16S rRNA Nucleotide Sequence Research Articles

Molecular characterization and pathogenicity of Francisella noatunensis subsp. orientalis isolated from cultured tilapia (Oreochromis sp.) in Taiwan

Francisella noatunensis subsp. orientalis is a causative agent of systemic granulomatous disease in tilapia. The present study was designed to understand the genetic and phenotypic diversities among Taiwanese Fno isolates obtained from tilapia (n=17) and green Texas cichlid (Herichthys cyanoguttatus) (n=1). The enzymatic profiles of the isolates were studied using the API ZYM system. Phylogenetic tree analysis of the 16S rRNA and housekeeping gene and pulsed-field gel electrophoresis (PFGE) were carried out to determine the genotypic characters of all isolates. The phylogenetic tree showed similarity of 99%-100% nucleotide sequences of 16S rRNA and housekeeping genes compared to the Fno references genes from GenBank database. Comparatively, the results revealed an identical profile of enzymatic and PFGE pattern which was distincted from that of F. philomiragia. To understand the pathogenicity, the isolates were intraperitoneal injected to tilapia the gross lesions were observed concomitant with natural outbreak. Median lethal dose upon Nile tilapia and red tilapia were 9.06 × 103 CFU/fish and 2.08 × 102 CFU/fish, respectively. Thus, our data provide understanding the epidemiology of Taiwanese Fno isolates, and help in development of future control and prevention.

Host-dependent symbiotic efficiency of Rhizobium leguminosarum bv. trifolii strains isolated from nodules of Trifolium rubens

Trifolium rubens L., commonly known as the red feather clover, is capable of symbiotic interactions with rhizobia. Up to now, no specific symbionts of T. rubens and their symbiotic compatibility with Trifolium spp. have been described. We characterized the genomic diversity of T. rubens symbionts by analyses of plasmid profiles and BOX–PCR. The phylogeny of T. rubens isolates was inferred based on the nucleotide sequences of 16S rRNA and two core genes (atpD, recA). The nodC phylogeny allowed classification of rhizobia nodulating T. rubens as Rhizobium leguminosarum symbiovar trifolii (Rlt). The symbiotic efficiency of the Rlt isolates was determined on four clover species: T. rubens, T. pratense, T. repens and T. resupinatum. We determined that Rlt strains formed mostly inefficient symbiosis with their native host plant T. rubens and weakly effective (sub-optimal) symbiosis with T. repens and T. pratense. The same Rlt strains were fully compatible in the symbiosis with T. resupinatum. T. rubens did not exhibit strict selectivity in regard to the symbionts and rhizobia closely related to Rhizobium grahamii, Rhizobium galegae and Agrobacterium radiobacter, which did not nodulate Trifolium spp., were found amongst T. rubens nodule isolates.

The new bacterial strain Paenibacillus sp. IB-1: A producer of exopolysaccharide and biologically active substances with phytohormonal and antifungal activities

The bacterial strain IB-1, which exhibits antagonism towards phytopathogens and stimulates the growth of agricultural plants, was isolated from the soil. Analysis of the cultural, morphological, physiological, and biochemical features and nucleotide sequences of the genes 16S rRNA and gyrB, as well as the fatty acid composition, made it possible to attribute the strain IB-1 to the genus Paenibacillus; however, the results did not provide an unambiguous conclusion on its species. The strain Paenibacillus sp. IB-1 possesses nitrogenase activity, the ability to synthesize indoleacetic acid and cytokinin-like compounds, and antagonistic activity towards phytopathogenic fungi, which indicates prospects for its use as a biological product for agricultural purposes. A high-viscous exopolysaccharide was isolated from the cultural fluid of Paenibacillus sp. IB-1. Based on the data from IR and NMR spectroscopy, it was shown to be a heteropolymer comprised of one to four linked α-L-guluronic acid and β-D-mannuronic acid residues. The exopolysaccharide was successfully tested as an adhesive for presowing treatment of barley and wheat seeds with biofungicides.

