基于28S rDNA基因序列研究十种臂尾轮虫的系统关系和分类地位

通过对角突臂尾轮虫、尾突臂尾轮虫、裂足臂尾轮虫、剪形臂尾轮虫、方形臂尾轮虫、壶状臂尾轮虫、红臂尾轮虫、镰形臂尾轮虫、萼花臂尾轮虫和十指臂尾轮虫等10种臂尾轮虫以及透明囊足轮虫、大肚须足轮虫和晶囊轮虫等其他3种轮虫的28S rDNA序列分析,使用MAGE软件构建这13种轮虫系统发生树(NJ树、ME树、UPGMA树和MP树),探讨了臂尾轮属、须足轮属、晶囊轮属和囊足轮属之间以及10种臂尾轮虫之间的系统关系.结果表明,本研究所涉及的轮虫28S rDNA序列差异百分比均值为30.15%,可作为分子标记应用于轮虫属间和属内种间系统关系研究;系统树均支持将十指臂尾轮虫作为一个独立的支系从臂尾轮属中分离出来;裂足臂尾轮虫应隶属臂尾轮属;壶状臂尾轮虫和红臂尾轮虫是两个独立的种;透明囊足轮虫与臂尾轮属亲缘关系较近.     

基于线粒体COⅠ基因序列探讨臂尾轮属的系统发生和几种轮虫的分类地位

通过对萼花臂尾轮虫、角突臂尾轮虫、壶状臂尾轮虫、十指臂尾轮虫、镰形臂尾轮虫、尾突臂尾轮虫和红臂尾轮虫等7种轮虫mtDNA COⅠ序列分析,并以透明囊足轮虫为外群,使用MEGA软件构建臂尾轮属轮虫系统发生树(ME树和NJ树)以探讨臂尾轮属的系统发生和该属中几个种类的分类地位.结果表明本研究所涉及的轮虫mtDNA COⅠ平均序列差异百分比为24%;在两个系统树中,淡水臂尾轮虫和海水臂尾轮虫均独立成枝;用两种方法得到的系统发生树均支持将十指臂尾轮虫作为一个独立的支系分离出来.壶状臂尾轮虫和红臂尾轮虫应属于不同的种;来自芜湖、墨西哥和美国的十指臂尾轮虫以及来自芜湖和澳大利亚的萼花臂尾轮虫应分别互为姐妹种.本研究还首次揭示了角突臂尾轮虫、尾突臂尾轮虫和镰状臂尾轮虫与其它臂尾轮虫间的系统关系.     

基于16S rDNA序列探讨十种臂尾轮虫的系统关系和分类地位

通过对角突臂尾轮虫、尾突臂尾轮虫、裂足臂尾轮虫、剪形臂尾轮虫、方形臂尾轮虫、壶状臂尾轮虫、红臂尾轮虫、镰肜臂尾轮虫、萼花臂尾轮虫和十指臂尾轮虫等十种臂尾轮虫和大肚须足轮虫的线粒体16s rDNA进行扩增和序列测序,结合Genebank 中十指臂尾轮虫的16S rDNA序列,使用MAGE软件构建了NJ(neighbor-joining method)树,使用mrbayes软件构建了贝叶斯树,探讨了十种臂尾轮虫之间的系统关系.结果表明,本研究所涉及的轮虫16S rDNA序列差异百分比均值为14.6%,可作为分子标记应用于轮虫属内种间系统关系研究;系统树均支持将十指臂尾轮虫、裂足臂尾轮虫隶属于臂尾轮属;壶状臂尾轮虫和红臂尾轮虫是两个独立的种.此外,还依据16S rDNA序列变异百分比推测了十种臂尾轮虫的分化时间.     

