基于微卫星标记的绿背山雀遗传结构分析(英文)

近些年来,核序列基因和微卫星标记被广泛地应用于物种遗传结构分析,然而,对于两者的有效性我们却知之甚少。本文以绿背山雀(Parus monticolus)为研究对象,探讨核序列基因和微卫星标记在解析物种遗传结构方面的有效性。绿背山雀中部和西南线粒体谱系分支在核序列基因上存在相应的遗传分化,而在微卫星数据上却不存在分化。两个谱系分支在核序列基因上的FST值是微卫星的4倍。我们认为等位基因大小异源同形(size homoplasy)可能是导致微卫星标记无法区分两个谱系分支的主要原因。我们的研究结果表明在探讨物种遗传结构,且当目标种群具有较大的有效种群数量时,核序列基因比微卫星标记具有更强的适用性。绿背山雀各线粒体谱系分支间的 FST值与等位基因数量不存在显著的相关性,该结果表明微卫星标记的有效性与等位基因的数量无关。RST值的变化范围比 FST值大,因此更不利于探讨种群间的遗传分化。与核序列基因相符,微卫星标记同样揭示了台湾支系的遗传独立性。台湾支系与大陆支系间的遗传分化可能属于严格的异域分化过程。     

莺总科鸟类的系统分类进展评述(英文)

本文评述了莺总科鸟类的系统分类,并重点探讨科间、属内系统发育关系以及种级分类地位。基于已发表的 DNA 序列数据,对马岛莺科(Bernieridae)和柳莺科(Phylloscopidae)重新进行了分析。大量的分子水平的研究让我们对鸟类系统发育的认识有了很大提高。但是,由于一些主要进化分支(科)的快速分化,使得这些科之间的相互关系依然不明确。同样,分子标记和(或)鸣声及其他生活史资料在大量研究中的应用,大大提高了种级水平的分类可靠性。我们推测,随着人们运用现代的整合方法对新类群的不断研究,物种数量会不断增加。     

鸱鸮科27种鸟类COⅠ序列变异及系统发育分析

鸱鸮科鸟类的种属分类仍存在一些争议。例如:渔鸮属和雪鸮属是否应并入雕鸮属,四川林鸮是一独立种还是长尾林鸮的一个亚种?线粒体DNA中细胞色素C氧化酶亚基I(CO I)基因是DNA条形编码的主要基因,被广泛应用于鸟类的物种鉴定和系统发育研究。通过测定中国产红角鸮、雕鸮和长尾林鸮C0 I基因的序列,并结合GenBank中鸱鸮科鸟类的同源序列,本文对27种鸟类进行了序列变异和系统发育分析。结果显示,鸱鸮科鸟类多数是种间变异大于种内变异,该科鸟类几乎都是同属的优先聚类。序列分歧和系统发育分析结果支持将渔鸮属和雪鸮属置于雕鸮属下。同时,相对于欧亚大陆长尾林鸮种群,日本的长尾林鸮较四川林鸮分歧更大。系统发育分析结果也支持四川林鸮是长尾林鸮的一个亚种的观点。研究为进一步明晰鸱鸮科鸟类的系统发育和分类提供了新的分子证据。     

基于线粒体基因cyt b和COI的莺科部分鸟类系统发育

雀形目(Passeriformes)莺科(Sylviidae)鸟类广泛分布于旧大陆,该科许多种类的系统发育关系一直存在争议.本研究采用分子系统学方法,对莺科11属37种鸟类的cyt b全基因序列和COI部分基因序列进行系统发育分析,构建TML和Bayesian系统发育树.结果显示,柳莺属(Phylloscopus)并非单系发生,(鹟)莺属(Seicercus)可能是其同类或其属下的一个类群;在柳莺属内,乌嘴柳莺(P.magnirostris)与极北柳莺(P. borealis)亲缘关系较近;黄腰柳莺(P.proregulus)、云南柳莺(P.yunnanensis)、橙斑翅柳莺(P. pulcher)及灰喉柳莺(P. maculipennis)亲缘关系较近;黄腹柳莺(P.occisinensis)、巨嘴柳莺(P.schwarzi)、棕眉柳莺(P. armandii)、叽喳柳莺(P. collybita)及褐柳莺(P.fuscatus)亲缘关系较近;树莺属(Cettia)并非单系发生,与拟鹅莺属(Abroscopus)和地莺属(Tesia)聚在一起;大苇莺属(Acrocephalus)为单性系;此外,林莺属(Sylvia)与绣眼鸟属(Zosterops)的亲缘关系、以及鹪莺属(Prinia)、缝叶莺属(Orthotomus)及扇尾莺属(Cisticola)三者间的亲缘关系也被支持.     

