Phylogenetic analyses of some genera in Oedipodidae （Orthoptera： Acridoidea） based on 16S mitochondrial partial gene sequences

Basedonthe 16S mitochondrial partial gene sequences of 29 genera, containing 26 from Oedipodidae and one each from Tanaoceridae, Pyrgomorphidae and Tetrigidae （as outgroups）, the homologus sequences were compared and phylogenetic analyses were performed. A phylogenetic tree was inferred by neighbor-joining （N J）. The results of sequences compared show that： （i） in a total of 574 bp of Oedipodidae, the number of substituted nucleotides was 265 bp and the average percentages ofT, C, A and G were 38.3%, 11.4%, 31.8% and 18.5%, respectively, and the content of A＋T （70.1%） was distinctly richer than that of C＋G （29.9%）; and （ii） the average nucleotide divergence of 16S rDNA sequences among genera of Oedipodidae were 9.0%, among families of Acridoidea were 17.0%, and between superfamilies （Tetrigoidea and Acridoidea） were 23.9%, respectively. The phylogenetic tree indicated： （i） the Oedipodidae was a monophyletic group, which suggested that the taxonomic status of this family was confirmed; （ii） the genus Heteropternis separated from the other Oedipodids first and had another unique sound-producing structure in morphology, which is the type-genus of subfamily Heteropterninae; and （iii） the relative intergeneric relationship within the same continent was closer than that of different continents, and between the Eurasian genera and the African genera, was closer than that between Eurasians and Americans.     

Molecular systematics analysis of Lymantria dispar based on 18S rRNA and cox1 mtDNA sequence data

The complete sequence of the 18 S ribosomal RNA gene(18S rRNA) from Lymantria dispar was cloned and analysed here. 18 S rRNA and mitochondrial cytochrome c oxidasel(cox1) gene sequences of Lymantria dispar were compared with homologous sequences of other nine insects from different orders. Analytic results showed that 18 S rRNA of these insects had two conserved domains and the second domain was an even more conserved region. The phylogenetic trees based on the full-length sequence and the second domain fragment of 18 S rRNA as well as sequence of cox1 from different orders indicated that Lepidoptera and Trichoptera, which belongs to Amphiesmenoptera, had a closer phylogenetic relationship and fewer differences were observed comparing with traditional taxonomic results.     

小鲵科线粒体16S rRNA基因序列分析及其系统发育

To study the phylogeny of Hynobiidae, we amplified DNA fragments of 470 bp 16S ribosomal RNA (16S rRNA) gene on mitochondrial DNA from Ranodon sibiricus and Ranodon tsinpaensis. PCR products were cloned into PMD18 T vector after purification. These sequences were determined and deposited in the GenBank (accession numbers: AY373459 for Ranodon sibiricus, AY372534 for Ranodon tsinpaensis). By comparing the nucleotide differences of 16S ribosomal RNA sequences among Liua shihi, Pseudohynobius flavomaculatus and Batrachuperus genus from GenBank database, we analyzed the divergences and base substitution among these sequences with the MEGA software. The molecular results support that B. tibetanus, B. pinchonii and B. karlschmidti are classified into three valid species. Liua shihi has closer phylogenetic relationships to Ranodon tsinpaensis than to other species. More our results reveal that Pseudohynobius flavomaculatus is not a synonym of Ranodon tsinpaensis. [Acta Zoologica Sinica 50 (3) : 464 - 469,2004].     

