分析提出了游走类纤毛虫科属及种间新的鉴定依据: 18S rDNA 的GC含量可用于游走类纤毛虫的科属区分,且与游走类纤毛虫的分化密切相关; 18S rDNA的遗传距离在游走类纤毛虫的不同阶元中具有一定的阈值范围,即通常种内遗传距离阈值范围为0.000-0.005,属种间阈值范围为0.005-0.150,当遗传距离大于0.150时,则达到了科间水平。     

扇贝核糖体ITS1和5S rDNA序列的遗传变异及亲缘关系分析

为探讨引进种岩扇贝(Crassadoma gigantea)及虾夷扇贝(Patinopecten yessoensis)、栉孔扇贝(Chlamys farreri)和海湾扇贝(Argopecten irradians)的遗传变异及其之间的亲缘关系, 研究对4种扇贝的核糖体ITS1序列及5S rDNA序列进行了测定, 获得了4种扇贝的ITS1和5S rDNA全序列以及18S rDNA的部分序列, 并进行了序列特征、遗传多样性、遗传距离及系统发育等相关分析。结果显示, 基于ITS1序列的单倍型数、核苷酸变异位点、单倍型多样性指数、核苷酸多样性指数及平均核苷酸差异数分别在2—6、1—12、0.425—0.800、0.00081—0.00486和0.366—2.442; 基于5S 序列的相应参数分别在2—5、1—14、0.533—0.841、0.00108—0.01058和0.533—5.726。4种扇贝均表现出较低的多样性水平(π<0.01)。此外, 4种扇贝基于2种方法计算的种间遗传距离分别在0.040—0.121和0.042—0.413, 均表现为岩扇贝与虾夷扇贝的遗传距离最近, 而岩扇贝与海湾扇贝遗传距离最远。并且系统进化树分析显示岩扇贝与虾夷扇贝独自聚为一支, 说明引进物种岩扇贝与虾夷扇贝的亲缘关系较近。研究结果将为岩扇贝的种质资源保护以及今后的遗传育种研究工作提供参考信息。     

基于18S rDNA遗传距离与GC含量对游走类纤毛虫的分子系统学研究（英文）

为了揭示游走类纤毛虫的系统发生,对寄生于淡水鱼类的车轮虫科中的6种车轮虫进行了18S rDNA的测序并获得了9个序列。采用了最大似然法(ML)与贝叶斯法(BI)对GenBank中所有游走类纤毛虫的18S rDNA序列进行了系统树的构建,并首次将SPSS与18S rDNA遗传距离结合分析了游走类纤毛虫的系统发生。研究结果进一步证实了车轮虫属(Trichodina)的非单系发生与小车轮虫属(Trichodinella)的有效性。此外,研究结合18S rDNA的GC含量与遗传距离分析提出了游走类纤毛虫科属及种间新的鉴定依据:18S rDNA的GC含量可用于游走类纤毛虫的科属区分,且与游走类纤毛虫的分化密切相关;18S rDNA的遗传距离在游走类纤毛虫的不同阶元中具有一定的阈值范围,即通常种内遗传距离阈值范围为0.000—4).005,属种间阈值范围为0.005—0.150,当遗传距离大于0.150时,则达到了科间水平。     

冬虫夏草培养子实体ITS,5.8S的分析及系统发育研究

对野生冬虫夏草的人工培养子实体的ITS1,ITS2和5.8S间区序列进行了克隆,并结合已有的序列对ITS1,ITS2和5.8S进行了系统发育分析。结果表明,人工培养子实体与中华被毛孢(Hirsutella sinensis L95DBM-1)具有96%的同源性,在NJ邻接树上形成明显的一支,与虫草属其它类群种间具有明显差异,表明分离培养的虫草子实体为中华被毛孢。遗传距离分析结果显示,培养虫草序列与已知虫草在种内也存在一定差异,可能暗示虫草种群在不同区域具有一定的遗传分化。     

从16S rRNA基因序列初步探讨拟壁钱属和壁钱属蜘蛛系统发生关系

拟壁钱属Oecobius和壁钱属Uroctea蜘蛛之间系统发生关系存在一定的争议.为从分子水平探讨两属间系统发生关系,本研究测定了5科6种蜘蛛的16S rRNA基因部分序列,并联合来自GenBank的8科8种蜘蛛16S rRNA基因序列数据重建分子系统树.结果表明,拟壁钱属和壁钱属间遗传距离(28.1%)明显大于复杂生殖器类(Entelegynae)蜘蛛属间遗传距离的平均值(22.9%);与目前大多数文献把拟壁钱属和壁钱属并在一个科的观点相反,本研究重建的系统发生树显示两属不是姊妹群.作者建议把拟壁钱属和壁钱属分别重新划回拟壁钱科Oecobiidae和壁钱科Urocteidae.系统发生树还验证了简单生殖器类(Haplogynae)蜘蛛、复杂生殖器类蜘蛛各自的单系性以及筛器类(Cribellate)蜘蛛的多系发生,同时本文的结果还对圆网蛛类(Orbiclariae)单系发生及RTA类群单系发生的有效性提出了质疑.     

