Multilocus sequence analysis of the genus Bradyrhizobium

The use of multilocus sequence analysis (MLSA) for the taxonomy of Bradyrhizobium was assessed. We compared partial sequences for atpD, recA, gyrB, rpoB and dnaK for a set of reference strains representing named species and genospecies, and a number of new isolates from Lupinus albus, Arachis hypogaea and Ornithopus compressus from Spain. The phylogenies of the individual genes were compared with previous DNA–DNA hybridization results. High hybridization values were well reflected, but intermediary hybridization values were less clearly apparent. However, the phylogeny of a concatenated dataset of the five genes did reflect all values and thus is more informative of overall genome similarity. Our results indicate that only for the genes gyrB, rpoB and dnaK there is a small gap between the interspecies sequence similarities and the intraspecies similarity, and therefore cut-off levels for species delineation cannot be set, although high sequence similarity (>99%) does permit identification. In a few instances, a reference strain did not group as expected for one of the five genes tested. This may be a result of horizontal gene transfer and recombination events occasionally involving housekeeping genes. This observation indicates it is best to consider more than one gene for taxonomic inferences. The majority of the new isolates from the three host species was identified as Bradyrhizobium canariense. Four strains from L. albus from León, Spain, formed a separate group close to Bradyrhizobium japonicum.     

Phylogenetic analysis of the pathogenic bacteria Spiroplasma penaei based on multilocus sequence analysis

A pathogenic Spiroplasma penaei strain was isolated from the hemolymph of moribund Pacific white shrimp, Penaeus vannamei. The shrimp sample originated from a shrimp farm near Cartagena, Colombia, that was suffering from high mortalities in ponds with very low salinity and high temperatures. This new emerging disease in a marine crustacean in the Americas is described as a systemic infection. The multilocus phylogenetic analysis suggests that S. penaei strain has a terrestrial origin. Evolutionary relationship trees, based on five partial DNA sequences of 16S rDNA, 23S rDNA, 5S rDNA, gyrB, rpoB genes and two complete DNA sequences of 16S-23S rDNA and 23S-5S rDNA intergenic spacer region, were reconstructed using the distance-based Neighboring-Joining (NJ) method with Kimura-2-parameter substitution model. The NJ trees based on all DNA sequences investigated in this study positioned S. penaei in the Citri-Poulsonii clade and corroborate the observations by other investigators using the 16S rDNA gene. Pairwise genetic distance calculation between sequences of spiroplasmas showed S. penaei to be closely related to Spiroplasma insolitum and distantly related to Spiroplasma sp. SHRIMP from China.     

The genus Allochromatium (Chromatiales Chromatiaceae) revisited: a study on its intragenic structure based on multilocus sequence analysis (MLSA) and DNA-DNA hybridization (DDH)

In this study, the taxonomic status of anoxygenic photosynthetic bacteria belonging to the genus Allochromatium is revisited. The inter- and intraspecies relationship of seven Allochromatium strains, including a set of well described type strains, were examined by DNA-DNA hybridization (DDH) and multilocus sequence analysis (MLSA) using segments of seven protein-coding genes. The re-sequencing of the 16S rRNA, the internal transcriber spacer (ITS), multi-gene analysis and DDH comparison indicated that both type strains Allochromatium vinosum DSM 180^T and Allochromatium minutissimum DSM 1376^T are closely related to each other forming an independent cluster together with the strains A. vinosum DSM 183 and DSM 1686. The internal comparison of members of this A. vinosum phylogroup showed values of DDH relatedness above 80% and concatenated sequence similarities (4744 bp) above 98%. In contrast, the MLSA scheme has identified A. vinosum strain BH-2 as a separate lineage. Strain BH-2 was first classified as a member of the species A. vinosum based on DDH comparison. However, this strain showed the lowest similarity values of the 16S rRNA gene and concatenated sequences, as well as amino acid identity (AAI) when compared to other Allochromatium strains, suggesting that strain BH-2 may represent a new species.     