秦岭细鳞鲑物种有效性的探讨

采用PCR技术获得细鳞鲑秦岭亚种25尾个体的线粒体DNA控制区598bp的序列,从Gene Bank下载黑龙江流域的尖吻细鳞鲑与钝吻细鳞鲑的同源序列,以近缘物种哲罗鲑为外类群,构建了中国细鳞鲑属的分子系统发育树,并结合形态特征讨论了细鳞鲑秦岭亚种的有效性。遗传结构分析显示,秦岭亚种与黑龙江流域两种细鳞鲑之间以及后两种细鳞鲑之间的遗传距离分别为:0.0202,0.0207,0.0199,明显大于细鳞鲑属种内遗传距离:0.0023,0.0033,0.0091。以邻接法(NJ)和最大简约法(MP)构建的系统发育树表明,秦岭地区的细鳞鲑单独构成一个单系群,而黑龙江流域的两种细鳞鲑构成一个大的支系。以上结论证明了秦岭细鳞鲑已经达到了亚种水平的分化,该研究的结果支持形态分类上秦岭亚种的分类地位。     

燕麦属种质资源遗传多样性及遗传演化关系ISSR分析

为了研究燕麦属（Avena）物种遗传多样性及遗传演化关系,利用ISSR分子标记对燕麦属16个种共47份种质资源进行分析。结果表明：燕麦属种间存在丰富的遗传多样性,种间遗传相似性系数（GS）在0.5092～0.9544之间,种内平均遗传相似性系数（GS）最高的A. sativa为0.9512,最低的A. lusitanica为0.7119,说明燕麦属物种间存在较大的遗传变异,而物种内的遗传变异会因物种的不同存在差异。聚类结果显示,47份种质资源根据基因组组成聚成4大类群,AC基因组燕麦与ACD基因组燕麦聚成一大类,说明2组亲缘较近;由AB基因组燕麦聚成的第Ⅱ大类和由As和Ad基因组燕麦聚成的第Ⅲ大类在一分枝上,说明As和Ad基因组燕麦与AB基因组燕麦亲缘关系较AC基因组燕麦更近;Cp基因组燕麦单独构成一类群,且与其他燕麦相似数较低,说明该基因组燕麦与其他燕麦亲缘关系较远。所试燕麦种质资源种内的遗传差异性和遗传关系与地理来源密切相关。     

牦牛线粒体DNA细胞色素b基因序列测定及其起源、分类地位研究

牦牛在牛亚科中的分类地位仍存在较大的分歧。根据普通牛线粒体基因组序列设计引物对家牦牛基因组进行PCR扩增和克隆测序，获得了家牦牛细胞色素6（Cytochromeb）基因的全长序列，并以羊亚科绵羊（Ovisaries）为外类群，对牛亚科代表性物种进行了系统发育分析。结果显示：牛亚科不同物种间线粒体细胞色素b基因的转换／颠换比值为4．9，突变未达到饱和状态；牦牛与牛属间的序列差异百分比为8．0％～8．6％，大于牦牛与美洲野牛间的序列差异百分比；系统发育分析发现家牦牛与野牦牛首先聚为一类，再与美洲野牛聚为一类；说明牦牛与美洲牦牛属间的遗传相似性较高，而与牛属间的遗传相似性较低，结果支持现在的家牦牛和野牦牛都是同一祖先原始牦牛的后代，推测两者分化时间大约为0．55百万年前；支持将牦牛划分为牛亚科牦牛属的观点，牦牛属包括家牦牛和野牦牛两个种。     

猕猴桃种质资源不同居群遗传多样性及遗传演化关系分析

应用 SSR分子标记研究10个云南野生猕猴桃居群的遗传多样性及遗传演化关系。结果表明,不同地理居群间的遗传多样性水平差异不大;居群间的遗传一致度在0.9899 ~ 0.9991之间,遗传分化系数(Gst)为 0.0011 ~ 0.1743。威信居群与彝良居群间的遗传一致度最高,遗传距离最小,亲缘关系最近;永善居群与云龙居群间的遗传一致度最低,遗传距离最大,亲缘关系最远。10个居群可分为两大类群,师宗、绥江、西畴和永善4个居群聚为类群 Ⅰ,麻栗坡、屏边、云龙、镇雄、彝良和威信6个居群聚为类群 Ⅱ,从进化上看,类群 Ⅰ 内4个居群的遗传演化关系可分为2个进化层次,师宗居群与绥江居群,以及西畴居群与永善居群间的遗传进化关系最近。类群 Ⅱ 内的遗传演化关系可分为 5 个进化层次,麻栗坡与屏边居群间的遗传进化关系最近,云龙与威信居群间的亲缘关系较远。本研究为加快云南猕猴桃属种质资源的研究利用提供参考。     