樟属植物ITS序列多态性分析

对樟科樟属(Cinnamomum Schaeffer) 17个代表样本的核糖体DNA内转录间隔区(nrDNA ITS)进行克隆测序。对获得的87条不同ITS序列的长度变异、GC含量、5.8S区二级结构的稳定性、遗传距离、进化模式以及系统发育关系进行了相关分析。研究结果显示, ITS序列在樟属植物内存在明显的多态性, 87条序列中的22条序列被鉴定为假基因序列, 其余65条序列为功能基因序列; 假基因序列采用中性进化模式, 变异明显大于功能序列。ITS序列在樟属植物中出现一致性进化不完全和假基因现象也可能发生在樟科其它类群中, 这可能是导致樟科植物ITS序列直接测序方式成功率低的重要原因。     

Phylogeny of Labidodemas and the Holothuriidae (Holothuroidea: Aspidochirotida) as inferred from morphology

The Holothuriidae is one of the three established families within the large holothuroid order Aspidochirotida. The approximately 185 recognized species of this family are commonly classified in five nominal genera: Actinopyga , Bohadschia , Holothuria , Pearsonothuria and Labidodemas. Maximum parsimony analyses on morphological characters, as inferred from type and nontype material of the five genera, revealed that Labidodemas comprises highly derived species that arose from within the genus Holothuria . The paraphyletic status of the latter, large (148 assumed valid species) and morphologically diverse genus has recently been recognized and is here confirmed and discussed. Nevertheless, we adopt a Darwinian or eclectic classification for Labidodemas , which we retain at generic level within the Holothuriidae. We compare our phylogeny of the Holothuriidae with previous classifications of its genera and subgenera, and make suggestions concerning possible systematic changes.  © 2005 The Linnean Society of London, Zoological Journal of the Linnean Society , 2005, 144 , 103−120.     

Predictable and unpredictable spawning events: in situ behavioural data from free-spawning coral reef invertebrates

Summary

We describe the spawning behaviour and some aspects of spawning periodicity in a diverse group of marine invertebrates, principally echinoderms, but including sponges, anthozoans, molluscs, and polychaetes. Our observations were made both opportunistically and on a systematic basis between 1978 and 1992 on the central and northern Great Barrier Reef. Spawning was predictable in some of the species observed, for example Bohadschia argus, Euapta godeffroyi, and Stichopus variegatus (Holothuroidea), which exhibited regular lunar and diel periodicity. Others, such as Holothuria coluber, Actinopyga lecanora, and Bohadschia graffei (Holothuroidea), Acanthaster planei (Asteroidea), Hyotissa hyotis and Arca spp. (Bivalvia) exhibited no clear lunar or diel periodicity in spawning behaviour. Mass heterospecific spawnings which involved several species, often from different phyla, were commonly observed. The species participating were usually those with unpredictable spawning patterns and while the species involved were diverse there were also occasions when spawning involved species from the same genera. Fertilization rates were measured in situ for the predictable spawner Bohadschia argus and were found to vary between 0–96% depending on the circumstances of the spawnings. Fertilization rates for the unpredictable spawners showed similar variability; Holothuria coluber and Actinopyga lecanora ranged from 9–83%.     

Ribosomal ITS Sequences Allow Resolution of Freshwater Sponge Phylogeny with Alignments Guided by Secondary Structure Prediction