Molecular Characterization of Anaplasma sp. in Free-Living Gray Brockets (Mazama gouazoubira).

Bacteria in the genus Anaplasma are responsible for diseases in animals and humans. Studies carried out in Brazil have demonstrated that Brazilian deer are able to act as hosts of agents in the family Anaplasmataceae and are possibly potential reservoirs of these pathogens. Molecular and phylogenetic studies have been carried out on samples of two gray brocket specimens (Mazama gouazoubira) from the city of Guarapuava, state of Paraná, Brazil, for the detection of Anaplasma sp. in these animals. Partial nucleotide sequences of the genes 16S rRNA and groESL were used for phylogenetic analyses and compared with other 13 and 17 partial sequences of the respective genes obtained in GenBank. These assessments showed topological incongruence among the trees generated in the phylogenetic analyses. Phylogenetic analysis based on the gene 16S rRNA of the genotypes amplified in the samples of this study was similar to those of A. bovis detected in dogs and wild deer in Japan, whereas studies carried out on gene groESL indicated proximity with sequences of Anaplasma sp. that were also isolated in deer in Japan and allocated in the same clade of partial sequences of A. phagocytophilum. As the 16S rRNA gene is highly conserved, with few polymorphic positions, it may show low reliability for studies on phylogenetic positioning. The present study detected an Anaplasma sp. genotype in two specimens of M. gouazoubira in southern Brazil, which may mean that this agent possibly circulates in deer populations, and demonstrated the need for studies related to the possible role of deer in enzootic cycles of Anaplasmataceae in Brazil.

Comparative evaluation of sequence analysis of 16S rRNA and rpoBgenes for identification of aquatic mycobacteria

The nucleotide sequences of partial 16S rRNA and bacterial RNA polymerase β-subunit (rpoB ) genes for 57 mycobacterial strains were determined. Compared to the 16S rRNA gene sequences, variable regions were scattered along the whole fragment sequence, indicating that the rpoB gene is more polymorphic. Unlike 16S rRNA sequences, species variation was observed within M. fortuitum strains. The topology of the rpoB-based phylogenetic tree was almost the same as that of the 16S rRNA sequence analysis. These results suggest that the rpoB gene is a highly conserved gene, and taxonomical studies based on this gene may be comparable with similar studies based on the 16S rRNA gene. The overall mean distance for rpoB -gene-based sequences was 2.5 times greater than that of the 16S rRNA gene for all 57 mycobacterial strains examined. However, some slowly growing mycobacteria could not be differentiated based on rpoB gene sequences. Moreover, a bootstrap value above 70% was observed for 13 nodes, while this value was 14 nodes in the case of 16S rRNA sequences. To the best of our knowledge, this is the first investigation evaluating the use of 16S rRNA and rpoB sequence analyses for identification of aquatic mycobacteria obtained from diverse geographical locations.

Comparative study of different molecular methods for typing of Acinetobacter baumannii clinical isolates from University Hospitals

Acinetobacter baumannii, a rod-shape Gram-negative bacterium, is an opportunistic pathogen causing diseases in humans. This bacterium has been recognized as one of the leading causes of nosocomial infection which occurs in hospital or hospital-like setting. The antibiotic resistance of A. baumannii could result from the heavy use of antibiotics and has been recognized as a threat to human health. However, prevention against the disease caused by A. baumannii is difficult due to variable host susceptibility against their infections. We isolated 53 bacterial strains from four different university hospitals in South Korea and identified 34 out of the 53 isolates as A. baumannii, based on the nucleotide sequence of 16S rRNA and gpi genes. For the subtyping of the clinical isolates, we used enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and multilocus sequencing typing (MLST) and compared the results. The result of ERIC-PCR showed that there are 14 distinct DNA fingerprint patterns in the 34 A. baumannii clinical isolates. For MLST analysis of the isolates, we amplified and sequenced seven housekeeping genes (gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD) from each isolate. Each unique allelic profile at the concatenated nucleotide sequences of the seven genes was assigned as a sequence type (ST) and six different STs (ST92, ST105, ST138, ST169, ST262, and ST357) were detected in the 34 A. baumannii clinical isolates. Among the six STs, ST138 was the most ubiquitous in the A. baumannii clinical isolates. To examine the regional distribution of the isolates, STs were clustered into clonal complexes based on their similarity to a previously registered central genotype and the clustering was verified by network phylogenetic analysis.