藏系绵羊mtDNA遗传结构及多样性研究

为了研究青海地区藏系绵羊遗传结构、多样性和母系起源,采用PCR扩增和直接测序方法获得了青海地区高原型、欧拉型、山谷型和青海黑藏羊8个群体共187个个体的mtDNA D-loop序列片段,对序列数据进行遗传多样性、遗传结构、系统发育和网络关系分析。结果显示,187个个体均获得长度为527bp的mtDNA D-loop序列片段,共定义了94个单倍型。青海地区高原型藏羊具有较高的遗传多样性,而欧拉型和青海黑藏羊遗传多样性相对较低,这与各类群藏系绵羊在青海地区的资源现状、分布范围相适应。种群间遗传分化及AMOVA分析均显示,欧拉型与其他类群间存在较显著的遗传分化,其余类群间分化不明显。系统发育分析表明青海地区藏系绵羊可能有3个母系起源,与以往研究相一致。     

剪股颖属植物遗传分化及系统关系的分子标记研究

剪股颖属植物形态变异大、倍性复杂、种间易于杂交,导致异名多、分类混乱。本研究采用4种分子标记技术,对包括匍茎剪股颖、巨序剪股颖及红顶草在内的7种剪股颖,共58个个体的遗传分化和系统关系进行研究。根据谱带清晰程度及多态性,筛选出9个RAPD引物、2个SSR引物、5个ISSR引物及1个SCAR标记,并建立了最适的PCR反应条件。采用最大简约法和距离法对7种剪股颖植物进行系统树分析,并用靴带检验法计算内部分支的支持率。启发式搜索得到的简约树和由UPGMA方法得到的表征树近乎相同,其中,匍茎剪股颖和红顶草构成单系类群并得到100%的置信支持。红顶草显示了匍茎剪股颖特异性特征谱带,但同时具有不同于匍茎剪股颖的遗传分化,支持将红顶草视为匍茎剪股颖一个变种的观点。对匍茎剪股颖与巨序剪股颖间的遗传分化进行了探讨。     

Molecular phylogenetic analysis of Onchocerca lupi and its Wolbachia endosymbiont

The morphology of Onchocerca lupi, responsible for canine ocular onchocercosis, is unique within the genus. Earlier analyses of the 5S ribosomal RNA gene spacer region sequence of the parasite and the 16S ribosomal RNA gene sequence of its Wolbachia endosymbiotic bacteria (Rickettsiales) supported the morphological and biological arguments that O. lupi is a distinct species. However, the exact phylogenetic position of O. lupi and its endosymbiont could not be unambiguously determined. Herein we report analyses based on the mitochondrial cytochrome oxidase I (COI) gene of the filarial species and the Wolbachia surface protein (wsp) and the bacterial cell-cycle ftsZ genes of their wolbachiae. Our results indicate that O. lupi separated from other Onchocerca spp. early in evolution. This is in line with the previous morphological analysis demonstrating that O. lupi is an atypical Onchocerca species showing both primitive and evolved characters. The phylogenetic trees generated for the COI sequences of filariae and the wsp and ftsZ sequences of their wolbachiae were congruent with each other, which supports the hypothesis that nematodes and their Wolbachia endobacteria share a long co-evolutionary history.     

Evidence of multiple divergent mitochondrial lineages within the southern African diplopod genus Bicoxidens Attems, 1928 (Spirostreptida)

Two recent studies have suggested that divergent mitochondrial lineages may be present within spirostreptid genera such as Bicoxidens Attems, 1928. Bicoxidens, similar to many other endemic soil invertebrates, exhibits low dispersal capabilities and strict microclimate habitat preferences, attributes that often lead to geographic isolation. Given that prolonged geographic isolation often lays the foundation for population genetic differentiation, genetic divergence and possibly speciation, there was good reason to suspect that Bicoxidens may consist of several distinct lineages. On this basis, the mitochondrial cytochrome c oxidase subunit 1 (COI) was used to reconstruct the phylogeny of Bicoxidens and reveal divergent lineages within the genus. Maximum likelihood and Bayesian inference analyses recovered a paraphyletic Bicoxidens phylogram with divergent lineages present in three species – B. friendi, B. flavicollis and B. brincki – suggesting high genetic diversity within the genus. Bayesian genetic cluster analyses suggested the presence of multiple distinct mitochondrial lineages within the genus with four identified in B. flavicollis alone. It was therefore concluded that the divergent lineages observed among Bicoxidens populations may suggest the presence of hidden species.     

