A new record of ponyfish <Emphasis Type="Italic">Deveximentum megalolepis</Emphasis> (Perciformes: Leiognathidae) in Beibu Gulf of China

A new record ponyfish, Deveximentum megalolepis Mochizuki and Hayashi, 1989, was documented based on its morphological characteristics and DNA barcode. Fifty specimens were collected from Beibu Gulf of China and identified as D. megalolepis by morphological characterization. The coloration, meristic traits, and morphometric measurements were consistent with previously published records. In general, it is a silver-white, laterally compressed and deep bodied ponyfish with 6–9 rows of scales on cheek; scale rows above lateral line 6–8; scale rows below lateral line 14–17. Mitochondrial cytochrome oxidase I subunit (COI) gene fragment was sequenced for phylogenetic analysis. There is no sequence variation of COI gene between the specimens collected in this study. The genetic distances between D. megalolepis and other congeneric species range from 3.6% to 14.0%, which were greater than the threshold for fish species delimitation. The COI sequence analysis also supported the validity of D. megalolepis at genetic level. However, the genetic distance between Chinese and Philippine individuals was about 1.2% and they formed two lineages in gene tree, which may be caused by the geographical distance.     

巨蛎属牡蛎DNA序列的比较及鉴定

【目的】了解巨砺属牡蛎的系统关系和种类区分。【方法】对我国沿海分布的7种巨砺属牡蛎(香港牡蛎、有明牡蛎、熊本牡蛎、岩牡蛎、艾氏牡蛎、太平洋牡蛎、葡萄牙牡蛎)的线粒体DNA片段(COI、12S、16S和tRNAs),以及核糖体RNA基因转录间隔子序列(ITS-1、ITS-2和ITS)进行分子系统学研究。【结果】无论种间或种内,tRNAs序列差异最大,亚种内TS1序列差异最大;tRNAs和COI序列较其它序列变异更快,种间变异与种内变异之间有明显的条形码间隙;12S、16S、ITS和ITS2的种间变异与种内变异无重叠及条形码间隙;ITS1的种间变异与种内变异出现重叠。在亚种层面,12S、ITS、ITS1和ITS2的亚种间变异与亚种内变异出现重叠;16S的亚种间变异与亚种内变异无重叠及条形码间隙;tRNAs与COI序列亚种间变异与亚种内变异之间有明显的条形码间隙,有利于区分亚种。【结论】tRNAs和COI序列可用于种及亚种的鉴定。     

Genetic identification of two species of Pleuronichthys by DNA barcoding

DNA barcoding is a new method for biological taxonomy, offering the ability to identify species from fragments in any life-history stage. Pleuronichthys cornutus and P. japonicus are two morphologically similar species. Pleuronichthys japonicus has never been found previously in China. However, in this study, we identified both species using DNA barcoding (cytochrome c oxidase subunit I (COI)), the mtDNA control region and cytochrome b. The results reveal that: 1) intraspecific variation in the DNA barcode ...     

Zooplankton community analysis in the Changjiang River estuary by single-gene-targeted metagenomics

DNA barcoding provides accurate identification of zooplankton species through all life stages. Single-gene-targeted metagenomic analysis based on DNA barcode databases can facilitate longterm monitoring of zooplankton communities. With the help of the available zooplankton databases, the zooplankton community of the Changjiang (Yangtze) River estuary was studied using a single-gene-targeted metagenomic method to estimate the species richness of this community. A total of 856 mitochondrial cytochrome oxidase subunit 1 (cox1) gene sequences were determined. The environmental barcodes were clustered into 70 molecular operational taxonomic units (MOTUs). Forty-two MOTUs matched barcoded marine organisms with more than 90% similarity and were assigned to either the species (similarity>96%) or genus level (similarity<96%). Sibling species could also be distinguished. Many species that were overlooked by morphological methods were identified by molecular methods, especially gelatinous zooplankton and merozooplankton that were likely sampled at different life history phases. Zooplankton community structures differed significantly among all of the samples. The MOTU spatial distributions were influenced by the ecological habits of the corresponding species. In conclusion, single-gene-targeted metagenomic analysis is a useful tool for zooplankton studies, with which specimens from all life history stages can be identified quickly and effectively with a comprehensive database.     

