中国绿水螅分子系统发生地位的初步探讨

目的:初步探讨中国绿水螅(Hydra sinensis)分子系统发生地位以及水螅属内部各类群系统发生关系。方法:采用酚-氯仿法提取中国绿水螅总DNA,扩增线粒体COI和16S r RNA基因片段并进行DNA序列测定,再利用Clustal及MEGA等生物信息学分析软件进行系统发生分析。结果:在本研究重建的所有系统发生树中,中国绿水螅始终与绿水螅Hydra viridissima的不同种群一起构成绿水螅单系群。同时,棕色水螅群的单系性被基于COI基因的NJ树以及基于16S r RNA基因的NJ树和ML树支持,唯独基于COI基因的ML树不支持棕色水螅群的单系发生。在基于COI基因的ML树中纤弱水螅族在系统树的基部独立为一支系,而绿水螅群和其他棕色水螅群水螅一起组成另一支系,提示纤弱水螅族水螅的系统发生地位值得进一步探讨。值得注意的是,根据本文的结果,棕色水螅群内3族的划分仍然有一定疑问。基于COI基因的NJ树和ML树支持普通水螅族、寡水螅族和纤弱水螅族各自族内的单系发生,但16S r RNA基因的NJ树和ML树中仅普通水螅族水螅聚为单系群,而寡水螅族和纤弱水螅族水螅各自并非单系发生。结论:把水螅属划分为绿水螅群及棕色水螅群有一定的合理性,但棕色水螅群内寡水螅族、普通水螅族和纤弱水螅族3族的划分还有待商榷。     

双壳纲贝类18S rRNA基因序列变异及系统发生

双壳纲贝类栖息于环境多变的海域,是一个形态学和生态学都具有多样性的类群,清晰而可靠的进化关系对于养殖与相关种类的管理具重要意义。然而,目前对双壳类宏观分子系统学研究的报道较少。研究用18S rRNA基因(18S)分析了双壳类3个亚纲贝类的系统发育关系。从GenBank下载帘蛤目、海螂目、贻贝目、胡桃蛤目、蚶目、珍珠贝目6个目94个种类的18S全/部分序列107个,通过ClustalX软件进行序列比对, 用MEGA4.1软件和PHyML软件计算遗传距离, 构建系统发育树, 研究了双壳类18S变异规律及其在系统发生研究中的应用。结果显示18S有插入/缺失序列, 存在长度多态性。序列比对显示有5段约30 70bp的保守区, 4段约130 550bp的高变区。碱基组成平均为T:24.4%, C:23.6%, A:24.5%, G:27.5%。G+C含量为51.1%。在1796个比对位点中, 变异位点占31.7%, 简约信息位点占24.0%。目内科间遗传距离为0.003 0.043, 目间遗传距离为0.026 0.093。NJ树和ML树显示贻贝目、珍珠贝目、胡桃蛤目、蚶目和海螂目的缝栖蛤科先分别聚为支持率很高(BPN=94 100)的单系支, 后聚为一大支(BPN=100)。蛤蜊科与帘蛤目的其他科分离形成一置信度很高的单系支(BPN=93)。帘蛤科种类聚为置信度较低(BPN=60)的一支。海螂目、帘蛤目的种类没能完全聚到所属支系, 彼此嵌套,缝栖蛤科的种类从海螂目中分离出来。18S资料揭示帘蛤目的蛤蜊科、海螂目的缝栖蛤科已经进化为独立的支系。     

