线粒体ND4-ND4L基因在黑腹果蝇种组中的进化特征

本实验对黑腹果蝇种组(melanogaster species group)中8个种亚组33个样品两个线粒体基因ND4和ND4L进行了测序,并分析了ND4基因的序列差异和碱基替换特点,发现近缘物种中存在很明显的转换倾向,而在远缘物种中由于重复替换导致转换数处于饱和状态,我们的实验数据证实了线粒体基因较核基因有较快的进化速度.最后根据D.melanogaster与D.yakuba的遗传距离推算了8个种亚组的分化时间,ananassae种亚组最先分化,然后依次是montium,melanogaster,ficsphia,eugracilis,elegans,suzukii和takahashii最后分化.     

以ND4L和ND4基因为标记探讨黑腹果蝇种组的系统发育关系

多年来的形态学、染色体组学以及DNA序列几个方面的研究均没有很好地阐明黑腹果蝇种组内的系统发育关系。本实验测定了33个样品的ND4和31个样品的ND4L基因序列,以D．obscuroides为外群,用最大简约法和Bayesian法分别构建进化树。结果表明两种方法构建的拓扑结构一致,而且大部分支系的支持率较高。整个黑腹果蝇种组分成三大谱系：1）montium种亚组;2）ananssae种亚组;3）Oriental种亚组（melanogaster、ficsphila、eugracilis、elegans、suzukii、takahashii）。montium是最早分化的种亚组。在第三谱系中,melanogaster分化得最早;然后依次是ficsphila,eugracilis,elegans;suzukii与takahashii为姐妹种亚组,最后分化。     

基于ND4和ND5基因序列分析的鳅超科鱼类系统发育关系

ND4和ND5是线粒体基因组中编码NADH脱氢酶亚基4和亚基5的两个蛋白质编码基因.该研究以鳅超科鱼类为研究对象,新测定了10个物种的ND4和ND5基因全序列以及中间的3个tRNA基因共212 bp的序列,结合从GenBank 下载的15个物种的15条序列进行序列比较和系统发育关系分析.结果显示:鳅超科鱼类ND4基因全长1380～1387 bp,以ATG为起始密码子,终止密码子为不完全终止信号;ND5基因全长1821～1839bp,同样起始密码子为ATG,终止密码子为TAA或TAG;ND4和ND5基因之间插入了3个tRNA基因,分别编码携带组氨酸、丝氨酸、亮氨酸的tRNA.ND4和ND5基因(包含3个tRNA基因)中A、T、G、C的平均含量分别为30.4%、27.3%、14.2%、28.1%,A+T(57.7%)的含量高于G+C(42.3%)的含量.转换与颠换比(Ti/Tv)平均值为1.586.选取斑马鱼和鲤鱼作为外类群,采用最大简约法(MP)、最大似然法(ML)和贝叶斯推断法(BI)进行系统发育树的重建.三种方法的系统发育分析结果都显示:花鳅亚科、条鳅亚科、沙鳅亚科、平鳍鳅科及Vaillantellidae分别构成单系;它们的系统发育关系为:(Vaillantellidae+(沙鳅亚科+(花鳅亚科+(条鳅亚科+平鳍鳅科).这与线粒体全基因组和某些核基因(如RAG1基因)的研究结果类似,且支持率较高,表明ND4和ND5基因用于鳅超科鱼类的系统发育分析是可行的;但是该研究的结果有别于其他线粒体基因的分析结果,如基于cytb和D-loop基因进行的系统发育分析表明,条鳅亚科和花鳅亚科聚为姐妹群,再和平鳍鳅科聚在一起.这种差异可能是由于使用的基因长度差异造成的,长度越长,信息量越大,所反映的系统发育结果可能更加接近真实情况.     

