Comparative genetic diversity of Pseudomonas stutzeri genomovars, clonal structure, and phylogeny of the species

A combined phylogenetic and multilocus DNA sequence analysis of 26 Pseudomonas stutzeri strains distributed within the 9 genomovars of the species has been performed. Type strains of the two most closely related species (P. balearica, former genomovar 6, and P. mendocina), together with P. aeruginosa, as the type species of the genus, have been included in the study. The extremely high genetic diversity and the clonal structure of the species were confirmed by the sequence analysis. Clustering of strains in the consensus phylogeny inferred from the analysis of seven nucleotide sequences (16S ribosomal DNA, internally transcribed spacer region 1, gyrB, rpoD, nosZ, catA, and nahH) confirmed the monophyletic origin of the genomovars within the Pseudomonas branch and is in good agreement with earlier DNA-DNA similarity analysis, indicating that the selected genes are representative of the whole genome in members of the species.     

基于rDNA ITS序列的油茶叶片内生真菌多样性研究(英文)

油茶是我国热带和亚热带地区广泛栽培的一类重要的经济树种,其种子可生产优质食用油(茶油)。为了比较全面地了解油茶的内生真菌多样性,采用基于rDNA ITS的免培养法从油茶叶片中提取了总DNA,再从总DNA样本中直接扩增了内生真菌核糖体RNA基因内转录间隔区序列(ITS),进而构建了ITS克隆文库。共计从50个克隆子中获得了22种不同的克隆序列,其中3个为嵌合体,7个归属为植物,其余克隆序列根据序列相似性和系统发育分析归为12个不同的分类操作单元(OTUs),全部为子囊菌,分属于4纲4目,其中Aspergillus属2种,Mycosphaerellaceae科1种,Sordariales目4种,Helotiales目5种,Aspergillus属真菌是优势菌。结果表明油茶叶片内生真菌的种类分布较广。     

Genome phylogeny based on short-range correlations in DNA sequences.

The surprising fact that global statistical properties computed on a genomewide scale may reveal species information has first been observed in studies of dinucleotide frequencies. Here we will look at the same phenomenon with a totally different statistical approach. We show that patterns in the short-range statistical correlations in DNA sequences serve as evolutionary fingerprints of eukaryotes. All chromosomes of a species display the same characteristic pattern, markedly different from those of other species. The chromosomes of a species are sorted onto the same branch of a phylogenetic tree due to this correlation pattern. The average correlation between nucleotides at a distance k is quantified in two independent ways: (i) by estimating it from a higher-order Markov process and (ii) by computing the mutual information function at a distance k. We show how the quality of phylogenetic reconstruction depends on the range of correlation strengths and on the length of the underlying sequence segment. This concept of the correlation pattern as a phylogenetic signature of eukaryote species combines two rather distant domains of research, namely phylogenetic analysis based on molecular observation and the study of the correlation structure of DNA sequences.     

An integrated morphological and molecular approach to a new species description in the Trypanosomatidae: the case of Leptomonas podlipaevi n. sp., a parasite of Boisea rubrolineata (Hemiptera: Rhopalidae)

Leptomonas podlipaevi n. sp., a new trypanosomatid species, is described herein based on light microscopic, ultrastructural, and molecular phylogenetic data. The organism is pleomorphic both in host and culture, with two predominant forms-a typical promastigote with a long flagellum and a shorter promastigote with a small or barely extending flagellum. Several spliced leader RNA repeat sequences obtained from the original cultures and the clonal lines representing two types of cells were all nearly identical. These sequences formed a tight cluster in the neighbor-joining tree well separated from other trypanosomatid species. Glyceraldehyde phosphate dehydrogenase gene sequences were determined for L. podlipaevi and 10 previously described trypanosomatid species. Molecular phylogenetic analysis has demonstrated that the new species is most closely related to Leptomonas seymouri and Leptomonas pyrrhocoris. The analysis has also highlighted the polyphyly of the genus Leptomonas.     

Genetic diversity, reproductive biology, and speciation in the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin.

