Comparison of groupings among members of the genus Aspergillus based on phylogeny and production of bioactive compounds

Three hundred sixty-six Aspergillus strains preserved at the National Institute of Technology and Evaluation (NITE) were compared as to phylogenetic relationships (11 species-clusters) based on the DNA sequences of the D1/D2 domains of LSU rRNA and ITS regions, including the 5.8S rRNA and biological activities of their secondary metabolites. The results showed relatively well correlation between the phylogenetic distribution and the production of bioactive compounds, especially, antimicrobial activities.     

Preliminary phylogenetic analysis of plastid-encoded genes from an anomalously pigmented dinoflagellate Gymnodinium mikimotoi (Gymnodiniales, Dinophyta)

We cloned and sequenced three plastid-encoded genes, psbA (encoding D1 protein), psaA (encoding P700 chlorophyll a apoprotein) and the small-subunit ribo-somal RNA (pl-SSU rRNA) from an anomalously pigmented dinoflagellate, Gymnodinium mikimotoi Miyake et Kominami ex Oda, with a plastid containing 19′-hexanoyloxyfucoxanthin, 19′-butanoyloxyfucoxanthin and fucoxanthin instead of peridinin as the major carot-enoids. Molecular phylogenetic trees based on the deduced amino acid sequences of D1 and P700 chlorophyll a apoprotein and nucleotide sequence of pl-SSU rRNA were then constructed separately. In the D1 tree, G. mikimotoi and typically pigmented dinofl age Nates harboring a peridinin type plastid were monophyletic and G. mikimotoi was positioned most basally within the dinoflagellate lineage. The dinoflagellate lineage was the sister group of heterokonts and the dinoflagellates/heterokonts lineage was clustered with the rhodophytes/cryptophyte lineage. In the P700 chlorophyll a apoprotein phylogenetic tree, G. mikimotoi was clustered with a rhodo-phyte, a cryptophyte and a heterokont. In the pl-SSU rRNA tree, G. mikimotoi and haptophytes constituted a monophyletic group associated with rhodophytes and heterokonts. These results, derived from the three phylogenetic analyses, support the hypothesis that the plastid of G. mikimotoi belongs to the rhodoplast lineage. Although we have previously demonstrated that D1 from peridinin type dinofl age Nates lacks a ‘C-terminus extension’ (which should be removed by proteolytic cleavage from the D1 precursor), the D1 from G. mikimotoi revealed a C-terminus extension that is different from those of other photosynthetic organisms with respect to the length of the amino acid residues.     

Evaluation of sequence alignments and oligonucleotide probes with respect to three-dimensional structure of ribosomal RNA using ARB software package

Background  

Availability of high-resolution RNA crystal structures for the 30S and 50S ribosomal subunits and the subsequent validation of comparative secondary structure models have prompted the biologists to use three-dimensional structure of ribosomal RNA (rRNA) for evaluating sequence alignments of rRNA genes. Furthermore, the secondary and tertiary structural features of rRNA are highly useful and successfully employed in designing rRNA targeted oligonucleotide probes intended for in situ hybridization experiments. RNA3D, a program to combine sequence alignment information with three-dimensional structure of rRNA was developed. Integration into ARB software package, which is used extensively by the scientific community for phylogenetic analysis and molecular probe designing, has substantially extended the functionality of ARB software suite with 3D environment.     

Phylogenetic Analysis of Ruminant Theileria spp. from China Based on 28S Ribosomal RNA Gene

Species identification using DNA sequences is the basis for DNA taxonomy. In this study, we sequenced the ribosomal large-subunit RNA gene sequences (3,037-3,061 bp) in length of 13 Chinese Theileria stocks that were infective to cattle and sheep. The complete 28S rRNA gene is relatively difficult to amplify and its conserved region is not important for phylogenetic study. Therefore, we selected the D2-D3 region from the complete 28S rRNA sequences for phylogenetic analysis. Our analyses of 28S rRNA gene sequences showed that the 28S rRNA was useful as a phylogenetic marker for analyzing the relationships among Theileria spp. in ruminants. In addition, the D2-D3 region was a short segment that could be used instead of the whole 28S rRNA sequence during the phylogenetic analysis of Theileria, and it may be an ideal DNA barcode.     