Burkholderia pyrrocinia CAB08106-4 균주를 이용한 마늘 흑색썩음균핵병의 생물학적 방제

Sclerotium cepivorum에 의한 흑색썩음균핵병은 마늘에 큰 피해를 주는 중요한 토양병이다. 충남 서산 및 태안의 마늘 재배농가 포장을 중심으로 2009년부터 3년간 조사한 결과, 흑색썩음균핵병은 4월 하순부터 발생을 시작하고, 5월 중순에 발병이 급격히 증가하여 5월 하순에는 최고 발병률을 나타내었다. 본 시험에 사용된 길항미생물 CAB008106-4 균주는 16S rRNA gene의 염기서열 분석결과 Burkholderia pyrrocinia로 확인되었다. B. pyrrocinia CAB08106-4 균주는 흑색썩음균핵병에 대하여 69.6%의 포장방제효과를 보였다. 이러한 결과로 B. pyrrocinia CAB08106-4는 마늘에 발생하는 흑색썩음균핵병에 대하여 효과적인 생물적 방제제로 활용이 가능함을 확인할 수 있었다. White rot caused by Sclerotium cepivorum was reported to be severe soil-born disease on garlic. Disease progress of white rot of garlic (Allium sativum L.) was investigated during the growing season of 2009 to 2011 at Taean and Seosan areas. The white rot disease on bulb began to occur from late April and peaked in late May. The antifungal bacteria, Burkholderia pyrrocinia CAB08106-4 was tested in field bioassay for suppression of white rot disease. As a result of the nucleotide sequence of the gene 16S rRNA, CAB008106-4 strain used in this study has been identified as B. pyrrocinia. B. pyrrocinia CAB080106-4 isolate suppressed the white rot with 69.6% control efficacy in field test. These results suggested that B. pyrrocinia CAB08106-4 isolate could be an effective biological control agent against white rot of garlic.

Isolation and characterization of Pseudomonas sp. strain capable of degrading diethylstilbestrol

Since diethylstilbestrol (DES) interrupts endocrine systems and generates reproductive abnormalities in both wildlife and human beings, methods to remove DES from the environments are urgently recommended. In this study, bacterial strain J51 was isolated and tested to effectively degrade DES. J51 was identified as Pseudomonas sp. based on its nucleotide sequence of 16S rRNA. The quinoprotein alcohol dehydrogenase and isocitrate lyase were identified to be involved in DES degradation by MALDI-TOF-TOF MS/MS analysis. In the presence of 40 mg/l DES, increase of the genes encoding quinoprotein alcohol dehydrogenase and isocitrate lyase in both RNA and protein levels was determined. The HPLC/MS analysis showed that DES was hydrolyzed to a major degrading metabolite DES-4-semiquinone. It was the first time to demonstrate the characteristics of DES degradation by specific bacterial strain and the higher degradation efficiency indicated the potential application of Pseudomonas sp. strain J51 in the treatment of DES-contaminated freshwater and seawater environments.

