中国大头蛙属3个种线粒体ND1基因全序列分析与亲缘关系

测定了大头蛙和脆皮大头蛙线粒体ND1基因全序列长度分别为978 bp和958 bp,(对应编码325和319个氨基酸)。对所测基因序列组分进行了分析,并与福建大头蛙同源序列进行比较发现,978个核苷酸位点中,有664个保守位点和多变位点294个。同时发现福建大头蛙与大头蛙该基因序列的同源性最高(核苷酸序列同源性为78.77%,氨基酸序列为92.62%)。基于ND1基因全序列的氨基酸和核苷酸两种数据形式,选用M ega3.1软件中的NJ法对大头蛙属3个种、黑斑蛙、泽陆蛙及外群中国大鲵共6条基因序列进行系统树重建分析,结果表明:所得的2个NJ树均将大头蛙属3个种聚于一支,其中大头蛙与福建大头蛙为姐妹群关系(自检值均高度支持),从而证实了大头蛙与福建大头蛙亲缘关系较近的观点。     

用线粒体tRNA基因探讨现存两栖动物三个目间系统发生关系

现存两栖类3个目的系统发生关系仍然没有统一意见,最广泛被接受的假说是单系起源,并且无尾类和有尾类为姐妹群关系而排斥蚓螈类(蛙类假说)。然而,这一假说一直存在争议。我们在测定了泽蛙线粒体基因组全序列的基础上,与已知其他的6种两栖类进行详细的比较分析,同时选择了11种高等脊椎动物的线粒体全基因序列,以硬骨鱼类作外群,用22个tRNA基因合并数据进行系统发生重建分析,结果表明MP、ML树都强力地支持现生两栖类动物为单系群,并且有尾目和蚓螈目为姐妹群关系。这个结果与蛙类假说是相矛盾的,与Bolt(1991)在形态学基础上提出的有尾类和蚓螈类为姐妹群关系的假说相一致,并得到建立在线粒体和核rRNA基因数据基础上的许多分子研究的支持。另外还探讨了本结果与前人的研究不一致的原因,以及利用线粒体全基因序列进行系统发生分析可能存在的偏差。     

探讨两栖纲三个目之间的系统发生关系

现存两栖纲分为3个目,然而它们的系统发生关系仍存在较大的争议.选择Genbank上25种两栖动物线粒体全序列,结合已测定的福建大头蛙(Lim nonectes fujianensis)线粒体基因组全序列,以天鹅和眼镜凯门鳄为外群,用线粒体tRNA基因的合并数据重建系统树.采用P AU P(version 4.0b10)软件构建MP和NJ树,tree-puzzle5.2构建ML树.结果显示,NJ树和ML树以较高的自引导值支持有尾目和蚓螈目为姐妹群,而MP树则支持无尾目和有尾目为姐妹群.     

基于12S rRNA基因序列探讨凹耳蛙(无尾目,蛙科)的分类地位

为探讨凹耳蛙Rana tormotus的分类地位,测定了凹耳蛙线粒体12S rRNA基因序列,并从GenBank中下载了蛙科33种蛙的同源序列进行分析,用BI法、NJ法和MP法构建分子系统树.结果表明:3种树拓扑结构基本一致,都显示凹耳蛙与长吻胡湍蛙Huia nasica亲缘关系最近,凹耳蛙与长吻胡湍蛙相聚后再与臭蛙Odorrana组成单系群,而与湍蛙Amolops亲缘关系较远.据此,支持将该蛙从湍蛙属划出,并建议归入臭蛙属.     

