Preliminary study on mitochondrial 16S rRNA gene sequences and phylogeny of flatfishes (Pleuronectiformes)

A 605 bp section of mitochondrial 16S rRNA gene from Paralichthys olivaceus, Pseudorhombus cinnamomeus, Psetta maxima and Kareius bicoloratus, which represent 3 families of Order Pleuronectiformes was amplified by PCR and sequenced to show the molecular systematics of Pleuronectiformes for comparison with related gene sequences of other 6 flatfish downloaded from GenBank. Phylogenetic analysis based on ge- netic distance from related gene sequences of 10 flatfish showed that this method was ideal to explore the rela- tionship between species, genera and families. Phylogenetic trees set-up is based on neighbor-joining, maximum parsimony and maximum likelihood methods that accords to the general rule of Pleuronectiformes evolution. But they also resulted in some confusion. Unlike data from morphological characters, P. olivaceus clustered with K. bicoloratus, but P. cinnamomeus did not cluster with P. olivaceus, which is worth further studying.

16S rRNA Gene-Based Analysis of Ileal Bacterial Community and Phylogeny in Nursing and Weaned Piglets

Weaning of piglets is generally considered as a stressor which changes intestinal ecosystem and leads to clinical implications. Microbiota inhabiting in small intestine （especially ileum） are assumed to promote health, but their functional properties are yet poody dascdbed. As indicated by the 16S rRNA gene sequences of ileal micrebiota in nursing piglets （at the age of 21 and 28 d） and 28-day-old weaned piglets （weaned at 21 d of age）, the microbiota were mainly comprised of gram-positive bacteria. There were 40 operational taxonomic units （OTUs） （from 171 clones） in the ileum of nursing piglets aged 21 d, 61 OTUs （from 194 clones） in the ileum of nursing piglets aged 28 d, and 56 OTUs （from 171 clones） in the ileum of weaned piglets aged 28 d. The flea of nursing piglets aged 21 d were dominantly occupied by Lactobacilli （87.7%） as well as Streptococ cus （ 3.5 % ）. Lactobacillus amy/ovorus （41.5 % ）, Lactobaci/lus sp. （ 19.3 % ）, Lactobaci/lus reuteri （ 12.3 % ）, Lactobacillus salivarius （ 9.4 % ） and L. mucosae （4.7%） were the predominant species among Lactobacil/L Similar results were obtained in the nursing piglets at 28 d of age ex- cept that Lactobaci/li decreased to 71.1% and Streptococcus increased to 21.1% significantly. Lactobacillus （52.0%） and Streptococcus （26.3%） were the two major groups in the ileum of weaned piglets aged 28 d. Lactobacillus amylovorus （31.6%） and Lactobaci/lus reuteri （ 16.4% ） was the two most important species in Lactobacillus. Therefore, Lactobacilli were predominant in the ileum of nursing and weaned piglets, and they had the highest diversity, followed by Streptococcus. The diversity of ileal microbiota was not different remarkably between the nursing piglets and the weaned piglets, but the composition changed significantly. These findings are helpful to understand ileal bacterial ecophysiology and further develop nutritional regimes to prevent or counteract complications during the weaning transition.

Phylogeny of the cuttlefishes (Mollusca: Cephalopoda) based on mitochondrial COI and 16S rRNA gene sequence data

To clarify cuttlefish phylogeny, mitochondrial cytochrome c oxidase subunit I (COI) gene and partial 16S rRNA geneare sequenced for 13 cephalopod species. Phylogenetic trees are constructed, with the neighbor-joining method.Coleoids are divided into two main lineages, Decabrachia and Octobrachia. The monophyly of the order Sepioidea,which includes the families Sepiidae, Sepiolidae and Idiosepiidae , is not supported. From the two families ofSepioidea examined, the Sepiolidae are polyphyletic and are excluded from the order. On the basis of 16S rRNA andamino acid of COI gene sequences data, the two genera (Sepiella and Sepia) from the Sepiidae can be distinguished, butdo not have a visible boundary using COI gene sequences. The reason is explained. This suggests that the 16S rDNAof cephalopods is a precious tool to analyze taxonomic relationships at the genus level, and COI gene is fitter at ahigher taxonomic level (i.e., family).