Molecular identification of Korean catfish (Siluriformes) based on two genetic markers

Eleven species of catfish (Siluriformes) found in Korean freshwater are economically important resources and include five endemic species. However, there are no studies on phylogenetic analysis of all catfish species in Korea at a molecular level. The species-level analysis of catfish species is usually carried out through morphological characters and controversial due to phenotypic variation. In this study, the partial sequences of 16S rRNA and cytochrome b mitochondrial genes were analyzed for species identification and phylogenetic relationships among 11 species of catfish from 10 different rivers in Korea. The nucleotide sequences of 16S rRNA and cytochrome b consisted of 587 and 441 nucleotide base pairs, respectively. Sequence analysis of both genes revealed that the 11 species fell into three distinct groups, which were genetically distinct from each other and exhibited identical phylogenetic resolution. Sequence divergences between congeneric species averaged 1.78% and 7.39% for 16S rRNA and cytochrome b, respectively. The phylogenetic relationships forming well-differentiated clades in the NJ, ML and BI trees were identical for both fragments. This research demonstrates that partial sequences of both the genes can efficiently identify the 11 species of catfish in Korea, indicating the usefulness of mtDNA-based approach in species identification.

Control of postharvest soft rot caused by Erwinia carotovora of vegetables by a strain of Bacillus amyloliquefaciens and its potential modes of action

Erwinia carotovora subsp. carotovora (Ecc), the causal agent of bacterial soft rot, is one of the destructive pathogens of postharvest vegetables. In this study, a bacterial isolate (BGP20) from the vegetable farm soil showed strong antagonistic activity against Ecc in vitro, and its twofold cell-free culture filtrate showed excellent biocontrol effect in controlling the postharvest bacterial soft rot of potatoes at 25 °C. The anti-Ecc metabolites produced by the isolate BGP20 had a high resistance to high temperature, UV-light and protease K. Based on the colonial morphology, cellular morphology, sporulation, and partial nucleotide sequences of 16S rRNA and gyrB gene, the isolate BGP20 was identified as Bacillus amyloliquefaciens subsp. plantarum. Further in vivo assays showed that the BGP20 cell culture was more effective in controlling the postharvest bacterial soft rot of green peppers and Chinese cabbages than its twofold cell-free culture filtrate. In contrast, the biocontrol effect and safety of the BGP20 cell culture were very poor on potatoes. In the wounds of potatoes treated with both the antagonist BGP20 and the pathogen Ecc, the viable count of Ecc was 31,746 times that of BGP20 at 48 h of incubation at 25 °C. But in the wounds of green peppers, the viable count of BGP20 increased 182.3 times within 48 h, and that of Ecc increased only 51.3 %. In addition, the treatment with both BGP20 and Ecc induced higher activity of phenylalanine ammonia-lyase (PAL) than others in potatoes. But the same treatment did not induce an increase of PAL activity in green peppers. In conclusion, the present study demonstrated that the isolate BGP20 is a promising candidate in biological control of postharvest bacterial soft rot of vegetables, but its main mode of action is different among various vegetables.

Co-producing iturin A and poly-γ-glutamic acid from rapeseed meal under solid state fermentation by the newly isolated Bacillus subtilis strain 3-10

The strain 3-10 was isolated from soil and identified as B. subtilis according to morphological and physiological characteristics and nucleotide sequence of 16S rRNA. It co-produced anti-fungal iturin A and fertilizer synergist of poly-γ-glutamic acid (γ-PGA) under solid state fermentation (SSF) with rapeseed meal. The co-production of iturin A and γ-PGA reached 5.3 and 51.3 g/kg-dry weight culture, respectively, and the number of viable cells reached 1.9 × 10(10) CFU/g-dry weight culture. In pot tests, the shoot length and dry weight of watermelon seedlings treated by the SSF culture improved by 48.0 and 30.8%, respectively compared to the control; and its biocontrol effect on watermelon fusarium wilt achieved 89.6%. These results highlight a novel strategy to exploit the low-cost and widely available rapeseed meal as dual-functional bio-organic fertilizer under SSF by B. subtilis.

