Concatenated Gene Sequences Research Articles

Kribbella jiaozuonensis sp. nov., a novel actinomycete isolated from soil.

A novel actinobacterium, designated strain NEAU-THZ27T, was isolated from soil collected from the Cornel peak in Jiaozuo, Henan Province, PR China and characterized using a polyphasic approach. Morphological and chemotaxonomic characteristics of the strain coincided with those of members of the genusKribbella. The results of 16S rRNA gene sequence analysis showed that strain NEAU-THZ27T belongs to the genus Kribbella and was most closely related to Kribbella podocarpi YPL1T (98.96 %), Kribbella karoonensis Q41T (98.89 %), Kribbella aluminosa HKI 0478T (98.86%) and Kribbella hippodromi S1.4T (98.85 %), similarities to other type strains of species of the genus Kribbella were found to be less than 98.7 %. Phylogenetic analyses using the 16S rRNA gene sequence and multilocus sequence analysis using the concatenated gene sequences of the gyrB, rpoB, recA, relA and atpD genes all showed that the strain formed a separate branch in the genus Kribbella. The cell wall contained ll-diaminopimelic acid as the major diamino acid and the whole-cell hydrolysates were ribose and glucose. The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol. The predominant menaquinone was MK-9(H4). Major fatty acids were iso-C16 : 0, iso-C14 : 0 and anteiso-C15 : 0, these chemotaxonomic data supported the affiliation of strain NEAU-THZ27T to the genus Kribbella. The DNA G+C content was 68.0 mol%. Furthermore, the strain could be clearly distinguished by concatenated gene genetic distances, the combination of DNA-DNA hybridization results and some phenotypic characteristics. Therefore, it is proposed that strain NEAU-THZ27T represents a novel species of the genus Kribbella, for which the name Kribbella jiaozuonensis sp. nov. is proposed. The type strain is NEAU-THZ27T (=CGMCC 4.7504T=DSM 105535T).

Cryobacterium tepidiphilum sp. nov., isolated from rhizosphere soil of lettuce (var. ramosa Hort.).

A Gram-stain positive, aerobic, rod-shaped bacterium, designated strain NEAU-85T, was isolated from rhizosphere soil of lettuce and characterised using a polyphasic approach. Strain NEAU-85T was found to be catalase positive, motile and able to grow at between 10 and 30°C. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain NEAU-85T belongs to the genus Cryobacterium and is closely related to the type strain Cryobacterium psychrotolerans JCM 13925T (98.5%) and also to Leifsonia kafniensis NCCB 100216T (97.6%). Multilocus sequence analysis using the concatenated gene sequences of the atpD, dnaK, recA, rpoB, sevY and ychF genes showed that strain NEAU-85T belongs to the genus Cryobacterium. The digital DNA-DNA hybridization values revealed that strain NEAU-85T is distinct from its close phylogenetic neighbour C. psychrotolerans. The menaquinones were identified as MK-10 and MK-11. The phospholipid profile was found to consist of diphosphatidylglycerol, phosphatidylglycerol, phospholipid, an unidentified glycolipid and an unidentified lipid. The major fatty acids were identified as anteiso-C15:0, anteiso-C15:1 and iso-C16:0. The genomic DNA G + C content of strain NEAU-85T was determined to be 68.9mol%. The DNA-DNA hybridization value between them was less than 70%. On the basis of phenotypic, genotypic and phylogenetic data, strain NEAU-85T can be concluded to represent a novel species of the genus Cryobacterium, for which the name Cryobacterium tepidiphilum sp. nov. is proposed. The type strain is NEAU-85T (= CCTCC AA 2018035T = JCM 32545T).

Pseudomonas urumqiensis sp. nov., isolated from rhizosphere soil of Alhagi sparsifolia.