Phylogeny of γ-proteobacteria inferred from comparisons of 3’ end 16S rRNA gene and 5’ end 16S-23S ITS nucleotide sequences

The phylogeny of γ-proteobacteria was inferred from nucleotide sequence comparisons of a short 232 nucleotide sequence marker. A total of 64 γ-proteobacterial strains from 13 Orders, 22 families, 40 genera and 59 species were analyzed. The short 232 nucleotide sequence marker used here was a combination of a 157 nucleotide sequence at the 3’ end of the 16S rRNA gene and a 75 nucleotide sequence at the 5’ end of the 16S-23S Internal Transcribed Spacer (ITS) sequence. Comparative analyses of the 3’ end of the 16S rRNA gene nucleotide sequence showed that the last 157 bp were conserved among strains from same species and less conserved in more distantly related species. This 157 bp sequence was selected as the first part in the construction of our nucleotide sequence marker. A bootstrapped neighbor-joining tree based on the alignment of this 157 bp was constructed. This 157 bp could distinguish γ-proteobacterial species from different genera from same family. Closely related species could not be distinguished. Next, an alignment of the 16S-23S ITS nucleotide sequences of alleles from same bacterial strain was performed. The first 75 bp at the 5’ end of the 16S-23S ITS was highly conserved at the intra-strain level. It was selected as the second part in the construction of our nucleotide sequence marker. Finally, a bootstrapped neighbor-joining tree based on the alignment of this 232 bp sequence was constructed. Based on the topology of the neighbour-joining tree, four major Groups, Group I to IV, were revealed with several sub-groups and clusters. Our results, based on the 232 bp sequence were, in general, in agreement with the phylogeny of γ-proteobacteria based on the 16S rRNA gene. The use of this 232 bp sequence as a phylogenetic marker presents several advantages over the use of the entire 16S rRNA gene or the generation of extensive phenotypic and genotypic data in phylogenetic analyses. First, this marker is not allele-dependant. Second, this 232 bp marker contains 157 bp from the 3’ end of the 16S rRNA gene and 75 bp from the 5’ end of the 16S-23S ITS. The 157 bp allows discrimination among distantly related species. Owing to its higher rate of nucleotide substitutions, the 75 bp adds discriminating power among closely related species from same genus and closely related genera from same family. Because of its higher percentage of nucleotide sequence divergence than the 16S rRNA gene, the 232 bp marker can better discriminate among closely related γ-proteobacterial species. Third, the method is simple, rapid, suited to large screening programs and easily accessible to most laboratories. Fourth, this marker can also reveal γ-proteobacterial species which may appear misassigned and for which additional characterization appear warranted.

Sequence Comparison of Mitochondrial 16S rRNA Gene between Two Sesarma Species

Partial sequences of the mitochondrial 16S rRNA gene were determined for two Sesarma species （S. haematocheir and S. pficata）, and the length of sequence of S. haematocheir and S. plicata was the same. Of the 533 nucleotides obtained, the A, T, G and C contents were similar, which were 198 bp （37.1%）, 206 bp （38.6%）, 84 bp （15.8%）, 45 bp （8.4%） and 200 bp （37.5%）, 205 bp （38.5%）, 81 bp （15.2%）, 47 bp （8.8%） respectively. There were 49 different sites between the two species, including 21 transition sites, 22 transversion sites and 6 deletion/insertion sites. Furthermore, 361 bp homologous fragment was analyzed to discuss the phylogenetic relationship of 20 Sesarma species. The results showed that AT content （78.6% - 82.9%） was higher than GC content and there were 91 vadable positions of 16S rRNA sequences among the 20 species. Among 3 Sesarma species in China, S. dehaani and S. haematocheir were closely related （d=0.0151）; and there were greater genetic distances between S. pficata and S. dehaani I S. haematocheir （d=0.0924/0.0923）. The average genetic distances of S. plicata , S. dehaani and S. haematocheir with the North American crabs of genus Sesarma were respectively 0.103 2 ± 0.008 9, 0.0783±0.0105 and 0.072 0± 0.011, which indicated that there were greater genetic differences between China and North American Sesarma crabs and they were monophyletic each other.

