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.     

Phylogenetic affiliations ofRhodoferax fermentans and related species of phototrophic bacteria as determined by automated 16S rDNA sequencing

16S rDNA sequences of strains ofRhodoferax fermentans were analyzed and compared with those of species of the generaRubrivivax andRhodocyclus. Approximately 1.5-kb fragments of 16S rDNA from crude cell lysates were amplified by the polymerase chain reaction (PCR) and sequenced directly by usingTth DNA polymerase with the linear PCR sequencing protocol, followed by on-line detection with an automated laser fluorescent DNA sequencer. Pairwise sequence comparisons and distance matrix tree analysis showed thatRhodoferax fermentans, Rubrivivax gelatinosus, andRhodocyclus species belong to three separate lineages within the beta subclass of theProteobacteria, thereby confirming the phylogenetic validity of the genusRhodoferax, as well as of the generaRubrivivax andRhodocyclus.     

Phylogenetic placement of the basidiomycetous yeastsKondoa malvinella andRhodosporidium dacryoidum,and the anamorphic yeastSympodiomycopsis paphiopedili by means of 18S rRNA gene sequence analysis

The 18S ribosomal RNA gene sequences of the basidiomycetous yeastsKondoa malvinella andRhodosporidium dacryoidum, and an anamorphic yeastSympodiomycopsis paphiopedili were determined. The 18S rRNA gene ofR. dacryodium IAM 13522 (ex type) revealed the presence of an intron-like region with a length of 404 nucleotides, which is presumably assigned to a group I intron. The phylogenetic tree, including 34 published reference sequences, was inferred from 1493 sites which could be unambiguously aligned. The molecular phylogeny, using the ascomycetes as an outgroup, divided the basidiomycetes into three major lineages. The first lineage was composed of the smut fungi (Ustilaginales), represented byUstilago maydis, U. hordei, andTilletia caries, includingS. paphiopedili. The second lineage included the type species of teliospore-forming yeast generaLeucosporidium, Rhodosporidium, andSporidiobolus, and the generaErythrobasidium andKondoa, both previously included in the Filobasidiaceae.Rhodosporidium dacryodium showed a close relationship withE. hasegawianum, which was backed by a high bootstrap support. The rust fungiCronartium ribicola andPeridermium harknessii were also included in this lineage. The last lineage was formed by the filobasidiaceous yeasts,Cystofilobasidium capitatum, Mrakia frigida, Filobasidium floriforme, andFilobasidiella neoformans, and the anamorphic yeastsBullera alba (the anamorph ofBulleromyces albus) andTrichosporon cutaneum. Members ofTremella and selected hymenomycetous genera were also included in this lineage.The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the following accession numbers D13459 (Rhodosporidium dacryodium), D13776 (Kondoa malvinella), and D14006 (Sympodiomycopsis paphiopedili).     

Molecular phylogeny of silk-producing insects based on 16S ribosomal RNA and cytochrome oxidase subunit I genes

We have examined the molecular-phylogenetic relationships between nonmulberry and mulberry silkworm species that belong to the families Saturniidae, Bombycidae and Lasiocampidae using 16S ribosomal RNA (16S rRNA) and cytochrome oxidase subunit I (coxI) gene sequences. Aligned nucleotide sequences of 16S rRNA andcoxI from 14 silk-producing species were used for construction of phylogenetic trees by maximum likelihood and maximum parsimony methods. The tree topology on the basis of 16S rRNA supports monophyly for members of Saturniidae and Bombycidae. Weighted parsimony analysis weighted towards transversions relative to transitions (ts, tv4) forcoxI resulted in more robust bootstrap support over unweighted parsimony and favours the 16S rRNA tree topology. Combined analysis reflected clear biogeographic pattern, and agrees with morphological and cytological data.     

Origins of Bradyrhizobium nodule symbionts from two legume trees in the Philippines