基于12S和16S rRNA序列的湍蛙属部分物种的系统发育关系

测定了湍蛙属 6个种共 10个种群 ,以及 4个外群种的线粒体 12S和 16SrRNA基因片段 ,比对后有94 0bp序列 ,发现 35 2个变异位点、 186个简约性位点。运用NJ法、MP法、ML法构建了系统关系树 ,各系统树一致表明内群为一单系群 ,分为两组 :第一组中 ,四川湍蛙两种群先聚合 ,再和棕点湍蛙聚为一支 ;第二组中 ,香港湍蛙和戴云湍蛙聚为一支 ,而香港大屿山离岛湍蛙种群首先与华南湍蛙相聚 ,再与武夷湍蛙构成姐妹支。研究结果表明 :香港地区增加 1种湍蛙分布 ;戴云湍蛙是一有效种 ;四川湍蛙的石棉和洪雅种群间遗传差异达到或超过其他种间的分歧水平。     

虎纹蛙及其它几种蛙科动物线粒体16S rRNA的空间结构分析

测定了虎纹蛙16S rRNA基因全序列,并从GeneBank上下载了5种蛙科动物同源序列,利用CLUSTAL与MEGA3.1软件比较了种间的序列差异,用RNAdraw软件推测分析二级结构的空间构象,并根据空间结构对这6种蛙科动物的分类地位进行讨论分析。结果表明：虎纹蛙与黑斑蛙的二级结构最为相似,与泽蛙结构大体一致,而与黑斑蛙同属的金线蛙则表现出较大的差异;凹耳蛙与上述几种差别较大;福建大头娃与这几种蛙科动物差距最为明显。空间结构分析与序列比较结果存在一定的差异。     

凹耳蛙MHCII类B基因第二外显子多态性分析

两栖类正经历全球范围内的种群衰退,很多两栖动物集群灭绝事件与环境病原体(如壶菌(Batrachochytrium dendrobatidis)的侵扰有关。MHC基因的表达产物在有颌脊椎动物免疫应答过程中起关键作用,其多态性通常与动物对疾病的抗性或易感性密切相关,因而被认为是研究动物适应性进化的最佳候选基因之一。本文对中国特有的无尾两栖动物凹耳蛙(Odorrana tormota)MHC II类B基因多态性进行初步研究。首先,利用1对通用引物扩增出凹耳蛙MHC II类B基因exon2长约180bp的DNA片段。在此基础上,利用ligation-mediated PCR进一步获取侧翼未知序列,序列拼接后长2,030bp,包含exon2以及intron1和intron2的部分序列。基于上述序列设计出凹耳蛙B基因exon2特异性引物(IIQ1BU/IIQ1BD),对该物种黄山种群32个样品进行PCR扩增和克隆测序,共获得34个不同的等位基因,等位基因序列核苷酸和氨基酸变异位点的比例分别为16.17%(33/204)和26.87%(18/67),大多数氨基酸变异位点位于推测的抗原结合位点(antigen binding sites,ABS)。每个样品包含2-5个等位基因,结合等位基因序列特征以及cDNA表达分析结果,推测凹耳蛙至少拥有3个可表达的B基因座位。与文献报道的蛙科其他物种比较后发现,尽管凹耳蛙目前的分布区非常狭窄,但其MHC II类B基因多态性明显高于蛙科其他动物。等位基因碱基替换模式提示凹耳蛙MHC II类B基因曾经历过强烈的正选择作用,ABS区的dN值显著大于dS(P<0.05),PAML软件包CODEML程序中不同模型的似然比检测(likelihood rate test)结果同样支持上述推论,贝叶斯经验贝叶斯路径(Bayesian Em-pirical Bayes)共检测出5个显著受正选择作用的氨基酸位点。贝叶斯系统树的拓扑结构显示,无尾两栖类不同科的等位基因分别形成单系群,但蛙科不同属的等位基因未能形成单系群,蛙属绿池蛙(Rana clamitans)的1个等位基因与臭蛙属凹耳蛙的部分等位基因享有共同的谱系关系,提示蛙科不同属间的B基因存在跨种多态性。     

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))).     

