Application of <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">parE</Emphasis> sequence similarity analyses for differentiation of species within the genus <Emphasis Type="Italic">Geobacillus</Emphasis>

The primary structures of the genes encoding the β-subunits of a type II topoisomerase (gyrase, gyrB) and a type IV topoisomerase (parE) were determined for 15 strains of thermophilic bacteria of the genus Geobacillus. The obtained sequences were used for analysis of the phylogenetic similarity between members of this genus. Comparison of the phylogenetic trees of geobacilli constructed on the basis of the 16S rRNA, gyrB, and parE gene sequences demonstrated that the level of genetic distance between the sequences of the genes encoding the β-subunits of type II topoisomerases significantly exceeded the values obtained by comparative analysis of the 16S rRNA gene sequences of Geobacillus strains. It was shown that, unlike the 16S rRNA gene analysis, comparative analysis of the gyrB and parE gene sequences provided a more precise determination of the phylogenetic position of bacteria at the species level. The data obtained suggest the possibility of using the genes encoding the β-subunits of type II topoisomerases as phylogenetic markers for determination of the species structure of geobacilli.     

Discrimination among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> H serotypes,serovars and strains based on 16S rRNA, <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">aroE</Emphasis> gene sequence analyses

Our aim was to investigate the capability of each of three genes, 16S rRNA, gyrB and aroE, to discriminate, first, among Bacillus thuringiensis H serotypes; second, among B. thuringiensis serovars from the same H serotype; and third, among B. thuringiensis strains from the same serovar. The 16S rRNA, gyrB and aroE genes were amplified from 21 B. thuringiensis H serotypes and their nucleotide sequences determined. Additional strains from four B. cereus sensu lato species were included for comparison purposes. These sequences were pair-wise compared and phylogenetic relationships were revealed. Each of the three genes under study could discriminate among B. thuringiensis H serotypes. The gyrB and aroE genes showed a discriminatory power among B. thuringiensis H serotypes up to nine fold greater than that of the 16S rRNA gene. The gyrB gene was retained for subsequent analyses to discriminate B. thuringiensis serovars from the same H serotype and to discriminate strains from same serovar. A total of 42 B. thuringiensis strains, which encompassed 25 serovars from 12 H serotypes, were analyzed. The gyrB gene nucleotide sequences were different enough as to be sufficient to discriminate among B. thuringiensis serovars from the same H serotype and among B. thuringiensis strains from the same serovar. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration of <Emphasis Type="Italic">Alnus acuminata</Emphasis> H.B.K. ssp. <Emphasis Type="Italic">acuminata</Emphasis> and its root nodulation by <Emphasis Type="Italic">Frankia</Emphasis>

A procedure for in vitro plant regeneration of Alnus acuminata from epicotyls with cotyledonary buds was developed using different media formulations with different growth regulators and carbon sources. The development of multiple buds on explants at the initiation step was obtained with MS at 1/2 strength with either 1 or 2M of BAP but not without it. Multiplication gave up to 15 elongating shoots by explant, the best medium being MS supplemented with vitamins from B5 medium, 1M of BAP and 87mM sucrose. Rooting of about 88% occurred in the medium MS with 83 mM sucrose and 1M IBA. Alnus acuminata did not developed well on WPM. Roots of in vitro propagated plants were nodulated by Alnus-infective Frankia. The root nodules show a typical alder root nodule anatomy and differentiation pattern and effectively fixed nitrogen. Rhamnaceae-infective Frankia did not nodulate in vitro cultivated Alnus acuminata suggesting that symbiotic recognition was not altered by in vitro regeneration of the plant.     

Phylogenetic placement of <Emphasis Type="Italic">Marasmiellus juniperinus</Emphasis>

Recent collections and the type specimen of Marasmiellus juniperinus, the type species of the genus, were examined. Phylogenetic placement, based on ribosomal large subunit (LSU) and internally transcribed spacer (ITS) sequences, is within the lentinuloid clade, nested among Gymnopus taxa. This placement dictates genus name usage and phylogenetic position of other putative species of Marasmiellus. The mating system is tetrapolar.     