Molecular Phylogeny of Arctic Microbes Using Metagenomic Approach

Metagenomic approach to the characterization of uncultured and mixed bacterial consortium has been used in this study for a few arctic micro-organisms. Arctic soil, collected from one spot at Ny Alesund in Svalbard was used as the source of such organisms and enriched in culture medium. Only psychrotrophic microbes were chosen which could grow from 5&degC to 20&degC. Total genomic DNA isolated from the consortium was used as template for amplifying the 16S rRNA genes using the conserved forward and reverse primers. The amplified mixture of 1.4 kb DNA was cloned in pGEMT Easy vector and individual 16S rDNA sequences were determined by automated sequencing. A phylogenetic tree was constructed using MEGA 5 software which enlists 19 isolates in the consortium. Most of these arctic bacteria were Bacillus species as these were isolated from a single spot.

相手蟹属两种蟹类线粒体16S rRNA基因序列的比较

测定了相手蟹属红螯相手蟹和褶痕相手蟹线粒体16S rRNA基因部分片段的序列，二者的序列长度相同，均为533bp，且A、T、D、C的含量相似，分别为198bp（37．1％），206bp（38．6％），84bp（15．8％），45bp（8．4％）和200bp（37．5％），205bp（38．5％），81bp（15．2％），47bp（8．8％）；二者的序列有49处差异，其中21个位点为转换、22个位点为颠换和6个缺失／插入位点。进一步对20种相手蟹属蟹类的长度为361bp的16S rRNA基因同源序列进行分析，发现AT的含量为78．6％～82．9％，明显高于GC的含量，且存有91个变异位点。从NJ树和遗传距离来看，在分布于中国的3种相手蟹中，无齿相手蟹和红螯相手蟹的亲缘关系最近（d=0．0151），而它们与褶痕相手蟹的亲缘关系则较远（d=0．0924／0．0923）。分布于中国的相手蟹和分布于北美的相手蟹之间存在着较大的遗传距离（差异），表明它们之间有着较远的亲缘关系，互为单系起源。

两种大眼蟹线粒体16S rRNA基因序列分析

测定了宽身大眼蟹和日本大眼蟹线粒体16SrRNA基因部分片段的序列,其长度均为517bp。二者的核苷酸序列A,T,G,C的含量相似:宽身大眼蟹分别为33.3%,35.6%,19.9%,11.2%;日本大眼蟹分别为35.2%,35.4%,18.4%,11.0%。不包括6处插入/缺失位点,两序列间有63个变异位点,核苷酸差异率为12.26%,其中转换32个、颠换31个,转换与颠换比约为1.0。对国内外大眼蟹的24条长度为415bp的16SrRNA基因同源序列进行分析,A+T的平均含量为71.7%,明显高于G+C的平均含量,且存有变异位点171个,简约信息位点137个。中国和日本的日本大眼蟹之间的核苷酸差异率为4.42%,表明二者已有明显的遗传分化;中国的日本大眼蟹与日本的万岁大眼蟹之间的核苷酸差异率仅为0.21%,表明二者有可能为同一物种。上述结果得到了系统发生树拓扑结构的支持。

Phylogenetic relationship of 16 Oedipodidae species （Insecta： Orthoptera） based on the 16S rRNA gene sequences

The sequences of the mitochondrial 16S rRNA gene of 16 Oedipodidae species were amplified and sequenced. All sequences were aligned and analyzed and the phylogenetic relationships were inferred. The properties of 16S gene in Oedipodidae showed typical patterns of many insects such as a high A＋T content and variable distance-dependent transition/transversion ratios. The 0.2 weight for sites of loops may be advisable for phylogeny reconstruction using the maximum parsimony method. The phylogenetic analysis results do not support the current subfamily classification systems of Oedipodidae. Bryodemellinae and Bryodeminae are closely related and should be merged as one subfamily. The status of Oedipodinae and Locustinae is also problematic.