沼蛙(Rana guentheri)种内遗传分化状况初步研究

本文测定了香港特别行政区内沼蛙(Rana guentheri)多个种群的线粒体16S rRNA基因部分DNA序列,并与广州及越南共4个沼蛙基因序列作对比,研究各地区种群遗传分化状况,研究表明三个地区之间的序列遗传分化为0．4％～1．1％,表明沼蛙mtDNA 16S rRNA基因进化速率比较低,其中香港沼蛙10个个体在mtDNA 16S rRNA基因上没有出现变异,共发现5种单倍型,其中香港与广州种群关系最近,越南河内种群与志玲种群关系最近。     

大头蛙Sox基因的克隆与序列分析

参考人SRY基因HMC—box的保守区序列,设计一对特异引物,采用PCR技术扩增了大头蛙的Sox基因,并对扩增产物进行了克隆与测序。结果在雌雄个体中均扩增出217bp的基因片段,与人对照相同。序列分析表明,大头蛙雌雄个体之间Sox序列没有差异,与人SOX基因的同源性达到88％,与其它各类动物的Sox基因也都有非常高的相似性。结果支持Sox基因在进化上十分保守的结论。     

从线粒体基因探讨中国大头蛙群的分类及其属内地位

Partial sequences of the mitochondrial 12S rRNA and 16S rRNA gene were determined for 8 populations of three species of Chinese Limnonectes, and aligned with the published sequences of Limnonectes from other parts of the world. When Nanorana parker, Paa boulengeri, Fejervarya limnocharis and Hoplobatrachus rugulosus was used as outgroup taxa (Accession Nos. AY158705, AY313685, AF206111, AF206491, AY322311). The sequences of the 12S rRNA and 16S rRNA genes totaled 950 nueleotide positions with gaps including 510 variable sites. We reconstructed phylogenetie trees using Clustal X 1.8, Mega 2.1 and PHYLIP 3.5e software, and using the maximum parsimony and maximum likelihood methods, respectively. Our analyses suggest that these fanged frogs from China are another monophyletie group in addition to the four monophyletie groups identified by previous studies. The Chinese Limnonectes were grouped into three elades (BCL 55% ). The first elade contains one species (BCL 100% ), from a population of Limnoneetes fragilis from Hainan Province. The second contains four individuals (BCL 100% ), i. e. two populations of Limnonectes kuhlii from Yunnan Province. The third contains one species (BCL 100% ), i. e. five populations of Limnonectes fujianensis from Fujian Province and 1 from Taiwan Province. The resulted phylogenetie trees indicate L. fragilis is basal to L. kuhlii L. fujianensis 。     

太湖新银鱼m tDNA COII和Cytb基因的克隆与序列分析

设计了5对特异性引物,扩增、拼接并测定出太湖新银鱼线粒体tRNAAsp-COII-tRNALys和tRNAGlu-Cytb-tRNAThr两段基因序列片段。基因定位和序列分析发现,太湖新银鱼线粒体COII基因全序列长度为691 bp,序列AT含量为52.80%,编码230个氨基酸;线粒体Cytb基因序列全长为1141 bp,AT含量为48.90%,它编码380个氨基酸。分别位于线粒体COII和Cytb基因两翼的4个tRNA基因(tRNAAsp、tRNALys、tRNAGlu和tRNAThr)同时被测定出来。将太湖新银鱼与有明银鱼、小齿日本银鱼的同源序列进行比对分析,并基于线粒体COII Cytb基因合并数据的核苷酸和氨基酸两种序列形式,以黑斑蛙为外群,对10种鱼类进行分子系统树的构建,结果一致表明:小齿日本银鱼与有明银鱼的亲缘关系近于太湖新银鱼;鲱科与鲑科的亲缘关系近于银鱼科鱼类;此外在本研究硬骨鱼类的4个科中,白鲟科作为原始而古老的类群,是在系统进化的过程中首先分化出来的一支。     

Mitochondrial evidence on the phylogenetic position of caecilians (Amphibia: Gymnophiona)