Beauveria bassiana, a mitosporic fungus used for the biological control of many insect species, is recognized as a "species complex" comprising genetically diverse lineages. Being predominantly asexual, mating tests cannot be applied to delimit species in this species complex. Genetic tests offer an indirect means of identifying species among isolates. To this end, molecular genetic analysis of a sample of B. bassiana isolates with 2 subsamples, 1 representing a worldwide collection and another from a localized epizootic population was carried out. DNA markers generated through AFLPs (amplified fragment length polymorphisms) and SSCPs (single-strand conformation poly morphisms) and nucleotide sequence data of different allelic forms of 3 genes (large and small subunits of rRNA and beta-tubulin) were evaluated. The B. bassiana isolates from the worldwide sample showed 11% overall similarity and no closely clustered groups. Phylogenetic trees generated from the AFLP and SSCP data of this sample resolved the different isolates into distinct phylogenetic lineages. In the epizootic B. bassiana population, prevalence of recombination was evident from random association of alleles in multilocus tests and lack of phylogenetic concordance among 3 gene genealogies. Thus, the worldwide sample of B. bassiana exhibits a predominantly clonal structure, hinting at species divergence leading to cryptic speciation with recombination being customary among isolates sharing a close ecological niche.     

Phylogeny of the European species of the genus Pellia (Hepaticae; Metzgeriales) based on the molecular data from nuclear tRNA(Leu)(CAA) intergenic sequences

Species of the genus Pellia are similar to such an extent that their proper recognition based on morphological and anatomical features is difficult. To solve this problem isozyme methods were developed. As a result of these studies new cryptic species were recognized and new hypotheses concerning phylogenetic relationship in Pellia were formed. To examine hypotheses derived from isozyme data we decided to study species of the genus Pellia at the DNA level. Total DNA from all Polish Pellia species was isolated. The taxonomic and phylogenetic relationships in Pellia were examined using intergenic spacer sequences between nuclear tRNA(Leu) genes organised in tandem arrays. PCR (polymerase chain reaction) amplification of tRNA(Leu) gene spacers produced fragments of different sizes in all species and their restriction analysis showed species-specific patterns. PCR products were cloned and sequenced. Nucleotide sequence data were used for phylogenetic reconstruction. Sequence analysis confirmed previous results based on isozyme studies that P. endiviifolia- species A and species B as well as P. epiphylla- species S and species N (having different isozyme multilocus genotypes) are separate sibling species. Our results also confirmed the allopolyploid character of the only polyploid species in Pellia, P. borealis which was formed by hybridization of P. epiphylla- species N and P. epiphylla- species S cryptic species.     

Metabolic profiling and phylogenetic analysis of medicinal Zingiber species: Tools for authentication of ginger (Zingiber officinale Rosc)

Phylogenetic analysis and metabolic profiling were used to investigate the diversity of plant material within the ginger species and between ginger and closely related species in the genus Zingiber (Zingiberaceae). In addition, anti-inflammatory data were obtained for the investigated species. Phylogenetic analysis demonstrated that all Zingiber officinale samples from different geographical origins were genetically indistinguishable. In contrast, other Zingiber species were significantly divergent, allowing all species to be clearly distinguished using this analysis. In the metabolic profiling analysis, the Z. officinale samples derived from different origins showed no qualitative differences in major volatile compounds, although they did show some significant quantitative differences in non-volatile composition, particularly regarding the content of [6]-, [8]-, and [10]-gingerols, the most active anti-inflammatory components in this species. The differences in gingerol content were verified by HPLC. The metabolic profiles of other Zingiber species were very different, both qualitatively and quantitatively, when compared to Z. officinale and to each other. Comparative DNA sequence/chemotaxonomic phylogenetic trees showed that the chemical characters of the investigated species were able to generate essentially the same phylogenetic relationships as the DNA sequences. This supports the contention that chemical characters can be used effectively to identify relationships between plant species. Anti-inflammatory in vitro assays to evaluate the ability of all extracts from the Zingiber species examined to inhibit LPS-induced PGE(2) and TNF-alpha production suggested that bioactivity may not be easily predicted by either phylogenetic analysis or gross metabolic profiling. Therefore, identification and quantification of the actual bioactive compounds are required to guarantee the bioactivity of a particular Zingiber sample even after performing authentication by molecular and/or chemical markers.     

Phylogenetic relationships among Frankia genomic species determined by use of amplified 16S rDNA sequences

Actinomycetes of the genus Frankia establish a nitrogen-fixing symbiosis with a large number of woody dicotyledonous plants. Hundreds of strains isolated from various actinorhizal plants growing in different geographical areas have recently been classified into at least nine genomic species by use of the DNA-DNA hybridization technique (M.P. Fernandez, H. Meugnier, P.A.D. Grimont, and R. Bardin, Int. J. Syst. Bacteriol. 39:424-429, 1989). A protocol based on the amplification and sequencing of 16S ribosomal DNA segments was used to classify and estimate the phylogenetic relationships among eight different genomic species. A good correlation was established between the grouping of strains according to their 16S ribosomal DNA sequence homology and that based on total DNA homology, since most genomic species could be characterized by a specific sequence. The phylogenetic tree showed that strains belonging to the Alnus infectivity group are closely related to strains belonging to the Casuarina infectivity group and that strains of these two infectivity groups are well separated from strains of the Elaeagnus infectivity group, which also includes atypical strains isolated from the Casuarina group. This phylogenetic analysis was also very efficient for classifying previously unclassified pure cultures or unisolatable strains by using total DNA extracted directly from nodules.     