Freshwater sponges include six extant families which belong to the suborder Spongillina (Porifera). The taxonomy of freshwater sponges is problematic and their phylogeny and evolution are not well understood. Sequences of the ribosomal internal transcribed spacers (ITS1 and ITS2) of 11 species from the family Lubomirskiidae, 13 species from the family Spongillidae, and 1 species from the family Potamolepidae were obtained to study the phylogenetic relationships between endemic and cosmopolitan freshwater sponges and the evolution of sponges in Lake Baikal. The present study is the first one where ITS1 sequences were successfully aligned using verified secondary structure models and, in combination with ITS2, used to infer relationships between the freshwater sponges. Phylogenetic trees inferred using maximum likelihood, neighbor-joining, and parsimony methods and Bayesian inference revealed that the endemic family Lubomirskiidae was monophyletic. Our results do not support the monophyly of Spongillidae because Lubomirskiidae formed a robust clade with E. muelleri, and Trochospongilla latouchiana formed a robust clade with the outgroup Echinospongilla brichardi (Potamolepidae). Within the cosmopolitan family Spongillidae the genera Radiospongilla and Eunapius were found to be monophyletic, while Ephydatia muelleri was basal to the family Lubomirskiidae. The genetic distances between Lubomirskiidae species being much lower than those between Spongillidae species are indicative of their relatively recent radiation from a common ancestor. These results indicated that rDNA spacers sequences can be useful in the study of phylogenetic relationships of and the identification of species of freshwater sponges.     

Molecular phylogenetic studies on the Diatrypaceae based on rDNA-ITS sequences

The order Diatrypales (Ascomycota) contains one single family, the Diatrypaceae. To obtain insight in the phylogenetic relationships within this family, the complete sequences of the ITS region (ITS1, 5.8S rRNA gene and ITS2) of 53 isolates from the five main genera in the family (Diatrype, Diatrypella, Cryptosphaeria, Eutypa and Eutypella) were determined and aligned for phylogenetic reconstruction. Sequence analysis revealed the presence of tandem repeated motifs 11 nucleotides-long, placed in homologous positions along the ITS1 region. Parsimony analysis established the existence of nine monophyletic groups and one branch with a single isolate of Eutypella quaternata. The phylogenetic relationships established by parsimony analysis did not correlate well with classical taxonomic schemes. None of the five genera included in this study was found to be monophyletic. The genera Diatrype, Eutypa and Cryptosphaeria each were divided into two groups. Isolates of Diatrype flavovirens appeared in a clade separated from the one that grouped Diatrype disciformis and the rest of Diatrype species. The Eutypa strains appeared distributed into two clades, one grouping Eutypa lata and related species (Eutypa armeniacae, Eutypa laevata, Eutypa petrakii), and another with the remaining species of the genus. Eutypella (excluding Eutypella quaternaria) appeared as an unstable monophyletic group, which was lost when the sequence alignment was subjected to neighbor-joining analysis. The genus Diatrypella was not associated with any monophyletic group, suggesting that the multisporate asci character has appeared several times during the evolution of the group. Overall, our study suggests the need to revise many of the concepts usually applied to the classification of members of the family.     

Sea Cucumbers Triterpene Glycosides, the Recent Progress in Structural Elucidation and Chemotaxonomy

Triterpene glycosides are characteristic metabolites of sea cucumbers (Holothurioidea, Echinodermata). Majority of the glycosides belong to holostane type (lanostane derivatives with 18(20)-lactone). Carbohydrate chains of these glycosides contain xylose, glucose, quinovose, 3-O-methylglucose and 3-O-methyl sylose. During the last 5 years, main investigations were focused on holothurians belonging to the order Dendrochirotida collected in the North Pacific, North Atlantic, Antarctic and in subtropical waters. The glycosides of holothurians belonging to the order Aspidochirotida have also been studied. The most uncommon structural features of carbohydrate chains of new glycosides were: (1) the presence of quinovose as fifth terminal monosaccharide unit and the presence of two quinovose residues; (2) the presence of glucose instead of common xylose as fifth terminal monosaccharide unit; (3) trisaccharide carbohydrate chain; (4) the presence of two 3-O-methylxylose terminal monosaccharide units; (5) the presence of sulfate group at C-3 of quinovose residue. New glycosides without lactone or with 18(16)-lactone and having shortened side chains have also been isolated. The presence of 17α and 12α-hydroxyls, which are characteristic for glycosides from holothurians belonging to the family Holothuriidae (Aspidochirotida) in glycosides of dendrochirotids confirms parallel and relatively independent character of evolution of glycosides. All three families belonging to the order Aspidochirotida: Holothuriidae, Stichopodidae and Synallactidae have similar and parallel trends in evolution of the glycosides carbohydrate chains, namely from non-sulfated hexaosides to sulfated tetraosides. Sets of aglycones in glycosides from holothurians belonging to the genus Cucumaria (Cucumariidae, Dendrochirotida) are specific for each species. The carbohydrate chains are similar in all representatives of the genus Cucumaria.     