A motile, Gram-stain-negative, fusiform-shaped bacterium, designated strain T3T, was isolated from rhizosphere soil of Alhagi sparsifolia, collected from Xinjiang, PR China. Strain T3T grew at 15-42 °C, pH 4-9 and 1-6 % (w/v) NaCl concentrations. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain T3T belonged to the genus Pseudomonas and showed highest similarity of 98.6 % to Pseudomonas azotifigens JCM 12708T, followed by Pseudomonas balearica DSM 6083T (97.8 %), Pseudomonas matsuisoli JCM 30078T (97.7 %), Pseudomonas furukawaii KF707T (97.7 %), Pseudomonas tarimensis CCTCC AB 2013065T (97.3 %) and Pseudomonas indica DSM 14015T (97.1 %). Analysis based on concatenated gene sequences of 16S rRNA, rpoB and gyrB further confirmed the phylogenetic assignment of strain T3T. The Genome-to-Genome Distance Calculator results for P. azotifigens JCM12708T and P. balearica DSM 6083T were 28.7±4.4% and 24.1±2.4 %, and the average nucleotide identity scores were 81.3 and 78.1 %. The major polar lipids of strain T3T were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The predominant quinone was Q-9. The major fatty acids comprised summed feature 8 (C18 : 1ω6c/C18 : 1ω7c; 37.7 %), summed feature 3 (C16 : 1ω6c/C16 : 1ω7c; 28.2 %), C16 : 0 (15.6 %), C12 : 0 (7.8 %), C10 : 03-OH (3.0 %) and C12 : 03-OH (2.6 %). The G+C content of the genomic DNA of the type strain was 65.3 mol%. It is obvious from the phylogenetic, phenotypic and chemotaxonomic data that strain T3T represents a novel species of the genus Pseudomonas, for which the name Pseudomonasurumqiensis sp. nov., is proposed. The type strain is T3T (=ACCC 60124T=JCM 32830T).

A New Subclade of Leptosphaeria biglobosa Identified from Brassica rapa.

Blackleg (Phoma stem canker) of crucifers is a globally important disease caused by the ascomycete species complex comprising of Leptosphaeria maculans and Leptosphaeria biglobosa. Six blackleg isolates recovered from Brassica rapa cv. Mizspoona in the Willamette Valley of Oregon were characterized as L. biglobosa based on standard pathogenicity tests and molecular phylogenetic analysis. These isolates were compared to 88 characterized L. biglobosa isolates from western Canada, 22 isolates from Australia, and 6 L. maculans isolates from Idaho, USA using maximum parsimony and distance analysis of phylogenetic trees generated from the ITS rDNA (internal transcribed spacer rDNA) sequence, and the actin and β-tubulin gene sequences. The L. biglobosa isolates derived from B. rapa collected in Oregon formed a separate subclade based on concatenated gene sequences or a single gene sequence, regardless of the analyses. Pathogenicity tests showed that these isolates failed to infect either resistant or susceptible B. napus cultivars, but caused severe symptoms on three B. rapa cultivars (Accession number: UM1113, UM1112, and UM1161), a B. oleracea var. capitata (cabbage) cultivar (Copenhagen Market), and two B. juncea cultivars (CBM, a common brown Mustard, and Forge). These findings demonstrated that the L. biglobosa isolates derived from a B. rapa crop in Oregon were genetically distinct from existing species of L. biglobosa, and constitute a new subclade, herein proposed as L. biglobosa ‘americensis’.

Assessing the Pathogenicity of Two Bacteria Isolated from the Entomopathogenic Nematode Heterorhabditis indica against Galleria mellonella and Some Pest Insects.