强光及高温诱发白化的中国绿水螅共生细菌多样性的特征

目的：探究强光及高温诱发白化的中国绿水螅共生细菌多样性的特征。方法：以光照强度2000 Lux和温度25℃条件为对照,分别在强光胁迫（6000 Lux,25℃）、高温胁迫（2000 Lux,33℃）以及强光和高温联合胁迫（6000 Lux,33℃）等3种条件下进行诱导中国绿水螅白化的实验。白化实验进行到21天后,采用对单个水螅细胞中的共生藻进行计数的方法检测绿水螅白化的程度,同时通过16S r DNA高通量测序的方法分析绿水螅共生细菌多样性及丰度变化。结果：在强光胁迫、高温胁迫及强光和高温联合胁迫下绿水螅体内共生藻数量均显著减少（P<0.05）。强光胁迫下绿水螅共生细菌中蓝细菌门（Cyanobacteria）的丰度上升,而在强光和高温联合胁迫下绿水螅共生细菌中不但蓝细菌门的丰度上升,绿硫菌门（Chlorobi）的丰度也明显提高。此外,KEGG富集分析结果表明发生白化的绿水螅共生细菌与亚油酸、类胡萝卜素及甜菜红色素等抗氧化剂的合成和代谢相关。结论：蓝细菌和绿硫菌的光合作用产物有可能被水螅宿主所利用、以弥补水螅宿主由于共生藻的减少带来的营养缺乏;强光和高温胁迫下绿水螅出现白化现象后共...     

鹳形目12种鸟类核c-mos 基因和线粒体12S rRNA基因序列分析及其系统发生研究

鹳形目鸟类的传统分类一直存在分歧,而近期的分子系统学研究大多只用单个基因,其结论的可信度需要进一步验证.本文通过核c-mos基因和线粒体12S rRNA基因序列分别和合并分析,采用分子系统学方法探讨了鹳形目6科12种鸟类的系统发生关系.文中测出鹳形目鸟类6种核c-mos基因的片断序列,结合来自Genebank的其他种类的c-mos和12S rRNA基因序列,分别经Clustal W软件对位排列后,以原鸡为外类群用最大似然法、邻接法和最大简约法建立系统树.系统树分析表明, 鹳形目6科之间的系统发生关系总结为：（鹭科,（（鹮科,美洲鹫科）,（鹳科,（鲸头鹳科,锤头鹳科））））.鹭科7个属之间的系统发生关系总结为：（麻（开鸟）属（夜鹭属（池鹭属（苍鹭属（中白鹭属（白鹭属,大白鹭属））））））.分别基于两个单基因的系统树有一定差异,而基于合并数据的系统树支持率和分辨率都高于基于单基因的系统树,表明使用在遗传上相对独立的分子数据合并建立系统树有较高的可信度和分辨率,是一种更好的研究方法.     

基于12S rRNA基因序列探讨崇安地蜥的分类地位

为探讨崇安地蜥Platyplacous sylvaticus的分类地位,测定了崇安地蜥线粒体12S rRNA基因全序列,并从GenBank中下载了东亚产10种草蜥、3种地蜥的同源序列进行分析,采用Mega V2.1软件的NJ法和ME法、PAUP4.0软件的MP法构建分子系统树.结果表明:崇安地蜥线粒体12S rRNA基因全序列(952 bp)中T、C、A、G碱基含量分别为23.1%、22.9%、35.9%、18.1%;与其它同源序列比对后有978 bp,发现321个位点出现变异,占总位点数32.8%,其中199个简约信息位点,为总变异位点的62%;转换/颠换之比平均为2.16.构建的分子系统树中,NJ树和ME树完全一致,与MP树略有差异.3种构树法中崇安地蜥与南台草蜥Takydromus sauteri、峨眉地蜥P.intermedius、先岛地蜥P.dorsalis均聚为一支,崇安地蜥与先岛地蜥亲缘关系最近.本实验结果支持将地蜥属并入草蜥属和取消地蜥亚属的观点.     