Distribution of <Emphasis Type="Italic">Hordoindoline</Emphasis> genes in the genus <Emphasis Type="Italic">Hordeum</Emphasis>

Hordoindoline (Hin) genes, which are known to comprise Hina, Hinb-1, and Hinb-2, are associated with grain hardness in barley. However, the interspecific variation in the Hin genes in the genus Hordeum has not been studied in detail. We examined the variation in Hin genes and used it to infer the phylogenetic relationships between the genes found in two H. vulgare subspecies (cultivated barley and H. vulgare subsp. spontaneum) and 10 wild relatives (H. bogdanii, H. brachyantherum, H. bulbosum, H. chilense, H. comosum, H. marinum, H. murinum, H. patagonicum, H. pusillum, and H. roshevitzii). The Hina and Hinb genes of these species were amplified by PCR. We found two Hinb genes in three wild species (H. bogdanii, H. brachyantherum, and H. roshevitzii) and preliminarily named them Hinb-A and Hinb-B. Cluster analysis showed that the 17 Hinb genes present in Hordeum formed two distinct clusters (named A and B). Seven Hinb genes were included in Cluster-A, and 10 Hinb genes were included in Cluster-B. All Hinb-A genes were included in Cluster-A, while all of the Hinb-B genes were included in Cluster-B. In contrast, the Hinb-1 and Hinb-2 genes in H. vulgare were included in Cluster-B. These results suggest that the Hinb genes duplicated during the early stages of diversification in the genus Hordeum. On the other hand, the Hinb-1 and Hinb-2 genes in H. vulgare seem to have been generated by a duplication of the Hinb gene after the split of the lineages leading to H. vulgare and H. bulbosum.     

Phylogenetic Studies of <Emphasis Type="Italic">Gordonia</Emphasis> Species Based on <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">secA1</Emphasis> Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.     

Phylogeny of <Emphasis Type="Italic">Litsea</Emphasis> and related genera (Laureae-Lauraceae) based on analysis of <Emphasis Type="Italic">rpb2</Emphasis> gene sequences

The relationship between Litsea and related genera is currently unclear. Previous molecular studies on these taxa using cpDNA and nrITS were unable to produce well-resolved phylogenetic trees. In this study, we explored the potential of the rpb2 gene as a source of molecular information to better resolve the phylogenetic analysis. Although rpb2 was believed to be a single-copy gene, our cloning results showed that most species examined possessed several copies of these sequences. However, the genetic distance among copies from any one species was low, and these copies always formed monophyletic groups in our molecular trees. Our phylogenetic analyses of rpb2 data resulted in better resolved tree topologies compared to those based on cpDNA or nrITS data. Our results show that monophyly of the genus Litsea is supported only for section Litsea. As a genus, Litsea was shown to be polyphyletic. The genera Actinodaphne and Neolitsea were resolved as monophyletic groups in all analyses. They were also shown to be sisters and closer to the genus Lindera than to the genus Litsea. Our results also revealed that the genus Lindera is not a monophyletic group.     

Phylogenetic analysis of smelts (Osmeridae) based on the variation of cytochrome <Emphasis Type="Italic">b</Emphasis> gene

Putative phylogenetic relationships between all smelt species inhabiting Russian waters were studied using RFLP and the data on divergence between nucleotide sequences of the mitochondrial gene encoding cytochrome b. All types of phylogenetic trees (NJ, MP, and Bayesian) displayed stable clustering into isolated groups corresponding to the division of the subfamily Osmerinae into three genera, Mallotus, Osmerus, and Hypomesus. It was demonstrated that the Mallotus phylum was the first to diverge from a hypothetical common ancestor of the smelts. Later, it divided into two clusters corresponding to the modern subspecies Mallotus villosus villosus and M. v. catervarius. The phylum of the genus Osmerus diverged later than the genus Mallotus and also divided into two clusters, one including O. mordax of the Pacific (Sea of Japan) and Arctic (Kara and Barents Seas) basins and the other, anadromous and resident ecotypes of the O. eperlanus from the Baltic Sea basin. The smelts of the genus Hypomesus is the youngest phylogenetic group among the taxa studied; it forms three individual clusters matching the species H. olidus, H. nipponensis, and H. japonicus. The proposed phylogenetic hypothesis is discussed from the standpoint of its compliance with the phylogenetic constructions based on the external morphological and osteological traits.     

