The Small-Subunit rRNA Gene Sequences of Venerids and the Phylogeny of Bivalvia

The complete nucleotide sequence of the 18S subunit of ribosomal DNA (rDNA) was determined for the venerid clams Callista chione (Pitarinae) and Venus verrucosa (Venerinae). Comparison of the new sequences with the published sequences of 1 annelid, 2 gastropods, 2 polyplacophorans, and 19 bivalves showed that when the annelids are used as outgroup the gastropods diverge from the bivalves, which form a cluster including the polyplacophorans. When the gastropods alone were compared with the bivalves, the latter split in two groups corresponding to the two subclasses of Heterodonta and Pteriomorpha. The former include two taxa that diverged early, Galeomma and Tridacna, while the Veneridae and Mactridae form two sister groups. In contrast to previous reports and in line with morphological data, the Ostreidae are included in the Pteriomorphia and form a monophyletic group. Received: 16 May 1998 / Accepted: 11 August 1998     

Phylogeny of Trichomonads Inferred from Small-Subunit rRNA Sequences

ABSTRACT. Small subunit (16S-like) ribosomal RNA sequences were obtained from representatives of all four families constituting the order Trichomonadida. Comparative sequence analysis revealed that the Trichomonadida are a monophyletic lineage and a deep branch of the eukaryotic tree. Relative to other early divergent eukaryotic assemblages the branching pattern within the Trichomonadida is very shallow. This pattern suggests the Trichomonadida radiated recently, perhaps in conjunction with their animal hosts. From a morphological perspective the Devescovinidae and Calonymphidae are considered more derived than the Monocercomonadidae and Trichomonadidae. Molecular trees inferred by distance, parsimony and likelihood techniques consistently show the Devescovinidae and Calonymphidae are the earliest diverging lineages within the Trichomonadida, however bootstrap values do not strongly support a particular branching order. In an analysis of all known 16S-like ribosomal RNA sequences, the Trichomonadida share most recent common ancestry with unidentified protists from the hindgut of the termite Reticulitermes flavipes. The position of two putative free-living trichomonads in the tree is indicative of derivation from symbionts rather than direct descent from some free-living ancestral trichomonad.     

Congruent Phylogenies of Most Common Small-Subunit rRNA and Dissimilatory Sulfite Reductase Gene Sequences Retrieved from Estuarine Sediments

The diversity of sulfate-reducing bacteria (SRB) in brackish sediment was investigated using small-subunit rRNA and dissimilatory sulfite reductase (DSR) gene clone libraries and cultivation. The phylogenetic affiliation of the most commonly retrieved clones for both genes was strikingly similar and produced Desulfosarcina variabilis-like sequences from the inoculum but Desulfomicrobium baculatum-like sequences from a high dilution in natural media. Related organisms were subsequently cultivated from the site. PCR bias appear to be limited (or very similar) for the two primersets and target genes. However, the DSR primers showed a much higher phylogenetic specificity. DSR gene analysis is thus a promising and specific approach for investigating SRB diversity in complex habitats.     

Phylogenetic Analysis of Cryptosporidium Parasites Based on the Small-Subunit rRNA Gene Locus

Biological data support the hypothesis that there are multiple species in the genus Cryptosporidium, but a recent analysis of the available genetic data suggested that there is insufficient evidence for species differentiation. In order to resolve the controversy in the taxonomy of this parasite genus, we characterized the small-subunit rRNA genes of Cryptosporidium parvum, Cryptosporidium baileyi, Cryptosporidium muris, and Cryptosporidium serpentis and performed a phylogenetic analysis of the genus Cryptosporidium. Our study revealed that the genus Cryptosporidium contains the phylogenetically distinct species C. parvum, C. muris, C. baileyi, and C. serpentis, which is consistent with the biological characteristics and host specificity data. The Cryptosporidium species formed two clades, with C. parvum and C. baileyi belonging to one clade and C. muris and C. serpentis belonging to the other clade. Within C. parvum, human genotype isolates and guinea pig isolates (known as Cryptosporidium wrairi) each differed from bovine genotype isolates by the nucleotide sequence in four regions. A C. muris isolate from cattle was also different from parasites isolated from a rock hyrax and a Bactrian camel. Minor differences were also detected between C. serpentis isolates from snakes and lizards. Based on the genetic information, a species- and strain-specific PCR-restriction fragment length polymorphism diagnostic tool was developed.     