基于核基因和叶绿体基因序列的杏属系统发育分析——探讨洪平杏的起源和亲缘关系

洪平杏(Armeniaca hongpingensis C. L. Li)是杏属的一个狭域分布种,基于形态观察被推测为杏(A.vulgaris Lam.)和梅(A. mume Sieb.)的天然杂交种,但目前尚无该种与杏、梅亲缘关系的分子系统学研究。本文以洪平杏的成株和实生苗以及包括杏、梅在内的6种(含1变种)杏属植物为研究材料,分别采用核基因(ITS和SBEI)和叶绿体基因(mat K和ycf1b)序列构建系统发育树,并采用mat K、ycf1b和SBEI基因序列构建单倍型网络图,探讨该物种与杏、梅及杏梅(A. mume Sieb. var. bungo Makino)之间的亲缘关系。基于核基因和叶绿体基因序列分别构建的系统发育树均显示,洪平杏的成株及其全部实生苗个体单独聚为一支,且具有较高的支持率(分别为99/79、71/81),独立于杏属其他种之外。而基于核基因ITS序列的系统发育分析结果表明,洪平杏除极少数成株与杏、杏梅聚为一支外,其余所有成株与实生苗聚为2大支(支持率分别为0.82和0.97),而没有克隆的与梅聚在一起。单倍型分析结果表明,该物种的成株与实生苗在SBEI和ycf1b基因序列中均未检测到杏或梅的单倍型,仅有少数(2/9)的实生苗个体在叶绿体mat K基因序列中检测到杏的单倍型。研究结果不支持将洪平杏定为杏和梅的天然杂交种的观点,推测洪平杏应为一个独立的物种,与杏之间的亲缘关系更近并且存在可检测到的基因流。     

基于线粒体16S rRNA基因探讨鹿科动物系统发生关系

鹿类动物的系统演化关系一直存在争议,特别是獐亚科的设立与否.通过测定獐的线粒体16S rRNA基因,并从GenBank获得鹿类另外14种动物的线粒体16S rRNA基因全序列,以水牛和绵羊作双外群,构建系统进化树,探讨鹿类动物系统发生关系及獐亚科的有效性.结果分析表明:(1)支持鹿科分为鹿亚科、麂亚科、獐亚科和美洲鹿亚科,麝科成立;(2)獐亚科有效,支持獐与原属美洲鹿亚科狍共同组成獐亚科;(3)毛冠鹿的分类地位还有待进一步确定.     

基于生物信息学对部分鸡形目鸟类系统发生关系的研究

本研究从GenBank下载了31种鸡形目鸟类、2种雁形目鸟类、1种戴胜目鸟类、1种佛法僧目鸟类和1种犀鸟目鸟类的线粒体16SrRNA基因的部分序列。经比对后序列长度为1384bp,其中有400个变异位点。A+T平均含量为54.2%,C+G平均含量为45.8%。贝叶斯系统发生分析揭示出鸡形目和雁形目分别形成单系。研究表明:(1)珠鸡科是鸡形目中较为原始的一个类群;(2)传统分类中的雉亚科和鹑亚科并非分别形成单系;(3)吐绶鸡科和松鸡科与雉科的亲缘关系较近;(4)孔雀属、Francolinus和Bambusicola之间的亲缘关系较近;山鹑属与多个雉亚科的亲缘关系较近;(5)雉科内各个属的属内分化主要发生于后中新世-更新世期间。     

从c-mos和12S rRNA基因序列探讨现生鸟类早期历史和三趾鹑类的系统地位

通常认为古腭型鸟类处在现生鸟类系统进化树的基部,最近的分子水平研究则认为今腭型鸟类中雀形目种类构成了现生鸟类中一个最古老的支系.本研究通过对现生鸟类中21目39种核c-mos基因和线粒体12S rRNA基因部分序列的分析,从分子角度对现生鸟类的早期进化及三趾鹑鸟类的系统发生进行了探讨.研究结果表明,鸡雁类是现生鸟类最古老的一个支系,现生鸟类的祖先并不是经白垩纪到第三纪大灭绝后残留下来的一些过渡性水鸟(transitional shorebirds).在现生鸟类中,今腭型鸟类为并系发生,古腭型鸟类为单系发生.三趾鹑类在系统发生中晚于鸡雁类和古腭型鸟类,早于今腭型鸟类中非鸡雁类鸟类与鹤形目鸟类的亲缘关系较远.建议将现生鸟类分为初鸟下纲和新鸟下纲2个下纲,三趾鹑类属新鸟下纲的三趾鹑目(Turniciformes).     