Two Distinct <Emphasis Type="Italic">Photobacterium</Emphasis> Populations Thrive in Ancient Mediterranean Sapropels

Eastern Mediterranean sediments are characterized by the periodic occurrence of conspicuous, organic matter-rich sapropel layers. Phylogenetic analysis of a large culture collection isolated from these sediments revealed that about one third of the isolates belonged to the genus Photobacterium. In the present study, 22 of these strains were examined with respect to their phylogenetic and metabolic diversity. The strains belonged to two distinct Photobacterium populations (Mediterranean cluster I and II). Strains of cluster I were isolated almost exclusively from organic-rich sapropel layers and were closely affiliated with P. aplysiae (based on their 16S rRNA gene sequences). They possessed almost identical Enterobacterial Repetitive Intergenic Consensus (ERIC) and substrate utilization patterns, even among strains from different sampling sites or from layers differing up to 100,000 years in age. Strains of cluster II originated from sapropels and from the surface and carbon-lean intermediate layers. They were related to Photobacterium frigidiphilum but differed significantly in their fingerprint patterns and substrate spectra, even when these strains were obtained from the same sampling site and layer. Temperature range for growth (4 to 33°C), salinity tolerance (5 to 100‰), pH requirements (5.5–9.3), and the composition of polar membrane lipids were similar for both clusters. All strains grew by fermentation (glucose, organic acids) and all but five by anaerobic respiration (nitrate, dimethyl sulfoxide, anthraquinone disulfonate, or humic acids). These results indicate that the genus Photobacterium forms subsurface populations well adapted to life in the deep biosphere.     

Divergent Evolution of Symbiotic Bacteria: Rhizobia of the Relic Legume <Emphasis Type="Italic">Vavilovia formosa</Emphasis> Form an Isolated Group within <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viciae</Emphasis>

Comparative sequence analysis of symbiotic genes (nodA, nodC, nodD, nifH), which are elements of accessory component of the rhizobial genome, demonstrated that the strains of Rhizobium leguminosarum bv. viciae, isolated from the nodules of a relic legume, Vavilovia formosa, the closest relative of hypothetical common ancestor of the tribe Fabeae, represented a group separated from the strains of R. leguminosarum bv. viciae, isolated from other representatives of this tribe (Vicia, Lathyrus, Pisum, Lens). No isolation was observed relative to the genes representing the core component of the rhizobial genome (16S rDNA, ITS, glnII) or relative to host specificity of the rhizobia. The data obtained suggest that sequence divergence of symbiotic genes marks the initial stage of sympatric speciation, which can be classified as the isolation of the relic “vaviloviae” symbiotype, a possible evolutionary precursor of the “viciae” biotype.     

Molecular Phylogenetic Relationships Between Prostanoid-Containing Okinawan Soft Coral (<Emphasis Type="Italic">Clavularia viridis</Emphasis>) and Nonprostanoid-Containing <Emphasis Type="Italic">Clavularia</Emphasis> Species Based on Ribosomal ITS Sequence

To study phylogenetic relationships among Okinawan soft corals of the genus Clavularia, the ribosomal internal transcribed spacer sequences of host corals and the 18S rDNA sequences of symbiotic algae were analyzed. The molecular phylogenetic trees of hosts showed that a prostanoid-containing species, Clavularia viridis, is deeply diverged from other species of Clavularia which do not biosynthesize the prostanoids as the main secondary metabolites. Comparison of their trees suggested poor phylogenetic concordance between hosts and symbionts.     

