Relationships between two Pseudoalteromonas strains isolated from the Canada Basin and the Southern Ocean using a polyphasic approach

The evolutionary relationships and taxonomic position of two marine planktonic bacterial strains BSw20211 and BSw10014, isolated from the Canada Basin and from the Southern Ocean, respectively, were determined using a polyphasic taxonomic approach. There was a close phylogenetic relationship between the two strains and most phenotypic properties were shared. Nonetheless, they were found to belong to different species of the genus Pseudoalteromonas on the basis of genotypic analyses. Findings were consistent with the suggestion that gyrB gene sequence comparison and DNA-DNA relatedness might better define phylogenetic relationships of bacteria at the species level. However, a cut-off value of 90% gyrB gene sequence similarity was not reliable for the differentiation of species within the genus Pseudoalteromonas.     

Phylogenetic analysis of cultivable bacteria isolated from Arctic sea-ice

Phylogenetic analysis based on 16S rDNA of 8 strains of cultivable bacteria isolated from Arctic sea-ice was studied.The results showed that strain BJ1 belonged to genus Planococcus,which was a genus of low mole percent G C gram-positive bacteria;strain BJ6 belonged to genus Burkholderia of β-proteobacteria and the rest 6 strain all belonged to γ-proteobacteria,of which strain BJ8 was a species of Pseudoalteromonas,strain BJ2-BJ5 and BJ7 were members of genus Psychrobacter.Phylogenetic analysis also indicated that bacteria of genus Psychrobacter of the isolates formed a relatively independent phylogenetic cluster in comparison with other bacteria belonged to genus Psychrobacter.     

Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

1　Introduction　PorphyraisthemainobjectforalgafarmingandplaysaveryimportantroleinChinesemarineindus tries.Recently ,therehasbeenagreatlossinPor phyracultivationduetothedegenerationoftheculti var,sothereisanincreasingdemandforgoodPor phyracultivars .Theprerequisiteofthetraditionalbreed ingandbioengineeringresearchofPorphyraistheconstructionofpurelines .Traditionally ,theclassifi cationofPorphyrawasaccordingtotheirmorphologi calcharacteristics .However ,mostmorphologicalfea turesofPorphyraar…     

A re-investigation of the bloom-forming unarmored dinoflagellate Karenia longicanalis (syn. Karenia umbella ) from Chinese coastal waters

The dinoflagellate genus K arenia is known for recurrent harmful blooms worldwide. However, species diversity of the genus is generally overlooked owing to the difficulty of identifying small unarmored dinoflagellates. We have established four clonal cultures of K arenia longicanalis isolated from the type locality, Hong Kong harbor(strain HK01) and other three locations along the Chinese coasts(strains YB01, DT01, and NJ01). The morphology of the strain was studied by light and scanning electron microscopy(LM and SEM) and the pigment composition analyzed by high-performance liquid chromatography. We provide the first molecular data of K. longicanalis based on the large subunit(LSU) rRNA gene sequence and internal transcribed spacer(ITS). The four strains showed identical LSU rDNA sequences with a similarity of 99.4% to the holotype of K arenia umbella(strain KUTN05) from Australia. In the ITS phylogeny, the sequence of K. umbella branched between the Chinese strains of K. longicanalis. A careful comparison of the morphology of K. longicanalis and K. umbella reveals the similarity in the diagnostic characters. Diff erences may appear due to the sample treatment for SEM. We conclude that K. umbella is a junior synonym of K. longicanalis.     

Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

The wild Porphyra yezoensis collected from the Qingdao coast was used to prepare protoplasts by enzyme digestion. The pure line was constructed by cultivating the protoplasts. The 18S rDNA of the P. yezoensis pure line was cloned and sequenced. Sequence analysis was executed for this sequence and other 22 sequences retrieved from GenBank. A phylogenetic tree was constructed using the neighbor-joining method. The results revealed a high diversity of 18S rDNA sequences in genus Porphyra and the considerable variation of 18S rDNA sequences in different strains of the same species P. yezoensis and P. tenera. Significant difference of 18S rDNA sequence was observed between P. yezoensis from Qingdao, China, and the two strains of P. yezoensis from Japan, but the three strains of P. yezoensis formed a stable clade in the phylogenetic tree. These results indicate the possibility of interspecies and intraspecies discrimination of Porphyra using the 18S rDNA sequences.     