Toward a Wolbachia Multilocus Sequence Typing System: Discrimination of Wolbachia Strains Present in Drosophila Species

Among the diverse maternally inherited symbionts in arthropods, Wolbachia are the most common and infect over 20% of all species. In a departure from traditional genotyping or phylogenetic methods relying on single Wolbachia genes, the present study represents an initial Multilocus Sequence Typing (MLST) analysis to discriminate closely related Wolbachia pipientis strains, and additional data on sequence diversity in Wolbachia. We report a new phylogenetic characterization of four genes (aspC, atpD, sucB, and pdhB), and provide an expanded analysis of markers described in previous studies (16S rDNA, ftsZ, groEL, dnaA, and gltA). MLST analysis of the bacterial strains present in 16 different Drosophila–Wolbachia associations detected four distinct clonal complexes that also corresponded to maximum-likelihood identified phylogenetic clades. Among the 16 associations analyzed, six could not be assigned to MLST clonal complexes and were also shown to be in conflict with relationships predicted by maximum-likelihood phylogenetic inferences. The results demonstrate the discriminatory power of MLST for identifying strains and clonal lineages of Wolbachia and provide a robust foundation for studying the ecology and evolution of this widespread endosymbiont.     

Bacteria Associated with Toxic Clonal Cultures of the Dinoflagellate <Emphasis Type="Italic">Ostreopsis lenticularis</Emphasis>

The marine toxic dinoflagellate Ostreopsis lenticularis has been implicated as the major vector in ciguatera seafood poisoning on the southwest coast of Puerto Rico. Studies have demonstrated that associated bacteria play a role in the ciguatoxin production and that different clonal cultures of O. lenticularis harbor different culturable bacteria. In this study, more than 125 associated bacteria from two toxic clonal cultures of O. lenticularis (no. 302 and no. 303) were analyzed utilizing polymerase chain reaction amplification of the partial small subunit ribosomal DNA (rRNA), denaturing gradient gel electrophoresis, and DNA sequencing. Approximately 50% of total bacteria identified in both cultures were a single species belonging to the Cytophaga-Flavobacter-Bacteroides complex. This bacterium was also found in six new O. lenticularis clonal cultures established 10 years after the original cultures used in this study and absent from a clonal culture of a different dinoflagellate species. The data presented here indicate a persistent and apparently specific association of this bacterium with O. lenticularis, which makes it a candidate involved in ciguatoxin production.     

Community composition, host range and genetic structure of the fungal entomopathogen Beauveria in adjoining agricultural and seminatural habitats

Although intensively investigated for biological control of insect pests, little is known about the ecology of the fungal entomopathogenic genus Beauveria in natural or agricultural habitats. In this study, we used molecular phylogenetic and genotypic information to infer species diversity, reproductive potential and genetic structure of Beauveria occurring within a single arable field and bordering hedgerow in Denmark. Isolates were sampled from cultivated field and hedgerow soils, from insects harbouring latent fungal infections, and from the phylloplanes of three plant species common in the hedgerow flora. A nuclear phylogeny of this local Beauveria assemblage resolved seven phylogenetic species, including (i) five phylogenetic species within Beauveria bassiana sensu stricto ; (ii) Clade C, a taxonomically uncharacterized species that is morphologically indistinguishable but phylogenetically distant from B. bassiana s.s. ; and (iii) Beauveria brongniartii. All seven species were present throughout the hedgerow habitat, including as infections in insects. Significantly, only B . bassiana s.s. phylogenetic species Eu_1 was isolated from tilled soils. Mating type polymerase chain reaction assays demonstrated that all five B. bassiana s.s. phylogenetic species possess bipolar outcrossing mating systems. Of these, only the Eu_1 population contained two mating types; however, a 31:2 skew in MAT1:MAT2 mating types suggests a low frequency of sexual reproduction in this population. The four remaining B. bassiana s.s. phylogenetic species were fixed for single mating types and these populations are evidently clonal. Multilocus microsatellite genotyping revealed polymorphism in all five phylogenetic species of B. bassiana s.s. ; however, all show evidence of clonal genetic structure.     

