Differences in structure and dynamics of Polynucleobacter communities in a temperate and a subtropical lake, revealed at three phylogenetic levels

The results of ecological studies investigating bacteria by cultivation-independent methods are expected to be influenced by the phylogenetic resolution of the applied molecular tools. This potential influence was investigated in a comparative community study on Polynucleobacter cluster bacteria (Betaproteobacteria) inhabiting the pelagic of the large, shallow, partially hypertrophic Taihu Lake located in subtropical East Asia, and the deep oligo-mesotrophic Lake Mondsee located in temperate Central Europe. The two contrasting habitats were sampled over the same period of 12 months. The community dynamics were investigated at three phylogenetic levels by fluorescent in situ hybridizations with a set of nested probes specific for the beta II clade (including the Polynucleobacter cluster), the genus-like monophyletic Polynucleobacter cluster, and four species-like subclusters. Subcluster B was the numerically dominating subcluster in both lakes over the investigation periods, but demonstrated different population dynamics in the two habitats. Interhabitat comparisons of the Polynucleobacter community structure in the two lakes in the study, and a previously investigated acidic pond indicated ecological diversification within the phylogenetically narrow Polynucleobacter cluster. These results could be obtained by help of the subcluster-specific probes, but would have been missed with probes of a lower phylogenetic resolution.     

Phylogeny of the genus Azospirillum based on 16S rDNA sequence

Abstract The 16S rDNA of 17 strains of Azospirillum , 14 assigned to one of the known species A. amazonense A. brasilense A. halopraeferens A. irakense and A. lipoferum , and the other three of uncertain taxonomic position, was sequenced after polymerase chain reaction amplification and analysed in order to investigate the phylogenetic relationships at the intra-generic and super-generic level. The phylogenetic analysis confirms that the genus Azospirillum constitutes a phylogenetically separate entity within the a subclass of Proteobacteria and that the five species are well defined. A. brasilense and A. lipoferum are closely related species and form one cluster together with A. halopraeferens ; the pair of species A. amazonense and A. irakense forms a second cluster in which Rhodospirillum centenum is also placed.     

Phylogenetic Analysis of the Genus Bifidobacterium and Related Genera Based on 16S rDNA Sequences

The 16S rRNA gene sequences were determined for type strains of 21 Bifidobacterium species. A phylogenetic tree was constructed using the determined sequences and sequences from DNA databases, which contain the sequences of 11 type strains of Bifidobacterium species and 11 strains of related genera. All species of the genus Bifidobacterium and Gardnerella vaginalis ATCC 14018 belonged to a cluster phylogenetically distinct from the other genera. The cluster was divided into two subclusters: subcluster 1 composed of most species of Bifidobacterium and G. vaginalis, and subcluster 2 consisting of two species, B. denticolens and B. inopinatum; both of which were isolated from human dental caries. In the genus Bifidobacterium, four groups of species are known to be moderately to highly related by DNA-DNA hybridization. The four groups of species exhibited more than 99% similarity among their 16S rDNA sequences within each group. These results indicated that species with around 99% or more similarity in their 16S rDNA sequences should be confirmed for species identities.     

Amplified rDNA restriction analysis and further genotypic characterisation of metal-resistant soil bacteria and related facultative hydrogenotrophs.

The level of genotypic relationship between czc+ soil bacteria mainly resistant to zinc (but also to various other metals), and related facultative hydrogenotrophs previously assigned to the genera Alcaligenes, Ralstonia, and Burkholderia was evaluated using ARDRA (Amplified Ribosomal DNA Restriction Analysis). The analysis included 44 strains isolated from harsh industrial environments in sediments, soils and wastes with high content of heavy metals. These strains were selected by their ability to grow in the presence of high concentrations of multiple heavy metals and to hybridise with czc or ncc probes. The czc operon confers resistance to cadmium, zinc and cobalt in strain Ralstonia eutropha CH34. The ncc operon confers resistance to nickel, cobalt and cadmium in strain 31A known as Alcaligenes xylosoxidans. The analysis showed a close phylogenetic clustering of the czc+ strains inside the Ralstonia genus despite of their different origins and that the Ralstonia genus contained also the hydrogenotrophs and some catabolic strains assigned to the genus Ralstonia eutropha, strains up to now registrated as CDC IV c-2 strains as well as reference strains belonging to Ralstonia solanacearum and Ralstonia pickettii. The ncc+ strains are phylogenetically less related to each other compared to the czc+ strains. This suggests that the tested czc+ strains and some of the ncc+ strains may be considered as belonging to the genus Ralstonia. Inside this major Ralstonia cluster, a subcluster gathers most of the czc+ isolates maybe giving a clue to define a new species. Besides, from 30 tested strains, 15 metal resistant strains of this subcluster proved to display the unusual mutator phenotype characteristic of the representative strain CH34.     