黄喉拟水龟体表溃疡病原菌SG_(24)的分类鉴定

从患溃疡的黄喉拟水龟病灶中分离到一株病原菌SG24。对菌株SG24进行了常规生理生化测定和ATBExpression半自动细菌鉴定仪鉴定,并测定16S rRNA序列,分析其与相关细菌相应序列的同源性,构建了系统进化树。普通细菌学方法结果显示菌株SG24为黏质沙雷氏菌(Serratia marcescens)。以沙雷氏菌属的16S rRNA基因序列设计一对引物进行PCR扩增,获得了菌株SG24大小约950 bp的16S rRNA部分基因片段,测序结果显示菌株SG24与黏质沙雷氏菌同类,与已登录的黏质沙雷氏菌(S.marcescensDQ207558)的16S rRNA同源性大于99%。综合以上分类鉴定结果,确定菌株SG24属于沙雷氏菌属的黏质沙雷氏菌。药物敏感试验结果表明:菌株SG24对链霉素、庆大霉素、壮观霉素、强力霉素、卡那霉素、阿米卡星、诺氟沙星、氧氟沙星和复方新诺明敏感。     

The Complete Sequence of Mitochondrial COⅡ Gene of Fenneropenaeus chinensis and Its Applicability as a Marker for Phylogenetic Analysis

The complete mitochondrial cytochrome oxidase subunit Ⅱ (COⅡ) gene of Penaeinae shrimp Fenneropenaeus chinensis was cloned and sequenced. The gene is 688 bp in length and codes for 229 amino acids. It shows 83.2%, 87.0% and 83.8% sequence similarity to Marsupenaeus Japonicus, Penaeus monodon and Farfantepenaeus notialis, respectively. The A+T content of the whole gene and that at the third position of codons are 64.7% and 78.2%, respectively. The phylogenetic relationship between F. chinensis and three other species representing genera Farfanatepenaeus, Marsupenaeus and Penaeus was analyzed. Results showed that the genetic distances among the four taxa ranged from 0.144 0 to 0.200 5, exceeding those estimated with COⅠ and partial 16S rRNA gene sequences among Marsupenaeus, Litopenaeus and Melicertus, and being therefore larger than the value among subgenera. It has been suggested that the COⅡ gene has a faster evolutionary rate than that of the COⅠ gene and partial 16S rRNA gene and could be used for phylogenetic analysis at genus or species level. The results of the present study indicated that Farfantepenaeus, Fenneropenaeus, Marsupenaeus and Penaeus are at a higher phylogenetic level than subgenus, which supports the opinion of the elevation of phylogenetic status of the four subgenera to genus level.     

Methods used to study bacterial diversity in the marine environment around Qingdao

Pollution has a considerable effect on biological communities, in terms of size and diversity of the populations. Yet, the precise consequences of human activity on microbial communities in the marine environment are poorly understood. Therefore, in an ongoing collaborative research programme between Heriot-Watt University and the Ocean University of Qingdao, bacteria were isolated in 1999 and 2000 from marine sediment, seawater, seaweed, fish and shellfish, taken from locations in Shandong Province adjacent to Qingdao. Sampling locations were comprised of industrial and aquacultural sites and a clean, control site. In order to analyse microbial diversity, a polyphasic approach was adopted for characterisation of these isolates, specifically through examination of key phenotypic traits, i.e. using Biolog GN MicroPlate^™ profiles, bacterial whole cell protein profiles and 16S and 23S rRNA gene sequences. These techniques yielded complex taxonomic data, which were subjected to statistical and cluster analyses. The application of these methods to studies of microbial communities is discussed.     

The Complete Sequence of Mitochondrial COII Gene of Fenneropenaeus chinensis and Its Applicability as a Marker for Phylogenetic Analysis

