基于mtDNA COⅠ和Cytb基因序列对南中国海不同海域波纹唇鱼群体遗传多样性的研究

研究以海南陵水、马来西亚、西沙、南沙4个海域共101尾波纹唇鱼作为研究对象, 通过线粒体DNA的COⅠ和Cytb基因序列分析方法对波纹唇鱼进行了遗传多样性研究。经PCR扩增、克隆与序列测定, 分别获得1560 bp COⅠ基因和1141 bp Cytb基因序列。两者多态性遗传参数统计显示, 101尾个体分别存在23 (COⅠ)和30 (Cytb)个变异位点, 分别检测出20 (COⅠ)和27 (Cytb)个单倍型, 总群体单倍型多样性(Hd)分别为0.629 (COⅠ)和0.755 (Cytb), 平均核苷酸差异数(K)分别为1.195 (COⅠ)和1.424 (Cytb), 核苷酸多样性指数(Pi)分别为0.00077 (COⅠ)和0.00126 (Cytb)。分子方差分析(AMOVA)结果分别为26.26% (COⅠ)和4.22% (Cytb)的变异来自群体间, 73.74% (COⅠ)和95.78% (Cytb)的变异来自群体内。同时, 两个基因的单倍型网络图呈星状放射结构, 不同地理来源的单倍型无明显分支, 呈交错分布, 没有体现地理差异性。研究初步认为, 波纹唇鱼的遗传多样性处于较低水平, 遗传分化存在但不显著, 该结果可为今后波纹唇鱼的种质资源保护工作提供必要的科学依据。       

基于线粒体Cytb 和COⅠ基因对珠江、海南和红河长臀鮠群体遗传结构分析

为了解3 个种群的野生资源状况, 并对3 个种群的物种有效性进行分析, 采用Cytb 和COⅠ基因序列测定技术, 分析了珠江水系、海南水系和越南红河水系长臀鮠种群的群体遗传结构及其变异, 结果发现, 三个群体平均遗传距离分别为0.003(cytb)和0.002(coⅠ); 94 尾个体分别存在35(Cytb)和13(COⅠ)个变异位点, 分别检测出24 (Cytb)和13 (COⅠ)个单倍型, 总群体单倍型多样性(Hd)分别为0.875 (Cytb)和0.787 (COⅠ), 平均核苷酸差异数(K)分别为3.194(Cytb)和1.328(COⅠ), 核苷酸多样性指数(Pi)分别为0.00295 (Cytb)和0.00213 (COⅠ)。珠江长臀鮠单倍型多样性(Hd)和核苷酸多样性(Pi)最高, 海南最低; 对长臀鮠群体进行历史动态分析,三个种群各自无明显扩张趋于稳定, 但将三个种群作为一个群体时发现长臀鮠可能在某一个时期产生了扩张。研究结果表明, 三个种群遗传距离显著小于临界值2%,应为同一个种; 长臀鮠野生资源较为贫乏, 且海南群体最为严重。     

日本三角涡虫河南八种群基于COⅠ基因片段的分子系统学研究

对采自河南4大山系8产地的日本三角涡虫线粒体DNA细胞色素C氧化酶Ⅰ亚基(COⅠ)基因进行PCR扩增和测序,用MEGA4.0、PAUP*4.0等程序对所得序列进行分析和系统树重建。结果表明,采自大别山系商城县的两个种群XYshangcheng1和XYshangcheng2的COⅠ片段序列同源性最高,为100%;而采自伏牛山系嵩县的种群LYsongxian与其它7种群的COⅠ片段序列同源性最低,在84.3%～89.0%之间。NJ树、MP树和贝叶斯树拓扑结构完全一致,从系统树上可以看出LYsongxian种群与其它7种群的亲缘关系最远;另外7种群又聚为2支。该聚类结果基本和各种群的山系分布相符合。同时结合基于Hsp70基因的分子系统学研究以及涡虫形态学、细胞遗传学等方面的证据初步推测:LYsongxian种群可能不是日本三角涡虫,而是三角涡虫属另外一个种,该推测是否正确尚待进一步的组织解剖学验证。     