Polyphyly in 16S rRNA-based LVTree Versus Monophyly in Whole-genome-based CVTree

We report an important but long-overlooked manifestation of low-resolution power of 16S rRNA sequence analysis at the species level, namely, in 16S rRNA-based phylogenetic trees polyphyletic placements of closely-related species are abundant compared to those in genomebased phylogeny. This phenomenon makes the demarcation of genera within many families ambiguous in the 16S rRNA-based taxonomy. In this study, we reconstructed phylogenetic relationship for more than ten thousand prokaryote genomes using the CVTree method, which is based on wholegenome information. And many such genera, which are polyphyletic in 16S rRNA-based trees, are well resolved as monophyletic clusters by CVTree. We believe that with genome sequencing of prokaryotes becoming a commonplace, genome-based phylogeny is doomed to play a definitive role in the construction of a natural and objective taxonomy.

Phylogenetic Relationships among 12 Species of Tetrigidae (Orthoptera:Tetrigoidea) Based on Partial Sequences of 12S and 16S Ribosomal RNA

Mitochondrial 12S and 16S ribosomal RNA genes sequences were sequenced using dye-labeled terminator on an ABI 377 automated sequencer in 11 individuals and 1 species' sequences were gained from GenBank,representing 6 genera of family Tetrigidae.The collated sequences were aligned using Clustal X version 1.81 and then,the sequence variability and heredity distances based on Kimura 2-parameter model were calculated using Mega 2.1.In obtained sequences (736 bp),the average A+T content is 73.9%,ranging from 71.2% to 77.5%;the overall G+C content is 26.1%,ranging from 22.5% to 28.8%.Based on alignment of the combined sequences,185 parsimony-informative sites were revealed in 755 available base pairs.Phylogenetic trees were reconstructed using NJ,MP and ML methods with Cylindraustralia kochii as outgroup.The results indicated that the monophyletic nature of Tetrix is questioned and suggest that T.tubercarina may be given tribal rank.Our results also show that Coptltettix huanjiangensis and C.gongshanensis are the same species,i.e.Coptltettix gongshanensis Zheng,and C.huanjiangensis is the synonyms of C.gongshanensis.

Prokaryote phylogeny meets taxonomy: An exhaustive comparison of composition vector trees with systematic bacteriology

We perform an exhaustive, taxon by taxon, comparison of the branchings in the composition vector trees (CVTrees) inferred from 432 prokaryotic genomes available on 31 December 2006, with the bacte-riologists' taxonomy-primarily the latest online Outline of the Bergey's Manual of Systematic Bacteri-ology. The CVTree phylogeny agrees very well with the Bergey's taxonomy in majority of fine branchings and overall structures. At the same time most of the differences between the trees and the Manual have been known to biologists to some extent and may hint at taxonomic revisions. Instead of demonstrating the overwhelming agreement this paper puts emphasis on the biological implications of the differences.

Assessment of a short phylogenetic marker based on comparisons of 3'end 16S rDNA and 5'end 16S-23S ITS nucleotide sequences on the genus Xanthomonas

A short phylogenetic marker previously used in the reconstruction of the Class γ-proteobacteria was assessed here at a lower taxa level, species in the genus Xanthomonas. This maker is 224 nucleotides in length. It is a combination of a 157 nucleotide sequence at the 3' end of the 16S rRNA gene and a 67 nucleotide sequence at the 5' end of the 16S-23S ITS sequence. A total of 23 Xanthomonas species were analyzed. Species from the phylogenetically related genera Xylella and Stenotrophomonas were included for com- parison purposes. A bootstrapped neighbor- joining phylogenetic tree was inferred from comparative analyses of the 224 bp nucleotide sequence of all 30 bacterial strains under study. Four major Groups were revealed based on the topology of the neighbor-joining tree, Group I to IV. Group I and II contained the genera Steno-trophomonas and Xylella, respectively. Group III included five Xanthomonas species: X. theicola, X. sacchari, X. albineans, X. transluscens and X. hyacinthi. This group of Xanthomonas species is often referred to as the hyacinthi group. Group IV contained the other 18 Xanthomonas species. The overall topology of the neighbor-joining tree was in agreement with currently accepted phylogenetic. The short phylogenetic marker used here could resolve species from three dif-ferent Xanthomonadacea genera: Stenotro-phomonas, Xylella and Xanthomonas. At the level of the Xanthomonas genus, distant spe-cies could be distinguished, and whereas some closely-related species could be distinguished, others were undistinguishable. Pathovars could not be distinguished. We have met the resolving limit of this marker: pathovars and very closely related species from same genus.