Larsenia salina gen. nov., sp. nov., a new member of the family Halomonadaceae based on multilocus sequence analysis

Two Gram-staining-negative, moderately halophilic bacteria, strains M1-18^T and L1-16, were isolated from a saltern located in Huelva (Spain). They were motile, strictly aerobic rods, growing in the presence of 3–25% (w/v) NaCl (optimal growth at 7.5–10% [w/v] NaCl), between pH 4.0 and 9.0 (optimal at pH 6.0–7.0) and at temperatures between 15 and 40 °C (optimal at 37 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that both strains showed the higher similarity values with Chromohalobacter israelensis ATCC 43985^T (95.2–94.8%) and Chromohalobacter salexigens DSM 3043^T (95.0–94.9%), and similarity values lower than 94.6% with other species of the genera Chromohalobacter, Kushneria, Cobetia or Halomonas. Multilocus sequence analysis (MLSA) based on the partial sequences of atpA, rpoD and secA housekeeping genes indicated that the new isolates formed an independent and monophyletic branch that was related to the peripheral genera of the family Halomonadaceae, Halotalea, Carnimonas and Zymobacter, supporting their placement as a new genus of the Halomonadaceae. The DNA–DNA hybridization between both strains was 82%, whereas the values between strain M1-18^T and the most closely related species of Chromohalobacter and Kushneria were equal or lower to 48%. The major cellular fatty acids were C_18:1ω7c/C_18:1ω6c, C_16:0, and C_16:1ω7c/C_16:1ω6c, a profile that differentiate this new taxon from species of the related genera. We propose the placement of both strains as a novel genus and species, within the family Halomonadaceae, with the name Larsenia salina gen. nov., sp. nov. The type strain is M1-18^T (= CCM 8464 = CECT 8192^T = IBRC-M 10767^T = LMG 27461^T).     

Marichromatium litoris sp. nov. and Marichromatium chrysaorae sp. nov. isolated from beach sand and from a jelly fish (Chrysaora colorata)

Three strains (JA349^T, JA553^T, JA439) of phototrophic sulphur bacteria were isolated from marine habitats of India. 16S rRNA gene sequence of the three strains clustered phylogenetically with members of the genus Marichromatium of the family Chromatiaceae belonging to the class Gammaproteobacteria. All the strains shared highest sequence similarity with the type strains of Marichromatium spp. (96-99% sequence similarity) and the new strains were characterized based on polyphasic taxonomy. Strains JA349^T and JA553^T can be distinguished from closest relative species of the genus Marichromatium with respect to distinct differences in cellular polar lipids, fatty acids and carbon/nitrogen sources utilization. Both strains were distinctly related (<50% based on DNA-DNA hybridization) with the type strains of the genus Marichromatium. Multilocus Sequence Analysis (MLSA) of the concatenated five protein coding genes (fusA, pufM, dnaK, recA, soxB) along with internal transcribed spacer (ITS; 16S-23S rRNA) had sequence similarity of less than 92% with the type strains of Marichromatium spp. Distinct phenotypic, chemotaxonomic and molecular differences allow the separation of strains JA349^T and JA553^T into new species of the genus Marichromatium for which, we propose the names Marichromatium litoris sp. nov. and Marichromatium chrysaorae sp. nov., respectively.     

Taxonomic evaluation of the Streptomyces hygroscopicus clade using multilocus sequence analysis and DNA-DNA hybridization, validating the MLSA scheme for systematics of the whole genus

Streptomyces hygroscopicus and related species are the most well known candidate producers of antibiotics and many other industrially and agronomically important secondary metabolites in the genus Streptomyces. Multilocus sequence analysis (MLSA) has shown to be a powerful and pragmatic molecular method for unraveling streptomycete diversities. In this investigation, a multilocus phylogeny of 58 representatives of the S. hygroscopicus 16S rRNA gene clade including S. violaceusniger and related species was examined. The result demonstrated that the MLSA data were helpful in defining members of the S. hygroscopicus clade, providing further evidence that the MLSA scheme of five housekeeping genes (atpD, gyrB, recA, rpoB and trpB) is a valuable alternative for creating and maintaining operational protocols for the Streptomyces species assignment. DNA-DNA hybridization (DDH) between strains with representative MLSA evolutionary distances, combined with previous data from S. griseus and S. albidoflavus clades, revealed a high correlation between MLSA and DDH, and sustains that the five-gene nucleotide sequence distance of 0.007 could be considered as the species cut-off for the whole genus. This significant correlation thus makes the MLSA scheme applicable to construction of a theory-based taxonomy for both ecology and bioprospecting of streptomycetes. Based on the MLSA and DDH data, as well as phenotypic characteristics, 10 species and three subspecies of the S. hygroscopicus clade are considered to be later heterotypic synonyms of eight genomic species, and Streptomyces glebosus sp. nov., comb. nov. (type strain CGMCC 4.1873(T)=LMG 19950(T)=DSM 40823(T)) and Streptomyces ossamyceticus sp. nov., comb. nov. (type strain CGMCC 4.1866(T)=LMG 19951(T)=DSM 40824(T)) are also proposed.     