The evolutionary history of PDR in <Emphasis Type="Italic">Brachypodium</Emphasis><Emphasis Type="Italic">distachyon</Emphasis> polyploids

The ATP-binding cassette transporter genes include the pleiotropic drug resistance (PDR) family found only in fungi and plants. These transporters transport toxic compounds across biological membranes. Here, we investigated the evolution of the PDR1 gene in Brachypodium distachyon, a widely distributed temperate grass species that belongs to the Poaceae (Gramineae) family, which also contains the domesticated cereal crops. Because this species has multiple ploidy levels, investigating PDR1 evolution in B. distachyon will offer insights into the formation and evolution of polyploidy. From 23 B. distachyon ecotypes, 39 PDR1 homologs were identified. All ecotypes had either one or two PDR1 copies. Based on restriction site analysis, the PDR1 homologs were classified as E or H type. All but one diploid and tetraploid ecotypes had only a single H type PDR1. All but one hexaploid ecotypes had both an E and a H type PDR1. Phylogenetic analysis revealed that each type formed a well-supported cluster. The two PDR1 types appeared to evolve differently. These different evolutionary patterns could indicate a difference in age between the two types or might indicate different mutation rates or selection pressures on the two types. The phylogenetic analysis also revealed that the hexaploid ecotypes shared a genomic origin for their E type PDR1, but there were multiple origins for hexaploid H type PDR1 homologs. Overall, the results suggest that tetraploid and hexaploid might be misnomers in B. distachyon and suggest a complex polyploidization history during B. distachyon evolution.     

Two new species of <Emphasis Type="Italic">Trichoderma</Emphasis> from Yunnan,China

Two new species of the fungal genus Trichoderma, Trichoderma compactum and Trichoderma yunnanense, isolated from rhizosphere of tobacco in Yunnan Province, China are described based on morphological characters and phylogenetic analyses of nucleotide sequences. Our DNA sequences included the internal transcribed spacer (ITS) regions of the rDNA cluster (ITS1 and ITS2), and partial sequences of the translation elongation factor 1-alpha (tef1) and a fragment of the gene coding for endochitinase 42 (ech42). The analyses show that T. compactum belongs to the Harzianum clade, and T. yunnanense belongs to the Hamatum clade.     

Phylogenetic study of Oryzoideae species and related taxa of the Poaceae based on <Emphasis Type="Italic">atpB</Emphasis>-<Emphasis Type="Italic">rbcL</Emphasis> and <Emphasis Type="Italic">ndhF</Emphasis> DNA sequences

Oryzoideae (Poaceae) plants have economic and ecological value. However, the phylogenetic position of some plants is not clear, such as Hygroryza aristata (Retz.) Nees. and Porteresia coarctata (Roxb.) Tateoka (syn. Oryza coarctata). Comprehensive molecular phylogenetic studies have been carried out on many genera in the Poaceae. The different DNA sequences, including nuclear and chloroplast sequences, had been extensively employed to determine relationships at both higher and lower taxonomic levels in the Poaceae. Chloroplast DNA ndhF gene and atpB-rbcL spacer were used to construct phylogenetic trees and estimate the divergence time of Oryzoideae, Bambusoideae, Panicoideae, Pooideae and so on. Complete sequences of atpB-rbcL and ndhF were generated for 17 species representing six species of the Oryzoideae and related subfamilies. Nicotiana tabacum L. was the outgroup species. The two DNA datasets were analyzed, using Maximum Parsimony and Bayesian analysis methods. The molecular phylogeny revealed that H. aristata (Retz.) Nees was the sister to Chikusichloa aquatica Koidz. Moreover, P. coarctata (Roxb.) Tateoka was in the genus Oryza. Furthermore, the result of evolution analysis, which based on the ndhF marker, indicated that the time of origin of Oryzoideae might be 31 million years ago.     

Phylogeny and phylogeography of the Mediterranean species of the parasitic ant genus <Emphasis Type="Italic">Chalepoxenus</Emphasis> and its <Emphasis Type="Italic">Temnothorax</Emphasis> hosts