The entomopathogenic nematodes Heterorhabditis are parasites of insects and are associated with mutualist symbiosis enterobacteria of the genus Photorhabdus; these bacteria are lethal to their host insects. Heterorhabditis indica MOR03 was isolated from sugarcane soil in Morelos state, Mexico. The molecular identification of the nematode was confirmed using sequences of the ITS1-5.8S-ITS2 region and the D2/D3 expansion segment of the 28S rRNA gene. In addition, two bacteria HIM3 and NA04 strains were isolated from the entomopathogenic nematode. The genomes of both bacteria were sequenced and assembled de novo. Phylogenetic analysis was confirmed by concatenated gene sequence datasets as Photorhabdus luminescens HIM3 (16S rRNA, 23S rRNA, dnaN, gyrA, and gyrB genes) and Pseudomonas aeruginosa NA04 (16S rRNA, 23S rRNA and gyrB genes). H. indica MOR03 infects Galleria mellonella, Tenebrio molitor, Heliothis subflexa, and Diatraea magnifactella larvae with LC50 values of 1.4, 23.5, 13.7, and 21.7 IJs/cm2, respectively, at 48 h. These bacteria are pathogenic to various insects and have high injectable insecticide activity at 24 h.

Species identification of processed sea cucumbers from Malaysian market based on concatenated gene sequences of mitochondrial rRNA genes

Species identification of sea cucumbers that have undergone body deformation due to extensive food processing, e.g., beche-de-mer, is difficult, especially with the copresence of cases of unlabelled or mislabelled sea cucumber-based products on the market. Therefore, a study was done to determine the species identities of processed sea cucumbers from selected Malaysian markets using concatenated gene sequences of non-protein-coding 12S and 16S mitochondrial rRNA genes. Phylogenetic analyses, based on the distance-based Neighbor Joining method and character-based methods, i.e., the Maximum Parsimony method, Maximum Likelihood method, and the Bayesian Analysis method, of 47 ingroup sequences, representing 37 processed sea cucumber specimens, 6 reference samples, and 4 additional specimens, suggested the presence of 3 main clusters, i.e., gamat family, consisting of genus Stichopus and genus Thelenota; and timun laut family, comprising family Holothuriidae. A number of 3 gamat species, i.e., Stichopus horrens, Stichopus vastus, and Thelenota anax, were recorded. Meanwhile, the specimens of Holothuria (Halodeima) atra, Holo-thuria (Halodeima) edulis, Holothuria (Metriatyla) lessoni, Holothuria (Merten-siothuria) leucospilota, and Holothuria (Metriatyla) scabra were the 5 timun laut species that are grouped under the family Holothuriidae. The outcomes of this study can be utilised by enforcement agencies to monitor and overcome the issues of species substitution and product mislabelling of processed sea cucumber products in Malaysian markets.

Complete mitochondrial genomes for three lizards (Anolis punctatus, Sceloporus woodi, and S. grammicus): a contribution to mitochondrial phylogenomics of Iguanoidea

The complete mitochondrial genomes of the Spotted Anole (Anolis punctatus), Florida Scrub Lizard (Sceloporus woodi), and Mesquite Lizard (S. grammicus) were assembled and annotated. Genomic DNA of A. punctatus was sequenced in one-sixth of a lane in an Illumina HiSeq while comparable sequencing data for S. woodi and S. grammicus were retrieved from the Short Read Archive (entries SRR1286259 and SRR1145766, respectively). We assembled these mitogenomes using MIRA, NovoPlasty, and/or MITObim software. The resulting whole and circularized mitogenome assemblies exhibited typical vertebrate sequence length and gene organization. We deposited these mitogenomes into public databases under accession numbers MK091854, BK010487, and BK010486, respectively. To verify the taxonomic identifications of our mitogenomes (as iguanian lizards), we conducted maximum likelihood analyses with 14 published mitogenomes from the lizard superfamily Iguanoidea and two outgroups. Our analysis of concatenated gene sequences from mitochondria confirmed the taxonomic identities of our mitogenomes to the genus level.