Development and evaluation of specific PCR primers targeting the ribosomal DNA-internal transcribed spacer(ITS) region of peritrich ciliates in environmental samples

Peritrich ciliates are highly diverse and can be important bacterial grazers in aquatic ecosystems. Morphological identifi cations of peritrich species and assemblages in the environment are time-consuming and expertise-demanding. In this study, two peritrich-specifi c PCR primers were newly designed to amplify a fragment including the internal transcribed spacer(ITS) region of ribosomal rDNA from environmental samples. The primers showed high specifi city in silico, and in tests with peritrich isolates and environmental DNA. Application of these primers in clone library construction and sequencing yielded exclusively sequences of peritrichs for water and sediment samples. We also found the ITS1, ITS2, ITS, D1 region of 28 S rDNA, and ITS+D1 region co-varied with, and generally more variable than, the V9 region of 18 S rDNA in peritrichs. The newly designed specifi c primers thus provide additional tools to study the molecular diversity, community composition, and phylogeography of these ecologically important protists in dif ferent systems.     

A new identification method for five species of oysters in genus Crassostrea from China based on high-resolution melting analysis

The high phenotypic plasticity in the shell of oysters presents a challenge during taxonomic and phylogenetie studies of these economically important bivalves. However, because DNA can exhibit marked differences among morphologically similar species, DNA barcoding offers a potential means for oyster identification. We analyzed the complete sequences of the cytochrome oxidase subunit I （COI） of five common Crassostrea species in China （including Hong Kong oyster C. hongkongensis, Jinjiang oyster C. ariakensis, Portuguese oyster C. angulata, Kumamoto oyster C. sikamea, and Pacific oyster C. gigas） and screened for distinct fragments. Using these distinct fragments on a high-resolution melting analysis platform, we developed an identification method that does not rely on species-specific PCR or fragment length polymorphism and is efficient, reliable, and easy to visualize. Using a single pair of primers （Oyster- COI-1）, we were able to successfully distinguish among the five oyster species. This new method provides a simple and powerful tool for the identification of oyster species.     

The Relationships Between Toxicity,Species and Populations in Nassarius Based on Toxin Detection and Multiple Gene Barcoding

Nassarius causes many poisonings in China and other Asian countries. The toxicity of Nassarius is related to species and populations. However, the molecular assignment of different toxic Nassarius samples is still unclear, which deteriorates the poisoning. Our study combined toxicity, toxin detection and multiple genes DNA barcoding to assign the various toxic Nassarius species and populations from China coast. The toxicity test indicated that different Nassarius species and different populations within one Nassarius species had different toxicity. Based on High Performance Liquid Chromatography, the toxin was confirmed to be TTX. The toxin content varied in different samples. The COI, 16 S and ITS sequences revealed different toxic Nassarius species as monophyletic clades. All the samples tested have been assigned to species level. However, the different toxic populations within N. succinctus, N. variciferus and N. semiplicata did not form separated clades, and the toxic and nontoxic species did not form two clades separately. Our study constructed the relationship between toxicity and taxonomy assignment of different toxic Nassarius samples based on toxicity detection and multiple gene DNA barcoding, which provides the basis for controlling poisoning and studying the toxin-resistance difference in Mollusca. It is also indicated that the toxicity of Nassarius is really related with species at the genetic level, but not absolutely related with populations.     

Integrating a DNA barcoding project with an ecological survey: a case study on temperate intertidal polychaete communities in Qingdao, China

In this study, we integrated a DNA barcoding project with an ecological survey on intertidal polychaete communities and investigated the utility of CO1 gene sequence as a DNA barcode for the classification of the intertidal polychaetes. Using 16S rDNA as a complementary marker and combining morphological and ecological characterization, some of dominant and common polychaete species from Chinese coasts were assessed for their taxonomic status. We obtained 22 haplotype gene sequences of 13 taxa, including 10 CO1 sequences and 12 16S rDNA sequences. Based on intra- and inter-specific distances, we built phylogenetic trees using the neighbor-joining method. Our study suggested that the mitochondrial CO1 gene was a valid DNA barcoding marker for species identification in polychaetes, but other genes, such as 16S rDNA, could be used as a complementary genetic marker. For more accurate species identification and effective testing of species hypothesis, DNA barcoding should be incorporated with morphological, ecological, biogeographical, and phylogenetic information. The application of DNA barcoding and molecular identification in the ecological survey on the intertidal polychaete communities demonstrated the feasibility of integrating DNA taxonomy and ecology.     