大壁虎不同地理居群的遗传变异与分化

以线粒体细胞色素b(Cytb)基因作为分子标记,对中国广西12个地区,以及越南和老挝大壁虎(Gekko gecko)进行序列测定,获得Cytb基因424bp的序列片段,共有7个单倍型。以白脊壁虎和沙虎为外群,用邻接法和最大简约法构建了大壁虎不同地理种群的系统发育关系,其结果显示中国广西4个不同单倍型黑大壁虎之间的平均遗传距离为0.20%—1.20%,越南红大壁虎与老挝红大壁虎之间的平均遗传距离为0.50%,广西宁明红大壁虎与越南红大壁虎和老挝红大壁虎之间平均遗传距离分别为1.70%和2.20%。广西黑大壁虎种群与红大壁虎种群之间的平均遗传距离为8.60%—9.50%,达到了亚种或种分化的差异。     

家鸭细胞色素C氧化酶Ⅲ（COⅢ）基因的克隆及序列分析

线粒体是存在于绝大多数真核细胞内的一种基本的、重要的细胞器,是细胞进行氧化磷酸化的场所。不同脊椎动物同源基因序列的比较显示,细胞色素b(Cytb)、细胞色素C氧化酶Ⅰ（COⅠ）、细胞色素C氧化酶Ⅱ（COⅡ）、细胞色素C氧化酶Ⅲ（COⅢ） 基因最保守,同源性最高,ATPase6、ATPase8基因、ND基因变异比较大。本试验以家鸭（家鸭起源于绿头鸭：Anas platyrhynchos）肝脏的线粒体DNA为模板,按照GenBank已经公布的潜鸭族（AF090337）的全序列及其绿头鸭的mtDNA部分序列（L22476、L16770、L22477）设计合成特异引物进行PCR扩增,克隆并测定了线粒体细胞色素C氧化酶Ⅲ亚基（COⅢ）的全序列784bp以及ATPase6基因的3'端和tRNA-Gly基因的5'端序列共934bp。 用DNAStar分析软件对家鸭与GenBank中7种禽类的COⅢ序列进行比较分析,显示家鸭与这些动物的COⅢ基因具有较高的同源性,与同科潜鸭属中的Aythya Americana的相似性 最高为90.6 %,与同目不同科的加拿大雁、小天鹅、白额雁的相似性分别为89%、88.6%、88.6%。根据家鸭与其他7种禽类的COⅢ基因序列相似性所建立的进化树,与传统的分类地位基本吻合。     

斑鳠细胞色素b基因的初步研究

斑鳠是珠江四大名鱼之一,目前资源量急剧下降,种质资源的保护迫在眉睫。测定了斑鳠线粒体细胞色素b基因的全序列,该序列全长1137bp,其中T、C、A、G四种碱基含量分别为31.6%、25.7%、29.6%、13.1%,A+T的含量(61.2%)显著高于G+C的含量(38.8%)。将测定序列(序列自编号:MGGD)与GenBank中的2个斑鳠序列(序列自编号:MGZJ、MGFJ)进行了比较,3个序列之间的序列差异为0.005-0.017,转换后的氨基酸序列间差异位点都仅为2个,反应出细胞色素b基因作为功能蛋白质编码基因序列相对比较保守。鳠属鱼类线粒体Cytb基因的转换大于颠换。研究为斑鳠渔业种质资源保护和品种选育提供了遗传方面的资料,斑鳠的群体结构有待进一步深入研究。     

松江鲈鱼Cytb基因序列分析及种质鉴定

采用PCR方法扩增四尾松江鲈鱼线粒体细胞色素b(mitochondrial cytochrome b,Cytb)基因序列,全序列长度为1 141 bp,并建立了松江鲈鱼的实时定量PCR检测方法对该物种进行种质鉴定。分析Cytb基因序列基本特征显示,Cytb基因在第3位密码子表现出明显的反G偏倚,显示出脊椎动物Cytb基因的共同特性。第2位密码子嘧啶的含量远高于嘌呤的含量,有5个位点发生转换,0个位点发生颠换。选取杜父鱼科(Cottidae)的10种鱼类,与松江鲈鱼进行序列分析,构建发育系统树并分析遗传距离,与鲈鱼的亲缘关系最远。以松江鲈鱼Cytb基因序列为靶基因,利用PRIMER EXPRESS2.0软件设计引物和Taqman探针,建立检测松江鲈鱼的定量PCR方法,特异性强,灵敏度高,检测限为3.20×102拷贝/反应。     