Molecular phylogenetic relationships of China Seas groupers based on cytochrome b gene fragment sequences

The classification and evolutionary relationships are important issues in the study of the groupers. Cytochrome b gene fragment of twenty-eight grouper species within six genera of subfamily Epinephelinae was amplified using PCR techniques and the sequences were analyzed to derive the phylogenetic relationships of the groupers from the China Seas. Genetic information indexes, including Kimura-2 parameter genetic distance and Ts/Tv ratios, were generated by using a variety of biology softwares. With Niphon spinosus, Pagrus major and Pagrus auriga as the designated outgroups, phylogenetic trees, which invoke additional homologous sequences of other Epinephelus fishes from GenBank, were constructed based on the neighbor-joining (NJ), maximum-parsimony (MP), maximum-likelihood (ML) and minimum-evolution (ME) methods. Several conclusions were drawn from the DNA sequences analysis: (1) genus Plectropomus, which was early diverged, is the most primitive group in the subfamily Epinephelinae; (2) genus Variola is more closely related to genus Cephalopolis than the other four genera; (3) genus Cephalopolis is a monophyletic group and more primitive than genus Epinephelus; (4) Promicrops lanceolatus and Cromileptes altivelis should be included in genus Epinephelus; (5) there exist two sister groups in genus Epinephelus.     

Molecular differentiation of the microgastrine species commonly found in paddy fields from Southeast Asia,with additional data on their phylogeny （Hymenoptera：Braconidae）

Partial DNA sequences of three genes, that is, mitochondrial large ribosomal subunit (16S), nuclear large ribosomal subunit (28S D2) and mitochondrial NADH1 dehydrogenase (NADH1) gene, were sequenced from different microgas trine species(Braconidae: Microgastrinae) collected fresh from paddy fields. The DNA sequences were used to determine the extent of sequence variation among species in order to evaluate the specific status of each species. Cladistic analysis was also used to infer a phylogenetic relationship among these species. The results showed that sequence divergence among species of the same genus Cotesia was much lower than those among different genera, such as Cotesia, Exoryza and Apanteles; the sequence similarity of 16S rDNA and NADH 1 genes between Cotesia sp. and C. chilonis was higher than that between C. sp. and C. ruficrus.Phylogenetic analyses suggested that four species of Cotesia were always grouped in the same clade regardless of using different analysis methods; Cotesia sp. and C. chilonis are more closely related to each other than to C. ruficrus, different from previous morphological results. Additionally, sequence analyses indicated that NADH1 gene has more parsimony informative characters than 28S rDNA D2 and 16S rDNA at the species-level analysis,indicating that NADH1 gene might be a useful marker for species-level analysis.     

Computational identification and evolutional analysis of Piwi subfamily in 11 Drosophila species

The complete genome sequences of 11 Drosophila species provide an opportunity to investigate the gene family evolution in closely related species. Drosophila Piwi subfamily, including three members, piwi, Aub and Ago3, has attracted increasing attention as it participates in the biogenesis of piRNA. Here, we identified 33 Piwi homologs from the genome of 11 Drosophila species. The full-length cDNA sequences ofpiwi and Aub genes were obtained by using New GENSCAN Web Server. The Ago3 homologs were difficult regarding full-length information because they had long introns. The genomic structure of Piwi subfamily genes are highly conserved among diverse Drosophila species. Insect piwi and Aub genes have long first introns. The average length of the first intron is 1 284 bp for piwi and 840 bp for Aub, which is much larger than those of other introns （93 bp for Piwi and 54 bp for Aub）. However, this phenomenon is not observed in mammalian piwi genes. We also found that there were abundant repeat sequences in both exons and introns of insect Ago3 genes. Due to recent insertions of long terminal repeat elements in four Drosophila species, part of the third introns exhibit higher conservation than adjacent exons and other introns. An evolutional tree created by Minimum Evolution method indicates that mammalian piwi genes are more closely related to the insect Ago3 Piwi subfamily.     

Analysis of ESTs and gene expression patterns of the poste-rior silkgland in the fifth instar larvae of silkworm, Bom-byx mori L.