Phylogeny of the grass family (Poaceae) from rpl16 intron sequence data

DNA sequence data from the chloroplast noncoding rpl16 intron are used to address phylogenetic relationships among the major lineages of the grass family, with particular emphasis on the highly heterogeneous subfamily Bambusoideae and the basal lineages. Thirty-five grass sequences representing all six currently recognized major groups of the family and one outgroup sequence were analyzed using both parsimony and distance methods. The phylogenetic analyses indicated: (1) Puelia, a traditionally isolated bambusoid genus, is the most basal lineage in the BOP clade (Bambusoideae, Oryzoideae, and Pooideae); (2) the bambusoid clade is a sister group to the pooid clade; and (3) the monophyletic oryzoid clade is well separated from the bambusoid clade. The study further confirmed the recognition of two primary groups in the grass family: the BOP clade and the PACC clade (Panicoideae, Arundinoideae, Chloridoideae, and Centothecoideae); it also provided further evidence that the traditional subfamily Bambusoideae is highly heterogeneous and phylogenetically unacceptable. The data support Streptochaeteae, Anomochloeae, and Phareae as the most basal lineages among the extant grasses. Within the BOP clade, oryzoids and pooids are confirmed as two monophyletic clades, but the bambusoid clade, including only the woody bamboo tribe Bambuseae and the herbaceous bamboo tribe Olyreae, is relatively weakly supported. The study also indicated that the chloroplast noncoding region sequence data could be useful in phylogenetic analysis at relatively high taxonomic levels.     

中国榕属榕亚属植物的系统发育初探

对分布于中国的榕亚属植物的15个种和2个变种(分别代表榕亚属的3个组)的nrDNA的ITS序列和系统发育关系进行分析。结果表明,榕亚属是非单系起源,其中榕组构成了一个独立的单系群,与无花果亚属有较近的亲缘关系,而与榕亚属其它组间并未表现出近缘关系。榕亚属的其它5个组构成一单系群并得到中度的支持率。来自中国的种类分别聚在相应的组内。但环纹榕组被分成2个单支,其中来自中国的大叶水榕(F.glaberrima)和环纹榕(F.annulata)构成一个分支,二者表现较近的亲缘关系。榕亚属的亚洲特有组印度榕组在ITS树中聚在环纹榕组的另一个分支中,但它与此组其它种的亲缘关系并不清楚。     

An evaluation of the monophyly of Massarina based on ribosomal DNA sequences

The monophyletic status of the genus Massarina was evaluated on the basis of phylogenetic analysis of the partial small subunit gene (SSU), internal transcribed spacers (ITS 1 & 2), and 5.8S gene sequences of the ribosomal DNA. Species of Massarina used in the study clustered into two distinct clades with high bootstrap support in trees generated from maximum parsimony, weighted parsimony, maximum likelihood, and neighbor-joining analyses. The hypothesis that Massarina species belong to a phylogenetically monophyletic group is rejected. Species with narrowly fusiform ascospores form a monophyletic clade with Lophiostoma, a genus highly similar in morphology. The five species currently accepted in Massarina with such spore morphology are here transferred into the genus Lophiostoma. Massarina species with broadly fusiform to ellipsoidal ascospores are retained as Massarina s. str., lectotypified by M. eburnea. Massarina walkeri is presently excluded from both Massarina and Lophiostoma. The transfer of M. papulosa to a new genus Oletheriostrigula is verified.     

Phylogenetic relationships of the anamorphic genus <Emphasis Type="Italic">Fusicladium</Emphasis> s. lat. as inferred by ITS nrDNA data

The genus Fusicladium s. lat. (incl. Pollaccia and Spilocaea) was phylogenetically analysed using ITS nrDNA sequences. Pollaccia and Spilocaea did not form monophyletic groups of their own, but were intermingled between Fusicladium species, together with which they formed a monophyletic clade. Thus, Pollaccia and Spilocaea should be included in a wider genus concept of Fusicladium, constituting a morphologically variable genus. Furthermore, all Venturia and Fusicladium isolates clustered together on the bases of available ITS data, providing support for the monophyly of the anamorphic genus Fusicladium and the teleomorphic genus Venturia. Within this clade several subclades can be recognized. All taxa on the host family Salicaceae were found in one subclade. Three other subclades comprised taxa on Rosaceae whereas taxa on other host families all clustered separately. Geographic specializations were not observed. Two examples of host switching could be demonstrated, but these were confined to instances involving host species belonging to the same family. Fusicladium convolvularum and F. effusum, two species with unknown teleomorphs, clustered within the Fusicladium/Venturia clade, supporting the correct placement of these taxa in Fusicladium. The placement of Pseudocladosporium hachijoense within the family Venturiaceae was also supported.