Multilocus sequence analysis for detection of finer genetic variation and phylogenetic interrelatedness in 16SrI group phytoplasma strains infecting different plants in China

The group 16SrI phytoplasmas are associated with severe diseases of many cash and ecological plants throughout the world. To date, the genetic variation and population structure of very closely related phytoplasma strains are still not fully understood in China. In this study, a multilocus sequence analysis (MLSA) scheme was developed using ten housekeeping genes (rp, tuf, secA, secY, ipt, dnaK, fusA, gyrB, pyrG and rpoB) fragments compared with 16S rDNA to analyze 18 phytoplasma strains infecting chinaberry, lettuce, mulberry, paulownia and periwinkle from ten provinces in China. The nucleotide site polymorphisms resolved all strains into 15 sequence types (STs), demonstrating extensive genetic diversity among the 16SrI group strain population. All the strains, classified in 16SrI-B and-D subgroup by 16S rDNA analysis, clustered into one clade and clearly differentiated into discrete subclades by phylogenetic analysis of the concatenated gene sequences. The 10 chinaberry witches’ broom (CWB) strains that were most closely related to two mulberry dwarf (MD) and hardly distinguished with 16S rDNA, were definitely split into four distinct clusters and 8 STs apparently congruent to their geographical locations. Two lettuce yellows (LY) strains in Sanming, Fujian province, China were more closely related to the onion yellows OY-M strain in Japan than the periwinkle virescence (PeV) and paulownia witches’ broom (PaWB) strains in China. The levels of variation in dnaK gene were higher than those in 16S rDNA and other genes tested. This MLSA is a promising approach for phytoplasma differentiation as well as for in depth examination of strain diversity and evolution of various 16Sr groups or subgroups.

Kribbella monticola sp. nov., a novel actinomycete isolated from soil.

A novel actinobacterium, designated strain NEAU-SW521T, was isolated from soil collected from Xianglu Mountain, Heilongjiang province, north PR China. The results of analysis of the 16S rRNA gene indicated that NEAU-SW521T represented a member of the genus Kribbella. The results of phylogenetic analyses using the 16S rRNA gene and multilocus sequence analysis using the concatenated gene sequences of the gyrB, rpoB, relA, recA and atpD genes all indicated that the strain formed a clade with Kribbella alba DSM 15500T (99.16 %), Kribbella ginsengisoli JCM 16928T (98.96 %), Kribbella catacumbae JCM 14968T (98.82 %), Kribbella sancticallisti JCM 14969T (98.62 %), Kribbella qitaiheensis NEAU-GQTH2-3T (98.61 %) and Kribbella koreensis JCM 10977T (98.47 %). The cell wall contained ll-diaminopimelic acid as the major diamino acid and the whole-cell hydrolysates were ribose, glucose and galactose. The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol and an unidentified phospholipid. The predominant menaquinone was MK-9(H4). Major fatty acids were iso-C16 : 0 and anteiso-C15 : 0. These chemotaxonomic data supported the affiliation of NEAU-SW521T to the genus Kribbella. The DNA G+C content was 67.8 mol%. Furthermore, the strain could be clearly distinguished by concatenated gene genetic distances, the combination of DNA-DNA hybridization results and some phenotypic characteristics. Therefore, it is proposed that NEAU-SW521T represents a novel species of the genus Kribbella, for which the name Kribbellamonticola sp. nov. is proposed. The type strain is NEAU-SW521T (=CGMCC 4.7465T=DSM 105770T).

Genetic diversity of glacier-inhabiting Cryobacterium bacteria in China and description of Cryobacterium zongtaii sp. nov. and Arthrobacter glacialis sp. nov.