基于12S rRNA基因的鹳形目系统发生关系

采用分子系统学的方法探讨鹳形目5个科之间的系统发生关系.文中测出鹳形目鸟类7种mtDNA 12SrRNA基因全序列,并结合来自Genbank的鹳形目另外7个物种及原鸡的同源区序列,经Clustal W软件对位排列后共1 009位点,包含405个变异位点,其中多态性位点381个,260个简约信息位点.基于上述序列数据,以原鸡为外群,使用距离邻接法、最大简约法、最大似然法及贝叶斯法分别重建了鹳形目5科14种的系统发生树.重建的系统发生树显示,内群中的14个种聚合为4支:鹮科构成第一支,聚在系统树的基部;锤头鹳科与鲸头鹳科聚为一支;鹭科和鹳科各自聚成一支.在比较不同建树方法的结果并进行合意树分析后认为:在鹳形目的系统发生中,鹮科可能是最早分化出的一支;锤头鹳科与鲸头鹳科之间的亲缘关系最近,它们祖先与鹭科、鹳科之间的分歧在时间上可能非常接近.鹳形目5个科之间的系统关系可以表示为:(鹮科,(鹭科,鹳科,(锤头鹳科,鲸头鹳科))).     

POLYPHYLY OF TETRASPORALEAN GREEN ALGAE INFERRED FROM NUCLEAR SMALL-SUBUNIT RIBOSOMAL DNA

Ultrastructural studies of tetrasporalean green algae have suggested the order is polyphyletic. These features, including the absolute orientation of the flagellar apparatus and the bi- versus quadriflagellated motile cell morphology, suggest that Chaetopeltis as well as a number of others, may be ancestral to a group that includes Tetraspora. In this study, we examine the phylogenetic relationships of selected tetrasporalean taxa based on analysis of 18S ribosomal RNA gene sequences. Results show that the tetrasporalean taxa are polyphyletic. Biflagellated genera group with biflagellated volvocalean taxa, whereas the quadriflagellated species compose a distinct monophyletic clade not closely related to the biflagellated taxa. In addition, tetrasporalean taxa group with other chlorophycean algal species with similar flagellar apparatus absolute orientation, but the quadriflagellated Tetrasporales do not appear to be ancestral to the entire Chlorophyceae. These results are concordant with previous conclusions drawn from ultrastructural data and further confirm the utility of (small-subunit) ribosomal RNA gene sequences to discern green algal evolutionary relationships.     

基于18S rRNA基因序列的直翅目主要类群系统发育关系研究

基于78种直翅目昆虫的18S rRNA基因全序列构建了直翅目各主要类群间的系统发育关系。本研究的结果支持直翅目的单系性,但不支持蝗亚目和螽亚目各自的单系性;直翅目下除蜢总科和蝗总科外各总科的划分多数与Otte系统相一致;蜢总科的单系性得不到支持;蝗总科的剑角蝗科、斑腿蝗科、斑翅蝗科、网翅蝗科和槌角蝗科5科均不是单系群,各物种间的遗传距离差异不大,应合并为一科,即蝗科;本研究支持将Otte系统中蚱总科和螽蟖总科下各亚科级阶元提升为科级阶元;18S rRNA基因全序列可以作为划分科级阶元的工具,当位于同一分支上互成姐妹群的类群间的遗传距离超过1%时,这几个类群属于不同的科;但由于其在进化上的保守性,18S rRNA基因只能用于纲目等高级阶元间关系的研究,而由其获得的总科以下阶元间的关系并不可靠。     

中国绿水螅抗坏血酸过氧化物酶基因的克隆、抗体制备及表达分析

为探讨中国绿水螅(Hydra sinensis)抗坏血酸过氧化物酶(Ascorbate peroxidase, APX)基因的起源及功能, 研究采用RACE方法克隆了中国绿水螅APX基因的全长cDNA序列。该cDNA序列总长1357 bp, 包括5′非编码区107 bp, 3′非编码区146 bp及开放阅读框(Open reading frame, ORF) 1104 bp, 共编码367个氨基酸, 预测蛋白质分子量为40.79 kD。BLAST结果表明中国绿水螅APX蛋白同源序列绝大部分来自植物界; 通过最大似然法(Maximum-likelihood)和贝叶斯分析(Bayesian inference)进行的系统发生分析显示植物界及动物界物种的APX序列各自形成单系群。把APX基因ORF全长序列克隆到原核表达质粒pET-GST中, 重组质粒转化E. coli BL21 (DE3)菌株, IPTG诱导后成功表达重组融合蛋白GST-APX, 再使用纯化的重组蛋白免疫新西兰兔制备多克隆抗体用于APX蛋白的免疫印迹分析(Western blotting assay, WB)。在不同光照时长梯度(光强度2000 lx, 每天分别光照0、4h、8h、12h、16h、20h及24h)下培养中国绿水螅30d, 实时定量PCR (Quantitative real-time PCR, qPCR)及WB检测结果均表明光照时间较长时(每天光照12h以上)绿水螅APX表达呈现一定程度的上调。在长时间光辐射下水螅体内共生绿藻连续进行光合作用所累积的大量活性氧能够扩散到水螅细胞内, 此时水螅体内表达上调的APX可能参与清除其细胞内的活性氧。     