基于线粒体ND4基因和核c-mos基因初步探讨广西拟水龟和艾氏拟水龟的系统发生

广西拟水龟和艾氏拟水龟的分类和系统发生多年来存在争议。通过测定广西拟水龟、艾氏拟水龟和黄喉拟水龟线粒体ND4基因和核c-mos基因部分序列,合并GenBank中拟水龟属其他物种的ND4基因和c-mos基因部分序列进行分析,从分子水平探讨广西拟水龟和艾氏拟水龟的系统发生。ND4基因数据分析发现NJ、MP和BI树中广西拟水龟与安南拟水龟的聚类分支相互交织聚为一支,二者种内遗传距离均为0.002～0.017,种间遗传距离为0.000～0.035,种间遗传距离明显小于同属内其他种间0.056～0.109的遗传距离,表明广西拟水龟与安南拟水龟可能为同一物种,可能是安南拟水龟的同种异名,或是作为安南拟水龟的一个亚种;NJ树、MP树和BI树均显示,艾氏拟水龟与黄喉拟水龟的位置和关系最为相近,二者间的遗传距离为0.020～0.035,明显小于拟水龟属其他物种间遗传距离,而明显大于同属各物种内遗传距离,艾氏拟水龟与黄喉拟水龟之间系统分类关系是介于种内与种间之间;乌龟、中华花龟、安南拟水龟等物种都是与拟水龟属中其他物种先聚成一支后再与同科的地龟属的地龟形成姐妹支,支持将乌龟属、花龟属和安南龟属并入拟水龟属的分类。c-mos基因数据分析发现,拟水龟属各物种间不存在明显的遗传距离,NJ树和MP树也未能对属中各物种的分子系统发生位置进行有效的界定,但在属及属以上阶元的分子分类系统中c-mos基因可以作为分类依据,并与线粒体基因数据有较好的一致性。     

长蠹科几种检疫性害虫的ND4基因序列及系统进化

长蠹科昆虫严重危害林木和仓贮物品。应用非损伤性DNA测序技术测定了来自不同国家的长蠹科害虫的线粒体DNA ND4 基因的部分序列。在获得的204 bp的序列中,5种昆虫的序列变异丰富,多数变异发生在密码子的第3位点上。用PAUP3.1.1数据分析软件构建了5个种的合意简约树。并将实验结果与形态学特征比较分析,探讨5个种及所在属的系统进化。结果表明：双钩异翅长蠹所在的异翅长蠹属分化最早,其次是竹大长蠹所在的大长蠹属、双棘长蠹和黑双棘长蠹所在的双棘长蠹属及红艳长蠹所在的钻木长蠹属。双棘长蠹和黑双棘长蠹隶属同一个属,遗传关系最近,分化最晚,与形态学研究结果相吻合。     

象甲科线粒体基因组特征及系统发育分析

昆虫线粒体基因组广泛应用于系统发育关系的重新建立、分子进化、谱系地理学及物种诊断等领域。为揭示象甲科昆虫线粒体全基因组序列的主要结构特征,探究其系统发育相关信息,为进化遗传学研究和分子标记选取等提供参考依据,本研究利用比较基因组学和生物信息学方法,对NCBI上已公布的35种象甲科物种线粒体全基因组序列进行了分析。结果显示:(1)象甲科tRNA基因存在排序及数目异常情况,不同物种中蛋白质编码基因和2种rRNAs排列相同,线粒体全基因组具有明显AT偏向;(2)COX1、ATP6、ND5、ND4、ND4L和ND1基因除标准三联密码子外,还存在特殊的起始密码子AAT、TTG和GTG;(3)13种蛋白质编码基因的进化速率顺序为COX3ATP8ND2ND5ND1ND4ND6ND4LND3ATP6CytbCOX1COX2;(4)13个蛋白编码基因和rRNAs基因中,ND5、rrnL、ND4和ND2基因变异位点数较高,可作为备选的分子标记;(5)各亚科的系统发育关系可能为(((小蠹亚科Scolytinae+长小蠹亚科Platypodinae)+(隐喙象亚科Cryptorhynchinae+魔喙象亚科Molytinae+象虫亚科Curculioninae)+((孢喙象亚科Cyclominae+粗喙象亚科Entiminae)+(隐颏象亚科Dryophthorinae+长小蠹亚科))),为象甲科的系统发育分析有提供参考。     