The fibroin gene expression pattern and regulation of the posterior silkgland were studied by means of expressed sequence tags (ESTs) using the first and fifth day larvae of the fifth instar of silkworm, Bombyx mori L (strain: C 108). The results showed that there were 911 repetitive ESTs and 1950 single sequences (Singlets) among total 2861 consentient sequences, which were spliced. 1335 sequences were identified and the other 1526 were unknown. 5560 sequences (55.89%) in the posterior silkgland cell of the silkworm were new ESTs without ho-mology with EST data published by Mita et al. The number of repetitive ESTs and single sequences from the first day larvae of the fifth instar was double more than that of the fifth day of the same instar in the silkworms. The unigenes which were more than 50 in repetitive EST size (contig size) came to only about 0.5% in total consentient sequences. There were significant differences between gene expression frequencies, and expressed genes were related to fibroin synthesis and its secretion and fibroin composition. Comparing the fifth day with the first day of the fifth instar, the genes-expressed quantity of fibroin heavy-chain gene was 18 fold higher, fibroin light-chain gene 9 fold and fibroin P52 gene 8 fold. 508 genes functioned for cellular component and 315 for enzyme after function tracing. These results implied that the gene expression of the first day was mainly for preparation for fibroin synthesis except for the growth of silkgland cells, and the gene expression of the fifth day of the fifth instar was mainly for synthesizing and excreting fibroin. Because the ratio of heavy chain, light chain and p25 of fibroin was not 6:6:1 as theoretically expected, or its special H-chain structure, the H-chain gene was not easy to detect through EST technique. Most of genes among total 2861 consentient sequences functioned for fibroin synthesis and secretion. This suggested the fibroin synthesis and secretion procedure of the posterior silkgland was more complex than the knowledge we have.     

Building a sequence map of the pig pan-genome from multiple de novo assemblies and Hi-C data

Pigs were domesticated independently in the Near East and China, indicating that a single reference genome from one individual is unable to represent the full spectrum of divergent sequences in pigs worldwide. Therefore, 12 de novo pig assemblies from Eurasia were compared in this study to identify the missing sequences from the reference genome. As a result, 72.5 Mb of nonredundant sequences(～3% of the genome) were found to be absent from the reference genome(Sscrofa11.1) and were defined as pan-sequences. Of the pan-sequences, 9.0 Mb were dominant in Chinese pigs, in contrast with their low frequency in European pigs. One sequence dominant in Chinese pigs contained the complete genic region of the tazarotene-induced gene 3(TIG3) gene which is involved in fatty acid metabolism. Using flanking sequences and Hi-C based methods, 27.7% of the sequences could be anchored to the reference genome. The supplementation of these sequences could contribute to the accurate interpretation of the 3D chromatin structure. A web-based pan-genome database was further provided to serve as a primary resource for exploration of genetic diversity and promote pig breeding and biomedical research.     

A new genus and three new species of miniaturized microhylid frogs from Indochina(Amphibia: Anura:Microhylidae: Asterophryinae)

We report on the discovery of a new genus of microhylid subfamily Asterophryinae from northern and eastern Indochina, containing three new species.Vietnamophryne Gen. nov. are secretive miniaturized frogs(SVL21 mm) with a mostly semi-fossorial lifestyle. To assess phylogenetic relationships, we studied 12 S rRNA – 16 S rRNA mt DNA fragments with a final alignment of 2 591 bp for 53 microhylid species. Morphological and osteological characters were analyzed using micro-CT scanning and used to describe the new genus. Results of phylogenetic analyses assigned the new genus into the mainly Australasian subfamily Asterophryinae as a sister taxon to the genus Siamophryne from southern Indochina. The three specimens collected from Gia Lai Province in central Vietnam, Cao Bang Province in northern Vietnam, and Chiang Rai Province in northern Thailand proved to be separate species, different both in morphology and genetics(genetic divergence 3.1%≤P≤5.1%). Our work provides further evidence for the "out of Indo-Eurasia"scenario for Asterophryinae, indicating that the initial cladogenesis and differentiation of this group of frogs occurred in the Indochina Peninsula. To date, each of the three new species of Vietnamophryne Gen. nov. is known only from a single specimen; thus,their distribution, life history, and conservation status require further study.     