Aim Geographic affinities were analysed for nodule bacteria (Bradyrhizobium sp. Jordan) associated with two legume trees indigenous to the Philippines: Pterocarpus indicus (Papilionoideae) and Wallaceodendron celebicum (Mimosoideae). Location Nodule bacteria from Luzon, the Philippines, were compared with reference strains from Central America, eastern North America, Japan, Korea, China and Australia. Methods Two PCR assays targetting length polymorphisms in the rRNA region were carried out on 96 Philippine bacterial isolates. A 496‐bp portion of the 23S rRNA gene was sequenced in 14 representative isolates. Eight strains were analysed in greater depth by sequencing portions of four other genes (16S rRNA [1410 bp], dnaK [603 bp], nifD [822 bp], recA [512 bp]), and phylogenetic trees were constructed by maximum parsimony, neighbour joining and maximum likelihood methods. Results Most of the Philippine Bradyrhizobium strains showed greater similarity to reference strains from Central America than to strains from other source regions included in the analysis. However, phylogenetic trees for the five genes had significantly conflicting topologies, suggesting that lateral gene transfer events had altered genealogical relationships at different loci. In particular, two Philippine strains resembled Bradyrhizobium strains from Central America or China for 16S rRNA, dnaK and recA sequences, but had nifD sequences that clustered with Australian strains (with bootstrap support values of 90–96%). Main conclusions The Philippines have been colonized by Bradyrhizobium strains from multiple source regions. Subsequent lateral gene transfer has resulted in the evolution of Bradyrhizobium strains that combine DNA segments of different geographic origin.     

枣食芽象甲线粒体基因组全序列测定与系统发育分析

【目的】从线粒体基因组水平上探讨枣食芽象甲Scythropus yasumatsui与近缘种的系统发育关系。【方法】利用Illumina MiSeq测序平台对枣食芽象甲线粒体基因组进行测序,对基因组序列进行拼装、注释和特征分析;利用贝叶斯法和最大似然法构建基于象甲科13个物种的线粒体基因组13个蛋白质编码基因核苷酸序列的系统发育树。【结果】结果表明,枣食芽象甲线粒体基因组全长为16 472 bp (GenBank登录号: MF807224),包含13个蛋白质编码基因、22个tRNA基因、2个rRNA基因和2个非编码控制区,37个基因的排列顺序与祖先昆虫的线粒体基因排列顺序一致。13个蛋白质编码基因的起始密码子为ATN,其中除了cob和nad1基因的完全终止密码子为TAG外,其余11个基因的完全终止密码子为TA(A)。22个tRNA基因中除了trnS1缺少DHU臂,反密码子由GCT变为TCT外,其余均能形成典型的三叶草结构。基于13个蛋白质编码基因序列构建的系统发育树结果显示,象甲科8个亚科系统发育关系为：(((隐喙象亚科(Cryptorhynchinae)+(象虫亚科(Curculioninae)+魔喙象亚科(Molytinae)))+长小蠹亚科(Platypodinae))+(粗喙象亚科(Entiminae)+Cyclominae亚科))+隐颏象亚科(Dryophthorinae)+小蠹亚科(Scolytinae))。【结论】在13种象甲科昆虫物种中,同属于粗喙象亚科的枣食芽象甲与南美果树象甲Naupactus xanthographus在系统发育树中聚为同一分支,表明基于线粒体基因组全序列的分子系统发育结果与传统的形态分类结果是一致的。     

Physicochemical and microbiological characteristics of groundwater from observation wells of a deep radioactive liquid waste repository

A radioactive liquid waste repository was found to be the habitat of a rich microbial community with a high catabolic potential. Groundwater from a depth of 162–189 m contained aerobic saprotrophic and anaerobic fermentative, sulfate-reducing, and denitrifying bacteria. Nitrate-reducing bacteria residing in this groundwater were isolated in pure cultures. Based on the results of their physiological studies, 16S rRNA sequencing, and phylogenetic analysis, the microorganisms isolated were ascribed to one phylogenetic branch, the γ-subclass of gram-negative bacteria. Among six isolates, four belonged to the genusAcinetobacter, whereas two others belonged to the generaComamonas andAeromonas. The data obtained indicate that the microflora of the repository can exert a certain effect on the chemical composition of the formation fluids and bearing rocks, as well as on the migration of radionuclides     