基于rDNAITS序列分析莲塘湖萼花臂尾轮虫两种形态型的分类地位

轮虫的周期变形是指种群内出现的轮虫形态随时间推移而发生的周期性变化,包括轮虫个体大小的变化、轮虫后棘刺或后侧棘刺的有无及长度的变化等。广泛存在的周期变形使轮虫的分类更加复杂。利用分子标记对不同形态型轮虫的遗传分化进行研究将有助于正确认识它们的分类地位。为此,研究对采自芜湖市莲塘湖水体中萼花臂尾轮虫(Brachionus calyciflorus)30个有棘刺型(Spined morphotype)的克隆(S1-S30)和18个无棘刺型(Unspined morphotype)的克隆(U1-U18)进行了rDNA ITS序列分析;以十指臂尾轮虫(B.patulus)为外群,构建了48个克隆的系统发生树(NJ、MP、ML和贝叶斯树)。结果表明,所测48个克隆共包括16个单元型。在ITS序列中,T、C、A、G碱基的平均含量分别为28.6%、18.7%、35.9%、16.8%,其中A+T含量为64.5%,C+G含量为35.5%。单元型U12与其他单元型间的序列差异百分比为26.2%-26.6%,平均为26.47%;其中,发生在ITS1、5.8S和ITS2区间的序列差异百分比分别为26.9%-27.8%、2.9%-3.5%和44.4%-45.0%,平均依次为27.27%、3.09%和44.48%。而其他单元型间平均序列差异百分比为0.41%。4个系统树均支持将48个克隆分为2个支系:无棘刺型克隆U12独成一支,无棘刺型的其余克隆与所有有棘刺型克隆构成另一支系。单元型U12与其他单元型应分别属于两个不同的姐妹种;但两种形态型并非不同的亚种或互为姐妹种,它们间的形态差异主要由表型可塑性引起。       

五种淡水轮虫种群增长参数的比较研究

在32℃培养条件下,实验研究了5种淡水常见浮游轮虫的种群增长参数。通过编制生命表计算其内禀增长率rm(h-1)、净生殖力(R0)和世代时间T(h)分别为:萼花臂尾轮虫-0.091、14.1和34.1;壶状臂尾轮虫-0.055、6.9和38.4;角突臂尾轮虫-0.035、4.9和50.9;臂尾水轮虫-0.038、4.3和46.1以及裂足臂尾轮虫-0.017、2.1和43.7。其中,萼花臂尾轮虫内禀增长率大,繁殖率高,可作为批量培养的首选种类。       

镜湖不同湖区沉积物中轮虫休眠卵萌发的比较研究

实验室内对镜湖沉积物中的轮虫休眠卵进行了萌发,共孵出轮虫47种,隶属于10科19属;其中大湖区沉积物中萌发出的轮虫40种,小湖区41种,两湖区共同种类34种.每毫升大湖区沉积物中休眠卵萌发出的轮虫平均数量为(1.1±0.1)个,显著高于小湖区的(0.4±0.0)个.在两湖区沉积物中,以3种孵化方式所萌发出的平均密度超过4.0ind./200mL的轮虫种类总计有6种,它们是多须伪前翼轮虫(Proalides tentaculates)、裂痕龟纹轮虫(Anuraeopsis fissa)、螺形龟甲轮虫(Keratella cochlearis)、裂足臂尾轮虫(Brachionus diversicornis)、臂三肢轮虫(Filinia brachiata)和萼花臂尾轮虫(Brachionus calyciflorus);其中前三种轮虫为两湖区所共有.共有的3种轮虫的孵出情况在两湖区沉积物间也存在着差异.在小湖区沉积物中,3种孵化方式下孵出的平均密度大于0.05ind./200mL的轮虫属是臂尾轮属、伪前翼轮属、龟甲轮属和龟纹轮属,而大湖区沉积物中孵出的平均密度较高的轮虫属还包括三肢轮属、巨头轮属(Cephalodella)和异尾轮属(Trichocerca);水体营养程度较高的大湖区沉积物中孵出的臂尾轮属、伪前翼轮属和龟甲轮属轮虫的密度均高于营养程度较低的小湖区.     

分子生物学技术在轮虫遗传多样性和系统发生研究中的应用

分子生物学技术,如等位酶分析、DNA序列分析、限制性片段长度多态性、随机扩增多态、重复DNA序列多态性,在轮虫研究中的应用始于上世纪70年代,研究领域主要是轮虫群体遗传学、生物变异与多样性和系统发生与进化,被研究的轮虫种类主要集中在臂尾轮虫属、晶囊轮虫属和蛭态轮虫的一些种类。着重介绍分子生物学技术在轮虫遗传学和系统学研究中的应用,并对今后的研究热点提出了展望。     