基于SSU序列的串珠藻目植物系统发育分析

通过18S rDNA基因(SSU)序列,构建了串珠藻目植物的系统发育关系.结果显示:SSU基因序列片段长度为1 871 bp,核苷酸变异位点有709个,占序列长度的38％;其中简约信息位点有169个,占序列长度的9％.用最大似然法、邻接法和贝叶斯法构建的系统树拓扑结构基本一致,都显示红索藻目的2个属独立于串珠藻目成单独分支,支持红索藻目的建立;胶串珠藻独立于其他串珠藻组植物,支持将其单独分组;数据同时支持将扭曲组和杂生组合并,建立Kumanoa属;但多芒组、绿色组、沼生组等因分子序列数据涉及的种类较少,其系统关系的确定还需要更多的证据.     

16S rRNA phylogenetic investigation of the candidate division "Korarchaeota"

The environmental distribution and phylogeny of "Korarchaeota," a proposed ancient archaeal division, was investigated by using the 16S rRNA gene framework. Korarchaeota-specific primers were designed based on previously published sequences and used to screen a variety of environments. Korarchaeota 16S rRNA genes were amplified exclusively from high temperature Yellowstone National Park hot springs and a 9 degrees N East Pacific Rise deep-sea hydrothermal vent. Phylogenetic analyses of these and all available sequences suggest that Korarchaeota exhibit a high level of endemicity.     

Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5′-Nuclease Assays

Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Quantification of specific genotypes has relied mainly on oligonucleotide hybridization to extracted rRNA or intact rRNA in whole cells. However, low abundance and cellular rRNA content limit the application of these techniques in aquatic environments. In this study, we applied a newly developed quantitative PCR assay (5′-nuclease assay, also known as TaqMan) to quantify specific small-subunit (SSU) rRNA genes (rDNAs) from uncultivated planktonic prokaryotes in Monterey Bay. Primer and probe combinations for quantification of SSU rDNAs at the domain and group levels were developed and tested for specificity and quantitative reliability. We examined the spatial and temporal variations of SSU rDNAs from Synechococcus plus Prochlorococcus and marine Archaea and compared the results of the quantitative PCR assays to those obtained by alternative methods. The 5′-nuclease assays reliably quantified rDNAs over at least 4 orders of magnitude and accurately measured the proportions of genes in artificial mixtures. The spatial and temporal distributions of planktonic microbial groups measured by the 5′-nuclease assays were similar to the distributions estimated by quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry.     

Pair-preferences: a Quantitative Measure of Regularities in Protein Sequences

Abstract

We present here the results obtained by applying several different methods to quantitatively measure regularities in protein sequences based on pair-preferences. We have studied the distribution of amino acid residues, singly as well as in pairs in a large data base and have attempted this task. We confirmed the existence of well-defined pair-preferences in proteins which were shown to be remarkably absent in simulated random sequences of similar amino acid distribution. The analysis of the sequences from the SWISS-PROT data base using simple statistical tests, Fourier analysis, fractal analysis and statistical thermodynamical tests were used to derive parameters to define a natural sequence. As a consequence of the existence of pair-preferences, parameters like fractal dimension (D), spectral exponent (β), scaling parameter (H) and entropy (statistical) were found to be characteristic for natural sequences. For a reference state we chose a randomised state devoid of any pair-preference. The pair-preferences qualified well to be used as quantitative measures of regularities in protein sequences.     

Small-Subunit rRNA Genotyping of Rhizobia Nodulating Australian Acacia spp.

The structure of rhizobial communities nodulating Acacia in southeastern Australia from south Queensland to Tasmania was investigated by a molecular approach. A total of 118 isolates from nodule samples from 13 different Acacia species collected at 44 sites were characterized by small-subunit (SSU) ribosomal DNA (rDNA) PCR-restriction fragment length polymorphism analysis. Nine rhizobial genomospecies were identified, and these taxa corresponded to previously described genomospecies (B. Lafay and J. J. Burdon, Appl. Environ. Microbiol. 64:3989–3997, 1998). Eight of these genomospecies belonged to the Bradyrhizobium lineage and accounted for 96.6% of the isolates. The remaining genomospecies corresponded to Rhizobium tropici. For analysis of geographic patterns, results were grouped into five latitudinal regions regardless of host origin. In each region, as observed previously for rhizobial isolates taken from non-Acacia legumes (Lafay and Burdon, Appl. Environ. Microbiol. 64:3989–3997, 1998), rhizobial communities were dominated by one or two genomospecies, the identities of which varied from place to place. Despite this similarity in patterns, the most abundant genomospecies for Acacia isolates differed from the genomospecies found in the non-Acacia-derived rhizobial collection, suggesting that there is a difference in nodulation patterns of the Mimosoideae and the Papilionoideae. Only two genomospecies were both widespread and relatively abundant across the range of sites sampled. Genomospecies A was found in all regions except the most northern sites located in Queensland, whereas genomospecies B was not detected in Tasmania. This suggests that genomospecies A might be restricted to the more temperate regions of Australia, whereas in contrast, genomospecies B occurs in different climatic and edaphic conditions across the whole continent. The latter hypothesis is supported by the presence of genomospecies B in southwestern Australia, based on partial SSU rDNA sequence data (N. D. S. Marsudi, A. R. Glenn, and M. J. Dilworth, Soil Biol. Biochem. 31:1229–1238, 1998).     