嗜盐古细菌的系统发育分析

用“Clustalw”和“PHYLIP”程序包分析嗜盐古细菌16S rRNA序列,建立了嗜盐古细菌的系统发育树。比较分析的结果进一步支持了以前的结论,即嗜盐古细菌在自然系统分类上应被分成嗜盐菌科的6个属。此系统发育分析方法不仅体现了在嗜盐古细菌属一级分类上的优势,而且还可能被用作一种相应于以《伯杰氏细菌系统分类手册》(第三卷)为基础的嗜盐古细菌种一级分类上的代换方法。实际上,这种系统发育分析方法比其他建立在表型特性基础上的分类系统更真实地反映了嗜盐古细菌内的亲缘关系。该方法的其他运算细节在本文中也进行了讨论。

斑鳠烂鳃病病原菌的研究

从池塘患烂鳃病的斑鳠(Mystus guttatus)亲鱼病灶中分离出一株毒力较强的致病菌-Mg2,经形态学观察、生理生化特性和16SrRNA基因序列分析鉴定,其主要特征为:菌体细长,无鞭毛和荚膜,革兰氏阴性,大小0.5×(6.5—11)μm,菌落黄色,边缘不整齐呈假树根状。氧化酶、过氧化氢酶阳性,分解酪素和明胶,硝酸盐还原阳性;不分解纤维素、几丁质、酪氨酸、七叶灵和淀粉,吲哚和葡萄糖产气阴性。对恩诺沙星、诺氟沙星、萘啶酸、红霉素、洁霉素敏感,菌株的16SrRNA基因序列分析结果表明:Mg2菌株与柱状黄杆菌聚类,基因序列的同源性在97.5%以上,综合生理生化、分子生物学两方面的分类鉴定结果,Mg2菌株应归类鉴定为柱状黄杆菌(FlavobacteriumColumnare)。

Biogeographical note on Antarctic microforae: Endemism and cosmopolitanism

This study deals with the biogeography of Antarctic microflora （Antarctica acts as best model to study microbial biogeography） such as cyanobacteria and selected halophiles with special emphasis on Halomonas variabilis and Bacillus licheniformis.Halophiles are known to be resistant not only to salt stress,but also to extreme temperature,pressure,and aridity and they are capable of surviving in harsh environments such as polar regions,deep-sea habitats,and deserts.Many microbes are known to be resistant to hostile environmental conditions,and are capable of surviving in harsh environments.Our group has isolated 444 strains belonging to 28 genera of halophiles from various environments around the world.The 16S rRNA gene sequences revealed that many of the isolated strains from geographically distant habitats having different environmental conditions,were closely related to each other,with some strains possessing 100％ identical sequences.Organisms possessing survival mechanism such as spore formation are usually ubiquitous.The genus Halomonas is represented by potentially endemic strains and the ubiquitous H.variabilis,while spore-forming B.licheniformis showed cosmopolitan distribution.One potentially endemic （moderate endemicity that is regional and/or continental distribution） strain was reported from Syowa station,East Antarctica,and Mario Zucchelli station,West Antarctica,which are geographically separated by 3000 km.Moreover,15 strains having 100％ similarity with B.licheniformis were considered cosmopolitans.The results of this work provide support for the middle-ground model that some microbes have moderate endemicity and others have cosmopolitan distribution.These results will contribute to a greater understanding of microbial biogeography with special emphasis on Antarctica.

