Molecular phylogeny of the Chinese ranids inferred from nuclear and mitochondrial DNA sequences

The phylogeny of representative species of Chinese ranids was reconstructed using two nuclear (tyrosinase and rhodopsin) and two mitochondrial (12S rRNA, 16S rRNA) DNA fragments. Maximum parsimony, Bayesian, and maximum likelihood analyses were employed.-

Molecular phylogeny of Rhacophoridae (Anura): A framework of taxonomic reassignment of species within the genera Aquixalus, Chiromantis, Rhacophorus, and Philautus

Phylogenetic relationships among representative species of the family Rhacophoridae were investigated based on 2904 bp of sequences from both mitochondrial (12S rRNA, 16S rRNA, the complete t-RNA for valine), and nuclear (tyrosinase, rhodopsin) genes. Max

Phylogenetic relationships among Rhacophorinae (Rhacophoridae, Anura, Amphibia), with an emphasis on the Chinese species

The partial nucleotide sequence of mitochondrial 12S and 16S rRNA genes was determined for 23 Chinese species of Rhaeophoridae (Amphibia: Anura), representing four of the eight recognized genera. Using Buergeriinae as the outgroup, phylogenetic analyses (

Molecular phylogeny of the yuhinas (Sylviidae : Yuhina): a paraphyletic group of babblers including Zosterops and Philippine Stachyris

Mitochondrial sequences (2,379 bp) from cytochrome b, ND3, 12s and 16s rRNA were analyzed in order to reconstruct the phylogenetic relationships within the yuhinas (Yuhina), including the chestnut-faced babbler Stachyris whiteheadi which is endemic to the

Re-examination of the phylogeny of Rhacophoridae (Anura) based on mitochondrial and nuclear DNA

The phylogenetic relationships among rhacophorid frogs are under dispute. We use partial sequences of three mitochondrial (12S rRNA, 16S rRNA, and cytochrome b) and three nuclear protein-coding (Rag-1 rhodopsin exon 1, and tyrosinase exon 1) genes from 57

中国鸟类的DNA分类及系统发育研究概述

鸟类分类是鸟类学其他研究领域的基础,近年来分子技术的发展,以及计算机技术的应用为鸟类分类学和鸟类系统演化研究提供了新的研究手段,给传统的系统分类研究带来了新的机遇.Tautz等于2002年首先提出运用DNA序列作为生物分类系统的主要平台,即DNA分类学(DNA Taxonomy).而Hebert等于2003年则首次提出了DNA条形码(DNA Barcoding)的概念,并对其物种分类和鉴定意义予以肯定,建议利用线粒体细胞色素C氧化酶亚单位Ⅰ(COI)的特定区段来做DNA条形编码的基础.在鸟类DNA分类方面,国内学者应用线粒体基因Cut b,COI,c-mos,c-myc,12s rRNA,16s rRNA,ND2,ND3,CR,RAG-1以及核基因myoglobin introⅡ等不同片段对很多类群进行了分类探讨和系统发育研究.但是主要集中在鸡形目及雀形目鸟类.中国是鸟类多样性极其丰富的国家,近年来很多亚种、种及以上分类阶元依然存在问题,因此,中国鸟类物种的分类地位、系统发育与演化关系等依然有很多问题等待深入研究.目前国内基于COI的鸟类分类及系统发育研究有了一些报道,但是真正的DNA条形码工作尚需继续、深入地开展.

Meiothermus rosaceus sp nov isolated from Tengchong hot spring in Yunnan, China

A rosy-pigmented Gram-negative, thermophilic bacterium with an optimum growth temperature of about 55degreesC was isolated from Tengchong hot springs in Yunnan province, China. Its growth scarcely occurred below 40degreesC or above 70degreesC. Phylogenetic and secondary structural analyses of 16S rRNA and DNA-DNA hybridization showed that the organism represented a new species of the genus Meiothermus. This new species could be distinguished easily from other species of the genus Meiothermus by the following phenotypic characteristics: rosy pigment, expanded body, sucrose and maltose were not utilized, gelatin and starch were not hydrolyzed. On the basis of the above data, the name Meiothermus rosaceus sp. nov. was proposed for the species represented by the strain RH9901(T)(CCTCC-AB200291). (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