Use of rapd for detecting and identifyingPorphyra (Bangiales, Rhodophyta)

Randomly amplified polymorphic DNA (RAPD) technique was applied to assess the genetic variations and phylogenetic relationships in 4 species ofPorphyra. The samples were collected from the coast of Canada, Vietnam, Zhoushan, Fujian and Qingdao in China. Amplifications with 20 primers were carried out under predetermined optimal reaction conditions (samples were first heated at 94°C for 5 min. and followed by 45 cycles of 1 min at 94°C, 1 min at 36°C, and 2 min at 72°C, then held at 72°C for 10 min). The amplified products were scored as present (1) or absent (0) for each DNA sample and an index of genetic similarity (F) was calculated by using Nei & Li’s matching coefficient method (1979). The value of (1−F) was used to quantify the genetic distances between species and construct a phylogenetic tree. The relationship indicated by the UPGMA and NJ cluster analysis on the values of the genetic distance is in good overall agreement with classical taxonomy. The obvious differences between natural and cultivated population ofP. haitanensi suggest that variation or hybridization with other species occurred during the culture. Contribution No. 100 from the Experimental Marine Biology Laboratory, IOCAS. Contribution No. 3289 from the Institute of Oceanology, Chinese Academy of Sciences. The research supported by the Foundation of the Chinese Academy of Sciences.     

Ulva and Enteromorpha (Ulvaceae, Chlorophyta) from two sides of the Yellow Sea: analysis of nuclear rDNA ITS and plastid rbcL sequence data

Ulvacean green seaweeds are common worldwide; they formed massive green tides in the Yellow Sea in recent years, which caused marine ecological problems as well as a social issue. We investigated two major genera of the Ulvaceae, Ulva and Enteromorpha, and collected the plastid rbcL and nuclear ITS sequences of specimens of the genera in two sides of the Yellow Sea and analyzed them. Phylogenetic trees of rbcL data show the occurrence of five species of Enteromorpha (E. compressa, E. flexuosa, E. intestinalis, E. linza and E. prolifera) and three species of Ulva (U. pertusa, U. rigida and U. ohnoi). However, we found U. ohnoi, which is known as a subtropical to tropical species, at two sites on Jeju Island, Korea. Four ribotypes in partial sequences of 5.8S rDNA and ITS2 from E. compressa were also found. Ribotype network analysis revealed that the common ribotype, occurring in China, Korea and Europe, is connected with ribotypes from Europe and China/Japan. Although samples of the same species were collected from both sides of the Yellow Sea, intraspecific genetic polymorphism of each species was low among samples collected worldwide.     

Spermatozoid life-span of two brown seaweeds, Saccharina japonica and Undaria pinnatifida, as measured by fertilization efficiency

During sexual reproduction of seaweeds, spermatozoid (sperm) discharge is triggered by chemical messengers (pheromones) released by the female gametes. The chemotactic ability of the sperm ensures fertilization success. Using unialgal male and female gametophyte material under designated standard gametogenesis testing (SGT) conditions, the potential life-span of the sperm of two seaweeds, Saccharina japonica and Undaria pinnatifida, was assessed by their ability to fertilize eggs. Results show that within 20-30 min after being discharged, sperm of both species could complete fertilization without an apparent decline in fertilization rate. Although fertilization rate 60–120 min after sperm discharge dropped significantly in both species, some sperm were viable enough to fertilize the eggs. In S. japonica, at 12°C, some sperm were able to fertilize eggs up to 12 h after discharge. In both species, egg discharge rates (EDR) in the male and female mixed positive controls were significantly higher than those of all the sperm-testing groups. Doubling the seeded male gametophytes of S. japonica in the SGT tests significantly increased the EDR, further confirming the effect of the presence of the male on the female in terms of facilitating egg discharge from oogonia.     

Use of species-specific PCR for the identification of 10 sea cucumber species

We developed a species-specific PCR method to identify species among dehydrated products of 10 sea cucumber species. Ten reverse species-specific primers designed from the 16S rRNA gene, in combination with one forward universal primer, generated PCR fragments of ca. 270 bp length for each species. The specificity of the PCR assay was tested with DNA of samples of 21 sea cucumber species. Amplification was observed in specific species only. The species-specific PCR method we developed was successfully applied to authenticate species of commercial products of dehydrated sea cucumber, and was proven to be a useful, rapid, and low-cost technique to identify the origin of the sea cucumber product.     