Satellite DNA as a phylogenetic marker: case study of three genera of the Murinae subfamily

Satellite DNA (satDNA) represent tens percent of any of the vertebrate genome. Still, a complete set of sat-DNA fragments is not determined for either species. It is known that some genus with species-specific modifications possess a satDNA characteristic for the genus. So, satDNA was used as a phylogenetic marker in some cases when precise satDNA fragment was cloned. We used the probe of the whole pericentromeric region and 4 cloned satDNA fragments of Mus musculus in order to consider probes value for phylogenesis of 3 Murinae genera. Fluorescent in situ hybridization (FISH) revealed similar pattern on metaphase spreads inside genus Mus, though some difference was noted. None of the satDNA fragment gave signal in the centromeric region on chromosomes from genera Sylvaemus and Apodemus. These data are in agreement with those on satDNA fragments in the genome determined by dot-blot hybridization: M musculus satDNA fragments are absent in the genomes of both remote genera while they are present in the genomes of the genera Mus, though in different amounts. SatDNA of each genera should be cloned for the phylogenetic purposes.     

Genotypic diversity of Acidovorax strains isolated from activated sludge and description of Acidovorax defluvii sp. nov.

Fluorescence in situ hybridization of activated sludge samples from a municipal wastewater treatment plant using oligonucleotide probes specific for Acidovorax demonstrated that these bacteria are highly abundant in this environment. For the targeted cultivation of representatives belonging to this genus, isolates grown on agar plates after serial dilution were screened by whole-cell hybridization with specific probes. The obtained strains clustered in two phylogenetic groups as determined by 16S rRNA gene sequence analyses. The isolates of one cluster were phylogenetically and genotypically closely related to A. delafieldii. In contrast, the strains of the other cluster were genotypically and phenotypically distinct from the hitherto known Acidovorax species. Therefore, a new species, Acidovorax defluvii sp. nov., was proposed for these strains. The main characteristics of the newly defined species are as follows: Gram-negative, motile or non-motile rods with rounded ends, often with large polyhydroxybutyrate granules. In broth cultures flocs are formed. Test for cytochrome oxidase is positive with all strains. The majority of strains is catalase positive and reduces nitrate. All strains are metabolically inactive against most carbohydrates and organic acids. Fatty acid patterns are typical for the genus Acidovorax. The guanine-plus-cytosine content of DNAs varies between 62 and 64 mol%. The type strain of A. defluvii is BSB411T (DSM 12644). A new 16S rRNA-targeted oligonucleotide probe reacting by in situ hybridization with all known Acidovorax species, including A. defluvii sp. nov., was designed.     

Phylogenetic position and in situ identification of ectosymbiotic spirochetes on protists in the termite gut

Phylogenetic relationships, diversity, and in situ identification of spirochetes in the gut of the termite Neotermes koshunensis were examined without cultivation, with an emphasis on ectosymbionts attached to flagellated protists. Spirochetes in the gut microbial community investigated so far are related to the genus Treponema and divided into two phylogenetic clusters. In situ hybridizations with a 16S rRNA-targeting consensus oligonucleotide probe for one cluster (known as termite Treponema cluster I) detected both the ectosymbiotic spirochetes on gut protists and the free-swimming spirochetes in the gut fluid of N. koshunensis. The probe for the other cluster (cluster II), which has been identified as ectosymbionts on gut protists of two other termite species, Reticulitermes speratus and Hodotermopsis sjoestedti, failed to detect any spirochete population. The absence of cluster II spirochetes in N. koshunensis was confirmed by intensive 16S ribosomal DNA (rDNA) clone analysis, in which remarkably diverse spirochetes of 45 phylotypes were identified, almost all belonging to cluster I. Ectosymbiotic spirochetes of the three gut protist species Devescovina sp., Stephanonympha sp., and Oxymonas sp. in N. koshunensis were identified by their 16S rDNA and by in situ hybridizations using specific probes. The probes specific for these ectosymbionts did not receive a signal from the free-swimming spirochetes. The ectosymbionts were dispersed in cluster I of the phylogeny, and they formed distinct phylogenetic lineages, suggesting multiple origins of the spirochete attachment. Each single protist cell harbored multiple spirochete species, and some of the spirochetes were common among protist species. The results indicate complex relationships of the ectosymbiotic spirochetes with the gut protists.     