Strategies for Amplification by Polymerase Chain Reaction of the Complete Sequence of the Gene Encoding Nuclear Large Subunit Ribosomal RNA in Corals

The nearly complete nuclear large subunit ribosomal RNA (LSU rRNA) gene in corals was amplified by primers designed from polymerase chain reaction (PCR) strategies. The motif of the putative 3′-terminus of the LSU rRNA gene was sequenced and identified from intergenic spacer (IGS) clones obtained by PCR using universal primers designed for corals. The 3′-end primer was constructed in tandem with the universal 5′-end primer for the LSU rRNA gene. PCR fragments of 3500 bp were amplified for octocorals and non-Acropora scleractinian corals. More than 80% of the Acropora LSU rRNA gene (3000 bp) was successfully amplified by modification of the 5′-end of the IGS primer. Analysis of the 5′-end of LSU rDNA sequences, including the D1 and D2 divergent domains, indicates that the evolutionary rate of the LSU rDNA differs among these taxonomic groups of corals. The genus Acropora showed the highest divergence pattern in the LSU rRNA gene, and the presence of a long branch of the Acropora clade from the other scleractinian corals in the phylogenetic tree indicates that the evolutionary rate of Acropora LSU rDNA might have accelerated after divergence from the common ancestor of scleractinian corals. Received February 17, 2000; accepted June 12, 2000.     

GENETIC VARIABILITY AND MOLECULAR PHYLOGENY OF DINOPHYSIS SPECIES (DINOPHYCEAE) FROM NORWEGIAN WATERS INFERRED FROM SINGLE CELL ANALYSES OF rDNA1

The objectives of this study were to determine rDNA sequences of the most common Dinophysis species in Scandinavian waters and to resolve their phylogenetic relationships within the genus and to other dinoflagellates. A third aim was to examine the intraspecific variation in D. acuminata and D. norvegica, because these two species are highly variable in both morphology and toxicity. We obtained nucleotide sequences of coding (small subunit [SSU], partial large subunit [LSU], 5.8S) and noncoding (internal transcribed spacer [ITS]1, ITS2) parts of the rRNA operon by PCR amplification of one or two Dinophysis cells isolated from natural water samples. The three photosynthetic species D. acuminata, D. acuta, and D. norvegica differed in only 5 to 8 of 1802 base pairs (bp) within the SSU rRNA gene. The nonphotosynthetic D. rotundata (synonym Phalacroma rotundatum[Claparède et Lachmann] Kofoid et Michener), however, differed in approximately 55 bp compared with the three photosynthetic species. In the D1 and D2 domains of LSU rDNA, the phototrophic species differed among themselves by 3 to 12 of 733 bp, whereas they differed from D. rotundata by more than 100 bp. This supports the distinction between Dinophysis and Phalacroma. In the phylogenetic analyses based on SSU rDNA, all Dinophysis species were grouped into a common clade in which D. rotundata diverged first. The results indicate an early divergence of Dinophysis within the Dinophyta. The LSU phylogenetic analyses, including 4 new and 11 Dinophysis sequences from EMBL, identified two major clades within the phototrophic species. Little or no intraspecific genetic variation was found in the ITS1–ITS2 region of single cells of D. norvegica and D. acuminata from Norway, but the delineation between these two species was not always clear.     

Genetic interrelationships of proteolyticClostridium botulinum types A,B, and F and other members of theClostridium botulinum complex as revealed by small-subunit rRNA gene sequences

The phylogenetic interrelationships of members of theClostridium botulinum complex of species was investigated by direct sequencing of their 16S rRNA genes. Comparative analysis of the 16S rRNA sequences demonstrated the presence of four phylogenetically distinct lineages corresponding to: i) proteolyticC. botulinum types A, B, and F, andC. sporogenes, ii) saccharolytic types B, E and F, iii) types C and D andC. novyi type A, and iv) type G andC. subterminale. The phylogenetic groupings obtained from the 16S rRNA were in complete agreement with the four divisions recognised within the species complex on the basis of phenotypic criteria.     