Systematics and evolution of the genus Torrubiella (Hypocreales, Ascomycota)

Torrubiella is a genus of arthropod-pathogenic fungi that primarily attacks spiders and scale insects. Based on the morphology of the perithecia, asci, and ascospores, it is classified in Clavicipitaceae s. lat. (Hypocreales), and is considered a close relative of Cordyceps s. 1., which was recently reclassified into three families (Clavicipitaceae s. str., Cordycipitaceae, Ophiocordycipitaceae) and four genera (Cordyceps s. str, Elaphocordyceps, Metacordyceps, and Ophiocordyceps). Torrubiella is distinguished morphologically from Cordyceps s. lat. mainly by the production of superficial perithecia and the absence of a well-developed stipitate stroma. To test and refine evolutionary hypotheses regarding the placement of Torrubiella and its relationship to Cordyceps s. lat., a multi-gene phylogeny was constructed by conducting ML and Bayesian analyses. The monophyly of Torrubiella was rejected by these analyses with species of the genus present in Clavicipitaceae, Cordycipitaceae, and Ophiocordycipitaceae, and often intermixed among species of Cordyceps s. lat. The morphological characters traditionally used to define the genus are, therefore, not phylogenetically informative, with the stipitate stromata being gained and/or lost several times among clavicipitaceous fungi. Two new genera (Conoideocrella, Orbiocrella) are proposed to accommodate two separate lineages of torrubielloid fungi in the Clavicipitaceae s. str. In addition, one species is reclassified in Cordyceps s. str. and three are reclassified in Ophiocordyceps. The phylogenetic importance of anamorphic genera, host affiliation, and stipitate stromata is discussed.     

Diversity of Micromonospora strains isolated from nitrogen fixing nodules and rhizosphere of Pisum sativum analyzed by multilocus sequence analysis

It was recently reported that Micromonospora inhabits the intracellular tissues of nitrogen fixing nodules of the wild legume Lupinus angustifolius. To determine if Micromonospora populations are also present in nitrogen fixing nodules of cultivated legumes such as Pisum sativum, we carried out the isolation of this actinobacterium from P. sativum plants collected in two man-managed fields in the region of Castilla and León (Spain). In this work, we describe the isolation of 93 Micromonospora strains recovered from nitrogen fixing nodules and the rhizosphere of P. sativum. The genomic diversity of the strains was analyzed by amplified ribosomal DNA restriction analysis (ARDRA). Forty-six isolates and 34 reference strains were further analyzed using a multilocus sequence analysis scheme developed to address the phylogeny of the genus Micromonospora and to evaluate the species distribution in the two studied habitats. The MLSA results were evaluated by DNA-DNA hybridization to determine their usefulness for the delineation of Micromonospora at the species level. In most cases, DDH values below 70% were obtained with strains that shared a sequence similarity of 98.5% or less. Thus, MLSA studies clearly supported the established taxonomy of the genus Micromonospora and indicated that genomic species could be delineated as groups of strains that share > 98.5% sequence similarity based on the 5 genes selected. The species diversity of the strains isolated from both the rhizosphere and nodules was very high and in many cases the new strains could not be related to any of the currently described species.     

Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing

In a previous report (Luyo-Acero et al., 2004), we demonstrated that cytochrome b (Cyt b) gene analysis is an effective method for classifying several isolates of the genus Leishmania; hence, we have further applied this method to other Leishmania species in an effort to enhance the accuracy of the procedure and to construct a new phylogenic tree. In this study, a total of 30 Leishmania and Endotrypanum WHO reference strains, clinical isolates from our patients assigned to 28 strains (human and non-human pathogenic species) and two species of the genus Endotrypanum were analyzed. The Cyt b gene in each sample was amplified by PCR, and was then sequenced by several primers, as reported previously. The phylogenic tree was constructed based on the results obtained by the computer software MEGA v3.1 and PAUP* v4.0 Beta. The present phylogenic tree was almost identical to the traditional method of classification proposed by Lainson and Shaw (1987). However, it produces the following suggestions: (1) exclusion of L. (Leishmania) major from the L. (L.) tropica complex; (2) placement of L.tarentolae in the genus Sauroleishmania; (3) L. (L.) hertigi complex and L. (V.) equatorensis close to the genus Endotrypanum; (4) L. (L.) enrietti, defined as L. (L.) mexicana complex, placed in another position; and (5) L. (L.) turanica and L. (L.) arabica are located in an area far from human pathogenic Leishmania strains. Cyt b gene analysis is thus applicable to the analyzing phylogeny of the genus Leishmania and may be useful for separating non-human pathogenic species from human pathogenic species.     