中国石爬鮡属鱼类的形态变异及物种有效性研究

石爬属鱼类的青石爬和黄石爬的物种界限一直不清楚。采用形态判别和线粒体 16SrRNA基因序列分析相结合的方法 ,分别研究分析了青石爬和黄石爬的物种划分、地理分化及遗传多态性。结果表明 :(1)区别青石爬和黄石爬的重要特征腹鳍起点至臀鳍起点的距离是否大于至鳃孔下角的距离 ,腹鳍相对位置 ,头部相对大小与上颌须的须状延长部分等相互之间有一定的相关性 ,但是在研究的样本中没有截然的界限 ,而是有较大的重叠 ,难以区分 ;(2 )从地埋分布看 ,金沙江不同支流的样本在上述特征上有一定的区别 ,但是没

PCR-DGGE Fingerprinting Analysis of Plankton Communities and Its Relationship to Lake Trophic Status

Plankton communities in eight lakes of different trophic status near Yangtze, China were characterized by using denatured gradient gel electrophoresis (DGGE). Various water quality parameters were also measured at each collection site. Following extraction of DNA from plankton communities, 16S rRNA and 18S rRNA genes were amplified with specific primers for prokaryotes and eukaryotes, respectively; DNA profiles were developed by DGGE. The plankton community of each lake had its own distinct DNA profile. The total number of bands identified at 34 sampling stations ranged from 37 to 111. Both prokaryotes and eukaryotes displayed complex fingerprints composed of a large number of bands: 16 to 59 bands were obtained with the prokaryotic primer set; 21 to 52 bands for the eukaryotic primer set. The DGGE-patterns were analyzed in relation to water quality parameters by canonical correspondence analysis (CCA). Temperature, pH, alkalinity, and the concentration of COD, TP and TN were strongly correlated with the DGGE patterns. The parameters that demonstrated a strong correlation to the DGGE fingerprints of the plankton community differed among lakes, suggesting that differences in the DGGE fingerprints were due mainly to lake trophic status. Results of the present study suggest that PCR-DGGE fingerprinting is an effective and precise method of identifying changes to plankton community composition, and therefore could be a useful ecological tool for monitoring the response of aquatic ecosystems to environmental perturbations.

Investigations into the perplexing interrelationship of the Genus Takifugu Abe, 1949 (Tetraodontiformes, Tetraodontidae)

The phylogenetic relationships within the genus Takifugu Abe, 1949 (Tetraodontiformes, Tetraodontidae) remain unresolved. Because of the use of Takifugu as model organisms, the resolution of these relationships is crucial for the interpretation of evolutionary trends in biology. Pufferfishes of this genus are comprised of a comparatively small number of species and are mainly distributed along the coastal region of the western part of the Sea of Japan and the coastline of China. Mitochondrial gene sequences were employed to test the phylogenetic hypotheses within the genus. Seventeen species of the genus were examined. Molecular phylogenetic trees were constructed using the maximum parsimony, neighbor-joining, maximum likelihood and Bayesian methods. Our hypothesis of internal relationships within the genus differs from previous hypotheses. Our results indicate that (1) the genus Takifugu is a monophyletic assemblage; (2) the genus is divided into 6 subgroups based on the molecular data; and (3) there is low genetic diversity among the species within this genus. In addition, speciation within Takifugu appears to be driven by hybridization and isolation by distribution. Our results also suggested that the taxonomy in the genus should be clarified based on both molecular and morphological data.

Redescription of Trypanosoma siniperca Chang 1964 from freshwater fish of China based on morphological and molecular data

During the parasite fauna investigation within 2005, the freshwater fish trypanosome Trypanosoma siniperca Chang 1964 was isolated from the blood of Mandarin carp (Siniperca chuatsi) from Niushan Lake, Hubei Province, central China. Blood trypomastigotes were observed only, and the density of infection was low. Light microscopy examinations of this material made it possible to study in detail the morphology of this parasite and redescribe it according to current standards. T. siniperca is characterized also on the molecular level using the sequences of SSU rRNA gene. Phylogenetic analyses based on these sequences allowed clearer phylogenetic relationships to be established with other fish trypanosomes sequenced to date.

New data on Myxobolus longisporus (Myxozoa : Myxobolidae), a gill infecting parasite of carp, Cyprinus carpio haematopterus, from Chinese lakes

The original description of Myxobolus longisporus Nie et Li, 1992, the species infecting gills of Cyprinus carpio haematopterus L., is supplemented with new data on the spore morphology and pathogenicity. Spores are elongate pyriform with pointed anterior end, 15.7 (15.5-16.5) mum long, 6.7 (6-8) mum wide and 5.5 mum thick. Sutural ridge is straight and narrow. Mucus envelope is lacking. Two equal-sized elongate pyriform polar capsules are 8.5 mum long and 2.5 mum wide with convergent long axes. Polar filament coiled perpendicularly to the long axis of the capsule makes 9 (8-10) turns. Posterior end of polar capsules exceeds mid-spore by 15-20%. Cyst-like plasmodia are localised in the gill secondary lamellae. The infection is described in adult big host specimens. Gross lesions manifested as dark red colouration of gill tissues were restricted to the ventral part of the first gill arches. Remarkable site specificity (apical part of secondary lamellae) was observed in the course of development of microscopic lesions. M. longisporus is characterised also on the molecular level using sequences of SSU rRNA gene. Phylogenetic analysis based on these sequences has allowed clearer phylogenetic relationships to be established with other species of the genus Myxobolus sequenced to date.