基于rDNA ITS序列分析莲塘湖萼花臂尾轮虫种群遗传结构的季节变化

对采自芜湖市莲塘湖水体中的萼花臂尾轮虫(Brachionus calyciflorus)夏季种群中的30个克隆(S1-S30)和秋季种群中的20个克隆(A1-A20)的rDNA ITS序列进行了分析;以十指臂尾轮虫B.patulus为外群,构建了50个克隆的NJ、MP和ME系统发生树。结果表明:三个系统树均支持将50个克隆分为两个支系;第一支系包括29个夏季采集的单元型(单元型S9除外)和1个秋季采集的单元型A2,第二个支系包括18个秋季采集的单元型(单元型A2除外)和1个夏季采集的单元型S9;两支系间ITS序列差异为5.1%-8.8%;它们应属于两个不同的姐妹种。夏季,莲塘湖水体中的萼花臂尾轮虫种复合体内以第一支系为主构成的姐妹种在相对丰度上占绝对优势,达29/30;而秋季则相反。上述结果表明:浅水湖泊中萼花臂尾轮虫种群密度的季节变动实际上主要在于各姐妹种相对丰度的季节变化;两姐妹种的种群遗传结构季节变化符合重叠模型。     

卜氏晶囊轮虫对萼花臂尾轮虫形态及种群生活史对策的短期和长期影响

初步探究了捕食者卜氏晶囊轮虫对其猎物萼花臂尾轮虫形态及种群生活史对策的短期和长期影响。结果表明,在含卜氏晶囊轮虫信号的环境中培养得到的萼花臂尾轮虫所产后代个体较对照组侧棘刺明显增长,体型增大,卵体积增大,净生殖率明显降低。短期接触捕食者信号的萼花臂尾轮虫种群与长期接触捕食者信号的萼花臂尾轮虫种群相比,具有较大的卵体积和较短的侧棘刺,同时具有显著较高的净生殖率。     

淮北煤矿塌陷区水域轮虫的初步研究

采用筛绢过滤法对淮北煤矿塌陷区水域轮虫进行了初步的研究,共检出轮虫11种,隶属于3亚目4科7属。臂尾轮科种类最多,占63．6％,萼花臂尾轮虫（B．calyciflorus）、壶状臂尾轮虫（B．urceus）、矩形龟甲轮虫（K.quadrata）为臂尾轮科（Brachionidae）的常见种。调查结果还表明：轮虫的种类与数量的多少与所在的水体的水质有一定的相关性。     

Intragenomic variation in the ITS rDNA region obscures phylogenetic relationships and inflates estimates of operational taxonomic units in genus Laetiporus

Regions of rDNA are commonly used to infer phylogenetic relationships among fungal species and as DNA barcodes for identification. These regions occur in large tandem arrays, and concerted evolution is believed to reduce intragenomic variation among copies within these arrays, although some variation still might exist. Phylogenetic studies typically use consensus sequencing, which effectively conceals most intragenomic variation, but cloned sequences containing intragenomic variation are becoming prevalent in DNA databases. To understand effects of using cloned rDNA sequences in phylogenetic analyses we amplified and cloned the ITS region from pure cultures of six Laetiporus species and one Wolfiporia species (Basidiomycota, Polyporales). An average of 66 clones were selected randomly and sequenced from 21 cultures, producing a total of 1399 interpretable sequences. Significant variation (≥ 5% variation in sequence similarity) was observed among ITS copies within six cultures from three species clades (L. cincinnatus, L. sp. clade J, and Wolfiporia dilatohypha) and phylogenetic analyses with the cloned sequences produced different trees relative to analyses with consensus sequences. Cloned sequences from L. cincinnatus fell into more than one species clade and numerous cloned L. cincinnatus sequences fell into entirely new clades, which if analyzed on their own most likely would be recognized as "undescribed" or "novel" taxa. The use of a 95% cut off for defining operational taxonomic units (OTUs) produced seven Laetiporus OTUs with consensus ITS sequences and 20 OTUs with cloned ITS sequences. The use of cloned rDNA sequences might be problematic in fungal phylogenetic analyses, as well as in fungal bar-coding initiatives and efforts to detect fungal pathogens in environmental samples.     

Differentiation and grouping of isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR compared with grouping on the basis of internal transcribed spacer sequences.