Anaplasma phagocytophilum in livestock and small rodents

To identify the prevalence of Anaplasma phagocytophilum in both wild rodents and domestic animals and to make clear the genetic characteristics of the agents from different animals in China, a total of 105 livestock and 159 small rodents were analyzed by real-time-PCR and sequence analysis. The prevalence rate was 6.7% (7/105) and 14.5% (23/159), respectively. The nucleotide sequences of 16S rRNA ( rrs) from the positive livestock and rodents were identical to each other. The phylogenetic analysis based on partial A. phagocytophilum p44ESup1 gene revealed that A. phagocytophilum identified in this study was placed on a separate clade distinct from those in other continents. These findings indicated A. phagocytophilum in rodents might be able to infect livestock and intensified the threats of anaplasmosis to livestock in the area. Further studies on public health significance of the agent are worth investigation in future.

Comparison of 16S rRNA, 23S rRNA andgyrB genes sequences in phylogenetic relationships ofShigella isolates from Iran

Phylogenetic analysis of about twenty isolates ofShigella spp. was carried out using the nucleotide sequences of 16S rRNA, 23S rRNA and DNA gyrase B (gyrB) genes which was determined by directly sequencing PCR fragments. The results showed that the similarity based on 16S rRNA for specimens belonging to the same species ofShigella flexneri, Shigella boydii, Shigella dysenteriae andShigella sonnei were 99.7, 99.6, 99.5 and 100%, the corresponding results based on 23S rRNA were 99.4, 99.7, 99.3 and 100%, and forgyrB were 99.4, 99.7, 99.1 and 100% respectively. Our results demonstrate thatgyrB sequence analysis is a fruitful approach to determine the phylogenetic relationships ofShigella isolates and may be an alternative to 16S rRNA analysis. In particular,gyrB analysis of bacteria is an effective means to classify closely related species, while 23S rRNA gene sequence data may be used for phylogenetic analysis at the species level.

Utilization of Root-Colonizing Bacteria to Protect Hot-Pepper Against Tobacco Mosaic Tobamovirus

Tobacco Mosaic Tobamovirus (TMV) is one of many important viruses infecting Solanaceous plants including hot pepper in Indonesia. To accomplish and improve the effectiveness of virus management, we used root-colonizing bacteria (rhizobacteria) isolated from healthy hot pepper. Eight rhizobacteria isolates were selected and their capacity in enhancing plant growth and inducing systemic resistance (ISR) against TMV in greenhouse trials were evaluated. The rhizobacteria was applied as seed treatment and soil drench. Bacterized-seedling showed a better growth vigor, fitness and a milder symptom than non-bacterized control plants. The protective effect of rhizobacteria was more pronounced after challenging inoculation by TMV, especially for plants treated by isolates I-6, I-16, and I-35. However, TMV accumulation was slightly affected by bacterial treatment. The rhizobacteria might improved ISR by increasing peroxidase enzyme activity but this depends on the species. Based on whole results, isolate I-35 was the potential plant growth promotion rhizobacteria (PGPR). The I-35 was identified as Bacillus cereus based on morphological characteristics and nucleotide sequences of 16S r-RNA.

Japanese Spotted Fever, South Korea

We describe the first case of Japanese spotted fever and the first isolate of spotted fever group rickettsia from a patient in South Korea. The isolated rickettsia from the patient was identified as Rickettsia japonica by analysis of the nucleotide sequences of 16S rRNA, gltA, ompA, ompB, and sca4 genes.

Molecular phylogeny of silk-producing insects based on 16S ribosomal RNA and cytochrome oxidase subunit I genes

We have examined the molecular-phylogenetic relationships between nonmulberry and mulberry silkworm species that belong to the families Saturniidae, Bombycidae and Lasiocampidae using 16S ribosomal RNA (16S rRNA) and cytochrome oxidase subunit I (coxI) gene sequences. Aligned nucleotide sequences of 16S rRNA and coxI from 14 silk-producing species were used for construction of phylogenetic trees by maximum likelihood and maximum parsimony methods. The tree topology on the basis of 16S rRNA supports monophyly for members of Saturniidae and Bombycidae. Weighted parsimony analysis weighted towards transversions relative to transitions (ts, tv4) for coxI resulted in more robust bootstrap support over unweighted parsimony and favours the 16S rRNA tree topology. Combined analysis reflected clear biogeographic pattern, and agrees with morphological and cytological data.