PHYLOGENY OF THE BATRACHOSPERMALES (RHODOPHYTA) INFERRED FROM rbcL AND 18S RIBOSOMAL DNA GENE SEQUENCES

The sequence data from the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase ( rbc L) gene and 18S ribosomal DNA (small subunit) of taxa in the freshwater rhodophyte order Batrachospermales were used to construct phylogenetic hypotheses. Taxa examined in this study represent four families, eight genera, and six sections of the genus Batrachospermum . In addition, Rhododraparnaldia oregonica Sheath, Whittick et Cole, was included in the analysis because it shares particular ultrastructural, reproductive, and morphological characteristics with members of the Batrachospermales and Acrochaetiales. The trees generated from each gene, as well as a combined data set, were largely congruent. Rhododraparnaldia consistently occurs on an early branch within the Acrochaetiales – Palmariales clade and does not appear to be a member of the Batrachospermales. In addition, Thorea violacea Bory de St. Vincent was not closely related to the other taxa of the Batrachospermales in all trees and hence the family Thoreaceae does not appear to be a natural grouping within this order. All other taxa analyzed, which are presently classified within this order, formed a monophyletic clade in most analyses. Psilosiphon scoparium Entwisle was not closely allied with the taxa of the Lemaneaceae, lending support to the newly proposed family Psilosiphonaceae. Sequence data from the remaining taxa of the Lemaneaceae support the concept of a derived monophyletic clade. The genus Batrachospermum appears to comprise many morphologically similar but distantly related taxa, which will need further investigation to resolve their taxonomic status. Tuomeya, Sirodotia and Nothocladus are retained at the generic level until further data are obtained.     

TAXONOMIC SIGNIFICANCE OF NUCLEUS-MICROBODY ASSOCIATIONS,SEGREGATED NUCLEOLI AND OTHER NUCLEAR FEATURES IN SIPHONOUS GREEN ALGAE1

On the basis of fine-structural features of nuclei the “true” siphonous green algae (Eusiphoniidae) are divided into three groups. In the Codium group nuclei are generally similar to those in most other algae. The Penicillus group is characterized by the association of microbodies with nuclei. In the Avrainvillea group nucleoli are segregated into a granular and a fibrillar component. This condition, known as macrosegregation, persists throughout the vegetative phase of the life history and may indicate a peculiar ribosomal RNA cycle. The Penicillus group corresponds to the order Caulerpales Feldmann. Avrainvillea and Cladocephalus probably constitute a new order. Blastophysa, although having a type of segregated nucleoli, is probably more closely related to Siphonocladales than to any order in Eusiphoniidae.     

伪指环虫、异钩虫和三钩虫的系统位置

采用单个虫体PCR扩增、序列测定与分析的方法,对锚首虫科中后吸器形态较特殊的3个属:伪指环虫属、异钩虫属和三钩虫属的28S rRNA基因5'端序列进行了研究,并采用PAUP软件构建了分子系统树.结果显示,异钩虫属、三钩虫属和伪指环虫属明显地嵌合于其他锚首虫属之间,进而明确了这3个属的分类地位,应归属锚首虫科.锚首虫科和指环虫科之间的关系则有待今后进一步研究.     