Molecular taxonomic relationships of Psoroptes and Chorioptes mites from China based on COI and 18S rDNA gene sequences

In this present study, the mitochondrial DNA gene cytochrome coxidase subunit I (COI) and the small subunit ribosomal RNA (18S rDNA) gene were used to determine the taxonomic relationships of Psoroptes and Chorioptes mites from China. The neighbor-joining and maximum-parasimony approach were used to evaluate the evolutionary relatedness among different hosts in the genera Psoroptes and Chorioptes. Phylogenetic analysis showed that Psoroptes cuniculi and Psoroptes natalensis may be two different species within the genus Psoroptes, and Chorioptes texanus and Chorioptes panda are different species within the genus Chorioptes.     

Molecular identification of polydorid polychaetes (Annelida: Spionidae): is there a quick way to identify pest and alien species?

The early detection and correct identification of polydorid polychaete species is essential as they are often encountered as invasive alien pests in aquaculture facilities or the intertidal where they may modify the ecosystem. Accurate identification is, however, often hampered by high levels of morphological similarity among species. This taxon will therefore benefit from the development of a library of sequences, such as COI barcodes, to aid identification. However, the universal primers for the cytochrome c oxidase I (COI) DNA barcoding marker has failed to consistently amplify this gene for polydorids, greatly hampering the development of such a library. We describe the development of unique PCR primers for the COI gene that work across four genera and nine species of polydorids. We also compared its efficacy with sequence data for mitochondrial cytochrome b and nuclear 18S rRNA, and a concatenated dataset consisting of all three markers. The nuclear 18S rRNA gene showed the least variation both intra- (0.0–1.2%) and interspecifically (0.6–4.3%), and was the most accurate for species identifications among the three markers. Although COI was characterised by higher intraspecific variation compared with Cyt b (0.0–14.5% and 0.0–4.2%, respectively), Cyt b showed considerably higher levels of interspecific variation (16.6–30.2%) compared with COI (2.2–20.7%). Of the two mitochondrial DNA markers, COI was actually less accurate for species identifications, having suggested two species within Boccardia pseudonatrix that was not supported by the other markers. Overall, the concatenated dataset yielded the most consistent intraspecific groupings, suggesting that this is the most accurate means of identifying polydorids using DNA sequence data. Thus, there may not be a quick and easy way to identify these species accurately using only molecular data.     

Phylogenetic comparison of Leucocytozoon spp. from wild birds of Japan

Eight species of Japanese birds were found to be infected with Leucocytozoon species using microscopic analysis. We used PCR and sequence analysis of the mitochondrial cytochrome b gene (cyt b) to compare the genetic background among these detected protozoa species. In 20 individuals of 22 samples, a single amplified band was detected from 6 of 8 bird species; 9 Japanese rock ptarmigans (Lagopus mutus japonicus), 4 large-billed crows (Corvus macrorhynchos), 2 carrion crows (C. corone), 2 scops owls (Otus scops), 1 Japanese grosbeak (Eophona personata), and 2 brown-eared bulbuls (Hypsipetes amaurotis), respectively. Phylogenetic analysis based on the partial cyt b sequences revealed that all Leucocytozoon isolates in Japan closely grouped with other Leucocytozoon species previously reported in the literature. Among the Japanese isolates, the phylogenetic tree suggested that L. lovati from the Japanese rock ptarmigan may be basal to the parasites found in other bird species. Our study is the first to identify the molecular relationships among Leucocytozoon parasites in the avifauna of Japan.     

Rapid diagnostic PCR assays for members of the Culicoides obsoletus and Culicoides pulicaris species complexes, implicated vectors of bluetongue virus in Europe

Biting midges of the Culicoides obsoletus Meigen and Culicoides pulicaris L. species complexes (Diptera: Ceratopogonidae) are increasingly implicated as vectors of bluetongue virus in Palearctic regions. However, predicting epidemiological risk and the spread of disease is hampered because whilst vector competence of Culicoides is expressed only in adult females, morphological identification of constituent species is only readily applicable to adult males and some species distinguishing traits have overlapping character states. Furthermore, adult males are typically rare in field collections, making characterisation of Culicoides communities impossible. Here we highlight the utility of mitochondrial cytochrome oxidase subunit I (COI) DNA sequences for taxonomic resolution and species identification of all species within C. obsoletus and C. pulicarus complexes. Culicoides were collected from 18 sites in the UK and Continental Europe, and identified to species level, or species complex level, based on morphological characters. The sample comprised four species from the C. obsoletus complex (n = 88) and five species from the C. pulicaris complex (n = 39). The DNA sequence of the 5' end of the COI gene was obtained from all individuals. Each member species formed a well-supported reciprocally monophyletic clade in a maximum likelihood phylogeny. Levels of DNA sequence divergence were sufficiently high between species to allow the design of species-specific PCR primers that can be used in PCR for identification of members of the C. pulicaris complex or in a multiplex PCR to identify members of the C. obsoletus complex. This approach provides a valuable diagnostic tool for monitoring species composition in mixed field collections of Culicoides.     