Phylogenetic Analysis of Bacterial Communities Associated with Leaves of the Seagrass Halophila stipulacea by a Culture-Independent Small-Subunit rRNA Gene Approach

Abstract The phylogenetic diversity of the bacterial community associated with leaves of the marine plant Halophila stipulacea in the northern Gulf of Elat was examined by 16S rRNA gene (rDNA) sequence analyses of a clone library. For 59 clones corresponding to 51 ARDRA (amplified rDNA restriction analysis) groups, the sequence of ∼1 kb was determined, and the fraction of the corresponding ARDRA groups of the leaf library was calculated. The class Proteobacteria was represented by 62.6% of the clone sequences. Most sequences originated from members of the γ-subclass (27.3%), affiliated with members of the genera Pseudomonas, Vibrio, Marinomonas, Oceanospirillum, and other marine groups. Affiliation to the α-subclass was determined for 24.2% of the sequences. They were related to the genera Hyphomonas, Roseobacter, Ruegeria, and Rhizobiaceae. Several α-proteobacterial sequences were distantly related to known sequences. Only 4% of the clone sequences were related to β-Proteobacteria. Additionally, 7.1% of the sequences possibly belonged to the class Proteobacteria, but branched deeply from known subclasses. Several sequences were affiliated to members of the orders Verrucomicrobiales and Planctomycetales, the Holophaga/Acidobacterium phylum, and chloroplasts of marine diatoms. Received: 20 March 1999; Accepted: 13 August 1999; Online Publication: 2 March 2000     

从rRNA基因序列探讨云斑蛛属Cyrtophora的系统发生地位

由于云斑蛛属Cyrtophora蜘蛛所织的网形态上较为特殊,所以该属分类地位长期以来有异议.为从分子水平探讨该属系统发生地位,本文对相关的11种蜘蛛线粒体12S rRNA基因和核18S rRNA基因部分序列进行了测定.基于12S rRNA和18S rRNA基因序列联合数据进行的分子系统发生分析结果表明云斑蛛属属于园蛛科,该结果提示在园蛛总科分类实践中以蛛网形态特征为分类依据时要慎重考虑其可靠性.     

Kinetic Bias in Estimates of Coastal Picoplankton Community Structure Obtained by Measurements of Small-Subunit rRNA Gene PCR Amplicon Length Heterogeneity

Marine bacterioplankton diversity was examined by quantifying natural length variation in the 5′ domain of small-subunit (SSU) rRNA genes (rDNA) amplified by PCR from a DNA sample from the Oregon coast. This new technique, length heterogeneity analysis by PCR (LH-PCR), determines the relative proportions of amplicons originating from different organisms by measuring the fluorescence emission of a labeled primer used in the amplification reaction. Relationships between the sizes of amplicons and gene phylogeny were predicted by an analysis of 366 SSU rDNA sequences from cultivated marine bacteria and from bacterial genes cloned directly from environmental samples. LH-PCR was used to compare the distribution of bacterioplankton SSU rDNAs from a coastal water sample with that of an SSU rDNA clone library prepared from the same sample and also to examine the distribution of genes in the PCR products from which the clone library was prepared. The analysis revealed that the relative frequencies of genes amplified from natural communities are highly reproducible for replicate sets of PCRs but that a bias possibly caused by the reannealing kinetics of product molecules can skew gene frequencies when PCR product concentrations exceed threshold values.     