华北地区中华蒙潮虫(甲壳动物亚门:等足目)种群遗传多样性研究

中华蒙潮虫Mongoloniscus sinensis(Dollfus,1901)隶属于甲壳动物亚门Crustacea等足目Isopoda潮虫亚目Oniscidea,中国特有种。为了探究中华蒙潮虫的种群遗传分化和系统进化关系,采用PCR对采自华北地区10个地理种群89只个体线粒体2个基因COⅠ和ND5进行联合分析。结果表明:1)中华蒙潮虫COⅠ部分基因长604 bp,ND5部分基因长615 bp,拼接序列长1 219 bp,T、C、A和G含量分别为41.0%、11.2%、30.8%和17.0%,具有显著的A+T偏倚;变异位点503个(占总核苷酸序列的41.3%),序列间的转换/颠换比值为2.8。2)89只个体共45种单倍型,单倍型多样性0.964,核苷酸多样性0.005 6,整体遗传多样性水平中等;单倍型H1、H15、H16、H21、H41为2～3个种群共享单倍型。3)联合基因(COⅠ+ND5)系统发育树表明,最早出现的是华北以北地区(山西大同、河北石家庄),最晚分化出的是华北以南地区(山西临汾、陕西西安未央区、河南新乡),演化路线为从北向南,个别种群单倍型未按地理来源形成明显的簇群。4)平均遗传分化指数为0.513,基因流为0.24;分子变异分析结果表明,种群的变异与分化主要来自种群内部,错配分布呈多峰,结合中性检验(Tajima's D=-1.429;Fu's F_s=6.499),发现中华蒙潮虫近期未经历扩张,但种群内部分化显著,增长平稳。本研究首次基于线粒体多基因联合分析了中华蒙潮虫种群遗传多样性。     

基于线粒体ND2基因的直翅目部分种类分子系统学发育研究

本文的研究目的是通过对直翅目部分种类的线粒体ND2基因进行分析,重建直翅目内部昆虫的系统发育关系,并探讨分子系统发育关系和传统分类结果的异同。基于80个物种ND2基因的研究结果显示直翅目ND2基因存在碱基偏向性A T含量平均为73%,第三位点A T含量79.9%最高,推测这与氨基酸变异有关。直翅目具有单系性,而蝗亚目内部的剑角蝗科、网翅蝗科、槌角蝗科和斑腿蝗科均不是单系群,锥头蝗科和瘤锥蝗科亲缘关系较近,这与Otte分类系统相一致,建议将锥头蝗科和瘤锥蝗科合并为一个科。癞蝗科的分类地位存在争议有待进一步深入的研究。     

基于线粒体ND2基因的中国斑翅蝗科部分种类分子系统学研究（直翅目：蝗总科）

本文的目的是通过对斑翅蝗科部分种类的线粒体ND2基因进行分析,重建斑翅蝗科昆虫的系统发育关系,并探讨分子系统发育关系和传统分类结果的异同。扩增并测定了我国斑翅蝗科10属16种蝗虫的线粒体ND2全基因1 023 bp的序列,对序列的碱基组成、转换颠换、系统发育信号等进行了分析。并基于ND2全基因序列数据,分别采用邻接法（NJ）、最简约法（MP）、最大似然法（ML）和贝叶斯法重建了10属16种蝗虫的系统发育关系。结果表明：斑翅蝗科蝗虫ND2全基因A+T含量平均为74.6%;痂蝗亚科和异痂蝗亚科没能得到区分,建议合并为一个亚科;而斑翅蝗亚科和飞蝗亚科的分类地位还存在争议。     