Characterization of the Agamid Lizard Genus Uromastyx from Eastern Sudan Based on Morphology,Karyotypes and Mitochondrial DNA

This study focuses on taxonomy of Uromastyx species. Morphologically, U. ocellata and U. ornata were identified and cytogenetically, 2n = 36 chromosome. 16S, cyt b and 12S genes fragmentamplification shows 597, 399 and 450 base pair respectively and more A-T than G-C base pair andmore A/C than T/G base contents. While, sequences blasting in GenBank, followed by construction of phylogenetic tree revealed, 16S and cry b sequences were similar to U. ocellata sequences, andclustered together in phylogenetic tree, in contrast, 12S sequences from same species were related tosubspecies U. ornata ornata sequences and clustered together in the phylogenetic tree. Therefore, the results of this study suggest more studies on Uromastyx species in Sudan.     

基于线粒体ND2基因的中国斑翅蝗科部分种类分子系统学研究（直翅目：蝗总科）

本文的目的是通过对斑翅蝗科部分种类的线粒体ND2基因进行分析,重建斑翅蝗科昆虫的系统发育关系,并探讨分子系统发育关系和传统分类结果的异同。扩增并测定了我国斑翅蝗科10属16种蝗虫的线粒体ND2全基因1 023 bp的序列,对序列的碱基组成、转换颠换、系统发育信号等进行了分析。并基于ND2全基因序列数据,分别采用邻接法（NJ）、最简约法（MP）、最大似然法（ML）和贝叶斯法重建了10属16种蝗虫的系统发育关系。结果表明：斑翅蝗科蝗虫ND2全基因A+T含量平均为74.6%;痂蝗亚科和异痂蝗亚科没能得到区分,建议合并为一个亚科;而斑翅蝗亚科和飞蝗亚科的分类地位还存在争议。     

利用线粒体16S rRNA基因全序列分析直翅目主要类群的系统发生关系

为了构建稳健的直翅目主要类群间的系统发生关系并探讨16S rRNA基因序列在构建直翅目昆虫不同分类阶元系统发生关系时的可行性、功效以及性能,文章测定了直翅目4总科9科18种昆虫的16S rRNA基因全序列,联合已知该基因全序列的其他40种昆虫,构建了直翅目主要类群之间的系统发生关系,并分析了16SrRNA基因全序列的系统发生性能和功效。结果表明,直翅目昆虫的16S rRNA基因全长平均为1 310 bp;除生活方式特化的蚤蝼总科和蝼蛄总科的地位无法确定外,直翅目其他主要类群系统发生关系比较稳定;蝗总科下除了斑翅蝗科和槌角蝗科外,剑角蝗科、斑腿蝗科、网翅蝗科都不是单系群,且用不同的方法构建的系统发生树中聚类情况完全一致,各科间遗传距离差异不大,建议将其合为一科;锥头蝗科、瘤锥蝗科和癞蝗科间的遗传距离差异也不大;在构建系统发生树时,16S rRNA基因环区的信息量要比茎区的大;16S rRNA基因可以构建可靠的直翅目属与种水平和目与亚目高级阶元的系统发生关系,但对科和总科阶元缺乏足够的分辨力。     

Variation patterns of the mitochondrial 16S rRNA gene with secondary structure constraints and their application to phylogeny of cyprinine fishes (Teleostei: Cypriniformes)