Two new species of the genus Munidopsis Whiteaves,1874(Crustacea:Anomura:Munidopsidae) from the Caroline Ridge,South of the Mariana Trench

The genus Munidopsis has high biodiversity in seamount habitats.In this study,two new Munidopsis species,Munidopsis ahyongi sp.nov.and Munidopsis carolinensis sp.nov.from the Caroline Ridge in the tropical western Pacific Ocean were described.M.ahyongi sp.nov.is very similar to a group of species in the M.serricornis complex,but can be readily distinguished from the related species in having the oblique posterior orbital margins,2 spines on the lateral carapace margin,and a relatively longer rostral median spine.M.carolinensis sp.nov.is different from two related species primarily in having oblique frontal margins,blunt outer orbital angles,straight lateral margins of the rostrum,scale-like rugae on sternites 5-7,and less spinose pereopod 1(chelipeds,P1) merus and carpus.Aside from the morphological comparisons,DNA barcode analysis and phylogenetic analysis based on the cytochrome c oxidase subunit I gene(COI) were employed for the identification of these new species.     

Comparative studies on spectral properties of R-phycoerythrin from the red seaweeds from Qingdao

The present paper reports on comparative studies of R-phycoerythrin (R-PE) from 30 species of the red seaweeds in Bangiophyceae and in Florideophyceae from Qingdao. The measured absorption spectra show that R-PE from these red seaweeds have two spectral types, namely type I having two absorption peaks and type II having three absorption peaks. 9 specise of a lower red seaweed belonging to Bangiophyceae are all type I R-PE. 3 species of a higher red seaweed belonging to Florideophyceae are also type I R-PE. The remaining 18 species are all type II R-PE. According to the distribution of two types of R-PE in red algae, an evolutionary trend from type I R-PE to type II R-PE, can be seen, but this evolutionary process is more complicated and without a definite line of demarcation between the two spectral types of R-PE. The occurrence of two types of R-PE in red algae is of taxonomic significance. Project supported by the Science Fund of the Chinese Academy of Sciences. Contribution No. 1357 from the Institute of Oceanology, Academia Sinica.     

Effects of DNA extraction and universal primers on 16S rRNA gene-based DGGE analysis of a bacterial community from fish farming water

Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extraction on the outcome of DGGE analysis. In this study, these effects were tested with 16S rRNA gene-based DGGE on a bacterial community from farming water samples. The results indicate that the number of discernable bands in the DGGE fingerprint differed with the primer pairs used; the bands produced by 63f/518r, 341f/926r and 933f/1387r primer pairs were obviously fewer than those by 968f/1401r. Also, we found that each DNA extraction method resulted in different community profiles, reflected by the number and intensity of bands in the DGGE fingerprint. Furthermore, the main bands (theoretically representing dominant bacteria) differed with the extraction methods applied. It is therefore believed that the effects of universal primers and DNA extraction should be given more attention and carefully chosen before performing an investigation into a new environment with DGGE.     

Distribution of dominant zooplankton species along a latitudinal gradient in China sea during spring

Dominant species of zooplankton community vary with latitude. Though China possesses a vast coastal area in northwestern Pacific, studies on the latitudinal dominant species gradient are rare. We collected zooplankton samples from Haizhou Bay(34.56?–35.19?N, 119.51?–120.30?E), Yueqing Bay(28.14?–28.38?N, 121.10?–121.21?E) and Dongshan Bay(23.65?–23.90?N, 117.45?–117.60?E) in May 2012 and May 2013 to preliminarily characterize the latitudinal dominant species distribution. All the samples were collected vertically using a 0.505 mm mesh plankton net with 0.8 m in mouth diameter from bottom to surface. Calanus sinicus, Aidanosagitta crassa, Labidocera euchaeta, Zonosagitta nagae, Acartia pacifica and Paracalanus parvus were found to be dominant. C. sinicus was the most dominant species and the unique one occurred in all three bays. With latitude decreasing, both the abundance and proportion of C. sinicus declined sharply. Cluster analysis showed that the 6 dominant species could be divided into 3 groups, based on their occurrences in the three bays. Our results suggested that the distribution of dominant species along the coast of China has a significant latitudinal gradient. C. sinicus which widely distributes in the coastal water of the northwestern Pacific can well adapt to the temperature at different latitudes. The high abundance in Haizhou Bay indicated that C. sinicus was an exemplary warm-temperate species, and more commonly occurs in the north of China seas. The ecological characteristics of dominant species change from warm-temperate type in high-latitudinal bays to warm water type in low-latitudinal bays.     