Laccate polypores of the Ganoderma lucidum species complex are widespread white rot fungi of economic importance, but isolates cannot be identified by traditional taxonomic methods. Parsimony analysis of nucleotide sequences from the internal transcribed spacers (ITS) of the ribosomal gene (rDNA) distinguished six lineages in this species complex. Each ITS lineage may represent one or more putative species. While some isolates have identical ITS sequences, all of them could be clearly differentiated by genetic fingerprinting using random amplified polymorphic DNA (RAPD). To investigate the suitability of RAPD markers for taxonomic identification and grouping of isolates of the G. lucidum complex, RAPD fragments (RAPDs) were used as phenotypic characters in numerical and parsimony analyses. Results show that data from RAPDS do not distinguish the same clades as ITS data do. Groupings based on analysis of RAPD data were very sensitive to the choice of the grouping method used, and no consistent grouping of isolates could be proposed. However, analysis with RAPDs did resolve several robust terminal clades containing putatively conspecific isolates, suggesting that RAPDs might be helpful for systematics at the lower taxonomic levels that are unresolved by ITS sequence data. The limitations of RAPDs for systematics are briefly discussed. The conclusion of this study is that ITS sequences can be used to identify isolates of the G. lucidum complex, whereas RAPDs can be used to differentiate between isolates having identical ITS sequences. The practical implications of these results are briefly illustrated.     

Variation in ribosomal and mitochondrial DNA sequences demonstrates the existence of intraspecific groups in Paramecium multimicronucleatum (Ciliophora, Oligohymenophorea)

This is the first phylogenetic study of the intraspecific variability within Paramecium multimicronucleatum with the application of two-loci analysis (ITS1-5.8S-ITS2-5'LSU rDNA and COI mtDNA) carried out on numerous strains originated from different continents. The species has been shown to have a complex structure of several sibling species within taxonomic species. Our analysis revealed the existence of 10 haplotypes for the rDNA fragment and 15 haplotypes for the COI fragment in the studied material. The mean distance for all of the studied P. multimicronucleatum sequence pairs was p=0.025/0.082 (rDNA/COI). Despite the greater variation of the COI fragment, the COI-derived tree topology is similar to the tree topology constructed on the basis of the rDNA fragment. P. multimicronucleatum strains are divided into three main clades. The tree based on COI fragment analysis presents a greater resolution of the studied P. multimicronucleatum strains. Our results indicate that the strains of P. multimicronucleatum that appear in different clades on the trees could belong to different syngens.     

Phylogenetic analysis of ribosomal RNA operons from uncultivated coastal marine bacterioplankton

Analyses of small subunit ribosomal RNA genes (SSU rDNAs) have significantly influenced our understanding of the composition of aquatic microbial assemblages. Unfortunately, SSU rDNA sequences often do not have sufficient resolving power to differentiate closely related species. To address this general problem for uncultivated bacterioplankton taxa, we analysed and compared sequences of polymerase chain reaction (PCR)-generated and bacterial artificial chromosome (BAC)-derived clones that contained most of the SSU rDNAs, the internal transcribed spacer (ITS) and the large subunit ribosomal RNA gene (LSU rDNA). The phylogenetic representation in the rRNA operon PCR library was similar to that reported previously in coastal bacterioplankton SSU rDNA libraries. We observed good concordance between the phylogenetic relationships among coastal bacterioplankton inferred from SSU or LSU rDNA sequences. ITS sequences confirmed the close intragroup relationships among members of the SAR11, SAR116 and SAR86 clades that were predicted by SSU and LSU rDNA sequence analyses. We also found strong support for homologous recombination between the ITS regions of operons from the SAR11 clade.     

海南岛臂尾轮属物种多样性

于2010年11月、2011年5月和8月调查了海南岛臂尾轮虫的多样性,共检出臂尾轮虫25种。其中,东洋界特有种3种:双叉异棘臂尾轮虫(Brachionus donneri bifurcus)、墨氏臂尾轮虫(B.murphyi)和黄氏臂尾轮虫(B.huangi);东洋界新纪录种1种,刻纹臂尾轮虫(B.sericus);对折臂尾轮虫(B.dimidiatus)、褶皱二叉臂尾轮虫(B.dichotomus reductus)和奇异方形臂尾轮虫(B.quadridentatus mirabilis)是中国的新纪录种或亚种。方形臂尾轮虫(B.quadridentatus quadridentatus)、萼花臂尾轮虫(B.calyciflorus)、镰形臂尾轮虫(B.falcatus)和角突臂尾轮虫(B.angularis)为常见种类。海南岛臂尾轮属种类多样性高于我国其他地区,并由广布种、全热带种和泛热带种类组成。海南岛与我国其他地区的臂尾轮虫种类组成Bray-Curtis距离指数随着纬度的增加而增加。     