线粒体 DNA 相关疾病的系统发育学研究

线粒体是细胞内提供能量的细胞器，并负责调节细胞的程序化死亡。因遗传 缺陷引起的线粒体功能障碍会导致ATP 合成障碍、能量产生不足而出现一系列 病症。线粒体DNA 相关疾病目前日益受到广泛的关注，然而在线粒体DNA 相 关疾病领域常用的病例对照法容易受到遗传背景、群体分层、数据质量等方面因 素的影响以致经常得到假阳性结果。系统发育分析方法有助于解决这些方面的问 题，因此我们以此分析方法开展了如下工作： 首先我们对线粒体DNA C1494T 突变及其所属单倍型类群和氨基糖苷类药 物性耳聋之间的关联进行了研究。之前有研究报道了两个中国氨基糖苷类药物性 耳聋的家系，经过对先征者的线粒体DNA 全序列测定在这两个家系中都发现了 C1494T 突变。我们采用系统发育方法分析这两个家系先征者的全序列后，发现 这两个个体都属于线粒体单倍型A。巧合的是，在我们之前的研究中，在一个来 自武汉的汉族样本WH6980 中也有C1494T 突变，而且该个体同样也属于单倍型 类群A。这不由得使人想到：C1494T 突变可能是单倍型类群A 中一个分支的界 定位点，或者，受母系遗传背景的影响，该突变偏好于在A 类群中发生。那么 很有可能单倍型类群A对能够引发氨基糖苷类药物性耳聋的C1494T 突变的发生 有促进作用。 为了验证这个假设，我们从三个省份随机选取了553 个正常个体来检测 C1494T 突变，以调查该突变在普通人群中的发生频率。另外，我们从1823 个中 国人样本中筛选出属于单倍型类群A 的111 个体，在这111 个个体中检测C1494T 突变，以调查该突变是否为单倍型类群A 特有的变异位点。我们的结果表明： 在553 个随机样本群中没有检测到C1494T 突变，这说明该突变是一个稀有的突 变，在正常人群中发生的频率极低。另外，在111 个属于单倍型类群A 的样本 群中，我们也没有检测到C1494T 突变的存在，这说明该突变并非单倍型类群A 特有的变异位点。经过对带有C1494T 突变的全序列进行综合的系统发育学分析 表明，这三条序列的C1494T 突变应是来源于同一次突变事件。同时，根据序列 之间共享变异位点的状况推断，两个耳聋家系具有很近的母系亲缘关系，我们推 测这两个家系的C1494T 突变是来源于一位共同的近期母系祖先。综合这些结果 表明，C1494T 突变的发生与单倍型类群即母系遗传背景没有相关性，是属于在人群中频率极低的散发性突变。 我们又将系统发育分析的方法应用于肿瘤相关的线粒体DNA 突变的研究 中。线粒体在细胞的自由基产生以及细胞凋亡中扮演重要角色；有研究报道线粒 体功能的缺陷能导致癌症的发生，同时又有很多报道指出癌变组织的线粒体基因 组存在异常。为探讨癌组织中线粒体基因组的变异情况及其在癌症的发生和发展 中所扮演的角色，我们选取乳腺癌早期患者为研究对象。 近年来，有大量的研究都报导了在癌组织中存在高频率高密度的线粒体 DNA 体细胞突变，并认为这些突变在肿瘤发生过程中可能具有功能相关性。但 这些研究存在着不可回避的问题，总结来讲归为三个方面：①数据质量差：很多 前面报道的突变数据，经系统发育分析，发现存在不少因样本交叉污染所造成的 假阳性突变。②所测片断太短。大多数研究只是检测了D-loop 区，只占了整个 基因组的约十六分之一，很难全面反映问题。③对照设置有问题，许多研究进行 简单的case-control 分析，忽略了不同个体间母系遗传背景的差异，导致大量假 阳性突变的产生。 针对以上问题的存在，我们基于系统发育分析的方法在中国的乳腺癌病人中 开展了一项研究。分别取得10 例乳腺癌早期患者的癌组织、癌旁组织、以及远 端正常组织；提取了总DNA，对每一份组织样品进行线粒体全基因组测序。这 样做有两个研究目的：第一是调查在严格的质量控制手段下，排除交叉污染所造 成的假阳性突变后，在癌症病人中是否仍然能观察到高频率高密度的线粒体 DNA 体细胞突变。第二个目的是调查中国乳腺癌患者线粒体DNA 体细胞突变情 况，为乳腺癌早期诊断提供有效的信息。 为了避免过去部分研究中出现的问题，我们采取了相应的措施。首先是在严 格的质量控制下，我们对同一个病人的三种不同组织，即癌组织，癌旁组织，正 常组织中的线粒体基因组进行全序列测定的方式来检测体细胞突变。同时将所测 得线粒体全基因组序列进行系统发育的分析；结果表明属于同一个病人的不同组 织都能忠实的聚到一支，而且每一条全序列都完整的带有所属单倍型特有的界定 位点，不存在任何样本交叉或者污染的情况；这样进一步确证了我们数据的可靠 性。同时经过这样的比较，可以非常清晰的筛选出在某一种组织类型中所发生的 体细胞突变。来自10 例病人的癌组织，癌旁组织和正常组织的29 条（7#患者远端正常组 织没有得到）线粒体基因组全序列中，我们只检测到了两个体细胞突变（T2275C 和A8601G）。这两个突变经PCR 克隆试验的验证，在远端正常组织中均不存在， 是真实的体细胞突变。突变A8601G 同时出现在3#患者的癌组织和癌旁组织中； 提示线粒体DNA 的体细胞突变可能先于细胞的癌变，这可能对乳腺癌的早期临 床诊断具有一定的意义。突变T2275C 只出现在6#患者的癌组织中，该突变位于 16S rRNA 基因非常保守的位点上，我们对该突变位点潜在的生物学作用作了进 一步的讨论。 在我们研究的病例中体细胞突变的比率要远远低于先前的报道；造成差异的 主要原因可能是因为我们采取了严格的质量控制手段。综合我们的研究结果表 明，先前所报道的乳腺癌组织中的线粒体基因体细胞突变的频率存在被高估的现 象。目前线粒体突变与肿瘤发生的关系还有待于更深入的研究，同时我们呼吁本 领域的研究要加强数据质量的可靠性。我们的研究表明，系统发育的分析方法在 线粒体DNA 相关疾病的研究中是有效的，因此我们推荐在线粒体DNA 相关疾 病的研究中进行推广。