Evolution and microsynteny of the apyrase gene family in three legume genomes

Apyrases have been suggested to play important roles in plant nutrition, photomorphogenesis, and nodulation. To help trace the evolution of these genes in the legumes—and possibly, the acquisition of new functions for nodulation—apyrase-containing BACs were sequenced from three legume genomes. Genomic sequences from Medicago truncatula, Glycine max and Lotus japonicus were compared to one another and to corresponding regions in Arabidopsis thaliana. A phylogenetic analysis of apyrase homologs from these regions and sequences from other legume species, as well as other plant families, identified a potentially legume-specific clade that contains a well-characterized soybean ( G. soja) apyrase, Gs52, as well as homologs from Dolichos, Lotus , Medicago and Pisum. Sister clades contain homologs from members of Brassicaceae, Solanaceae, Poaceae and Fabaceae. Comparisons of rates of change at synonymous and nonsynonymous sites in the Gs52 and sister clades show rapid evolution in the potentially legume-specific Gs52 clade. The genomic organization of the apyrase-containing BACs shows evidence of gene duplication, genomic rearrangement, and gene conversion among Gs52 homologs. Taken together, these results suggest a scenario of local apyrase gene duplication in an ancestor of the legumes, followed by functional diversification and increased rates of change in the new genes, and further duplications in the Galegae (which include the genera Medicago and Pisum). The study also provides a detailed comparison of genomic regions between two model genomes which are now being sequenced ( M. truncatula and L. japonicus), and a genome from an economically important legume species ( G. max).Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by A. Kondorosi     

Compensatory substitutions and the evolution of the mitochondrial 12S rRNA gene in mammals

12S ribosomal RNA (rRNA) gene sequences from a suite of mammalian taxa (13 placentals, 4 marsupials, 1 monotreme), for which phylogenetic relationships are well established based on independent criteria, were employed to study the evolution of this gene. Phylogenetic analysis of 12S sequences produces a phylogeny that agrees with expectations. Base composition provides evidence for directional symmetrical substitution pressure in loops; in stems, base composition is much more even. Rates of nucleotide substitution are lower in stems than loops. Patterns of nucleotide substitution show an overall preference for transitions over transversions, with this difference more profound in stems than loops. Among different transversion pathways, there is a wide range of transformation frequencies. An analysis of compensatory substitutions shows that there is strong evidence for their occurrence and that a weighting factor of 0.61 should be applied in phylogenetic analyses to account for the dependence of mutations at stem positions relative to positions where changes are independent. Among stem variables (i.e., stem length, interaction distance, substitution rates, G+C content, and the percentage of bases that are paired), several significant correlations were discovered, but stem length and interaction distance are uncorrelated with other variables.      

Establishment of the phylogenetic relationships between the microbial producers of cyclodextrin glucanotransferases using complete amino acid sequences

A phylogenetic tree was constructed on the basis of the amino acid sequences of the known cyclodextrin glucanotransferases (CDGTs), including those deduced from the nucleotide sequences ofBacillus sp. strain 6.6.3 andPaenibacillus macerans IB-7 genes encoding α- and β-CDGTs. The tree clearly demonstrates the existence of distinct phylogenetic groups of CDGT-producing microorganisms and the divergence of the α-, β-, and γ-CDGT produced by microorganisms from the generaBacillus, Paenibacillus, Brevibacillus, andThermoanaerobacter from a common ancestor, whereas the CDGT ofKlebsiella pneumoniae is independent and results from the convergence of different ancestors. The degree of homology of the leader peptide sequences of CDGTs may serve as a criterion of intraspecies relatedness between CDGT-producing microorganisms.     

ITS sequences and speciation on far easternIndigofera (Leguminosae)

The internal transcribed spacer (ITS) regions of 18–26S nuclear ribosomal DNA was sequenced to address phylogenetic relationships and to measure the extent of differentiation among six species of the Far EasternIndigofera. ITS 1 had 230–240 base pairs (bp) long while ITS 2 had 211–213 bp long. The 5.8S rRNA coding region was 161 bp long. Sequence divergence calculated by Kimura's two parameter method between species ranged from 0.00 to 13.49%. A single most parsimonious tree was produced from 77 variable nucleotide sites, which had a consistency index of 0.97 and a retention index of 0.83. ITS sequence data suggested that the continental species ofI. kirilowii (2n=16) is diverged from the common ancestor of other species at first, and then the island species ofI. decora (2n=48) andI. venulosa and the Korean Peninsular species ofI. grandiflora (2n=16) andI. koreana (2n=32) are diverged from the ancestor. The molecular data supports that the speciation in the Far EasternIndigofera occurred with polyploidization from continental ancestor to peripheral peninsular and island species.     

Complete mitochondrial genome sequence of the Indian pipistrelle Pipistrellus coromandra (Vespertilioninae)

We characterized the complete mitogenome of Pipistrellus coromandra (Indian pipistrelle) for comparative analysis of mitogenomes and for resolving the phylogenetic relationship of four tribes in the subfamily Vespertilioninae. The mitogenome size of P. coromandra was 17,153?bp, with a control region and a typical set of 37 mitochondrial genes. The nucleotide composition of the P. coromandra mitogenome showed an AT bias with a nucleotide composition of 33.5% A, 30.7% T, 13.3% G, and 22.5% C. The mitochondrial protein-coding genes in P. coromandra use the standard start codon (ATN), two stop codons (TAA and AGA), and two incomplete stop codons (TA- and T--). The intertribal relationship of four tribes was highly resolved from the phylogenetic analysis of mitogenome sequences.     