基于线粒体 CO玉及 Cyt b 基因的7种龙虾类分子系统关系

运用PCR法扩增湛江沿海海域7种龙虾的线粒体COⅠ和Cytb基因并对其序列进行分析,以分析7种龙虾的分子系统关系。从7种龙虾中扩增到的COⅠ基因片段长度均为650bp,共存在224个核苷酸位点变异,变异率为35.22%,简约信息位点161个;扩增到的Cytb基因片段长度均为536bp,共存在148个核苷酸位点变异,变异率为29.48%,简约信息位点66个。所有的扩增序列中,没有发现碱基的缺失以及插入,序列中的转换大于颠换,且碱基替换多发生于密码子的第3位。以帝加洛真龙虾(Palinurus delagoae)、普通真龙虾(Palinurus elephas)、吉氏真龙虾(Palinurus gilchristi)为外群,对COⅠ和Cytb两个基因序列采用最大简约法(maximum parsimony,MP)和贝叶斯推论法(bayesian inference,BI)构建龙虾类分子系统树。结果显示:日本龙虾和密毛龙虾亲缘关系比较近,而其它5种龙虾与真龙虾属的3种关系较近,与传统的分类存在一定分歧。     

基于线粒体细胞色素b基因序列的雀形目18种鸟系统发育关系

采用线粒体细胞色素b(cytochrome b)基因,利用DNA测序的方法研究了雀形目18种鸟类的分子系统发育关系。通过序列分析比对,运用邻接法和最大似然法构建了雀形目18种鸟类的系统发育树,结果表明,燕雀类和类的分歧程度达到了科级水平,建议分别独立成科;长尾山雀可从山雀科中分离出来,单列成科;柳莺归入柳莺亚科(Phylloscopinae),树莺归入大苇莺亚科(Acrocephalinae)。Cytb基因的各主要分支中存在着相对恒定的分子钟,根据鸟类mtDNA的cytb进化速度大约是每百万年1.6%,得到18种鸟的科间分歧时间在10.5百万年左右,科内分歧在9.0百万年左右。     

基于线粒体基因Cytb和COI的蝗科中五亚科分子系统发育关系分析（直翅目: 蝗总科）（英文）

本研究基于Cytb 基因和COI基因的部分序列来推断17种蝗虫之间的系统发育关系。这17种蝗虫均采自国内,代表了蝗科(Acrididae)5个亚科：黑蝗亚科(Melanoplinae)、斑腿蝗亚科(Catantopinae)、刺胸蝗亚科(Cyrtacanthacridinae)、斑翅蝗亚科(Oedipodinae)和大足蝗亚科(Gomphocerinae)。采用联合序列方法进行分析,结果显示：Cytb 和COI联合序列长度为1 998 bp,其中A和T总含量为72.13%,G和C总含量为27.87%。联合序列共包含了889个保守位点,1 109个变异位点,在这些变异位点中有838个简约信息位点。系统发生树采用邻接法（NJ）、最大简约法（MP）和最大似然法（ML）进行构建。使用蜢总科的变色乌蜢Erianthus versicolor 和 Erianthus sp. 两个种作为外群。结果表明：大足蝗亚科和斑腿蝗亚科的单系性没有得到支持。斑翅蝗亚科内部各种聚成一个大支,在本研究中该亚科的单系性得到支持,与前人的研究结论相同。大足蝗亚科、斑腿蝗亚科、刺胸蝗亚科和黑蝗亚科这4科关系非常近,可以考虑将其合并为一个亚科。同时,我们发现基于Cytb和COI基因联合序列推断蝗科内各亚科间的系统发生关系并不十分可靠。     