A molecular analysis of some Eastern Atlantic grouper from the Epinephelus and Mycteroperca genus

Mitochondrial cytochrome b (397 bp) and 16S rDNA (516 bp) sequences analysis was used to investigate the phylogenetic relationships among some Eastern Atlantic Epinephelinae species. Six species of Epinephelus (E. aeneus, E. caninus, E. costae, E. haifensis, E. marginatus and E. tauvina) and two species of Mycteroperca (M. rubra and M. fusca) were analysed. Neighbour-joining and maximum-parsimony analysis support the paraphyletic grouping of the Epinephelus and Mycteroperca analysed. The maximum pairwise nucleotide divergence value in cyt b among all taxa was 0.196 between E. aeneus and E. marginatus and the minimum value was 0.006 between E. costae and M. rubra. Meanwhile, in 16S sequence analysis, the maximum value is 0.093 between E. aeneus and E. tauvina and the minimum value is 0.011 between E. marginatus and M. rubra. Molecular clock estimates for the species suggest a divergence time of 20-24 mya, which coincides with the Miocene period. A molecular analysis was also conducted, using other Epinephelinae sequences from GenBank in order to improve our understanding of the phyletic status of the Epinephelus and Mycteroperca species analysed.     

Description of Idiomarina insulisalsae sp. nov., isolated from the soil of a sea salt evaporation pond,proposal to transfer the species of the genus Pseudidiomarina to the genus Idiomarina and emended description of the genus Idiomarina

A halophilic, aerobic Gram-negative bacterium, designated strain CVS-6^T, was isolated from a sea salt evaporation pond on the Island of Sal in the Cape Verde Archipelago. Phylogenetic analysis of the 16S rRNA gene sequence revealed a clear affiliation of the organism with members of the family Idiomarinaceae. Sequence similarities between CVS-6^T and the type strains of the species of the genera Pseudidiomarina and Idiomarina ranged from 93.7% to 96.9%. The major isoprenoid quinone was ubiquinone 8 (Q-8). The major cellular fatty acids were 15:0 iso (21.8%), 17:0 iso (12.5%), 17:1 iso ω9c (10.7%), and 16:1 ω7c (10.6%). The DNA G+C content was 51.6 mol%. The species represented by strain CVS-6^T could be distinguished from the species of the genera Pseudidiomarina and Idiomarina; however, it was not possible to distinguish both genera from each other using the phenotypic or chemotaxonomic characteristics examined. Consequently, we propose that the species classified in the genus Pseudidiomarina should be transferred to the genus Idiomarina. We also propose that, on the basis of physiological and biochemical characteristics, strain CVS-6^T (=LMG 23123=CIP 108836) represents a new species which we name Idiomarina insulisalsae.     

Ultrastructural characterization and multilocus sequence analysis (MLSA) of ‘Candidatus Rickettsiella isopodorum’, a new lineage of intracellular bacteria infecting woodlice (Crustacea: Isopoda)

The taxonomic genus Rickettsiella (Gammaproteobacteria; Legionellales) comprises intracellular bacteria associated with a wide range of arthropods including insects, arachnids and crustaceans. The present study provides ultrastructural together with genetic evidence for a Rickettsiella bacterium in the common rough woodlouse, Porcellio scaber (Isopoda, Porcellionidae), occurring in Germany, and shows that this bacterium is very closely related to one of the same genus occurring in California that infects the pill bug, Armadillidium vulgare (Isopoda, Armadillidiidae). Both bacterial isolates displayed the ultrastructural features described previously for crustacean-associated bacteria of the genus Rickettsiella, including the absence of well-defined associated protein crystals; occurrence of the latter is a typical characteristic of infection by this type of bacteria in insects, but has not been reported in crustaceans. A molecular systematic approach combining multilocus sequence analysis (MLSA) with likelihood-based significance testing demonstrated that despite their distant geographic origins, both bacteria form a tight sub-clade within the genus Rickettsiella. In the 16S rRNA gene trees, this sub-clade includes other bacterial sequences from woodlice. Moreover, the bacterial specimens from P. scaber and A. vulgare are found genetically or morphologically different from each of the four currently recognized Rickettsiella species. Therefore, the designation ‘Candidatus Rickettsiella isopodorum’ is introduced for this new lineage of isopod-associated Rickettsiella bacteria.     