Cryobacterium and Arthrobacter are members of Actinobacteria, and are often found in cold environments. In this study, 48 Cryobacterium strains, including 9 type strains and 39 new isolates collected from glaciers in China were subjected to multilocus sequence analysis (MLSA). Phylogenetic analysis revealed that Cryobacterium comprised four cold-adapted clusters. Also, 19 potential novel Cryobacterium species were found using 0.065 as the cut-off point of genetic distance between the concatenated gene sequences. Additionally, three Cryobacterium strains (TMN-42T, TMN-39-1 and TMB1-8) and two Arthrobacter strains (HLT2-12-2T, TMN-18) isolated from glaciers were subjected to taxonomic analysis. Based on 16S rRNA gene sequences, MLSA data and average nucleotide identity (ANI) values, they represented a novel Cryobacterium species and a novel Arthrobacter species. Specifically, strain TMN-42T was most closely related to the type strains of Cryobacterium arcticum and Cryobacterium psychrotolerans with 83.79% and 77.78% ANI values, respectively. The ANI values between strain HLT2-12-2T and its closely relatives Arthrobacter psychrochitiniphilus GP3T and Arthrobacter alpinus S6-3T were 76.66% and 77.94%, respectively. Therefore, we propose two novel species, Cryobacterium zongtaii sp. nov. (TMN-42T=CGMCC 1.9695T=NBRC 111591T) and Arthrobacter glacialis sp. nov. (HLT2-12-2T=CGMCC 1.10025T=NBRC 113092T).

Definition of two new symbiovars, sv. lupini and sv. mediterranense, within the genera Bradyrhizobium and Phyllobacterium efficiently nodulating Lupinus micranthus in Tunisia

In this study, a polyphasic approach was used to analyze three representative strains (LmiH4, LmiM2 and LmiT21) from a collection of six previously described strains isolated in Tunisia from root nodules of Lupinus micranthus. The phylogenetic analysis of the concatenated rrs, recA and glnII genes showed that strain LmiH4 had 100% concatenated gene sequence identity with the type strain Bradyrhizobium retamae Ro19T. Similarly, strain LmiM2 shared 100% concatenated gene sequence identity with the species Bradyrhizobium valentinum LmjM3T. However, strain LmiT21 showed an identical concatenated gene sequence with reference strain Phyllobacterium sophorae CCBAU03422T. The recA-glnII concatenated protein-coding genes used produced incongruent phylogenies compared with 16S rDNA phylogeny. The nodC gene analysis showed that the strains were phylogenetically divergent to the Bradyrhizobium symbiovars defined to date, and represented two new symbiovars. Plant infection analysis revealed that the three strains showed moderate host range and symbiotic specificities.Based on their symbiotic characteristics, we propose that the three strains isolated from Lupinus micranthus nodules belong to two new symbiovars, with the first denominated lupini within the two species Bradyrhizobium valentinum (type strain LmiM2) and B. retamae (type strain LmiH4), and the second denominated mediterranense within the species P. sophorae (type strain LmiT21).

Analyses of separate and concatenated cox1 and 18S rRNA gene sequences indicate that the bat piroplasm Babesia vesperuginis is phylogenetically close to Cytauxzoon felis and the 'prototheilerid' Babesia conradae.

Babesia vesperuginis is the only piroplasm known to infect bats. Unlike most members of the genus Babesia, it is probably transmitted by a soft tick species (i.e. Argas vespertilionis). Recently, two studies have been conducted to clarify the phylogenetic status of this species, and both agreed on placing it into a basal position among Babesia sensu stricto (s.s.). However, several important groups of piroplasms were not included in the already reported phylogenetic trees of B. vesperuginis isolates. Therefore, the aim of the present study was to amplify an approx. 950-bp fragment of the cytochrome c oxidase subunit 1 (cox1) gene of B. vesperuginis from A. vespertilionis specimens, and to compare its sequences with those from other piroplasmid groups in a broader phylogenetic context. Sequence comparisons focusing on either 18S rRNA or cox1 genes, as well as phylogenetic analyses involving separate and concatenated 18S rRNA and cox1 sequences indicate that B. vesperuginis is more closely related to the phylogenetic group of Theileriidae than to Babesia s.s. In particular, B. vesperuginis clustered closest to Cytauxzoon felis and the 'prototheilerid' B. conradae. The results of this study highlight that B. vesperuginis is a unique and taxonomically important species, which should be included in future studies aimed at resolving the comprehensive phylogeny of Piroplasmida.