Molecular epidemiology and evolutionary genetics of Leischmania parasites

In order to illustrate the relevance of the concepts and methods of evolutionary genetics in the understanding of the epidemiology of pathogenic agents, we develop in this paper the case of the Leishmania, a genus of parasitic protozoa. An extensive study of various natural populations of Leishmania in different countries (Old and New World) was carried out by using Multilocus Enzyme Electrophoresis (MLEE) and Random Amplified Polymorphic DNA fingerprinting (RAPD) as genetic markers. The data have been interpreted in evolutionary genetic terms. The main benefit of this approach has been to better define the concept of species in the genus Leishmnania, on rigorous phylogenetic bases. As a matter of fact, a sound taxonomical background is a prerequisite for any epidemiological approach. Since the biological concept of species is difficult or impossible to apply for most pathogenic microorganisms, we recommend relying on criteria of both phylogenetic discreteness and of epidemiological/medical relevance to describe new species of Leishmania. Through this approach, for example, we have shown that the species status of L. ( V.) perzzl.ianza can be supported. On the contrary, we have been unable to clearly distinguish L. (V.) panamensis from L. (V.) guyanensis with genetic tools. Additionally, we have shown that the epidemiological inferences based on a limited set of genetic markers can be misleading. As a matter of fact, we have demonstrated that a collection of L. (L.) infantum stocks identified as zymodeme 'MON 1' by other authors present additional genetic heterogeneity and do not correspond to a distinct 'Discrete Typing Unit' DTU, and are actually polyphyletic. Lastly, in the samples that were conveniently designed, we have confirmed that Leishmania parasites have a basically clonal population structure. As the clonal model specifies it, occasional bouts of genetic exchange remain nevertheless possible. Telling comparisons are drawn with the evolutionary genetics of other pathogens Trypanosoma cruzi and Trypanosoma congolense.     

Correlated evolution of self-incompatibility and clonal reproduction in Solanum (Solanaceae)

It has been suggested that clonality provides reproductive assurance in cross-fertilizing species subject to pollen limitation, relieving one of the main selective pressures favoring the evolution of self-fertilization. According to this hypothesis, cross-fertilizing species subject to pollen limitation should often be clonal. Here, we investigated the association between clonality and a genetic mechanism enforcing outcrossing, self-incompatibility, in Solanum (Solanaceae). We collected self-incompatibility and clonality information on 87 species, and looked for an association between these two traits. To account for the contribution of shared evolutionary history to this association, we incorporated phylogenetic information from chloroplast (NADH dehydrogenase subunit F) sequence data. We found that self-incompatibility is strongly associated with clonal reproduction: all self-incompatible species reproduce clonally, while the absence of clonality is widespread among self-compatible taxa. The observed correlation persists after taking into account shared phylogenetic history, assumptions about the evolutionary history of self-incompatibility, uncertainty associated with phylogeny estimation, and associations with life history (annual/perennial). Our results are consistent with the hypothesis that clonality provides reproductive assurance, and suggest that the consequences of clonal growth in the evolution of plant reproductive strategies may be more significant than previously thought.     

Genetic characterization of Trypanosoma cruzi natural clones from the state of Paraíba, Brazil

Eighteen Trypanosoma cruzi stocks from the state of Paraíba, Brazil, isolated from man, wild mammals, and triatomine bugs were studied by multilocus enzyme electrophoresis and random primed amplified polymorphic DNA. Despite the low number of stocks, a notable genetic, genotypic, and phylogenetic diversity was recorded. The presence of the two main phylogenetic subdivisions, T. cruzi I and II, was recorded. The strong linkage disequilibrium observed in the population under survey suggests that T. cruzi undergoes predominant clonal evolution in this area too, although this result should be confirmed by a broader sample. The pattern of clonal variation does not suggests a recent origin by founder effect with a limited number of different genotypes.     

叶绿体基因infA-rpl36区域在小麦族物种中的序列变异分析

利用小麦叶绿体基因组中infA-rpl36区域的序列设计引物, 对小麦族(Triticeae)的12个二倍体和多倍体的物种进行了PCR扩增和序列测定, 获得了长度为584～603 bp的12条DNA序列。序列分析表明, 供试物种在infA-rpl36基因间隔区的核苷酸变异明显高于基因编码区。基因编码区核苷酸序列同源性高达97%, 表明了目标片段具有高度的保守性。但在5个物种的infA编码区出现了较大的插入、缺失突变, 导致推导的氨基酸序列也发生了很大的变化, 证实了infA基因是叶绿体基因组中最活跃的基因之一, 而rpl36基因的变异较小, 说明不同叶绿体基因的进化速度是不同的。基于测定序列建立的种系树分析发现, 多倍体物种中间偃麦草(Thinopyrum intermedium)具有多种不同的细胞质起源, 与核基因组一样在进化上较为复杂。     