MLSA barcoding of Marichromatium spp. and reclassification of Marichromatium fluminis (Sucharita et al., 2010) as Phaeochromatium fluminis gen. nov. comb. nov

Thirty-one members of the genus Marichromatium were analysed based on multilocus sequence analysis (MLSA) of four concatenated protein-coding genes (fusA, pufM, dnaK, recA) along with the internal transcribed spacer (ITS; 16S-23S rRNA) region and 16S rRNA gene. The restriction patterns obtained from the in silico analysis of the concatenated sequences were good barcodes for the identification of Marichromatium spp. Distinct phenotypic, chemotaxonomic and molecular differences allowed the separation of Marichromatium fluminis JA418(T) into a new genus in the family Chromatiaceae, for which we propose the name Phaeochromatium fluminis gen. nov. comb. nov.     

Evolutionary divergence of the paralogs Methoprene tolerant (Met) and germ cell expressed (gce) within the genus Drosophila

Juvenile hormone (JH) signaling underpins both regulatory and developmental pathways in insects. However, the JH receptor is poorly understood. Methoprene tolerant (Met) and germ cell expressed (gce) have been implicated in JH signaling in Drosophila. We investigated the evolution of Met and gce across 12 Drosophila species and found that these paralogs are conserved across at least 63 million years of dipteran evolution. Distinct patterns of selection found using estimates of dN/dS ratios across Drosophila Met and gce coding sequences, along with their incongruent temporal expression profiles in embryonic Drosophila melanogaster, illustrate avenues through which these genes have diverged within the Diptera. Additionally, we demonstrate that the annotated gene CG15032 is the 5′ terminus of gce.In mosquitoes and beetles, a single Met-like homolog displays structural similarity to both Met and gce, and the intron locations are conserved with those of gce. We found that Tribolium and mosquito Met orthologs are assembled from Met- and gce-specific domains in a modular fashion. Our results suggest that Drosophila Met and gce experienced divergent evolutionary pressures following the duplication of an ancestral gce-like gene found in less derived holometabolous insects.     

The anamorphic genus Monotosporella (Ascomycota) from Eocene amber and from modern Agathis resin

The anamorphic fungal genus Monotosporella (Ascomycota, Sordariomycetes) has been reco-vered from a piece of Early Eocene Indian amber, as well as from the surface of extant resin flows in New Caledonia. The fossil fungus was obtained from the Tarkeshwar Lignite Mine of Gujarat State, western India, and was part of the biota of an early tropical angiosperm rainforest. The amber inclusion represents the second fossil record of Sordariomycetes, as well as the first fossil of its particular order (either Savoryellales or Chaetosphaeriales). The fossil fungus is distinguished from extant representatives by possessing both short conidiophores and small two-septate pyriform conidia, and is described as Monotosporella doerfeltii sp. nov. Inside the amber, the anamorph is attached to its substrate, which is likely the degraded thallus of a cladoniform lichen. The extant New Caledonian species is assigned to Monotosporella setosa. It was found growing on semi-solidified resin flows of Agathis ovata (Araucariaceae), and is the first record of Monotosporella from modern resin substrates.     

Towards a new classification of the Arthoniales (Ascomycota) based on a three-gene phylogeny focussing on the genus Opegrapha

A multi-locus phylogenetic study of the order Arthoniales is presented here using the nuclear ribosomal large subunit (nuLSU), the second largest subunit of RNA polymerase II (RPB2) and the mitochondrial ribosomal small subunit (mtSSU). These genes were sequenced from 43 specimens or culture isolates representing 33 species from this order, 16 of which were from the second largest genus, Opegrapha. With the inclusion of sequences from GenBank, ten genera and 35 species are included in this study, representing about 18 % of the genera and ca 3 % of the species of this order. Our study revealed the homoplastic nature of morphological characters traditionally used to circumscribe genera within the Arthoniales, such as exciple carbonization and ascomatal structure. The genus Opegrapha appears polyphyletic, species of that genus being nested in all the major clades identified within Arthoniales. The transfer of O. atra and O. calcarea to the genus Arthonia will allow this genus and family Arthoniaceae to be recognized as monophyletic. The genus Enterographa was also found to be polyphyletic. Therefore, the following new combinations are needed: Arthonia calcarea (basionym: O. calcarea), and O. anguinella (basionym: Stigmatidium anguinellum); and the use of the names A. atra and Enterographa zonata are proposed here. The simultaneous use of a mitochondrial gene and two nuclear genes led to the detection of what seems to be a case of introgression of a mitochondrion from one species to another (mitochondrion capture; cytoplasmic gene flow) resulting from hybridization.