Physiological versatility of the genus Rhodocista

A new purple bacterium (strain T4), capable of heterotrophic aerobic and phototrophic anaerobic growth, was isolated from waste water of a noodle factory near Hanoi, Vietnam. A comparison of 16S rDNA sequences revealed its association with the genus Rhodocista. The isolate, tentatively named "Rhodocista hanoiensis", forms cysts after growth on butyrate-containing plates at 42 degrees C. The vegetative cells form short (under aerobic conditions) or long curve-shaped rods. In contrast to other species of this genus T4 does not need any supplines (growth factors, not synthesized by the organisms). Comparative studies of T4 with Rhodocista centenaria (Rhodospirillum centenum) and Rhodocista pekingensis revealed a remarkable physiological versatility regarding nutrient spectra and survival properties of this genus.     

Non-sulfate-reducing, syntrophic bacteria affiliated with desulfotomaculum cluster I are widely distributed in methanogenic environments

The classical perception of members of the gram-positive Desulfotomaculum cluster I as sulfate-reducing bacteria was recently challenged by the isolation of new representatives lacking the ability for anaerobic sulfate respiration. For example, the two described syntrophic propionate-oxidizing species of the genus Pelotomaculum form the novel Desulfotomaculum subcluster Ih. In the present study, we applied a polyphasic approach by using cultivation-independent and culturing techniques in order to further characterize the occurrence, abundance, and physiological properties of subcluster Ih bacteria in low-sulfate, methanogenic environments. 16S rRNA (gene)-based cloning, quantitative fluorescence in situ hybridization, and real-time PCR analyses showed that the subcluster Ih population composed a considerable part of the Desulfotomaculum cluster I community in almost all samples examined. Additionally, five propionate-degrading syntrophic enrichments of subcluster Ih bacteria were successfully established, from one of which the new strain MGP was isolated in coculture with a hydrogenotrophic methanogen. None of the cultures analyzed, including previously described Pelotomaculum species and strain MGP, consumed sulfite, sulfate, or organosulfonates. In accordance with these phenotypic observations, a PCR-based screening for dsrAB (key genes of the sulfate respiration pathway encoding the alpha and beta subunits of the dissimilatory sulfite reductase) of all enrichments/(co)cultures was negative with one exception. Surprisingly, strain MGP contained dsrAB, which were transcribed in the presence and absence of sulfate. Based on these and previous findings, we hypothesize that members of Desulfotomaculum subcluster Ih have recently adopted a syntrophic lifestyle to thrive in low-sulfate, methanogenic environments and thus have lost their ancestral ability for dissimilatory sulfate/sulfite reduction.     

Phylogenetic Position and In Situ Identification of Ectosymbiotic Spirochetes on Protists in the Termite Gut

Phylogenetic relationships, diversity, and in situ identification of spirochetes in the gut of the termite Neotermes koshunensis were examined without cultivation, with an emphasis on ectosymbionts attached to flagellated protists. Spirochetes in the gut microbial community investigated so far are related to the genus Treponema and divided into two phylogenetic clusters. In situ hybridizations with a 16S rRNA-targeting consensus oligonucleotide probe for one cluster (known as termite Treponema cluster I) detected both the ectosymbiotic spirochetes on gut protists and the free-swimming spirochetes in the gut fluid of N. koshunensis. The probe for the other cluster (cluster II), which has been identified as ectosymbionts on gut protists of two other termite species, Reticulitermes speratus and Hodotermopsis sjoestedti, failed to detect any spirochete population. The absence of cluster II spirochetes in N. koshunensis was confirmed by intensive 16S ribosomal DNA (rDNA) clone analysis, in which remarkably diverse spirochetes of 45 phylotypes were identified, almost all belonging to cluster I. Ectosymbiotic spirochetes of the three gut protist species Devescovina sp., Stephanonympha sp., and Oxymonas sp. in N. koshunensis were identified by their 16S rDNA and by in situ hybridizations using specific probes. The probes specific for these ectosymbionts did not receive a signal from the free-swimming spirochetes. The ectosymbionts were dispersed in cluster I of the phylogeny, and they formed distinct phylogenetic lineages, suggesting multiple origins of the spirochete attachment. Each single protist cell harbored multiple spirochete species, and some of the spirochetes were common among protist species. The results indicate complex relationships of the ectosymbiotic spirochetes with the gut protists.     