The mitochondrial 16S ribosomal RNA (rRNA) gene sequences from 93 cyprinid fishes were examined to reconstruct the phylogenetic relationships within the diverse and economically important subfamily Cyprininae. Within the subfamily a biased nucleotide composition (A>T, C>G) was observed in the loop regions of the gene, and in stem regions apparent selective pressures of base pairing showed a bias in favor of G over C and T over A. The bias may be associated with transition-transversion bias. Rates of nucleotide substitution were lower in stems than in loops. Analysis of compensatory substitutions across these taxa demonstrates 68% covariation in the gene and a logical weighting factor to account for dependence in mutations for phylogenetic inference should be 0.66. Comparisons of varied stem-loop weighting schemes indicate that the down-weightings for stem regions could improve the phylogenetic analysis and the degree of non-independence of stem substitutions was not as important as expected. Bayesian inference under four models of nucleotide substitution indicated that likelihood-based phylogenetic analyses were more effective in improving the phylogenetic performance than was weighted parsimony analysis. In Bayesian analyses, the resolution of phylogenies under the 16-state models for paired regions, incorporating GTR + G + I models for unpaired regions was better than those under other models. The subfamily Cyprininae was resolved as a monophyletic group, as well as tribe Labein and several genera. However, the monophyly of the currently recognized tribes, such as Schizothoracin, Barbin, Cyprinion + Onychostoma lineages, and some genera was rejected. Furthermore, comparisons of the parsimony and Bayesian analyses and results of variable length bootstrap analysis indicates that the mitochondrial 16S rRNA gene should contain important character variation to recover well-supported phylogeny of cyprinid taxa whose divergences occurred within the recent 8 MY, but could not provide resolution power for deep phylogenies spanning 10-19 MYA.     

几种蚊虫线粒体DNA-16SrRNA序列及其相互关系的研究

测定我国尖音库蚊复合组4亚种（尖音库蚊、淡色库蚊、致倦库蚊和骚扰库蚊）、三带喙库蚊、白纹伊蚊和中华按蚊的线粒体DNA 16S rRNA基因（mtDNA-16S rRNA)序列,发现我国尖音库蚊复合组4亚种mtDNA-16S rRNA序列基本一致,长度为555bp(554bp),GC含量为25．41％。该序列与其他3种蚊虫在种间存在差异,与三带喙库蚊的种间差异为0．54％;与白纹伊蚊的种间差异为5．77％;与中华按蚊的种间差异为9．62％。分子系统关系分析表明该序列与传统分类系统的同属或同亚科种类近似性相一致。     

Reconstruction of phylogeny of locusts from the family Acrididae (Orthoptera) based on analysis of nucleotide sequences of 16S ribosome RNA gene in mitochondria

The sequences of two mitochondrial 16S RNA gene fragments (137- and 174-bp in size) were determined in nine grasshopper species belonging to three Acrididae subfamilies. Phylogenetic reconstruction was performed using the sequences of twelve grasshopper species and the cricket Acheta domesticus sequence as an outgroup (some data were purchased from the GeneBank Data Library (NCBI). In the phylogenetic tree, the Acridinae and Locustinae formed compact groups. Annexpected position of Celes scalozubovi (Locustinae) within the subfamily Acridinae indicated its vague phylogeny. The Catantopinae species lied close to the base of the Acridinae. Almost all branches of phylogenetic trees were strongly (55-100%) supported by bootstrap analysis.     

The phylogenetic relationships of cyanobacteria inferred from 16S rRNA,gyrB, rpoC1 and rpoD1 gene sequences

Phylogenetic analysis of cyanobacteria was carried out using the small subunit rRNA (16S rRNA), DNA gyrase subunit B (gyrB), DNA-dependent RNA polymerase gamma subunit (rpoC1) and a principal sigma factor of E. coli sigma(70) type for DNA-dependent RNA polymerase (rpoD1) gene sequences of 24 strains which contained 5 subgroups of cyanobacteria-3 strains of the Chroococcales, 5 strains of the Pluerocapsales, 7 strains of the Oscillatoriales, 7 strains of the Nostocales and 2 strains of the Stigonematales. Degenerated PCR primers of gyrB, rpoC1 and rpoD1 genes were designed using consensus amino acid sequences registered in GenBank. The phylogenetic positions of cyanobacteria were resolved through phylogenetic analysis based on 16S rDNA, gyrB, rpoC1 and rpoD1 gene sequences. Phylogenies of gyrB, rpoC1 and rpoD1 support 16S rRNA-based classification of cyanobacteria. Interestingly, phylogenies from amino acid sequences deduced from gyrB and combined amino acid sequences deduced from rpoC1 and rpoD1 genes strongly support that of 16S rRNA, but the branching pattens of the trees based on 16S rDNA, GyrB, rpoC1, rpoD1 and combined amino acid sequences deduced from rpoC1 and rpoD1 were not congruent. In this study, we showed the correlation among phylogenetic relationships of 16S rDNA, gyrB, rpoC1 and rpoD1 genes. The phylogenetic trees based on the sequences of 16S rDNA, GyrB, rpoC1, rpoD1 and the combined amino acid sequences deduced from rpoC1 and rpoD1 showed that the lateral gene transfer of rRNA might be suspected for Synechocystis sp. PCC 6803.     