中国特有果蝇 Drosophila curviceps 种亚组在Drosophila immigrans种组中的种系发生地位

果蝇immigrans种组中的curviceps种亚组是1992年新建立的中国特有果蝇类群。该种亚组中的物种主要分布在中国大陆和台湾。目前除了形态学水平的研究外,还没有其他证据支持建立该种亚组的合理性及其起源和种系发生地位。为了在DNA分子水平上探讨果蝇curviceps种亚组在果蝇immigrans种组中的种系发生地位,从而为今后更深入地研究中国特有果蝇,甚至为果蝇亚属的进化遗传学提供理论依据,测定了immigrans种组5个种亚组（nasuta、immigrans、hypocausta、quadrilineata、curviceps)中12个代表物种的rDNA的ITS1和部分Adh基因的序列。其中ITS1序列的长度为513－587bp,共有191个信息位点;Adh基因片段的长度在714－747bp之间,共99个信息位点。考虑到单个分子提供的信息较少,将两个分子的序列综合起来,组成一个较长的复合序列。分别根据ITS1,Adh和两个分子的复合序列排比（Alignment)结果,和最大简约法和邻接法构建分子系统树,其中根据复合序列构建的系统树与形态学研究结果最为一致。分子树显示curviceps种亚组的特种确定单独形成一个分枝,为种亚组级的分类阶元,支持了形态学将其建立为一个新种亚组。根据Kimura距离,估算了复合分子的替换速率约为每百万年1．48％,进而计算出5个种亚组的分 歧年代。结合各物种的地理分布,推测了immigrans种组的进化历史：curviceps种亚组与quadrilineata种亚组的亲缘关系最近,主要分布在中国南部的温带地区。它们之间的分歧时间大约为3．4百万年,是最年轻的两个种亚组。主要分布在苏门答腊及附近的热带地区的hypocausta种亚组的物种是最早分化出来的,与其他种亚组的分歧时间约为9．2百万年。该结果与形态学和生物地理学研究相吻合。值得一提是的,目前归属仍存在争议的物种D.neohypocausta,在分子系统树中与hypocausta种亚组的物种相距较远,而与immiagrasn种亚组的关系较近,但分枝置信度较低（＜50％）。由于还缺乏其他方面的证据,因此D.neohypocausta的归属有待今后的研究来作定论。     

Horizontal transfer of hobo transposable elements within the Drosophila melanogaster species complex: evidence from DNA sequencing.

The hobo family of transposable elements, one of three transposable-element families that cause hybrid dysgenesis in Drosophila melanogaster, appears to be present in all members of the D. melanogaster species complex: D. melanogaster, D. simulans, D. mauritiana, and D. sechellia. Some hobo-hybridizing sequences are also found in the other members of the melanogaster subgroup and in many members of the related montium subgroup. Surveys of older isofemale lines of D. melanogaster suggest that complete hobo elements were absent prior to 50 years ago and that hobo has recently been introduced into the species by horizontal transfer. To test the horizontal transfer hypothesis, the 2.6-kb XhoI fragments of hobo elements from D. melanogaster, D. simulans, and D. mauritiana were cloned and sequenced. The DNA sequences reveal an extremely low level of divergence and support the conclusion that the active hobo element has been horizontally transferred into or among these species in the recent past.     

Neutral and Non-Neutral Evolution of Drosophila Mitochondrial DNA

To test hypotheses of neutral evolution of mitochondrial DNA (mtDNA), nucleotide sequences were determined for 1515 base pairs of the NADH dehydrogenase subunit 5 (ND5) gene in the mitochondrial DNA of 29 lines of Drosophila melanogaster and 9 lines of its sibling species Drosophila simulans. In contrast to the patterns for nuclear genes, where D. melanogaster generally exhibits much less nucleotide polymorphism, the number of segregating sites was slightly higher in a global sample of nine ND5 sequences in D. melanogaster (s = 8) than in the nine lines of D. simulans (s = 6). When compared to variation at nuclear loci, the mtDNA variation in D. melanogaster does not depart from neutral expectations. The ND5 sequences in D. simulans, however, show fewer than half the number of variable sites expected under neutrality when compared to sequences from the period locus. While this reduction in variation is not significant at the 5% level, HKA tests with published restriction data for mtDNA in D. simulans do show a significant reduction of variation suggesting a selective sweep of variation in the mtDNA in this species. Tests of neutral evolution based on the ratios of synonymous and replacement polymorphism and divergence are generally consistent with neutral expectations, although a significant excess of amino acid polymorphism within both species is localized in one region of the protein. The rate of mtDNA evolution has been faster in D. melanogaster than in D. simulans and the population structure of mtDNA is distinct in these species. The data reveal how different rates of mtDNA evolution between species and different histories of neutral and adaptive evolution within species can compromise historical inferences in population and evolutionary biology.     