Phylodynamics and evolutionary epidemiology of African swine fever p72-CVR genes in Eurasia and Africa.

African swine fever (ASF) is a complex infectious disease of swine that constitutes devastating impacts on animal health and the world economy. Here, we investigated the evolutionary epidemiology of ASF virus (ASFV) in Eurasia and Africa using the concatenated gene sequences of the viral protein 72 and the central variable region of isolates collected between 1960 and 2015. We used Bayesian phylodynamic models to reconstruct the evolutionary history of the virus, to identify virus population demographics and to quantify dispersal patterns between host species. Results suggest that ASFV exhibited a significantly high evolutionary rate and population growth through time since its divergence in the 18th century from East Africa, with no signs of decline till recent years. This increase corresponds to the growing pig trade activities between continents during the 19th century, and may be attributed to an evolutionary drift that resulted from either continuous circulation or maintenance of the virus within Africa and Eurasia. Furthermore, results implicate wild suids as the ancestral host species (root state posterior probability = 0.87) for ASFV in the early 1700s in Africa. Moreover, results indicate the transmission cycle between wild suids and pigs is an important cycle for ASFV spread and maintenance in pig populations, while ticks are an important natural reservoir that can facilitate ASFV spread and maintenance in wild swine populations. We illustrated the prospects of phylodynamic methods in improving risk-based surveillance, support of effective animal health policies, and epidemic preparedness in countries at high risk of ASFV incursion.

The zoogeography of extant rhabdopleurid hemichordates (Pterobranchia : Graptolithina), with a new species from the Mediterranean Sea

The early origin and evolutionary radiation of graptolites (Hemichordata:Pterobranchia) is a story told almost entirely in the fossil record, but for four extant species of the genus Rhabdopleura Allman, 1869. Here we report the discovery of a fifth species, Rhabdopleura recondita, sp. nov., at a depth range of 2–70m from the Adriatic and Ionian Seas, always associated with bryozoans in coralligenous habitats. This is the first pterobranch record in Italian waters, and the second in the Mediterranean Sea. The new species is characterised by: (1) tubaria with smooth creeping tubes adherent to the inside of empty bryozoan zooecia; (2) erect outer tubes with a graptolite, fusellar-like organisation; and (3) zooids that extend from a black stolon, which is free from the creeping tube. Each of the paired feeding arms has two rows of tentacles that do not extend to the arm tip. The distal ends of the arms, the collar and the cephalic shield are replete with black granules. Phylogenetic analyses of individual and concatenated gene sequences of mitochondrial 16S rDNA and nuclear 18S rDNA support the validity of R. recondita as a new species. Finally, we discuss the global biogeographic and habitat distributions of the extant Rhabdopleura representatives. http://zoobank.org/urn:lsid:zoobank.org:pub:82C6A51E-F8F4-44AF-AD8F-16873BE80D03

Salinivibrio kushneri sp. nov., a moderately halophilic bacterium isolated from salterns