The complete nucleotide sequence (17,005 bp) of the mitochondrial genome of the caecilian Typhlonectes natans (Gymnophiona, Amphibia) was determined. This molecule is characterized by two distinctive genomic features: there are seven large 109-bp tandem repeats in the control region, and the sequence for the putative origin of replication of the L strand can potentially fold into two alternative secondary structures (one including part of the tRNA(Cys)). The new sequence data were used to assess the phylogenetic position of caecilians and to gain insights into the origin of living amphibians (frogs, salamanders, and caecilians). Phylogenetic analyses of two data sets-one combining protein-coding genes and the other combining tRNA genes-strongly supported a caecilian + frog clade and, hence, monophyly of modern amphibians. These two data sets could not further resolve relationships among the coelacanth, lungfishes, and tetrapods, but strongly supported diapsid affinities of turtles. Phylogenetic relationships among a larger set of species of frogs, salamanders, and caecilians were estimated with a mitochondrial rRNA data set. Maximum parsimony analysis of this latter data set also recovered monophyly of living amphibians and favored a frog + salamander (Batrachia) relationship. However, bootstrap support was only moderate at these nodes. This is likely due to an extensive among-site rate heterogeneity in the rRNA data set and the narrow window of time in which the three main groups of living amphibians were originated.     

Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of the bell-ring frog, Buergeria buergeri (family Rhacophoridae)

In this study we determined the complete nucleotide sequence (19,959 bp) of the mitochondrial DNA of the rhacophorid frog Buergeria buergeri. The gene content, nucleotide composition, and codon usage of B. buergeri conformed to those of typical vertebrate patterns. However, due to an accumulation of lengthy repetitive sequences in the D-loop region, this species possesses the largest mitochondrial genome among all the vertebrates examined so far. Comparison of the gene organizations among amphibian species (Rana, Xenopus, salamanders and caecilians) revealed that the positioning of four tRNA genes and the ND5 gene in the mtDNA of B. buergeri diverged from the common vertebrate gene arrangement shared by Xenopus, salamanders and caecilians. The unique positions of the tRNA genes in B. buergeri are shared by ranid frogs, indicating that the rearrangements of the tRNA genes occurred in a common ancestral lineage of ranids and rhacophorids. On the other hand, the novel position of the ND5 gene seems to have arisen in a lineage leading to rhacophorids (and other closely related taxa) after ranid divergence. Phylogenetic analysis based on nucleotide sequence data of all mitochondrial genes also supported the gene rearrangement pathway.     

大黄鱼16SrRNA和18SrRNA基因的测定与序列分析

利用多对引物,扩增并测定出大黄鱼16SrRNA基因和18SrRNA基因的部分序列,其长度分别为1202bp和1275bp,16SrRNA基因序列的GC含量为46．12％,18SrRNA基因的Gc含量为53．oo％。将大黄鱼16SrRNA基因序列与GenBank中15种硬骨鱼类的同源序列结合,同时将其18SrRNA基因序列与GenBank中9种脊索动物的同源序列相结合,运用软件获得各自序列间差异百分比,转换和颠换数值等信息。基于这两种基因序列,利用NJ法和BI法,分别构建16种硬骨鱼类和10种脊索动物的分子系统树。18SrRNA构建的系统树包括三大支,一支为哺乳类、鸟类和爬行类共6个物种,一支为两栖类的1个物种,另一支为2种硬骨鱼类。16SrRNA构建的系统树显示大黄鱼所在的石首鱼科与鲈科和盖刺鱼科亲缘关系较近。此外还讨论了这两个基因的序列特征。     

Taxonomic relationships among Turkish water frogs as revealed by phylogenetic analyses using mtDNA gene sequences

We assessed taxonomic relationships among Turkish water frogs through estimation of phylogenetic relationships among 62 adult specimens from 44 distinct populations inhabiting seven main geographical regions of Turkey using 2897 bp sequences of the mitochondrial Cytb, 12S rRNA and 16S rRNA genes with equally-weighted parsimony, likelihood, and Bayesian methods of inference. Monophyletic clade (Clade A) of the northwesternmost (Thrace) samples is identified as Pelophylax ridibundus. The other clade (Clade B) consisted of two monophyletic subclades. One of these contains specimens from southernmost populations that are regarded as an unnamed species. The other subclade consists of two lineages, of which one corresponds to P. caralitanus and another to P. bedriagae. Taxonomic relationships of these two species are discussed and recognition of P. caralitanus as a subspecies of P. bedriagae is proposed.     