铃蟾属(Bombina)的分子系统学研究

本研究采用直接测序的方法，研究了铃蟾属（两栖纲：无尾目：铃蟾科）的 分子系统发育关系及系统学问题。我们测定了我国铃蟾属5 个种共22 个样本的 线粒体DNA 部分片段的序列，包括12S rRNA， 16S rRNA， ND4-tRNALEU 以 及细胞色素b 共四个基因片段，欧洲铃蟾、花铃蟾以及B. pachypus 的相应序列 通过GenBank 获得。利用MrBayes 3.0b4 和 PAUP* 4.0b10 软件对系统发育关系 进行了重建（贝叶斯推断法，最大简约法，最大似然法和邻近法），分析结果表 明，铃蟾属可分为两大支系，一支包括利川铃蟾及中国西南部铃蟾（微蹼铃蟾、 强婚刺铃蟾、大蹼铃蟾）；另一支包括东方铃蟾，花铃蟾、欧洲铃蟾以及Bombina pachypus，从分子水平上支持了铃蟾属内不同亚属的划分。我们的结果拒绝东方 铃蟾起源于大蹼铃蟾的假设，同时表明：相对于东方铃蟾，欧洲铃蟾与花铃蟾间 的关系更近。利川铃蟾的有效地位得到支持，中国西南部3 种铃蟾 (微蹼铃蟾、 强婚刺铃蟾、大蹼铃蟾)在所有系统树中形成一个单系，利川铃蟾与之形成姐妹 群。根据我国西南部3 种铃蟾间的很小的遗传差异，以及以往的形态学研究和核 型研究的证据，我们认为将微蹼铃蟾、强婚刺铃蟾归入大蹼铃蟾可能更为合适。 分化时间估计表明，铃蟾属两个亚属的分化发生在3.08–9.16 MYA，我们推测秦 岭的抬升以及同时期青藏高原的上升是铃蟾属内产生亚属分化的主要原因。