Cytochrome b5 expression in gonadectomy-induced adrenocortical neoplasms of the domestic ferret (Mustela putorius furo)

Whereas the adrenal glands of healthy ferrets produce only limited amounts of androgenic steroids, adrenocortical neoplasms that arise in neutered ferrets typically secrete androgens or their derivative, estrogen. The 17,20-lyase activity of cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450c17) must increase to permit androgen biosynthesis in neoplastic adrenal tissue. We screened ferret adrenocortical tumor specimens for expression of cytochrome b(5) (cyt b(5)), an allosteric regulator that selectively enhances the 17,20-lyase activity of P450c17. Cyt b(5) immunoreactivity was evident in 24 of 25 (96%) adrenocortical adenomas/carcinomas from ferrets with signs of ectopic sex steroid production. Normal adrenocortical cells lacked cyt b(5), which may account for the low production of adrenal androgens in healthy ferrets. Other markers characteristic of gonadal somatic cells, such as luteinizing hormone receptor, aromatase, and GATA4, were coexpressed with cyt b(5) in some of the tumors. We concluded that cyt b(5) is upregulated during gonadectomy-induced adrenocortical neoplasia and is a marker of androgen synthetic potential in these tumors.     

Molecular evidence for a novel encapsulated genotype of Trichinella from Patagonia, Argentina

At present, Trichinella spiralis is the only species of this genus reported from South America. Herein, we detail a molecular analysis of a new encapsulated isolate of muscle larvae of Trichinella, found in a mountain lion (Puma concolor) coming from the Patagonia, Argentina. We studied three DNA regions previously probed to be useful for the identification of all eleven recognized Trichinella genotypes: expansion segment 5 (ES5), cytochrome c-oxidase subunit I (COI) and 5S ribosomal DNA intergenic spacer region (5S ISR). BLAST searches with these DNA sequences showed that the mitochondrial and nuclear ribosomal regions most closely resemble other Trichinella sequences available in GenBank. However, they did not exactly match any of the eleven recognized genotypes. The phylogenetic analysis from COI and 5S ISR sequences showed that the mountain lion isolate is grouped with encapsulated members, in concordance with morphological data. Furthermore, this new isolate was located at the base of the encapsulated genotypes, signifying that it is an old genotype that could have emerged earliest in this group. These data strongly suggest that this isolate from the Patagonia represents the twelfth genotype (T12) described in the genus Trichinella. Nevertheless, further studies are necessary to adequately establish this isolate as a unique genotype.     

The Anatomy and Venom-Emitting Mechanism of the Globiferous Pedicellariae of the Urchin Parechinus Angulosus (Leske) With Notes on Their Behaviour

The freshwater snails belonging to the genus Melanoides Olivier, 1804 are widespread across tropical regions of the world and endemic species have evolved in the African Lakes Malawi, Mweru and Tanganyika. The endemic Melanoides species of Lake Malawi have been investigated several times during the last century, due to their large conchological variation, but no unambiguous answer regarding the number of species has been given. The phylogenetic relationship between morphs or genetic clones of Melanoides in Lake Malawi was inferred by phylogenetic analyses of DNA sequence data from the mitochondrial genes 16S and COI. Additional sequences from GenBank were included to investigate the relationship to other morphs from different parts of the world. For the first time, a putative secondary structure was developed for a partial region of 16S in this genus to identify the variability of the secondary structure in stems and loops. The molecular analyses indicated that several genetic clones exist in Lake Malawi and that M. tuberculata is a paraphyletic taxon. It is not clear from the results whether invasions or dispersals account for the complex situation in Lake Malawi. The basal position of M. admirabilis, endemic to Lake Tanganyika, in the inferred phylogeny indicates that Africa might be the origin of the genus. The results further indicate that three major clades of Melanoides, consisting of several genetic clones, are present in Lake Malawi; one clade consisting of invasive M. tuberculata, another of native M. tuberculata and a third consisting of the M. polymorpha- complex. It appears as if the unique development of morphs within the Melanoides genus in Lake Malawi has evolved primarily by divergence of genetic clones instead of species differentiation.