Detection of hyperthermophilic archaea of the genus Desulforococcus by hybridization with oligonucleotide probes

Based on the analysis of nucleotide sequences of 16S rRNA, oligonucleotide probes were designed for the detection and identification of representatives of the genus Desulfurococcus (kingdom Crenarchaeota of the domain Archaea). The detection procedure included obtaining of PCR products on DNA isolated from pure cultures, enrichments, or natural samples with a Crenarchaeota-specific primer pair designed: Cren 7F (5'-TTCCGGTTGATCCYGCCGGACC-3') and Cren 518R (5'-GCTGGTWTTACCGCGGCGGCTGA-3'). The PCR products were hybridized with Dig-11-dUTP-labeled oligonucleotide probes targeting the genus Desulfurococcus (Dco 198, 5'-CGTTAACYCCYGCCACACC-3) and its species D. mobilis (Dco_mob 198, 5'-CGTTAACCCCTGCCACACC-3') and D. amylolyticus (Dco_amy 198, 5'-CGTTAACCCCCGCCACACC-3'). With the use of these primers and probes, four new strains isolated from hydrotherms of Kamchatka and Kunashir Island were identified as members of the species Desulfurococcus amylolyticus. Desulfurococcus representatives were detected in several natural samples, including a sample taken from a marine hydrotherm at the Kunashir Island; this demonstrates that representatives of this genus occur not only in terrestrial but also in marine environments.

Identification and characterization of cytopathogenic Mycoplasma hyorhinis from swine farms with a history of abortions.

A virus-like cytopathic agent isolated from swine farms with a history of recurrent abortion episodes was investigated. We employed a differential display reverse transcription-polymerase chain reaction (ddRT-PCR) to obtain genetic information of the cytopathic agent. Partial nucleotide sequence (527 bp) obtained from differentially displayed PCR fragments showed 88.7% similarity with the 23S rRNA gene of Mycoplasma hyopneumoniae. Unexpectedly, the 5' portion (1-333 bp) of the sequence shared 96.1% similarity with 5' untranslated region (UTR) of human prostate tumor inducing gene 1 (PTI-1). Cytopathic effects and extranuclear DNA fluorescence were no longer observed when BM-cyclin was added in the culture medium, suggesting that BM-cyclin sensitive mycoplasma-like organisms caused the cell death. Further evidence supporting the cytopathic agent as a mycoplasma-like organism was obtained by the capability of (3)H-thymidine and (3)H-uridine incorporation, a single peak in buoyant density gradient profile (1.20-1.24 g/ml), and ultrastructural morphology. Unlike M. hyopneumoniae, the organism was not propagated in Friis medium. Nucleotide sequence of 16S rRNA obtained from the cytopathic agent showed 0.8-1.0% divergences with other M. hyorhinis strains, suggesting that the newly isolated cytopathogenic swine mycoplasma was a variant form of M. hyorhinis. Striking homology between a portion of the 23S rRNA gene of M. hyorhinis and 5' UTR of human PTI-1 implicated that M. hyorhinis might potentially be related to the evolution of human PTI-1.

Phylogenetic study of the genus Oceanospirillum based on 16S rRNA and gyrB genes: emended description of the genus Oceanospirillum, description of Pseudospirillum gen. nov., Oceanobacter gen. nov. and Terasakiella gen. nov. and transfer of Oceanospirillum jannaschii and Pseudomonas stanieri to Marinobacterium as Marinobacterium jannaschii comb. nov. and Marinobacterium stanieri comb. no.