青藏高原特有种-藏扇穗茅叶绿体基因组测序及序列分析

藏扇穗茅（Littledalea tibetica）是禾本科（Poaceae）雀麦族（Bromeae）中一个具有重要生态价值的多年生高山特有种,主要分布于青藏高原及其毗邻地区。本文采用基于第二代高通量测序平台的Illumina MiSeq技术,对青藏高原特有种—藏扇穗茅进行了叶绿体基因组测序,首次建立了雀麦族物种的标准测序流程;同时,以其近缘物种—黑麦草（Lolium perenne）的叶绿体基因组序列作为参考,组装获得它的叶绿体基因组序列。结果表明,藏扇穗茅叶绿体基因组序列全长136 852 bp,GC含量为38.5%,呈典型的四段式结构,其中大（LSC）、小（SSC）单拷贝区大小分别为80 970和12 876 bp,反向互补重复区（IR）大小为21 503 bp,共注释得到141个基因,包含95个蛋白编码基因、38个tRNA基因和8个rRNA基因,主要分布于大单拷贝区和小单拷贝区。同时,基于藏扇穗茅和其它30种禾本科植物叶绿体基因全序列构建的系统发育树显示,藏扇穗茅与早熟禾亚科中小麦族植物亲缘关系较近。     

Relationship of the genusCordyceps and related genera, based on parsimony and spectral analysis of partial 18S and 28S ribosomal gene sequences

A molecular phylogenetic study of selected species of three sub-genera of the genusCordyceps was undertaken, along with representatives of the generaAkanthomyces, Aschersonia, Gibellula, Hymenostilbe, Hypocrella, Nomuraea andTorrubiella, to examine their inter-relationship. Phylogenetic analyses of the data indicated that the Clavicipitaceae form a monophyletic group within the Hypocreales, while the monophyly ofCordyceps was not supported. Four clades were identified:Cor. militaris/Cor. pseudomilitaris; Cor. iranginesis/Cor. sphecocephala; Cor. intermedia/Cor. capitata; andCor. cylindrica/Nom. atypicola. The sub-genusNeocordyceps was shown to be monophyletic while the sub-generaEucordyceps andOphiocordyceps do not form monophyletic groups. The genusHypocrella appeared monophyletic, and radiated after the formation of the generaCordyceps, andTorrubiella. Akanthomyces arachnophilus andGi. pulchra, anamorphs ofTorrubiella species, formed a distinct clade that was separate from one formed by the scale insect pathogens,To. luteorostrata andPaecilomyces cinnamomeus, suggesting that this genus may be polyphyletic.     

Evaluation of different partial 16S rRNA gene sequence regions for phylogenetic analysis of microbiomes

Operational taxonomic units (OTUs) are conventionally defined at a phylogenetic distance (0.03—species, 0.05—genus, 0.10—family) based on full-length 16S rRNA gene sequences. However, partial sequences (700 bp or shorter) have been used in most studies. This discord may affect analysis of diversity and species richness because sequence divergence is not distributed evenly along the 16S rRNA gene. In this study, we compared a set each of bacterial and archaeal 16S rRNA gene sequences of nearly full length with multiple sets of different partial 16S rRNA gene sequences derived therefrom (approximately 440-700 bp), at conventional and alternative distance levels. Our objective was to identify partial sequence region(s) and distance level(s) that allow more accurate phylogenetic analysis of partial 16S rRNA genes. Our results showed that no partial sequence region could estimate OTU richness or define OTUs as reliably as nearly full-length genes. However, the V1-V4 regions can provide more accurate estimates than others. For analysis of archaea, we recommend the V1-V3 and the V4-V7 regions and clustering of species-level OTUs at 0.03 and 0.02 distances, respectively. For analysis of bacteria, the V1-V3 and the V1-V4 regions should be targeted, with species-level OTUs being clustered at 0.04 distance in both cases.     