The complete mitochondrial cytochrome oxidase subunit II (COII) gene of Penaeinae shrimp Fenneropenaeus chinen- sis was cloned and sequenced. The gene is 688 bp in length and codes for 229 amino acids. It shows 83.2%, 87.0% and 83.8% sequence similarity to Marsupenaeus japonicus, Penaeus monodon and Farfantepenaeus notialis, respectively. The A T content of the whole gene and that at the third position of codons are 64.7% and 78.2%, respectively. The phylogenetic relationship between F. chinensis and three other species representing genera Farfanatepenaeus, Marsupenaeus and Penaeus was analyzed. Results showed that the genetic distances among the four taxa ranged from 0.144 0 to 0.200 5, exceeding those estimated with COI and partial 16S rRNA gene sequences among Marsupenaeus, Litopenaeus and Melicertus, and being therefore larger than the value among subgenera. It has been suggested that the COII gene has a faster evolutionary rate than that of the COI gene and partial 16S rRNA gene and could be used for phylogenetic analysis at genus or species level. The results of the present study indicated that Farfantepenaeus, Fenneropenaeus, Marsupenaeus and Penaeus are at a higher phylogenetic level than subgenus, which supports the opinion of the elevation of phylogenetic status of the four subgenera to genus level.     

The Complete Sequence of Mitochondrial COⅡ Gene of Fenneropenaeus chinensis and Its Applicability as a Marker for Phylogenetic Analysis

The complete mitochondrial cytochrome oxidase subunit Ⅱ （COⅡ） gene of Penaeinae shrimp Fenneropenaeus chinensis was cloned and sequenced. The gene is 688 bp in length and codes for 229 amino acids. It shows 83.2%, 87.0% and 83.8% sequence similarity to Marsupenaeus Japonicus, Penaeus monodon and Farfantepenaeus notialis, respectively. The A＋T content of the whole gene and that at the third position of codons are 64.7% and 78.2%, respectively. The phylogenetic relationship between F. chinensis and three other species representing genera Farfanatepenaeus, Marsupenaeus and Penaeus was analyzed. Results showed that the genetic distances among the four taxa ranged from 0.144 0 to 0.200 5, exceeding those estimated with COⅠ and partial 16S rRNA gene sequences among Marsupenaeus, Litopenaeus and Melicertus, and being therefore larger than the value among subgenera. It has been suggested that the COⅡ gene has a faster evolutionary rate than that of the COⅠ gene and partial 16S rRNA gene and could be used for phylogenetic analysis at genus or species level. The results of the present study indicated that Farfantepenaeus, Fenneropenaeus, Marsupenaeus and Penaeus are at a higher phylogenetic level than subgenus, which supports the opinion of the elevation of phylogenetic status of the four subgenera to genus level.     

Screening and molecular classification of low-temperature protease from Antarctic microorganism and its characterization

107 strains producing protease were screened from 260 strains of Antarctic psychrophilic bacteria, among which proteolytic activity of five strains was more than 45 U ml^-1. The 16S rRNA gcne sequences homology and phylogcnetic analysis of five Antarctic psychrophillc bacteria showed that NJ276, NJS-9, NJ16-70,NJ345 belonged tO the described genus Pseudoalteromonas and NJ341 belonged to the genus Colwellia. The growth and the protease characteristic of four Antarctic psychrophilic bacteria had been studied, and the result showed that the 6ptimal temperature for growth and protease-produeing of four strains was about 10℃. Their growth and protease-produeing were still high during incubatlng 2-5 days. The maximum proteolytic activity occurred at pH 9 for four Antarctic psychrophilic bacteria. The optimal temperature of protease action of both strains NJ276 and NJ5-9 was about 50℃, however, the optimal temperature of protease aetlon of both strains NJ341 and NJ345 was about 40 ℃, and their proteolytic activity under 0℃ exhibited nearly 30% of the maximum activity, but their thermal stabilities were weaker. These results indicated that proteases from NJ341 and NJ345 were low-temperature proteases.     

Preliminary Identification of Three New Isolates in Genus Alexandrium(Dinophyceae) from China Sea Area

1 Introduction Dinoflagellate Alexandrium has received intensive at-tention in recent years, as it is a valuable bioactive com-pound resource and related to harmful algal bloom (HAB)which has negative impact on marine ecology systemaquiculture, human health and tourism (Hallegraeff andBolch, 1992; Smayda, 1997; Vale and Antonia de MSampayo, 2001). However, not all of the species in genusAlexandrium produce toxin (Wendy et al., 2001), so rapididentification and clarification of the phyloge…     

Molecular phylogeny and evolution of Scomber (Teleostei: Scombridae) based on mitochondrial and nuclear DNA sequences