大黄鱼16SrRNA和18SrRNA基因的测定与序列分析

利用多对引物,扩增并测定出大黄鱼16SrRNA基因和18SrRNA基因的部分序列,其长度分别为1202bp和1275bp,16SrRNA基因序列的GC含量为46．12％,18SrRNA基因的Gc含量为53．oo％。将大黄鱼16SrRNA基因序列与GenBank中15种硬骨鱼类的同源序列结合,同时将其18SrRNA基因序列与GenBank中9种脊索动物的同源序列相结合,运用软件获得各自序列间差异百分比,转换和颠换数值等信息。基于这两种基因序列,利用NJ法和BI法,分别构建16种硬骨鱼类和10种脊索动物的分子系统树。18SrRNA构建的系统树包括三大支,一支为哺乳类、鸟类和爬行类共6个物种,一支为两栖类的1个物种,另一支为2种硬骨鱼类。16SrRNA构建的系统树显示大黄鱼所在的石首鱼科与鲈科和盖刺鱼科亲缘关系较近。此外还讨论了这两个基因的序列特征。     

基于16S rRNA和HSP60基因序列分析粘细菌孢囊杆菌亚目形态分类的种属之间的亲缘关系

[目的]利用16S rRNA和HSP60基因分子标记分析鉴定形态分类特征不稳定的粘细菌种属.[方法]利用粘细菌的传统分离纯化方法从土壤中分离粘细菌,根据菌株的形态特征进行分类,PCR方法扩增菌株的16S rRNA和HSP60基因序列并进行系统发育关系分析.[结果]根据形态特征,分离得到的15株粘细菌菌株归入孢囊杆菌亚目(Cystobacterineae)的2个科3个属.其中11株粘细菌具有典型的所在种属的子实体结构,而菌株0085-4、0121-3、NM03和Myx9736的子实体结构发生了不同程度退化.15株粘细菌的16S rRNA基因序列的相似性在95.4%到99.5%之间.而HSP60基因序列差异较大.[结论]在属水平上,粘细菌形态分类特征和16S rRNA基因系统进化关系具有很好的一致性;在揭示粘细菌种间系统发育关系中,HSP60基因序列更为适用.     

Bacterial diversity based on 16S rRNA and gyrB genes at Yinshan mine, China

The diversity of bacterial communities at three sites impacted by acid mine drainage (AMD) from the Yinshan Mine in China was studied using comparative sequence analysis of two molecular markers, the 16S rRNA and gyrB genes. The phylogenetic analyses retrieved sequences from six classes of bacteria, Nitrospira, Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Acidobacteria, and Actinobacteria, as well as sequences related to the plastid of the cyanobacterium Cyanidium acidocaldarium and also some unknown bacteria. The results of phylogenetic analyses based on gyrB and 16S rRNA were compared. This confirmed that gyrB gene analysis may be a useful tool, in addition to the comparative sequence analysis of the 16S rRNA gene, for the analysis of microbial community compositions. Moreover, the Mantel test showed that the geochemical characteristics, especially the pH value and the concentration of iron, strongly influenced the composition of the microbial communities.     

Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence

API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.