Ten Gram-strain-negative, facultatively anaerobic, moderately halophilic bacterial strains, designated AL184T, IB560, IB563, IC202, IC317, MA421, ML277, ML318, ML328A and ML331, were isolated from water ponds of five salterns located in Spain. The cells were motile, curved rods and oxidase and catalase positive. All of them grew optimally at 37°C, at pH 7.2–7.4 and in the presence of 7.5% (w/v) NaCl. Based on phylogenetic analyses of the 16S rRNA, the isolates were most closely related to Salinivibrio sharmensis BAGT (99.6–98.2% 16S rRNA gene sequence similarity) and Salinivibrio costicola subsp. costicola ATCC 35508T (99.0–98.1%). According to the MLSA analyses based on four (gyrB, recA, rpoA and rpoD) and eight (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA and topA) concatenated gene sequences, the most closely relatives were S. siamensis JCM 14472T (96.8–95.4% and 94.9–94.7%, respectively) and S. sharmensis DSM 18182T (94.0–92.6% and 92.9–92.7%, respectively). In silico DNA–DNA hybridization (GGDC) and average nucleotide identity (ANI) showed values of 23.3–44.8% and 80.2–91.8%, respectively with the related species demonstrating that the ten isolates constituted a single novel species of the genus Salinivibrio. Its pangenome and core genome consist of 6041 and 1230 genes, respectively. The phylogeny based on the concatenated orthologous core genes revealed that the ten strains form a coherent phylogroup well separated from the rest of the species of the genus Salinivibrio. The major cellular fatty acids of strain AL184T were C16:0 and C18:1. The DNA G+C content range was 51.9–52.5mol% (Tm) and 50.2–50.9mol% (genome). Based on the phylogenetic-phylogenomic, phenotypic and chemotaxonomic data, the ten isolates represent a novel species of the genus Salinivibrio, for which the name Salinivibrio kushneri sp. nov. is proposed. The type strain is AL184T (=CECT 9177T=LMG 29817T).

Kribbella podocarpi sp. nov., isolated from the leaves of a yellowwood tree (Podocarpus latifolius).

An endophytic actinobacterial strain was isolated from a yellowwood tree growing on the slope of Devil's Peak, Cape Town, South Africa. Analysis of the 16S rRNA gene showed that the strain belongs to the genus Kribbella. Phylogenetic analyses using the 16S rRNA gene and multilocus sequence analysis using the concatenated gene sequences of the gyrB, rpoB, relA, recA and atpD genes showed that strain YPL1T is closely related to the type strains of Kribbella karoonensis and Kribbella shirazensis. DDH experiments showed that strain YPL1T is a distinct genomic species from its close phylogenetic relative, K. karoonensis Q41T. Physiological comparisons further showed that strain YPL1T is phenotypically distinct from the type strains of Kribbella jejuensis, Kribbella aluminosa, K. karoonensis, K. shirazensis and Kribbella swartbergensis. Strain YPL1T is thus presented as the type strain of a novel species, for which the name Kribbella podocarpi sp. nov. (=DSM 29424T=NRRL B-65063T), is proposed.

Wing Morphometry and Genetic Variability Between Culex coronator and Culex usquatus (Diptera: Culicidae), Two Sibling Species of the Coronator Group.

Culex coronator Dyar and Knab and Culex usquatus Dyar belong to the Coronator Group of the subgenus Culex. Culex coronator and Cx. usquatus are widespread and sympatric throughout their distribution range, which includes Brazil. Morphological identification of these species is based primarily on the characteristics of the male genitalia; females are indistinguishable using the qualitative characteristics employed in identification keys. The primary objective of this study was to distinguish females of Cx. coronator from those of Cx. usquatus employing both wing geometric morphometrics, and DNA sequences (NADH5, COI, Hunchback, and CAD). Additionally, we employed the isolation with migration model (IMa) to evaluate: 1) the migration rates and 2) the divergence time, between Cx. coronator and Cx. usquatus. Specimens were captured in Pariquera-Açu and Cananéia south-eastern São Paulo, Ribeira Valley, Brazil. Canonical variate analysis (CVA) demonstrated two groups in the morphospace. The accuracy of species recognition was moderate (82.6%) for Cx. coronator and low (60.8%) for Cx. usquatus. Bayesian analyses of concatenated gene sequences recovered from specimens of Cx. coronator separated the species into three lineages (herein referred to as Culex coronator A, B, and C), whereas Cx. usquatus specimens clustered into a single lineage. Lineages A and B of Cx. coronator intermixed with specimens of Cx. usquatus, and one specimen of Cx. coronator clustered into the Cx. usquatus lineage. The IMa analysis indicated that the divergence of Cx. coronator and Cx. usquatus is a slow process, with some degree of gene flow between the two species.