Molecular phylogeny,diagnostics, and diversity of plant‐parasitic nematodes of the genus Hemicycliophora (Nematoda: Hemicycliophoridae)

The genus Hemicycliophora (Nematoda: Hemicycliophoridae) contains 132 valid species of plant‐parasitic nematodes, collectively known as ‘sheath nematodes’. Hemicycliophora spp. are characterized morphologically by a long stylet with rounded basal knobs and a cuticular sheath, present in juvenile and adult stages. Populations of 20 valid and 14 putative species of Hemicycliophora and Loofia from several countries were characterized morphologically using light (LM) and scanning electron microscopy (SEM) and molecularly using the D2‐D3 segments of 28S rRNA and internal transcribed spacer (ITS) rRNA gene sequences. LM and SEM observations provided new details on the morphology of these species. PCR‐restriction fragment length polymorphisms (PCR‐RFLPs) of the D2‐D3 of 28S rDNA were proposed for identification of the species. Phylogenetic relationships within populations of 36 species of the genus Hemicycliophora using 102 D2‐D3 of 28S rDNA and 97 ITS rRNA gene sequences as inferred from Bayesian analysis are reconstructed and discussed. Ancestral state reconstructions of diagnostic characters (body and stylet length, number of body annuli, shape of vulval lip and tail), using maximum parsimony and Bayesian inference, revealed that none of the traits are individually reliable characters for classifying the studied sheath nematode. The Shimodaira–Hasegawa test rejected the validity of the genus Loofia. This is the most complete phylogenetic analysis of Hemicycliophora species conducted so far. © 2014 The Linnean Society of London     

酒鬼酒酿造环境异常球菌属菌株的生物学特征及系统发育分析

研究对分离自馥郁香型白酒酒鬼酒制曲和发酵车间空气的8株异常球菌属(Deinococcus)菌株的特征及系统发育分析进行研究。采用添加体积分数10%酒鬼酒混合浸汁的营养琼脂培养基分离,挑选并纯化与异常球菌属菌株表型相似的菌株,考察其相关生理生化特征,并进行基于16S rRNA基因序列的系统发育分析。通过表型特点筛选,得到23株异常球菌属疑似菌株。系统发育分析表明,23株菌株中有8株属于异常球菌属,归属于该属的4个种(D.radiotolerans、D.daejeonensis、D.ficus和D.yunweiensis)。形态观察和生理生化实验表明,这些异常球菌属菌株在内生孢子、革兰染色、过氧化氢酶实验、生长温度范围、生长pH范围、多聚物水解等特征方面,与它们系统发育关系最为密切的典型菌株之间存在不同程度的差异。     

Rhodotorula himalayensis sp. nov., a novel psychrophilic yeast isolated from Roopkund Lake of the Himalayan mountain ranges, India

Twenty-five psychrophilic yeasts were isolated from the soil of Roopkund Lake, Himalayas, India. Two colony morphotypes were identified and representatives of ‘morphotype 1’ were identified as Cryptococcus gastricus. Representatives of ‘morphotype 2’, namely 3A^T, 4A, 4B and Rup4B, showed similar phenotypic properties and are identical with respect to the nucleotide sequence of the ITS1-5.8S rRNA gene-ITS2 region and D1/D2 domain of the 26S rRNA gene. The sequence of D1/D2 domain of 3A^T shows 97.6–98.8% similarity with Rhodotorula psychrophila CBS10440^T, Rhodotorula glacialis CBS10437^T and Rhodotorula psychrophenolica CBS10438^T and in the neighbour-joining phylogenetic tree strains; 3A^T, 4A, 4B and Rup4B form a cluster with Rhodotorula glacialis and Rhodotorula psychrophila. Strains 3A^T, 4A, 4B and Rup4B also differ from their nearest phylogenetic relatives in several biochemical characteristics such as in assimilation of d-galactose, l-sorbose, maltose, citrate, d-glucuronate and creatinine. Thus, based on the phylogenetic analysis and the phenotypic differences 3A^T, 4A, 4B and Rup 4B are assigned the status of a new species of Rhodotorula for which the name Rhodotorula himalayensis sp. nov. is proposed with 3A^T as the type strain (=CBS10539^T =MTCC8336^T). GenBank/EMBL accession numbers for (partial) 18SrRNA gene-ITS1-5.8S rRNA gene-ITS2-26S rRNA gene (partial) sequences of Rhodotorula himalayensis sp. nov. 3A^T is AM410635.     