A molecular phylogenetic analysis of the genus Scomber was conducted based on mitochondrial (COI, Cyt b and control region) and nuclear (5S rDNA) DNA sequence data in multigene perspective. A variety of phylogenetic analytic methods were used to clarify the current taxonomic Classification and to assess phylogenetic relationships and the evolutionary history of this genus. The present study produced a well-resolved phylogeny that strongly supported the monophyly of Scomber. We confirmed that S. japonicus and S. colias were genetically distinct. Although morphologically and ecologically similar to S. colias, the molecular data showed that S. japonicus has a greater molecular affinity with S. australasicus, which conflicts with the traditional taxonomy. This phylogenetic pattern was corroborated by the mtDNA data, but incompletely by the nuclear DNA data. Phylogenetic concordance between the mitochondrial and nuclear DNA regions for the basal nodes Supports an Atlantic origin for Scomber. The present-day geographic ranges of the species were compared with the resultant molecular phylogeny derived from partition Bayesian analyses of the combined data sets to evaluate possible dispersal routes of the genus. The present-day geographic distribution of Scomber species might be best ascribed to multiple dispersal events. In addition, our results suggest that phylogenies derived from multiple genes and long sequences exhibited improved phylogenetic resolution, from which we conclude that the phylogenetic reconstruction is a reliable representation of the evolutionary history of Scomber.     

Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum

The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.     

Molecular Identification of Dried Shellfish Products Sold on the Market Using DNA Barcoding

The dried shellfish products with rich nutrients and low-calorie content are favorite food in China, especially in coastal areas. However, the species of dried shellfish products in the market are usually unknown, as the taxonomic features were removed during the production process. This study described the application of DNA barcoding technique to the identification of 100 dried shellfish(scallop, squid, octopus and cuttlefish) products in markets. Samples were authenticated by comparing mitochondrial cytochrome oxidase subunit I(COI) gene and 16 S ribosomal RNA(16 S rRNA) gene sequences with public reference taxonomic databases. The results showed that all the 100 products can be identified at species level. Sixty four scallop adductor products were processed using the bay scallop, Argopecten irradians, and one was from Portuguese oyster, Crassostrea angulata. All the 27 squid, 2 cuttlefish and 6 octopus products were produced by the Jumbo flying squid, Dosidicus gigas. The neighbour-joining tree is in agreement with the results of DNA barcoding analysis. The 64 scallop samples formed one A. irradians cluster, leaving Sca65 clustered with the reference oyster sequence C. angulata(MH997922). All the 35 cephalopod(squid, octopus and cuttlefish) samples formed a D. gigas cluster. This investigation revealed a low variety of dried shellfish products sold on the market, and highlighted the high rate of mislabeling and species substitution. Our present work provides a practical method for tracing and authenticating shellfish products.     

A preliminary phylogenetic analysis of Luidia (Paxillosida: Luidiidae) from Chinese waters with cytochrome oxidase subunit I (COI) sequences

Luidia Forbes (Paxillosida: Luidiidae) are common soft bottom sea stars with 49 described species. Because of substantial morphological diversity, the taxonomy of the genus is complex and hasn’t been resolved definitely. In order to resolve general taxonomic issues, and determine species boundaries and phylogenetic relationships within the genus Luidia, the sequences of cytochrome oxidase subunit I (COI) gene from 24 specimens of Luidia, belonging to eight taxa in Chinese waters, were studied. Three sequences of two species in genus Luidia from GenBank were used to analyze the phylogenetic relationships. The molecular phylogeny exhibited three main clades, each with strong bootstrap support: Clade A including Luidia quinaria from the Sea of Japan; Clade B including seven nominal species (L. quinaria von Martens, L. yesoensis Goto, L. changi Liu, Liao and Li, L. orientalis Fisher, L. avicularia Fisher, L. longispina Sladen and L. hardwicki Gray) from Chinese waters; and Clade C including L. maculata Müller & Troschel from Chinese waters. Our molecular phylogeny results support the morphological Quinaria-Group and Alternata-Group assigned by Döderlein. Seven nominal species we sampled do not exhibit genetic distances that are large enough to recognize them as separate species. Cryptic species may exist in’ Luidia quinaria’ from the Yellow Sea and the Sea of Japan. Meaningful morphological characters need further investigation in Luidia.     