Closely related species of Drosophila can contain different libraries of middle repetitive DNA sequences

Very few of the middle repetitive DNA sequences found in Drosophila melanogaster are present in all of the species of the D. melanogaster subgroup. One member of the subgroup, D. erecta, lacks most of the families of repetitive elements from D. melanogaster (including copia and 412) but possesses many other families not present in the D. melanogaster genome. Other species in the subgroup possess some families in these two species and lack others. From this we conclude that most individual middle repetitive families are highly unstable components of the Drosophila genome over short periods of evolutionary time.     

Evolution of Polypyrimidines in Drosophila

We surveyed 101 different Drosophila species for the presence of a particular highly repetitive DNA sequence containing long tracts of polypyrimidine/polypurine DNA, first found in D. melanogaster. Out of 55 tested species in the melanogaster group, only the sibling species D. simulans and D. mauritiana, as well as one distant relative in the ananassae subgroup, D. varians, contained the same sequence. All four of these species have long pyrimidine tracts as shown by acid hydrolysis of labelled DNA. All four species have the same sequence, bu the amount of this polypyrimidine/polypurine DNA varies greatly. Four other species in the hydei subgroup were found to contain a polypyrimidine/polpurine sequence, with an oligonucleotide composition different from that of D. melanogaster. This polypyrimidine DNA varies from as much as 10% of the total DNA in D. nigrohydei, to as little as 0.4% in D. neohydei. The long pyrimidine tracts in the hydei subgroup are often more than a thousand nucleotides in length, representing exceedingly homogeneous repetitious sequences.--These results show a rapid but discontinuous pattern of evolution for polypyrimidine/polypurine DNA . These sequences are not species specific, yet closely related species have greatly different amounts of polypyrimidines. Drastic changes occur in the amounts of these satellite type DNA sequences, as if the sequence had no continuous selective advantage in evolution. The implications of these results with regard to the general function and evolution of satellite DNA are discussed.     

Divergence and heterogeneity of the histone gene repeating units in the Drosophila melanogaster species subgroup

The repeating units of the histone gene cluster containing the H1, H2A, H2B and H4 genes were amplified by PCR from the Drosophila melanogaster species subgroup, i.e., D. yakuba, D. erecta, D. sechellia, D. mauritiana, D. teissieri and D. orena. The PCR products were cloned and their nucleotide sequences of about 4.6-4.8kbp were determined to elucidate the mechanism of molecular evolution of the histone gene family. The heterogeneity among the histone gene repeating units was 0.6% and 0.7% for D. yakuba and D. sechellia, respectively, indicating the same level of heterogeneity as in the H3 gene region of D. melanogaster. Divergence of the genes among species even in the most closely related ones was much greater than the heterogeneity among family members, indicating a concerted mode of evolution for the histone gene repeating units. Among the species in the D. melanogaster species subgroup, the histone gene regions as well as 3rd codon position of the coding region showed nearly the same GC contents. These results suggested that the previous conclusion on analysis of the H3 gene regions, the gene family evolution in a concerted fashion, holds true for the whole histone gene repeating unit.     

The molecular evolution of the alcohol dehydrogenase and alcohol dehydrogenase-related genes in the Drosophila melanogaster species subgroup

The DNA sequences of the Adh genes of three members of the Drosophila melanogaster species subgroup have been determined. This completes the Adh sequences of the eight species of this subgroup. Two species, D. yakuba and D. teissieri, possess processed Adh pseudogenes. In all of the species of the subgroup, a gene of unknown function, Adhr, is located about 300 bp 3' to Adh. Although this gene is experiencing a higher rate of synonymous substitution than Adh, it is more constrained at the amino acid level. Phylogenetic relationships between all eight members of the melanogaster subgroup have been analyzed using a variety of methods. All analyses suggested that the D. yakuba and D. teissieri pseudogenes have a single common ancestor, rather than evolving independently in each species, and that D. melanogaster is the sister species to D. simulans, D. sechellia, and D. mauritiana. The evolutionary relationships of the latter three species remain equivocal.      