We analysed the phylogenetic and phylogeographic relationships of four Mediterranean species of the rare slave-making ant genus Chalepoxenus and eleven of its about 20 Temnothorax host species by sequencing the mitochondrial Cytochrome Oxidase I and II genes. Neighbour-Joining, Maximum Parsimony and Bayesian analyses based on 1320 bp indicate that the genus Chalepoxenus constitutes a monophylum. In all three analyses, C. kutteri from Southwest Europe and the workerless, “degenerate slavemaker” C. brunneus from North Africa form a monophyletic group. C. muellerianus and C. tauricus, distributed in Southern Europe and Ukraine, respectively, form a monophylum in the Neighbour-Joining and the Maximum Parsimony analysis. In our limited set of only 11 of several hundred Temnothorax species, T. flavicornis forms the sister group of Chalepoxenus. Our study further indicates paraphyly of the genus Temnothorax with respect to Chalepoxenus. Moreover, the results suggest that speciation in this slave-making genus is possibly caused by the formation of host races as different Chalepoxenus species use different hosts, and some samples seem to cluster by host species rather than by geographical distance. Received 5 September 2006; revised 17 March 2007; accepted 23 March 2007.     

<Emphasis Type="Italic">Nif</Emphasis>H-based studies on azotobacterial diversity in cotton soils of India

In order to promote the use of Azotobacter inoculants for cotton crop, a complete characterization of soil isolates of Azotobacter, isolated and screened on the basis of physiological properties, from four different cotton–wheat cropping regions of India was carried out, and their genetic diversity determined by RFLP (restriction fragment length polymorphism) analysis of the functional gene nifH. Genetic analysis of these isolates depicted a similarity coefficient of ≥80% among them, suggesting that though the isolates were obtained from different cotton soils of India, still they have large commonality in the nifH gene and constituted a homogeneous nifH population.     

Isolation of bacteria of the genus <Emphasis Type="Italic">Variovorax</Emphasis> from the <Emphasis Type="Italic">Thioploca</Emphasis> mats of Lake Baikal

Three strains of gram-negative bacteria were isolated from the mats of colorless sulfur bacteria Thioploca (Lake Baikal). The cells of new strains are motile with peritrichous flagella. Bacteria are aerobic, obligate chemoorganoheterotrophs growing within the pH range of 3.0–8.8 with the optimum at 8.3 and within the temperature range of 5–42°C with the optimum at 28°C. The cells contained menaquinones MK-8 H2 as the major component, as well as MK-7 H2 (less than 15%), while the content of ubiquinone Q8 was at least an order of magnitude lower. The G+C content of DNA in the new strains varied from 67.4 to 69.9 mol %. The level of DNA-DNA hybridization between the strains ranged from 80 to 94%, indicating that all the isolates belonged to one species. Analysis of the 16S rRNA gene nucleotide sequences of the type strain (Gen-Bank HQ400611) revealed close homologues among the known species of the genus Variovorax: 98% resemblance with the type strains of the species V. paradoxus, V. soli, V. ginsengisoli, and V. boronicumulans and 96% similarity with the type strain of V. dokdonensis. However, since the isolates differed significantly in the composition of fatty acids and isoprenoid quinones from the nearest neighbors in the phylogenetic tree, they cannot be related implicitly to the known species.     

Use of <Emphasis Type="Italic">rpoB</Emphasis> sequences and rep-PCR for phylogenetic study of <Emphasis Type="Italic">Anoxybacillus</Emphasis> species

This study was conducted to investigate the applicability of rpoB, which encodes the β subunit of RNA polymerase, to be used as an alternative to 16S rRNA gene sequence similarity analysis in the thermophilic genus Anoxybacillus. Partial rpoB sequences were generated for the 14 type strains of Anoxybacillus species and 6 other strains of four Anoxybacillus species. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The rpoB gene was found to provide a better resolution for Anoxybacillus species, with lower interspecies sequence similarities. The rpoB sequence similarity analysis permitted a more accurate discrimination of the species within the Anoxybacillus genus than the more commonly used 16S rRNA gene. Furthermore, rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP-, ERIC-, and BOX-PCR) were employed for the specimens of genus Anoxybacillus. Through comparison of the three methods, it was found that the BOX-PCR method generated more informative results than REP-PCR for the studied strains; BOX-PCR profiles were more distinct for the different strains, including a higher number of bands. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (rep-PCR) constitute a suitable molecular approach for the validation and maintenance of taxonomy within the Anoxybacillus genus. The results of this study show that rpoB and rep-PCR provide rapid and reliable methods for molecular typing of Anoxybacillus species.