Phylogenetically diverse group of native bacterial symbionts isolated from root nodules of groundnut (Arachis hypogaea L.) in South Africa

Groundnut is an economically important N​2-fixing legume that can contribute about 100–190kgNha−1 to cropping systems. In this study, groundnut-nodulating native rhizobia in South African soils were isolated from root nodules. Genetic analysis of isolates was done using restriction fragment length polymorphism (RFLP)-PCR of the intergenic spacer (IGS) region of 16S-23S rDNA. A total of 26 IGS types were detected with band sizes ranging from 471 to 1415bp. The rhizobial isolates were grouped into five main clusters with Jaccard's similarity coefficient of 0.00–1.00, and 35 restriction types in a UPGMA dendrogram. Partial sequence analysis of the 16S rDNA, IGS of 16S rDNA-23S rDNA, atpD, gyrB, gltA, glnII and symbiotic nifH and nodC genes obtained for representative isolates of each RFLP-cluster showed that these native groundnut-nodulating rhizobia were phylogenetically diverse, thus confirming the extent of promiscuity of this legume. Concatenated gene sequence analysis showed that most isolates did not align with known type strains, and may represent new species from South Africa. This underscored the high genetic variability associated with groundnut Rhizobium and Bradyrhizobium in South African soils, and the possible presence of a reservoir of novel groundnut-nodulating Bradyrhizobium and Rhizobium in the country.

Prototheca tumulicola sp. nov., a novel achlorophyllous, yeast-like microalga isolated from the stone chamber interior of the Takamatsuzuka Tumulus

Two achlorophyllous microalgal strains were isolated from the soil and white moldy colony collected inside the stone chamber of the Takamatsuzuka Tumulus in Japan. Phylogenetic analyses of the small subunit ribosomal RNA (SSU rRNA) and Dl/D2 large subunit ribosomal RNA (LSU rRNA) gene sequences, and concatenated gene sequences of the SSU and D1/D2 LSU rRNA genes indicated that our two isolates were the members of the non-photosynthetic, yeast-like microalgal Chlorellaceous genus Prototheca (Chlorellales, Trebouxiophyceae, Chlorophyta) but well distinguished from known species. Based on phenotypic and genotypic characteristics, isolates T6713-13-10T and T61213-7-11 are proposed to represent a novel species in Prototheca, P. tumulicola, with the type strain JCM 31123T (isolate T6713-13-10T).

Microsymbiont diversity and phylogeny of native bradyrhizobia associated with soybean (Glycine max L. Merr.) nodulation in South African soils

The genetic diversity and identification of slow- and fast-growing soybean root nodule bacterial isolates from different agro-climatic regions in Mpumalanga, Limpopo and Gauteng Provinces of South Africa were evaluated. The 16S-rDNA-RFLP analysis of 100 rhizobial isolates and eight reference type strains placed the isolates into six major clusters, and revealed their site-dependent genomic diversity. Sequence analysis of single and concatenated housekeeping genes (atpD, glnII and gyrB), as well as the symbiotic gene nifH captured a considerably higher level of genetic diversity and indicated the dominance of Bradyrhizobium diazoefficiens and Bradyrhizobium japonicum in Mpumalanga, Limpopo and Gauteng Provinces. Gene sequence similarities of isolates with type strains of Bradyrhizobium ranged from 97.3 to 100% for the 16S rDNA, and 83.4 to 100% for the housekeeping genes. The glnII gene phylogeny showed discordance with the other genes, suggesting lateral gene transfer or recombination events. Concatenated gene sequence analysis showed that most of the isolates did not align with known type strains and might represent new species from South Africa. This underscores the high genetic variability associated with soybean Bradyrhizobium in South African soils, and the presence of an important reservoir of novel soybean-nodulating bradyrhizobia in the country. In this study, the grouping of isolates was influenced by site origin, with Group I isolates originating from Limpopo Province and Groups II and III from Mpumlanga Province in the 16S rDNA-RFLP analysis.