Phylogeography and population structure of the saker falcon (Falco cherrug) and the influence of hybridization: mitochondrial and microsatellite data

Microsatellite as well as sequence analysis of the mitochondrial control region were applied to infer phylogeography and population genetic structure of the saker falcon (Falco cherrug). Furthermore, we compared the patterns of mitochondrial haplotypes with the variation of microsatellite alleles among the species of the hierofalcon complex (F. cherrug, Falco rusticolus, Falco biarmicus, Falco jugger) to test hypotheses on population history. Historical samples from museum specimens of F. cherrug were analysed together with samples from contemporary populations to investigate possible influences of hybrid falcons escaped from falconry on the genetic composition. In the mitochondrial DNA analysis, none of the four species represents a monophyletic group. Moreover, there are no clearly defined groups of haplotypes corresponding to taxonomic entities. In the microsatellite analysis most of the variation is shared between species and no clear differentiation by private alleles is found. Yet, with a Bayesian clustering method based on allele frequencies, a differentiation of F. cherrug, F. rusticolus and two geographic groups of F. biarmicus was detected. Results from both nuclear and mitochondrial markers are compatible with the previously postulated 'Out of Africa' hypothesis assuming an African origin of the hierofalcons. From an ancestral African population, F. cherrug, F. rusticolus and F. jugger split off in separate waves of immigration into Eurasia and South Asia. A combination of evolutionary processes, including incomplete lineage sorting as well as hybridization, may be responsible for the currently observed genetic patterns in hierofalcons.     

Molecular phylogeny of silk-producing insects based on 16S ribosomal RNA and cytochrome oxidase subunit I genes

We have examined the molecular-phylogenetic relationships between nonmulberry and mulberry silkworm species that belong to the families Saturniidae, Bombycidae and Lasiocampidae using 16S ribosomal RNA (16S rRNA) and cytochrome oxidase subunit I (coxI) gene sequences. Aligned nucleotide sequences of 16S rRNA andcoxI from 14 silk-producing species were used for construction of phylogenetic trees by maximum likelihood and maximum parsimony methods. The tree topology on the basis of 16S rRNA supports monophyly for members of Saturniidae and Bombycidae. Weighted parsimony analysis weighted towards transversions relative to transitions (ts, tv4) forcoxI resulted in more robust bootstrap support over unweighted parsimony and favours the 16S rRNA tree topology. Combined analysis reflected clear biogeographic pattern, and agrees with morphological and cytological data.     

从细胞色素b基因序列变异分析中国鲇形目鱼类的系统发育

采用PCR技术获得中国鲇形目鱼类11科24属27个代表种类细胞色素b基因1138bp全序列,比较分析了来自北美洲、非洲的部分鲇形目鱼类同一基因序列,并选取脂鲤目、鲤形目和鲱形目鱼类作外类群,采用Bayesian方法和最大简约法(MP)构建分子系统树。结果表明：(1)鲇形目鱼类细胞色素b基因序列中,与脂鲤目、鲤形目以及鲱形目鱼类相比存在3bp的缺失;(2)鲇形目鱼类各科代表种类形成一单系群;(3)两种建树方法均支持铫科、粒鲇科和钝头鮠科形成一单系群;而胡子鲇科、刀鲇科、海鲇科、鮰科、长臀鮠科、鲢科、鲇科、棘脂鲿科、鲿科形成一大的单系群;但鳗鲇科的系统位置两种建树方法没有取得一致结果;而其中长臀鲍科与北美的鮰科形成姐妹群,胡子鲇、鮰科、鲇科、鲿科和鮡科是较明显的单系群。     

Out of Africa? Phylogenetic relationships between Falco biarmicus and the other hierofalcons (Aves: Falconidae)