4科热带作物系统发育分析

以4科（大戟科、豆科、芭蕉科、番木瓜科）的29种热带作物为研究对象,采用叶绿体16～23 S基因间隔序列,对这29种植物的系统发育进行了分析.结果表明,这29种植物的16～23 S基因间隔区序列同源性很高,保守性较强;构建系统发育树表明,这29种植物进化完全符合恩格勒（1897）分类系统,序列变异程度较低;进一步分析了11个木薯品种间的差异,木薯种内差异很小,只有SC8与其他几个品种稍有差异,但不显著.结果表明,叶绿体16～23 S基因间隔序列虽可区别这4科29种植物,但该序列较长,序列变异程度较低,并不能作为候选DNA条形码之一.

Isolation and identification of bacteria associated with the surfaces of several algal species

We conducted this study to assess the diversity of bacteria associated with the surfaces of algae based on 16S rDNA sequence analyses.Twelve strains of bacteria were obtained from the surfaces of the following four species of algae:Gracilaria textorii,Ulva pertusa,Laminaria japonica,and Polysiphonia urceolata.The isolated strains of bacteria can be divided into two groups:Halomonas and Vibrio,in physiology,biochemical characteristics and 16S rDNA sequence analyses.The phylogenetic tree constructed based on 16S rDNA sequences of the isolates shows four obvious clusters,Halomonas venusta,Vibrio tasmaniensis,Vibrio lentus,and Vibrio splendidus.Isolates from the surface of P.urceolata are more abundant and diverse,of which strains P9 and P28 have a 16S rDNA sequence very similar(97.5%-99.8%) to that of V.splendidus.On the contrary,the isolates from the surfaces of G.textorii,U.pertusa and L.japonica are quite simple and distribute on different branches of the phylogenetic tree.In overall,the results of this study indicate that the genetic relationships among the isolates are quite close and display a certain level of host species specificity,and alga-associated bacteria species are algal species specific.

Isolation and diversity analysis of heterotrophic bacteria associated with sea anemones

A study was undertaken to investigate the heterotrophic bacterial flora associated with the sea anemones. Samples of the sea anemones Anthopleura midori were collected from the coast of Weihai and bacteria were isolated from these samples. Additionally, high numbers of viable bacteria were obtained from the celom wall and surface of anemone, the community of cultivable bacteria was very diverse. As a result of this isolation, 60 strains were obtained, 56 of them were selected for identification and characterization by 16S rRNA gene sequence analysis and limited phenotypic testing. Among these isolates, 16 strains were phylogenetically related to members of the genus Pseudoalteromonas and neighboring taxa. Other isolates included members of the genera Colwcllia, Vibrio, Acinetobactcr, Pseudomonas, Endozoicomonas, Roseovarius, Paraeoceus, Loktanella, Leisingcra, Sulfitobacter, Bacillus, Staphylococcus, Plantibacter, Microbaeterium, Micro- coccus, Joostella, Psychroserpens, Cellulophaga, Krokinobaeter, Polaribacter and Psychrobaeter. Seven potential novel species were found. Among 60 strains, 17 of them can produce proteolytic exoenzyme, 20 can produce lipolytic exoenzyme. Strain NQ8 has strong antagonistic effects on some Vibrio strains. This study demonstrates that the culturable fraction of bacteria from the sea anemones Anthopleura midori is diverse and appears to possess much potential as a source for the discovery of novel bioactive materials.

The Archaea Community Associated with Lava-Formed Gotjawal Forest Soil in Jeju, Korea

The abundance and diversity of archaeal assemblages were analyzed in soils collected from Gyorae Gotjawal forest, Jeju, Korea. Gotjawal soil refers to soil derived from a lava-formed forest, characterized by high organic matter content, fertility, and poor rocky soil. Using domain-specific primers, archaeal 16S rRNA gene sequences were PCR amplified for clone library construction, and a total of 185 archaeal clones were examined. The archaeal clones were affiliated with the phyla Thaumarchaeota (96.2%) and Euryarchaeota (3.8%). The most abundant thaumarchaeal group (90.3% of the clones) was the group I.1b clade, which includes soil ammonia-oxidizing archaea. The unique characteristics of Gotjawal soil, including basalt morphology, vegetation, and groundwater aquifer penetration, may be reflected in the archaeal community composition. Further study is necessary to understand the unique factors of Gotjawal soils that influence archaeal abundance, composition, and diversity.