Phylogenetic analysis of Glomeromycota by partial LSU rDNA sequences

We analyzed the large subunit ribosomal RNA (rRNA) gene [LSU ribosomal DNA (rDNA)] as a phylogenetic marker for arbuscular mycorrhizal (AM) fungal taxonomy. Partial LSU rDNA sequences were obtained from ten AM fungal isolates, comprising seven species, with two new primers designed for Glomeromycota LSU rDNA. The sequences, together with 58 sequences available from the databases, represented 31 AM fungal species. Neighbor joining and parsimony analyses were performed with the aim of evaluating the potential of the LSU rDNA for phylogenetic resolution. The resulting trees indicated that Archaeosporaceae are a basal group in Glomeromycota, Acaulosporaceae and Gigasporaceae belong to the same clade, while Glomeraceae are polyphyletic. The results support data obtained with the small subunit (SSU) rRNA gene, demonstrating that the LSU rRNA gene is a useful molecular marker for clarifying taxonomic and phylogenetic relationships in Glomeromycota.     

Evolution of fragmented mitochondrial ribosomal RNA genes inChlamydomonas

The fragmented mitochondrial ribosomal RNAs (rRNAs) of the green algaeChlamydomonas eugametos andChlamydomonas reinhardtii are discontinuously encoded in subgenic modules that are scrambled in order and interspersed with protein coding and tRNA genes. The mitochondrial rRNA genes of these two algae differ, however, in both the distribution and organization of rRNA coding information within their respective genomes. The objectives of this study were (1) to examine the phylogenetic relationships between the mitochondrial rRNA gene sequences ofC. eugametos andC. reinhardtii and those of the conventional mitochondrial rRNA genes of the green alga,Prototheca wickerhamii, and land plants and (2) to attempt to deduce the evolutionary pathways that gave rise to the unusual mitochondrial rRNA gene structures in the genusChlamydomonas. Although phylogenetic analysis revealed an affiliation between the mitochondrial rRNA gene sequences of the twoChlamydomonas taxa to the exclusion of all other mitochondrial rRNA gene sequences tested, no specific affiliation was noted between theChlamydomonas sequences andP. wickerhamii or land plants. Calculations of the minimal number of transpositions required to convert hypothetical ancestral rRNA gene organizations to the arrangements observed forC. eugametos andC. reinhardtii mitochondrial rRNA genes, as well as a limited survey of the size of mitochondrial rRNAs in other members of the genus, lead us to propose that the last common ancestor ofChlamydomonas algae contained fragmented mitochondrial rRNA genes that were nearly co-linear with conventional rRNA genes.     

Sequence and Phylogenetic Analysis of SSU rRNA Gene of Five Microsporidia

The complete small subunit rRNA (SSU rRNA) gene sequences of five microsporidia including Nosema heliothidis, and four novel microsporidia isolated from Pieris rapae, Phyllobrotica armta, Hemerophila atrilineata, and Bombyx mori, respectively, were obtained by PCR amplification, cloning, and sequencing. Two phylogenetic trees based on SSU rRNA sequences had been constructed by using Neighbor-Joining of Phylip software and UPGMA of MEGA4.0 software. The taxonomic status of four novel microsporidia was determined by analysis of phylogenetic relationship, length, G+C content, identity, and divergence of the SSU rRNA sequences. The results showed that the microsporidia isolated from Pieris rapae, Phyllobrotica armta, and Hemerophila atrilineata have close phylogenetic relationship with the Nosema, while another microsporidium isolated from Bombyx mori is closely related to the Endoreticulatus. So, we temporarily classify three novel species of microsporidia to genus Nosema, as Nosema sp. PR, Nosema sp. PA, Nosema sp. HA. Another is temporarily classified into genus Endoreticulatus, as Endoreticulatus sp. Zhenjiang. The result indicated as well that it is feasible and valuable to elucidate phylogenetic relationships and taxonomic status of microsporidian species by analyzing information from SSU rRNA sequences of microsporidia.     