Phylogeny and evolution of the Drosophila nasuta subgroup based on mitochondrial ND4 and ND4L gene sequences

The sequences of the mitochondrial ND4 gene (1339 bp) and the ND4L gene (290 bp) were determined for all the 14 extant taxa of the Drosophila nasuta subgroup. The average A + T content of ND4 genes is 76.5% and that of ND4L genes is 83.5%. A total of 114 variable sites were scored. The ND4 gene sequence divergence ranged from 0 to 5.4% within the subgroup. The substitution rate of the ND4 gene is about 1.25% per million years. The base substitution of the genes is strongly transition biased. Neighbor-joining and parsimony were used to construct a phylogeny based on the resultant sequence data set. According to these trees, five distinct mtDNA clades can be identified. D. niveifrons represents the most diverged lineage. D. sulfurigaster bilimbata and D. kepulauana form two independent lineages. The other two clades are the kohkoa complex and the albomicans complex. The kohkoa complex consists of D. sulfurigaster sulfurigaster, D. pulaua, D. kohkoa, and Taxon-F. The albomicans complex can be divided into two groups: D. nasuta, D. sulfurigaster neonasuta, D. sulfurigaster albostrigata, and D. albomicans from Chiangmai form one group; and D. pallidifrons, Taxon-I, Taxon-J, and D. albomicans from China form the other group. High genetic differentiation was found among D. albomicans populations. Based on our phylogenetic results, we hypothesize that D. niveifrons diverged first from the D. nasuta subgroup in Papua New Guinea about 3.5 Mya. The ancestral population spread to the north and when it reached Borneo, it diversified sequentially into the kohkoa complex, D. s. bilimbata, and D. kepulauana. About 1 Mya, another radiation occurred when the ancestral populations reached the Indo-China Peninsula, forming the albomicans complex. Discrepancy between morphological groupings and phylogenetic results suggests that the male morphological traits may not be orthologous.     

Evolution of gypsy endogenous retrovirus in the Drosophila obscura species group

The Ty3/gypsy family of retroelements is closely related to retroviruses, and some of their members have an open reading frame resembling the retroviral gene env. Sequences homologous to the gypsy element from Drosophila melanogaster are widely distributed among Drosophila species. In this work, we report a phylogenetic study based mainly on the analysis of the 5' region of the env gene from several species of the obscura group, and also from sequences already reported of D. melanogaster, Drosophila virilis, and Drosophila hydei. Our results indicate that the gypsy elements from species of the obscura group constitute a monophyletic group which has strongly diverged from the prototypic D. melanogaster gypsy element. Phylogenetic relationships between gypsy sequences from the obscura group are consistent with those of their hosts, indicating vertical transmission. However, D. hydei and D. virilis gypsy sequences are closely related to those of the affinis subgroup, which could be indicative of horizontal transmission.     

Basal relationships in the Drosophila melanogaster species group

The Drosophila melanogaster species group is a popular model for evolutionary studies due to its morphological and ecological diversity and its inclusion of the model species D. melanogaster. However, phylogenetic relationships among major lineages within this species group remain controversial. In this report, the phylogeny of 10 species representing each of the well-supported monophyletic clades in the melanogaster group was studied using the sequences of 14 loci that together comprise 9493 nucleotide positions. Combined Bayesian analysis using gene-specific substitution models produced a 100% credible set of two trees. In the strict consensus of these trees, the ananassae subgroup branches first in the melanogaster species group, followed by the montium subgroup. The remaining lineages form a monophyletic clade in which D. ficusphila and D. elegans branch first, followed by D. biarmipes, D. eugracilis, and the melanogaster subgroup. This strongly supported phylogeny resolves most basal relationships in the melanogaster species group, and provides a framework that can be extended in the future to encompass more species.