The phylogenetic relationships of Oceanospirillum strains were analysed by using the nucleotide sequences of 16S rRNA and gyrB genes. Results from sequence analysis demonstrated that the Oceanospirillum core group consisted of four species, Oceanospirillum linum, Oceanospirillum maris, Oceanospirillum beijerinckii and Oceanospirillum multiglobuliferum, with enough distance to separate them as different species. However, four other Oceanospirillum species occupied taxonomic positions separate from the Oceanospirillum core group: Oceanospirillum jannaschii, Oceanospirillum japonicum and Oceanospirillum kriegii in the gamma-Proteobacteria and Oceanospirillum pusillum in the alpha-Proteobacteria. Oceanospirillum jannaschii clustered with Marinobacterium georgiense, Pseudomonas iners and Pseudomonas stanieri on the basis of phylogenetic analysis of 16S rRNA and gyrB genes. The other three species did not cluster with known genera. Also, the sequence similarity values of the gyrB genes between the three subspecies of Oceanospirillum maris and those between the two subspecies of Oceanospirillum beijerinckii were above 99%. The close relationships between the subspecies of Oceanospirillum maris and of Oceanospirillum beijerinckii were further supported by similar physiological properties and high DNA-DNA hybridization values, suggesting that these subspecies should not be regarded as valid. From these results, Oceanospirillum sensu stricto should be defined to consist of Oceanospirillum linum, Oceanospirillum maris, Oceanospirillum beijerinckii and Oceanospirillum multiglobuliferum. We propose to create the following new genera: Pseudospirillum gen. nov. for Oceanospirillum japonicum as Pseudospirillum japonicum comb. nov.; Oceanobacter gen. nov. for Oceanospirillum kriegii as Oceanobacter kriegii comb. nov.; and Terasakiella gen. nov. for Oceanospirillum pusillum as Terasakiella pusilla comb. nov. The transfer is proposed of Oceanospirillum jannaschii and Pseudomonas stanieri to Marinobacterium as Marinobacterium jannaschii comb. nov. and Marinobacterium stanieri comb. nov. Furthermore, Pseudomonas iners should be reclassified as a strain of Marinobacterium georgiense. Finally, the subspecies of Oceanospirillum maris (O. maris subsp. maris, O. maris subsp. hiroshimense and O. maris subsp. williamsae) and Oceanospirillum beijerinckii (O. beijerinckii subsp. beijerinckii and O. beijerinckii subsp. pelagicum) should be combined as Oceanospirillum maris and Oceanospirillum beijerinckii, respectively.

Mitochondrial and nuclear rRNA based copepod phylogeny with emphasis on the Euchaetidae (Calanoida)

Phylogenetic relationships within the copepod family Euchaetidae and between representatives of three copepod orders (Calanoida, Harpacticoida, and Poecilostomatoida) were investigated using partial nucleotide sequences of the mitochondrial 16S rRNA and the nuclear 28S rRNA genes. DNA isolation, polymerase chain reaction, cloning, and DNA sequencing techniques were customized for these crustaceans. Our results support the monophyly of each copepod order, but in contrast to traditional morphology-based phylogenies of copepod orders, the Poecilostomatoida are basal to the Calanoida and Harpacticoida on our DNA-based phylogenetic tree. Phylogenetic trees generated by maximum parsimony, neighbor-joining, and maximum-likelihood analyses support the classification of the genera Euchaeta and Paraeuchaeta in the family Euchaetidae; results, however, suggest that Euchaetaacuta Giesbrecht is more closely related to species of the genus Paraeuchaeta than to those of Euchaeta, although limited taxon sampling may be partially responsible for this result. Phylogenetic mapping using the most parsimonious 16S tree suggests that the morphological synapomorphies distinguishing the genus Euchaeta evolved independently twice during the history of the Euchaetidae. Further, phylogenetic mapping suggests that the most recent common ancestor of the Euchaetidae and the Aetideidae was a deep-living, vertically migrating copepod, and that a bathypelagic, vertically migrating lifestyle characteristic of Paraeuchaeta is an ancestral trait of the family Euchaetidae which was lost apomorphically by Euchaeta. The application of a molecular clock suggests that the sibling species Euchaeta rimana Bradford and Euchaeta marina (Prestandrea) diverged due to the emergence of the Panamanian land bridge.