DISCONTINUOUS MITOCHONDRIAL AND CHLOROPLAST LARGE SUBUNIT RIBOSOMAL RNAs AMONG GREEN ALGAE: PHYLOGENETIC IMPLICATIONS1

To provide insights into the occurrence, evolution, and phylogenetic distribution of discontinuous mitochondrial and chloroplast large subunit ribosomal RNAs (LSU rRNAs) among green algae, we surveyed 12 taxa representing three classes of green algae: the Chlorophyceae, Pleurastrophyceae, and Micromonadophyceae (sensu Mattox and Stewart 1984). We present evidence that discontinuous mitochondrial and chloroplast LSU rRNAs are quite widespread among green algae. Mitochondrial LSU rRNAs appear discontinuous in zoosporic chlorophycean lineages displaying a clockwise or directly opposed configuration in their flagellar apparatus, as well as in chlorococcalean autosporic taxa phylogenetically related to them, but are continuous among zoosporic green algal lineages with a counterclockwise flagellar apparatus configuration, as well as among chlorococcalean autosporic taxa phylogenetically related to them. Chloroplast LSU rRNAs appear discontinuous in all of the lineages investigated. Discontinuous mitochondrial LSU rRNA represents a molecular trait that might have originated at or near the base of Chlorophyceae, whereas discontinuous chloroplast LSU rRNA might have developed very early in the evolutionary history of the green algal group itself. We suggest, therefore, that the presence of discontinuous mitochondrial but not chloroplast LSU rRNA can be used as an additional character in assessing phylogenetic affiliations among green algae.     

USING rbcL SEQUENCES TO TEST HYPOTHESES OF CHLOROPLAST AND THALLUS EVOLUTION IN CONJUGATING GREEN ALGAE (ZYGNEMATALES, CHAROPHYCEAE)

Sequences for the Rubisco large subunit (rbcL) gene were used to test hypotheses about the evolution of chloroplast shape and thallus type in genera of two families of conjugating green algae (Zygnematales): the Mesotaeniaceae (saccoderm desmids, mostly unicellular) and the Zygnemataceae (strictly filamentous). Unicellular (u) and filamentous (f) genera exhibit a series of three similar chloroplast shapes: ribbonlike (e.g. Spirotaenia [u], Spirogyra [f], and Sirogonium [f], laminate (e.g. Mesotaenium [u] and Mougeotia [f]), and twin-stellate (e.g. Cylindrocystis [u] and Zygnema [f]. Two conflicting phylogenetic hypotheses have been proposed: 1) families are polyphyletic constructs drawn from three lineages, each with unicellular and filamentous taxa characterized by a specific chloroplast shape; or 2) unicells form one monophyletic lineage (Mesotaeniaceae) and filaments form another (Zygnemataceae), with some chloroplast shapes independently derived. The rbcL data strongly refute hypothesis 2 (monophyly of the two traditional families) and support hypothesis 1 in part. Parsimony, maximum likelihood, and neighbor-joining analyses of the rbcL data strongly support monophyly of a clade containing taxa with ribbonlike chloroplasts and, to a lesser extent, monophyly of a second clade of the four genera with the other two chloroplast shapes. Two saccoderm genera (Roya, curved laminate chloroplasts; Netrium, cucumber-shaped chloroplasts) are not members of either of these clades, but they are included in a monophyletic Zygnematales .     

THE OCCURRENCE AND PHYLOGENETIC SIGNIFICANCE OF PUTATIVE PLACENTAL TRANSFER CELLS IN THE GREEN ALGA COLEOCHAETE

Following fertilization, zygotes of the green alga Coleochaete orbicularis, which are retained on the haploid thallus, first enlarge, then become covered with a layer of vegetative cells. Light microscopy and high-voltage electron microscopy revealed the presence of localized wall ingrowths in vegetative cells adjacent to zygotes. These covering cells resemble the gametophytic placental transfer cells of embryophytes in their morphology, location, and time of development. If Coleochaete cells with wall protuberances function as do placental transfer cells of embryophytes, their presence is evidence that photosynthates may be transported between haploid thallus cells and zygotes. Thus, a nutritional relationship between different phases of the life cycle, similar to that which occurs in embryophytes, may also have evolved in green algae. This first report of putative placental transfer cells in a green alga supports Bower's (1908) ideas concerning the origin of land plant sporophytes and alternation of generations. The presence or absence of cells with wall ingrowths in several species of Coleochaete was correlated with estimates of zygote-plant area ratios.