Primers are designed for amplification and direct sequencing of ITS region of rDNA from Myxomycetes

Four new primers were designed, based on comparison of Physarum polycephalum sequences retrieved from Genbank (primers PHYS-5 and PHYS-4) and our own sequences (primers PHYS-3 and PHYS-2), to amplify the ITS regions of rDNA, including the 5.8S gene segment from Lamproderma species. Sequencing analysis shows that Lamproderma contains ITS1-5.8S-ITS2 regions of approximately 900 bp, which is similar in size to most eukaryotes. However, the corresponding region in another common myxomycete, Fuligo septica, is more than 2000 bp due to the presence of large direct-repeat motifs in ITS1. Myxomycete rDNA ITS regions are interesting both as phylogenetic markers in taxonomic studies and as model sequences for molecular evolution.     

Phylogenetic analysis of Allium subg. Melanocrommyum infers cryptic species and demands a new sectional classification

Allium subgenus Melanocrommyum (Alliaceae) from Eurasia comprises about 150 mostly diploid species adapted to arid conditions. The group is taxonomically complicated with different and contradictory taxonomic treatments, and was thought to include a considerable number of hybrid species, as the taxa show an admixture of assumed morphological key characters. We studied the phylogeny of the subgenus, covering all existing taxonomic groups and their entire geographic distribution. We analyzed sequences of the nuclear rDNA internal transcribed spacer region (ITS) for multiple individuals of more than 100 species. Phylogenetic analyses of cloned and directly sequenced PCR products confirmed the monophyly of the subgenus, while most sections were either para- or polyphyletic. The splits of the large sections are supported by differences in the anatomy of flower nectaries. ITS data (i) demand a new treatment at sectional level, (ii) do not support the hypotheses of frequent gene flow among species, (iii) indicate that multiple rapid radiations occurred within different monophyletic groups of the subgenus, and (iv) detected separately evolving lineages within three morphologically clearly defined species (cryptic species). In two cases these lineages were close relatives, while in Allium darwasicum they fall in quite different clades in the phylogenetic tree. Fingerprint markers show that this result is not due to ongoing introgression of rDNA (ITS capture) but that genome-wide differences between both lineages exist. Thus, we report one of the rare cases in plants where morphologically indistinguishable diploid species occurring in mixed populations are non-sister cryptic species.     

Phylogenetic utility of indels within ribosomal DNA and beta-tubulin sequences from fungi in the Rhizoctonia solani species complex

The genus Rhizoctonia consists of a diverse assemblage of anamorphic fungi frequently associated with plants and soil throughout the world. Some anamorphs are related with teleomorphs (sexual stage) in different taxonomic classes, orders, and families. The fungus may exist as pathogen, saprophyte, or mycorrhizal symbiont and shows extensive variation in characteristics such as geographic location, morphology, host specificity, and pathogenicity. In this study, phylogenetic analyses were performed in the Rhizoctonia solani species complex with individual and combined data sets from three gene partitions (ITS, LSU rDNA, and beta-tubulin). To explore whether indels were a source of phylogenetically informative characters, single-site indels were treated as a new state, while indels greater than one contiguous nucleotide were analyzed by including them as ambiguous data (Coding A); excluding them from the analyses (Coding B), and with three distinct codes: multistate for different sequence (Coding C); multistate for different length (Coding D) and different characters for each distinct sequence (Coding E). Results suggest that indels in noncoding regions contain phylogenetic information and support the fact that the R. solani species complex is not monophyletic. Six clades within R. solani (teleomorph=Thanatephorus) representing distinct anastomosis groups and five clades within binucleate Rhizoctonia (teleomorph=Ceratobasidium) were well supported in all analyses. The data suggest that clades with representatives of R. solani fungi belonging to anastomosis groups 1, 4, 6, and 8 should be recognized as phylogenetic species.