A phylogenetic reconsideration of suctorian ciliates (Protista,Ciliophora, Phyllopharyngea) based on small subunit rRNA gene sequences

Compared with other ciliated protozoa, molecular studies of phylogenetic relationships within the subclass Suctoria are rare. In this work, phylogenetic analyses focusing on this group were performed based on all data available. In addition, the small subunit ribosomal RNA (SSU rRNA) genes of three suctorian ciliates (Acineta compressa, Acineta tuberosa and Paracineta limbata) were newly sequenced. Furthermore, the putative secondary structures of the variable region 2 of the SSU rRNA gene were predicted and compared within the Suctoria. Our results show that (i) there is support for the monophyly of the subclass Suctoria, which is a sister clade to the cyrtophorids; (ii) based on combined morphologic and molecular features, we propose the following evolutionary routine within the Suctoria: Exogenina – Evaginogenina – Endogenina; (iii) the similarities of the secondary structures of the V2 region and the SSU rRNA gene sequences within the subclass Suctoria are consistent with the branching of the phylogenetic lineages.     

Molecular phylogeny of an unidentified <Emphasis Type="Italic">Haliphthoros</Emphasis>-like marine oomycete and <Emphasis Type="Italic">Haliphthoros milfordensis</Emphasis> inferred from nuclear-encoded small- and large-subunit rRNA genes and mitochondrial-encoded cox2 gene

The SSU rRNA, LSU rRNA, and cox2 genes of an unidentified Haliphthoros-like marine oomycete (NJM0034) and Haliphthoros milfordensis (NJM0131) were sequenced, and their phylogenetic relationships are analyzed and discussed. All phylogenetic trees showed that NJM0034 and NJM0131 were branched before separation of the two main saprolegnian and peronosporalean clades. These data suggest that the clear phylogenetic separation of those marine oomycete endoparasites from the two main oomycete clades. Excepting the LSU rRNA gene tree, NJM0034 and Haliphthoros spp. did not form a monophyletic group. On the other hand, H. milfordensis NJM0131 clustered with H. philippinensis SANK 15178, not with H. milfordensis NJM9434 in the cox2 amino acid sequence (COII) tree. This result strongly suggests that a taxonomic reinvestigation of the genus Haliphthoros should be considered.     

Implications of complete nuclear large subunit ribosomal RNA molecules from the harmful unarmored dinoflagellate Cochlodinium polykrikoides (Dinophyceae) and relatives

The D1/D2 domains of large subunit (LSU) rDNA have commonly been used for phylogenetic analyses of dinoflagellates; however, their properties have not been evaluated in relation to other D domains due to a deficiency of complete sequences. This study reports the complete LSU rRNA gene sequence in the causative unarmored dinoflagellate Cochlodinium polykrikoides, a member of the order Gymnodiniales, and evaluated the segmented domains and secondary structures when compared with its relatives. Putative LSU rRNA coding regions were recorded to be 3433 bp in length (49.0% GC content). A secondary structure predicted from the LSU and 5.8S rRNAs and parsimony analyses showed that most variation in the LSU rDNA was found in the 12 divergent (D) domains. In particular, the D2 domain was the most informative in terms of recent evolutional and taxonomic aspects, when compared with both the phylogenetic tree topologies and molecular distance (approximately 10 times higher) of the core LSU. Phylogenetic analysis was performed with a matrix of LSU DNA sequences selected from domains D2 to D4 and their flanking core sequences, which showed that C. polykrikoides was placed on the same branch with Akashiwo sanguinea in the “GPP” complex, which is referred to the gymnodinioid, peridinioid and prorocentroid groups. A broad phylogeny showed that armored and unarmored dinoflagellates were never clustered together; instead, they were clearly divided into two groups: the GPP complex and Gonyaulacales. The members of Gymnodiniales were always interspersed with peridinioid, prorocentroid and dinophysoid forms. This supports previous findings showing that the Gymnodiniales are polyphyletic. This study highlights the proper selection of LSU rDNA molecules for molecular phylogeny and signatures.     