The phylogeographic history of the lanner falcon ( Falco biarmicus ) and the phylogenetic relationships among hierofalcons ( F. biarmicus , Falco cherrug , Falco jugger and Falco rusticolus ) were investigated using mitochondrial (mt) DNA sequences. Of the two non-coding mt sections tested, the control region (CR) appeared more suitable as phylogenetic marker sequence compared with the pseudo control region (ΨCR). For the comprehensive analysis samples from a broad geographic range representing all four hierofalcon species and their currently recognized subspecies were included. Moreover, samples of Falco mexicanus were analysed to elucidate its phylogenetic relationships to the hierofalcons. The sequence data indicate that this species is more closely related to Falco peregrinus than to the hierofalcons. In the DNA-based trees and in the maximum parsimony network all hierofalcons appear closely related and none of the species represents a monophyletic group. The close relationships among haplotypes suggest that the hierofalcon complex is an assemblage of morphospecies not yet differentiated in the genetic markers used in the present study and that the radiation of the four hierofalcon species took place rather recently. Based on the high intraspecific diversity found within F. biarmicus we assume an African origin of the hierofalcon complex. The observed pattern of haplotype distribution in the extant species may be due to incomplete lineage sorting of ancestral polymorphisms, and interspecific gene flow through hybridization.     

Molecular phylogeny of Pamphagidae (Acridoidea,Orthoptera) from China based on mitochondrial cytochrome oxidase II sequences

Abstract Phylogenetic relationships of Pamphagidae were examined using cytochrome oxidase subunit II (COII) mtDNA sequences (684 bp). Twenty‐seven species of Acridoidea from 20 genera were sequenced to obtain mtDNA data, along with four species from the GenBank nucleotide database. The purpose of this study was analyzing the phylogenetic relationships among subfamilies within Pamphagidae and interpreting the phylogenetic position of this family within the Acridoidea superfamily. Phylogenetic trees were reconstructed using neighbor‐joining (NJ), maximum parsimony (MP) and Bayesian inference (BI) methods. The 684 bp analyzed fragment included 126 parsimony informative sites. Sequences diverged 1.0%–11.1% between genera within subfamilies, and 8.8%–12.3% between subfamilies. Amino acid sequence diverged 0–6.1% between genera within subfamilies, and 0.4%–7.5% between subfamilies. Our phylogenetic trees revealed the monophyly of Pamphagidae and three distinct major groups within this family. Moreover, several well supported and stable clades were found in Pamphagidae. The global clustering results were similar to that obtained through classical morphological classification: Prionotropisinae, Thrinchinae and Pamphaginae were monophyletic groups. However, the current genus Filchnerella (Prionotropisinae) was not a monophyletic group and the genus Asiotmethis (Prionotropisinae) was a sister group of the genus Thrinchus (Thrinchinae). Further molecular and morphological studies are required to clarify the phylogenetic relationships of the genera Filchnerella and Asiotmethis.     

Phylogeographic analysis of Pimoidae (Arachnida: Araneae) inferred from mitochondrial cytochrome c oxidase subunit I and nuclear 28S rRNA gene regions

Using mitochondrial DNA cytochrome c oxidase subunit I and nuclear DNA 28S rRNA data, we explored the phylogenetic relationships of the family Pimoidae (Arachnida: Araneae) and tested the North America to Asia dispersal hypothesis. Sequence data were analysed using maximum parsimony and Bayesian inference. A phylogenetic analysis suggested that vicariance, instead of dispersal, better explained the present distribution pattern of Pimoidae. Times of divergence events were estimated using penalized likelihood method. The dating analysis suggested that the emergence time of Pimoidae was approximately 140 million years ago (Ma). The divergence time of the North American and Asian species of Pimoa was approximately 110 Ma. Our phylogenetic hypothesis supports the current morphology‐based taxonomy and suggests that the cave dwelling might have played an important role in the speciation of pimoids in arid areas.     