Natural relationship between bacteroides and flavobacteria.

Comparisons among 16S rRNA sequences from various eubacteria reveal a natural relationship between the bacteroides (represented by the Bacteroides fragilis sequence) and a phylogenetic unit that comprises the flavobacteria, cytophagae, flexibacteria, and others (represented by the Flavobacterium heparinum sequence). Although the relationship is not a close one, it is, nevertheless, specific. rRNAs from these two organisms are not only closer to one another in overall sequence than they are to outgroup species (such as Bacillus subtilis, Escherichia coli, Desulfovibrio desulfuricans, and Agrobacterium tumefaciens), but they show common idiosyncrasies (i.e., derived characteristics) in both rRNA sequences and higher-order structures.     

Endozoicomonas elysicola gen. nov., sp. nov., a gamma-proteobacterium isolated from the sea slug Elysia ornata

A Gram-negative, strictly aerobic, rod-formed bacterium, strain MKT110(T), was isolated from a mollusk, the sea slug Elysia ornata collected in seawater off the coast of Izu-Miyake Island, Japan at a depth of 15m. Comparative 16S rRNA gene sequences analysis indicated that the isolate MKT110(T) constituted a novel lineage in gamma-proteobacteria related to the genera Zooshikella, Oceanospirillum, Microbulbifer, Marinobacter, Saccharospirillum and Pseudomonas. The strain MKT110(T) was closely related to the clones from marine sponge Halichondria okadai (AB054136, AB054161) and the coral Pocillopora damicornis (AY700600, AY700601). The phylogenetic tree based on the 16S rRNA gene sequences showed that MKT110(T) and four clones formed a sub-lineage related to the genus Zooshikella, with a bootstrap value of 100%. MKT110(T) required salt for its growth and was mesophilic. The bacterium contained 16:1omega7c, 16:0 and 14:0 as major cellular fatty acids, and 3-OH 14:0, 3-OH 10:0 and 3-OH 12:0 as major hydroxy fatty acids. The DNA base composition of the isolate was 50.4 mol% G+C. The major quinone was Q-9. The bacterium is distinguished from currently recognized bacterial genera based on phylogenetic and phenotypic features and should be classified in a novel genus for which the name Endozoicomonas elysicola gen. nov., sp. nov. is proposed. (type strain MKT110(T)=IAM 15107(T)=KCTC 12372(T); GenBank accession no. AB196667).     

Molecular Evidence for the Early History of Living Amphibians

The evolutionary relationships of the three orders of living amphibians (lissamphibians) has been difficult to resolve, partly because of their specialized morphologies. Traditionally, frogs and salamanders are considered to be closest relatives, and all three orders are thought to have arisen in the Paleozoic (>250 myr). Here, we present evidence from the DNA sequences of four mitochondrial genes (2.7 kilobases) that challenges the conventional hypothesis and supports a salamander–caecilian relationship. This, in light of the fossil record and distribution of the families, suggests a more recent (Mesozoic) origin for salamanders and caecilians directly linked to the initial breakup of the supercontinent Pangaea. We propose that this single geologic event isolated salamanders and archaeobatrachian frogs on the northern continents (Laurasia) and the caecilians and neobatrachian frogs on the southern continents (Gondwana). Among the neobatrachian frog families, molecular evidence supports a South American clade and an African clade, inferred here to be the result of mid-Cretaceous vicariance.