DNA barcoding assessment of green macroalgae in coastal zone around Qingdao,China

An assessment with assistance of DNA barcoding was conducted on green macroalgae in coastal zone around Qingdao, China, during the period of April–December, 2011. Three markers were applied in molecular discrimination, including the plastid elongation factor tufA gene, the internal transcribed spacer (ITS) region of the ribosomal cistron and rubisco large subunit gene 3′ regions (rbcL-3P). DNA barcoding discriminated 8 species, excluding species of genus Cladophora and Bryopsis due to failures in amplification. We ascertained and corrected 4 species identified by morphological methods for effectively assisting the classification. The gene tufA presented more advantages as an appropriate DNA marker with the strongest amplification success rate and species discrimination power than the other two genes. The poorest sequencing success largely handicapped the application of ITS. Samples identified by tufA and rbcL as Ulva flexuosa were clustered into the clade of U. prolifera by ITS in the neighbor-joining tree. Confusion with discrimination of the complex of U. linza, U. procera and U. prolifera (as the LPP complex) still existed for the three DNA markers. Based on our results, rbcL is recommended as a preferred marker for assisting tufA to discriminate green macroalgae. In distinguishing green-tide-forming Ulva species, the free-floating sample collected from the green tide in 2011 was proved to be identical with U. prolifera in Yellow Sea for ITS and rbcL genes. This study presents a preliminary survey of green macroalgae distributed in the coastal area around Qingdao, and proves that DNA barcoding is a powerful tool for taxonomy of green macroalgae.     

Identification of two Skeletonema costatum-like diatoms by fluorescence in situ hybridization

A harmful algae bloom (HAB) is a dense aggregation of algae in a marine or aquatic environment that can result in significant environmental problems. To forecast the occurrence of HAB, development of a rapid and precise detection method is urgently required. In this study, two Skeletonema costatum-like diatoms (SK-1 and SK-2), were identified morphologically under a light microscope, and detected using fluorescent in situ hybridization (FISH). Strain SK-1 was isolated from a frequently HAB affected area of the East China Sea, and strain SK-2 from an aquatic farm in Qingdao, China. Fluorescent DNA probes were designed that were complementary to the ITS sequence (including 5.8S rDNA) of strain SK-1. After hybridization, strong green fluorescence was observed in cells of strain SK-1 under an epifluorescence microscope; however, no such fluorescence was observed with strain SK-2, which indicates that probes hybridized only the DNA of the target strain, SK-1, in species-specific manner, and that the two strains do not belong to a same species. This finding was confirmed by ITS sequence analysis. The FISH technique used in this study was sensitive, simple, and rapid, and is a promising tool for detecting target HAB species in natural environments.     

Intraspecific genetic analysis, gamete release performance, and growth of Sargassum muticum （Fucales, Phaeophyta） from China

Sargassum muticum is one of the most abundant and conspicuous native macroalgae species on the northern coasts of China. It often forms large-scale seaweed beds in subtidal zones. This investigation was designed to understand the intraspecific genetic relationships of this alga based on samples collected from four northern coastal sites of China, and to evaluate gamete release and growth capacity in laboratory conditions. The nuclear internal transcribed spacer 2 （ITS2） sequences of 16 samples from four locations were identical. Based on cox3 gene and partial rbcLS operon sequences, intraspecific genetic variability was detected with three and two ribotypes, respectively. Temperature, not irradiance, was shown to significantly affect gamete release and fertilization. Elevated temperature and irradiance enhanced the growth of germlings and vegetative branchlets. Maximum growth rate of germlings was detected at 18-24℃and an irradiance of 60-100 μmol photons/（m^2.s）. Under ambient conditions （12-25℃ and 60-125 gmol photons/ （m2.s））, relative growth rate of young branchlets could reach 7.5%/d.