A new family of Alteromonadaceae fam. nov., including the marine proteobacteria species Alteromonas, Pseudoalteromonas, Idiomarina i Colwellia

The taxonomic position of the marine genera Alteromonas, Pseudoalteromonas, Idiomarina, and Colwellia within the gamma subclass of the class Proteobacteria were specified on the basis of their phenotypic, genotypic, and phylogenetic characteristics. Gram-negative aerobic bacteria of the genera Alteromonas, Pseudoalteromonas, and Idiomarina and facultatively anaerobic bacteria of the genus Colwellia were found to form a phylogenetic cluster with a 16S rRNA sequence homology of 90% or higher. The characteristics of these genera presented in this paper allow their reliable taxonomic identification. Based on the analysis of our experimental data and analyses available in the literature, we propose to combine the genera Alteromonas, Pseudoalteromonas, Idiomarina, and Colwellia into a new family, Alteromonadaceae fam. nov., with the type genus Alteromonas.     

Hidden generic diversity in Neotropical birds: molecular and anatomical data support a new genus for the "Scytalopus"indigoticus species-group (Aves: Rhinocryptidae)

The genus Scytalopus is a species-rich and taxonomically complicated component of the Neotropical avian family Rhinocryptidae. Probably because Scytalopus is a superficially uniform assemblage, its monophyly has not been seriously questioned. We investigated phylogenetic relationships of a representative set of species in the genus using nuclear and mitochondrial DNA sequences as well as anatomical data, and provided the first test of its presumed monophyly by including in the analyses its hypothesized closest relatives (the genera Myornis, Eugralla, and Merulaxis) as well as most rhinocryptid genera. We found strong support for the paraphyly of the genus Scytalopus, with the Scytalopus indigoticus species-group forming a clade with Merulaxis. A well-supported clade including the genera Eugralla, Myornis, and the remaining Scytalopus was also recovered. Because these results were recovered independently and with strong support using mitochondrial and nuclear data, and were entirely consistent with anatomical data, we erect a new genus for the S.indigoticus species-group. These findings illustrate the importance of formally testing hypotheses of monophyly even for well-accepted groups of Neotropical birds.     

A new basal Carnivoramorphan (Mammalia) from the ‘Bridger B’ (Black’s Fork member,Bridger Formation,Bridgerian Nalma,middle Eocene) of Wyoming,USA

Abstract: A new genus and species of basal non‐Viverravidae Carnivoramorpha, Dawsonicyon isami, is named and described. This new taxon is based upon DMNH 19585, an almost complete skeleton, which was collected from the Black’s Fork Member (informal ‘Bridger B’ subunit) of the Bridger Formation in southwestern Wyoming, USA. The specimen is incorporated into an existing craniodental data matrix, and the associated phylogenetic analyses support the identification of this species as a new basal carnivoramorphan. This new taxon is dentally compared to all known genera of non‐viverravid basal carnivoramorphans, as well as with all known species of the problematic genus Miacis. Postcrania are compared in detail with other described specimens of non‐viverravid basal carnivoramorphans and more generally with known postcrania of viverravids. Preliminary functional interpretations of a scansorial locomotor mode are offered for this new taxon. Its implications for the diversity of middle Eocene basal carnivoramorphans is briefly discussed, including expansion of the already high diversity in the Black’s Fork Member of the Bridger Formation (at least 11 species in 8 genera).     

Culturable Actinobacteria from the Marine Sponge Hymeniacidon perleve: Isolation and Phylogenetic Diversity by 16S rRNA gene-RFLP Analysis

A total of 106 actinobacteria associated with the marine sponge Hymeniacidon perleve collected from the Yellow Sea, China were isolated using eight different media. The number of species and genera of actinobacteria recovered from the different media varied significantly, underlining the importance of optimizing the isolation conditions. The phylogenetic diversity of the actinobacteria isolates was assessed using 16S rRNA gene amplification–restriction fragment length polymorphism (RFLP) analysis of the 106 strains with different morphologies. The RFLP fingerprinting of selected strains by HhaI-digestion of the 16S rRNA genes resulted in 11 different patterns. The HhaI-RFLP analysis gave good resolution for the identification of the actinobacteria isolates at the genus level. A phylogenetic analysis using 16S rRNA gene sequences revealed that the isolates belonged to seven genera of culturable actinobacteria including Actinoalloteichus, Micromonospora, Nocardia, Nocardiopsis, Pseudonocardia, Rhodococcus, and Streptomyces. The dominant genus was Streptomyces, which represented 74% of the isolates. Three of the strains identified are candidates for new species.     