Independent gene evolution in the potato actin gene family demonstrated by phylogenetic procedures for resolving gene conversions and the phylogeny of angiosperm actin genes

Summary Nine different actin DNA sequences were isolated from the common potato,Solanum tuberosum, and the nucleotide sequence of five actin loci and of two allelic variants are presented. Unlike the wide variation in intron position among animal actin genes, the potato actin genes have three introns situated in the same positions as reported for all other angiosperm actin genes. Using a novel combination of analytical procedures (G-test and compatibility analysis), we could not find evidence of frequent large or small nonreciprocal exchanges of genetic material between the sequenced loci, although there were a few candidates. Resolution of such gene conversion events and the quantification of independence of gene evolution in multigene families is critical to the inference of phylogenetic relationships. Comparison with actin genes in other angiosperm species suggests that the actin multigene family can be divided into a number of subfamilies, evolved by descent rather than gene conversion, which are of possible functional origin, with one major subfamily diversification occurring before the divergence of monocots and dicots. The silent rate of nucleotide substitution was estimated to be similar to that suggested for a number of other plant nuclear genes, whereas the replacement rate was extremely slow, suggestive of selective constraints.     

a molecular phylogeny of apiaceae subfamily Apioideae: evidence from nuclear ribosomal DNA internal transcribed spacer sequences

Phylogenetic relationships among 40 New World and Old World members of Apiaceae subfamily Apioideae, representing seven of the eight tribes and eight of the ten subtribes commonly recognized in the subfamily, were inferred from nucleotide sequence variation in the internal transcribed spacer (ITS) regions of 18-26S nuclear ribosomal DNA. Although the sequences are alignable, with only 11% of sites excluded from the analyses because of alignment ambiguity, divergence values in pairwise comparisons of unambiguous positions among all taxa were high and ranged from 0.5 to 33.2% of nucleotides in ITS 1 and from 0 to 33.2% of nucleotides in ITS 2. Average sequence divergence across both spacer regions was 18.4% of nucleotides. Phylogenies derived from ITS sequences estimated using neighbor-joining analysis of substitution rates, and maximum likelihood and parsimony methods give trees of essentially similar topology and indicate that: (1) there is little support for any existing system of classification of the subfamily that is based largely on morphological and anatomical features of the mericarp; (2) there is a major phylogenetic division within the subfamily, with one clade comprising the genus Smyrnium and those taxa belonging to Drude's tribes Dauceae, Scandiceae, and Laserpitieae and the other clade comprising all other examined taxa; and (3) the genera Arracacia, Coaxana, Coulterophytum, Enantiophylla, Myrrhidendron, Prionosciadium, and Rhodosciadium, all endemic to Mexico and Central America, comprise a clade but their relationships to other New World taxa are equivocal. A phylogeny derived from parsimony analysis of chloroplast DNA rpoC1 intron sequences is consistent with, but considerably less resolved than, relationships derived from these ITS regions. This study affirms that ITS sequences are useful for phylogenetic inference among closely related members of Apioideae but, owing to high rates of nucleotide substitution, are less useful in resolving relationships among the more ancestral nodes of the phylogeny.     

Sequence and characterisation of a ribosomal RNA operon fromAgrobacterium vitis

One of the four ribosomal RNA operons (rrnA) from theAgrobacterium vitis vitopine strain S4 was sequenced.rrnA is most closely related to therrn operons ofBradyrhizobium japonicum andRhodobacter sphaeroides and carries an fMet-tRNA gene downstream of its 5S gene, as in the case ofR. sphaeroides. The 16S rRNA sequence of S4 differs from theA. vitis K309 type strain sequence by only one nucleotide, in spite of the fact that S4 and K309 have very different Ti plasmids. The predicted secondary structure of the S4 23S rRNA shows several features that are specific for the alpha proteobacteria, and an unusual branched structure in the universal B8 stem. The 3 ends of the three otherrrn copies of S4 were also cloned and sequenced. Sequence comparison delimits the 3 ends of the four repeats and defines two groups:rrnA/rrnB andrrnC/rrnD.     

Intervening Sequences in 16S rRNA Genes of Campylobacter sp.: Diversity of Nucleotide Sequences and Uniformity of Location

We found and sequenced intervening sequences (IVSs) in the PCR-amplicons of 16S rRNA genes of 3 strains of Campylobacter rectus, 2 strains of C. curvus and 2 strains of C. sputorum. The lengths of the IVSs were 140 to 233 bp. The IVSs of C. rectus were identical and had a sequence homology of 55 to 79% against those of C. curvus and C. helveticus. The IVSs of C. sputorum were 97.9-100% homologous but poorly homologous to the other IVSs. In spite of the diversities of the lengths and the nucleotide sequences, all of the IVSs were located at the same position in the 16S rRNA genes.