Molecular taxonomic identification of Dacus and Ceratitis species from Sub-Saharan Africa using mitochondrial haplotypes

Several different taxa within the genera Dacus and Ceratitis (Diptera: Tephritidae) are important agricultural pests in Sub-Saharan Africa. Although the status of many of these taxa as distinct species and their phylogenetic relationships is unclear, it is clear that these pests use a wide range of host plants and are highly invasive. The great potential for economic damage inflicted by these pests requires the ability to make accurate and reliable taxonomic identification of specimens. However, many limitations and uncertainties are encountered when these species are examined using traditional approaches based on morphological identification techniques. We describe here the amplification and analysis of DNA sequences from the mitochondrial cytochrome oxidase II (COII)-tRNA(lys)-tRNA(ASP) genes from individuals of various Dacus and Ceratitis species and populations from Sub-Saharan Africa. The variation detected in the DNA sequences of these individuals is used both for clarification of their taxonomic status and the analysis of phylogenetic relationships of these taxa.     

The relative contributions of recombination and point mutation to the diversification of bacterial clones

Low levels of recombination in bacterial species have often been inferred from the presence of linkage disequilibrium between the alleles at different loci in the population. However, significant linkage disequilibrium is inevitable in organisms that divide by binary fission, and recombinational replacements must be very frequent, compared to point mutation, to dissipate disequilibrium. Recent studies using data from multilocus sequence typing indicate that, in many species, recombinational replacements contribute more greatly to clonal diversification than do point mutations and, in some species, recombination has been sufficient to eliminate any phylogenetic signal from gene trees. Recent efforts to improve understanding of the extent and impact of homologous recombination in the diversification of bacterial clones are discussed.     

Identification of triploid individuals and clonal lines in Carassius auratus complex using microsatellites

The Carassius auratus complex in natural populations includes diploid triploid and polyploidy individuals. Diploid individuals belong to the species Carassius auratus whereas triploid and polyploidy individuals are from the subspecies Carassius auratus gibelio. Triploid individuals are all female and reproduce clonally by gynogenesis. Therefore the Carassius auratus complex is an ideal system for studying evolution of unisexual reproduction. Identification of triploid individuals and clonal lines is the first step towards understanding of the evolution of unisexual clonal lines. We examined the ability of 10 microsatellites in identifying triploid individuals in 94 individuals from Japan and China. In 40 confirmed triploid individuals and eight confirmed diploid individuals, all triploid and diploid individuals can be identified by genotyping 10 microsatellite, and four triploid clonal lines were identified. Using the 10 microsatellites we genotyped 46 adult individuals (40 females and six males) from a natural population in China and found that all six males were diploid whereas the majority of females (36 of 40) were triploid and three triploid clonal lines were detected. In 18 diploid individuals from China, all individuals showed different genotypes, suggesting there is no diploid clonal line in diploid crucian carp. A phylogenetic analysis of 94 individuals from China and Japan showed that triploid individuals and clonal lines have originated recurrently.     

Molecular phylogenetics and evolution of the endemic Hawaiian genus Adenophorus (Grammitidaceae)

Recent studies of the phylogeny of several groups of native Hawaiian vascular plants have led to significant insights into the origin and evolution of important elements of the Hawaiian flora. No groups of Hawaiian pteridophytes have been subjected previously to rigorous phylogenetic analysis. We conducted a molecular phylogenetic analysis of the endemic Hawaiian fern genus Adenophorus employing DNA sequence variation from three cpDNA fragments: rbcL, atpbeta, and the trnL-trnF intergenic spacer (IGS). In the phylogenetic analyses we employed maximum parsimony and Bayesian inference. Bayesian phylogenetic inference often provided stronger support for hypothetical relationships than did nonparametric bootstrap analyses. Although phylogenetic analyses of individual DNA fragments resulted in different patterns of relationships among species and varying levels of support for various clades, a combined analysis of all three sets of sequences produced one, strongly supported phylogenetic hypothesis. The primary features of that hypothesis are: (1) Adenophorus is monophyletic; (2) subgenus Oligadenus is paraphyletic; (3) the enigmatic endemic Hawaiian species Grammitis tenella is strongly supported as the sister taxon to Adenophorus; (4) highly divided leaf blades are evolutionarily derived in the group and simple leaves are ancestral; and, (5) the biogeographical origin of the common ancestor of the Adenophorus-G. tenella clade remains unresolved, although a neotropical origin seems most likely.