Neotropical Emerald moths: a review of the genera (Lepidoptera: Geometridae, Geometrinae)

The classification of the Neotropical genera of the Geometrinae is reviewed and 38 genera are recognized (including one nomen dubium). Seven generic synonyms are newly established and the genus Gnathosocia is described as new. Other changes established in this work include 1 species synonym and 36 new or reinstated combinations. A key to genera, based on external features, is provided for their identification. External features and genitalia of representatives of each genus are illustrated. All the 506 known Neotropical species and subspecies of Geometrinae are catalogued and details of type material, examined extensively, are given.     

Phylogenetic validation of the genera Angomonas and Strigomonas of trypanosomatids harboring bacterial endosymbionts with the description of new species of trypanosomatids and of proteobacterial symbionts

We comparatively examined the nutritional, molecular and optical and electron microscopical characteristics of reference species and new isolates of trypanosomatids harboring bacterial endosymbionts. Sequencing of the V7V8 region of the small subunit of the ribosomal RNA (SSU rRNA) gene distinguished six major genotypes among the 13 isolates examined. The entire sequences of the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes were obtained for phylogenetic analyses. In the resulting phylogenetic trees, the symbiont-harboring species clustered as a major clade comprising two subclades that corresponded to the proposed genera Angomonas and Strigomonas. The genus Angomonas comprised 10 flagellates including former Crithidia deanei and C. desouzai plus a new species. The genus Strigomonas included former Crithidia oncopelti and Blastocrithidia culicis plus a new species. Sequences from the internal transcribed spacer of ribosomal DNA (ITS rDNA) and size polymorphism of kinetoplast DNA (kDNA) minicircles revealed considerable genetic heterogeneity within the genera Angomonas and Strigomonas. Phylogenetic analyses based on 16S rDNA and ITS rDNA sequences demonstrated that all of the endosymbionts belonged to the Betaproteobacteria and revealed three new species. The congruence of the phylogenetic trees of trypanosomatids and their symbionts support a co-divergent host-symbiont evolutionary history.     

A new genus of Paramacronychiinae (Diptera: Sarcophagidae), argued from a genus-level cladistic analysis

Primorya gen.n. is described as a new genus of the flesh fly subfamily Paramacronychiinae, including the type species and sole member Primorya ussuriensis sp.n. from the Russian Far East. Arguments for erecting a new genus are obtained from a phylogenetic analysis of all paramacronychiine genera. Goniophyto suenagai (Kurahashi), comb.n. is transferred from Wohlfahrtiodes Villeneuve and included in a broadened concept of Goniophyto Townsend, and Agria cicadina (Kato) , comb.n. is transferred from Angiometopa Brauer & Bergenstamm. A key to all paramacronychiine genera is provided.     

Phenotype of FAECB (Facility for Automated Experiments in Cell Biology) Chinese hamster ovary mutants with minimal UV-sensitivity

Mitochondrial DNA (mtDNA) haplogroup U, defined by the polymorphism 12308A>G, may constitute a risk factor for an occipital stroke in migraine. We therefore identified 14 patients with an occipital stroke and with 12308A>G. We determined complete mtDNA coding region sequence for the patients and for population controls by conformation sensitive gel electrophoresis (CSGE) and direct sequencing. Sequence information was used to construct a phylogenetic network of mtDNA haplogroups U and K, which was found to be composed of subclusters U2, U4, U5 and a new subcluster U7, as well as cluster K. Five patients with a migrainous stroke belonged to subcluster U5 (P=0.006; Fisher's exact test). Many unique mutations were found among the patients with an occipital stroke including two tRNA mutations that have previously been suggested to be pathogenic. Analysis of mtDNA sequences by CSGE and comparison of the sequences through phylogenetic analysis greatly enhances the identification of mtDNA clusters in population and detection of mtDNA mutations in patients.