Effect of Primers Hybridizing to Different Evolutionarily Conserved Regions of the Small-Subunit rRNA Gene in PCR-Based Microbial Community Analyses and Genetic Profiling

Genetic profiling techniques of microbial communities based on PCR-amplified signature genes, such as denaturing gradient gel electrophoresis or single-strand-conformation polymorphism (SSCP) analysis, are normally done with PCR products of less than 500-bp. The most common target for diversity analysis, the small-subunit rRNA genes, however, are larger, and thus, only partial sequences can be analyzed. Here, we compared the results obtained by PCR targeting different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of the bacterial 16S rRNA gene with primers hybridizing to evolutionarily conserved flanking regions. SSCP analysis of single-stranded PCR products generated from 13 different bacterial species showed fewer bands with products containing V4-V5 (average, 1.7 bands per organism) than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the additional bands (>1 per organism) were caused by intraspecies operon heterogeneities or by more than one conformation of the same sequence. Community profiles, generated by PCR-SSCP from bacterial-cell consortia extracted from rhizospheres of field-grown maize (Zea mays), were analyzed by cloning and sequencing of the dominant bands. A total of 48 sequences could be attributed to 34 different strains from 10 taxonomical groups. Independent of the primer pairs, we found proteobacteria (α, β, and γ subgroups) and members of the genus Paenibacillus (low G+C gram-positive) to be the dominant organisms. Other groups, however, were only detected with single primer pairs. This study gives an example of how much the selection of different variable regions combined with different specificities of the flanking “universal” primers can affect a PCR-based microbial community analysis.     

鹳形目12种鸟类核c-mos 基因和线粒体12S rRNA基因序列分析及其系统发生研究

鹳形目鸟类的传统分类一直存在分歧,而近期的分子系统学研究大多只用单个基因,其结论的可信度需要进一步验证.本文通过核c-mos基因和线粒体12S rRNA基因序列分别和合并分析,采用分子系统学方法探讨了鹳形目6科12种鸟类的系统发生关系.文中测出鹳形目鸟类6种核c-mos基因的片断序列,结合来自Genebank的其他种类的c-mos和12S rRNA基因序列,分别经Clustal W软件对位排列后,以原鸡为外类群用最大似然法、邻接法和最大简约法建立系统树.系统树分析表明, 鹳形目6科之间的系统发生关系总结为：（鹭科,（（鹮科,美洲鹫科）,（鹳科,（鲸头鹳科,锤头鹳科））））.鹭科7个属之间的系统发生关系总结为：（麻（开鸟）属（夜鹭属（池鹭属（苍鹭属（中白鹭属（白鹭属,大白鹭属））））））.分别基于两个单基因的系统树有一定差异,而基于合并数据的系统树支持率和分辨率都高于基于单基因的系统树,表明使用在遗传上相对独立的分子数据合并建立系统树有较高的可信度和分辨率,是一种更好的研究方法.     

Quantitative Comparisons of 16S rRNA Gene Sequence Libraries from Environmental Samples

To determine the significance of differences between clonal libraries of environmental rRNA gene sequences, differences between homologous coverage curves, C_X(D), and heterologous coverage curves, C_XY(D), were calculated by a Cramér-von Mises-type statistic and compared by a Monte Carlo test procedure. This method successfully distinguished rRNA gene sequence libraries from soil and bioreactors and correctly failed to find differences between libraries of the same composition.     

Microvariation Artifacts Introduced by PCR and Cloning of Closely Related 16S rRNA Gene Sequences

A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a mutational hot spot, and cloning of heteroduplexes and chimeras. These data may partially explain the high degree of microheterogeneity typical of sequence clusters detected in environmental clone libraries.     

基于涉案兽类12S rRNA基因的NCBI数据库序列可靠性分析

本研究以常用于动物种属鉴定的12S rRNA基因位点为研究对象,利用所测得的17种常见涉案兽类12S rRNA基因部分片段序列及NCBI数据库中下载的该物种DNA序列及其近缘物种DNA序列,构建系统进化树。根据进化树的聚类情况,判断NCBI数据库中的相关基因序列或物种名称的正确性,并对其中错误序列的登陆号进行标记,以防对后续涉案动物的准确鉴定造成影响。分别从17种常见涉案兽类(共26份样本)中提取线粒体DNA,并利用通用引物扩增线粒体DNA上的12S rRNA基因部分片段并进行测序分析。通过NCBI数据库的Blast比对功能,筛选出与本研究物种同源性由高到低的物种,并从NCBI基因数据库中下载此类近缘物种的12S rRNA基因序列共351条,利用MEGA7.0软件构建该物种及其近缘物种系统进化树。通过比对发现NCBI中登录号为KP202279等3个序列所对应物种拉丁名错误。登录号为AY184436等11个序列所对应物种拉丁名可能存在疑问。GenBank中某些物种拉丁名有同种异名现象。因此,NCBI数据库数据可靠性有待进一步验证,只能作为涉案物种鉴定的参考数据之一,可借助构建系统进化树等方法来确认其结果的准确性。