Re-examining the phylogeny of clinically relevant Candida species and allied genera based on multigene analyses

Yeasts of the artificial genus Candida include plant endophytes, insect symbionts, and opportunistic human pathogens. Phylogenies based on rRNA gene and actin sequences confirmed that the genus is not monophyletic, and the relationships among Candida species and allied teleomorph genera are not clearly resolved. Protein-coding genes have been useful to resolve taxonomic positions among a broad range of fungi. Over 70 taxa of the genus Candida and its allied sexually reproducing genera were therefore selected, and their phylogenetic relationships were investigated using nuclear sequences of the largest subunit and second largest subunit of RNA polymerase II gene, actin, the second subunit of the mitochondrial cytochrome oxidase gene, and D1/D2 LSU rRNA gene. The DNA sequences were analysed by maximum parsimony and Bayesian inference, resulting in the recognition of six major phylogenetic groups (A-F). Group A contains six facultative pathogenic Candida species, which seem to have derived from nonpathogenic species, while Group B contains species of Clavispora, Metschnikowia, and Pichia guilliermondii. Species of Debaryomyces form an independent group C that is related to groups A and B. Pichia fermentans and other environmental species are concentrated in Group D. Group E, containing Pichia anomala, may be a sibling to group F, which is represented by the Saccharomyces species complex.     

AFLPs resolve phylogeny and reveal mitochondrial introgression within a species flock of African electric fish (Mormyroidea: Teleostei)

Estimating species phylogeny from a single gene tree can be especially problematic for studies of species flocks in which diversification has been rapid. Here we compare a phylogenetic hypothesis derived from cytochrome b (cyt b) sequences with another based on amplified fragment length polymorphisms (AFLP) for 60 specimens of a monophyletic riverine species flock of mormyrid electric fishes collected in Gabon, west-central Africa. We analyze the aligned cyt b sequences by Wagner parsimony and AFLP data generated from 10 primer combinations using neighbor-joining from a Nei-Li distance matrix, Wagner parsimony, and Dollo parsimony. The different analysis methods yield AFLP tree topologies with few conflicting nodes. Recovered basal relationships in the group are similar between cyt b and AFLP analyses, but differ substantially at many of the more derived nodes. More of the clades recovered with the AFLP characters are consistent with the morphological characters used to designate operational taxonomic units in this group. These results support our hypothesis that the mitochondrial gene tree differs from the overall species phylogeny due at least in part to mitochondrial introgession among lineages. Mapping the two forms of electric organ found in this group onto the AFLP tree suggests that posteriorly innervated electrocytes with nonpenetrating stalks have independently evolved from anteriorly innervated, penetrating-stalk electrocytes at least three times.     

Comparing the performance of multiple mitochondrial genes in the analysis of Australian freshwater fishes

In this study, four mitochondrial genes (cytochrome oxidase I, ATPase, cytochrome b and control region) were amplified from most of the fish species found in the fresh waters of south-eastern Queensland, Australia. The performance of these different gene regions was compared in terms of their ability to cluster fish families together in a neighbour-joining tree, both individually by gene and in all combinations. The relative divergence rates of each of these genes were also calculated. The three coding genes (cytochrome oxidase I, ATPase and cytochrome b) recovered similar number of families and had broadly similar divergence rates. ATPase diverged a little more quickly than cytochrome oxidase I and cytochrome b slightly more slowly than cytochrome oxidase I. All two-gene combinations recovered the same number of families. Results from the control region were much more variable, and, although generally possessing more diversity than the other regions, were sometimes less variable.     

Identification of mosquito bloodmeals using mitochondrial cytochrome oxidase subunit I and cytochrome b gene sequences

Abstract.  Primer pairs were designed and protocols developed to selectively amplify segments of vertebrate mitochondrial cytochrome oxidase subunit 1 (COI) and cytochrome b (Cyt b ) mtDNA from the bloodmeals of mosquitoes (Diptera: Culicidae). The protocols use two pairs of nested COI primers and one pair of Cyt b primers to amplify short segments of DNA. Resultant sequences are then compared with sequences in GenBank, using the BLAST function, for putative host identification. Vertebrate DNA was amplified from 88% of our sample of 162 wild-caught, blood-fed mosquitoes from Oregon, U.S.A. and GenBank BLAST searches putatively identified 98% of the amplified sequences, including one amphibian, seven mammalian and 14 avian species. Criteria and caveats for putative identification of bloodmeals are discussed.