Gel microbead cultivation with a subenrichment procedure can yield better bacterial cultivability from a seawater sample than standard plating method

A gel microbead (GMD) cultivation method was employed to cultivate microorganisms from an amphioxus breeding zone in Qingdao, P. R. China. The culture results were compared with those by standard plating method. In the GMD-based method, the microcolony-forming GMDs were sorted by fluorescence-activated cell sorting (FACS). To further get pure cultures, a subsequent enrichment culture and a streaking purification procedure were conducted on marine R2A medium. Eighty bacterial strains isolated by the GMD-based method were randomly selected for sequencing. These isolates belonged to Alphaproteobacteria (33%), Gammaproteobacteria (44%), Bacteroidetes (11%), Actinobacteria (5%), Firmicutes (5%), Epsilonproteobacteria (1%), and Verrucomicrobia (1%), the last two groups being usually difficult to culture. The 16S rRNA gene sequences revealed a diverse community with 91.1%-100% of the bacterial rRNAs similarities. Thirteen strains were sharing 16S rRNA gene sequence which was less than 97% similar to any other rRNA genes currently deposited in TYP16S database. Seventy isolates derived from the standard plating method fell into 4 different taxonomic groups: Alphaproteobacteria (9%), Gammaproteobacteria (81%), Bacteroidetes (7%) and Firmicutes (3%) with a 16S rRNA gene sequence similarities between 95.8%-100%, in which only 3 strains were sharing 16S rRNA gene sequence of less than 97%. The results indicated that the GMD-based method with subenrichment culture yielded more taxonomic groups and more novel microbial strains, including members of previously rarely cultured groups, when compared with the standard plating method, and that this method markedly improved the bacterial cultivability.     

Monophyly or polyphyly? Possible conflict between morphological and molecular interpretations of the wellknown genus Zoothamnium(Ciliophora,Peritrichia)

In this paper,we explore possible conflict between morphological and molecular interpretations of phylogenetic relationships within the well-known peritrichous genus Zoothamnium. On the basis of morphological evidence,for a long time this genus has been believed to be a well-defined monophyletic taxon. Nonetheless,Z oothamnium exhibits higher genetic diversity than the gross morphology of its species. Here,we used all available genetic information for the small subunit of ribosomal RNA(SSU r RNA) and internal transcribed spacer region(ITS1-5.8S-ITS2) for this genus to reconstruct phylogenies for four datasets(SSU rRNA,ITS1,ITS2,and ITS1-5.8S-ITS2) and a combined dataset(SSU rRNA+ITS1-5.8SITS2) using different phylogenetic methods and with consideration of the secondary structure of the genes. Confidence in phylogenetic tree selection was assessed with the approximately unbiased test. The molecular results showed both that Zoothamnium is more likely to be polyphyletic,and morphologically similar genera Zoothamnopsis and Myoschiston were always nested among Zoothamnium species. Accordingly,as with some other groups of ciliates,to understand more fully the correct phylogeny of Zoothamnium there remains a need for additional data from both morphological and molecular studies,covering additional Zoothamnium spp. and members of closely related genera(e.g. Zoothamnopsis,Myoschiston,and Epistylis).     

Phylogenetic relationships within the genus Aspidisca (Protozoa, Ciliophora, Euplotida) revealed by ITS1-5.8S-ITS2 region sequences

The internal transcribed spacer regions (ITS1 and ITS2) and 5.8S rRNA genes were sequenced in six populations of four Aspidisca species, namely A. leptaspis, A. orthopogon, A. magna and A. aculeata. Phylogenetic trees were constructed by means of Bayesian inference (BI), Maximum Parsimony (MP), Neighbor-Joining (NJ), and Maximum Likelihood (ML) to assess the inter- and intra-species relationships within the genus Aspidisca. All trees show similar topologies with stable supports and indicate that: (1) four well known groups, i.e., Oligotrichia, Stichotrichia, Choreotrichia and Hypotrichia, are distinctly outlined within the class Spirotrichea, and all are monophyletic other than Hypotrichia; (2) members of Aspidisca can be distinguished well, based on the ITS1-5.8S-ITS2 region sequences, and A. leptaspis and A. magna shared a closer relationship than other species; (3) Aspidisca and Euplotes branch early in the subclass Hypotrichia. To compare the phylogenetic relationships based on different genes, SSU rRNA trees were also constructed with nearly the same species inclusion, which revealed different topologies of inter-species, inter-genera and inter-subclasses.