Molecular data and the evolutionary history of dinoflagellates

We have sequenced small-subunit (SSU) ribosomal RNA (rRNA) genes from 16 dinoflagellates, produced phylogenetic trees of the group containing 105 taxa, and combined small- and partial large-subunit (LSU) rRNA data to produce new phylogenetic trees. We compare phylogenetic trees based on dinoflagellate rRNA and protein genes with established hypotheses of dinoflagellate evolution based on morphological data. Protein-gene trees have too few species for meaningful in-group phylogenetic analyses, but provide important insights on the phylogenetic position of dinoflagellates as a whole, on the identity of their close relatives, and on specific questions of evolutionary history. Phylogenetic trees obtained from dinoflagellate SSU rRNA genes are generally poorly resolved, but include by far the most species and some well-supported clades. Combined analyses of SSU and LSU somewhat improve support for several nodes, but are still weakly resolved. All analyses agree on the placement of dinoflagellates with ciliates and apicomplexans (=Sporozoa) in a well-supported clade, the alveolates. The closest relatives to dinokaryotic dinoflagellates appear to be apicomplexans, Perkinsus, Parvilucifera, syndinians and Oxyrrhis. The position of Noctiluca scintillans is unstable, while Blastodiniales as currently circumscribed seems polyphyletic. The same is true for Gymnodiniales: all phylogenetic trees examined (SSU and LSU-based) suggest that thecal plates have been lost repeatedly during dinoflagellate evolution. It is unclear whether any gymnodinialean clades originated before the theca. Peridiniales appear to be a paraphyletic group from which other dinoflagellate orders like Prorocentrales, Dinophysiales, most Gymnodiniales, and possibly also Gonyaulacales originated. Dinophysiales and Suessiales are strongly supported holophyletic groups, as is Gonyaulacales, although with more modest support. Prorocentrales is a monophyletic group only in some LSU-based trees. Within Gonyaulacales, molecular data broadly agree with classificatory schemes based on morphology. Implications of this taxonomic scheme for the evolution of selected dinoflagellate features (the nucleus, mitosis, flagella and photosynthesis) are discussed.     

MORPHOLOGICAL AND 16S rRNA GENE EVIDENCE FOR RECLASSIFICATION OF THE PARALYTIC SHELLFISH TOXIN PRODUCING APHANIZOMENON FLOS‐AQUAE LMECYA 31 AS APHANIZOMENON ISSATSCHENKOI (CYANOPHYCEAE)1

A taxonomic reevaluation of the paralytic shellfish toxin (saxitoxins) producing cyanobacterium Aphanizomenon flos‐aquae Ralfs ex Born. & Flah. LMECYA31 was done using morphology and 16S rRNA gene sequences. We found that strain LMECYA31 was incorrectly identified as Aph. flos‐aquae based on (a) lack of bundle formation in trichomes, (b) shape of terminal cells in the trichomes, (c) lower similarity (<97.5%) in the 16S rRNA gene sequences relative to those of Aph. flos‐aquae, and (d) comparison within a phylogenetic tree of 16S rRNA gene sequences. The shape of the terminal trichome cells and the shape and size of the vegetative cell, heterocyst, and akinete in strain LMECYA31 match characters of Aph. issatschenkoi (Ussachew) Proschkina‐Larvernko. 16S rRNA gene sequences and phylogenetic clusters constructed from 16S rRNA gene sequences support our conclusion that strain LMECYA31 should be Aph. issatschenkoi.