Phylogenetic analyses of four species of Ulva and Monostroma grevillei using ITS, rbc L and 18S rDNA sequence data

Chlorophyta species are common in the southern and northern coastal areas of China. In recent years, frequent green tide incidents in Chinese coastal waters have raised concerns and attracted the attention of scientists. In this paper, we sequenced the 18S rDNA genes, the internal transcribed spacer (ITS) regions and the rbcL genes in seven organisms and obtained 536-566 bp long ITS sequences, 1 377-1 407 bp long rbcL sequences and 1 718-1 761 bp long partial 18S rDNA sequences. The GC base pair content was highest in the ITS regions and lowest in the rbcL genes. The sequencing results showed that the three Ulva prolifera (or U. pertusa) gene sequences from Qingdao and Nan’ao Island were identical. The ITS, 18S rDNA and rbcL genes in U. prolifera and U. pertusa from different sea areas in China were unchanged by geographic distance. U. flexuosa had the least evolutionary distance from U. californica in both the ITS regions (0.009) and the 18S rDNA (0.002). These data verified that Ulva and Enteromorpha are not separate genera.     

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.     

Antioxidant system responses in two co-occurring green-tide algae under stress conditions

Green tides have occurred every year from 2007 to 2014 in the Yellow Sea. Ulva prolifera(Müller) J. Agardh has been identified as the bloom-forming alga,co-occurring with U. intestinalis. We observed distinct strategies for both algal species during green tides. U. prolifera exhibited a high abundance initially and then decreased dramatically,while U. intestinalis persisted throughout. The antioxidant system responses of these two macroalgae were compared in the late phase of a green tide(in-situ) and after laboratory acclimation. Lipid peroxidation and antioxidant system responses differed significantly between the two. Malondialdehyde and hydrogen peroxide contents increased significantly in-situ in U. prolifera,but not in U. intestinalis. In U. prolifera,we observed a significant decrease in total antioxidant ability(T-AOC),antioxidant enzymes(SOD and Apx),and non-enzyme antioxidants(GSH and As A) in-situ. U. intestinalis showed the same pattern of T-AOC and SOD,but its Gpx,Apx,and GSH responses did not differ significantly. The results suggest that U. prolifera was more susceptible than U. intestinalis to the harsh environmental changes during the late phase of a Yellow Sea green tide. The boom and bust strategy exhibited by U. prolifera and the persistence of U. intestinalis can be explained by differences in enzyme activity and antioxidant systems.     

Analysis of expressed sequence tags from the Ulva prolifera (Chlorophyta)

In 2008, a green tide broke out before the sailing competition of the 29th Olympic Games in Qingdao. The causative species was determined to be Enteromorpha prolifera (Ulva prolifera O. F. Müller), a familiar green macroalga along the coastline of China. Rapid accumulation of a large biomass of floating U. prolifera prompted research on different aspects of this species. In this study, we constructed a nonnormalized cDNA library from the thalli of U. prolifera and acquired 10 072 high-quality expressed sequence tags (ESTs). These ESTs were assembled into 3 519 nonredundant gene groups, including 1 446 clusters and 2 073 singletons. After annotation with the nr database, a large number of genes were found to be related with chloroplast and ribosomal protein, GO functional classification showed 1 418 ESTs participated in photosynthesis and 1 359 ESTs were responsible for the generation of precursor metabolites and energy. In addition, rather comprehensive carbon fixation pathways were found in U. prolifera using KEGG. Some stress-related and signal transduction-related genes were also found in this study. All the evidences displayed that U. prolifera had substance and energy foundation for the intense photosynthesis and the rapid proliferation. Phylogenetic analysis of cytochrome c oxidase subunit I revealed that this green-tide causative species is most closely affiliated to Pseudendoclonium akinetum (Ulvophyceae).     

The appearance of Ulva laetevirens (Ulvophyceae,Chlorophyta) in the northeast coast of the United States of America

Introduced species may outcompete or hybridize with native species, resulting in the loss of native biodiversity or even alteration of ecosystem processes. In this study, we reported an alien distromatic Ulva species, which was found in an embayment (Holly Pond) connected with Long Island Sound, USA. The morphological and anatomical observations in combination with molecular data were used for its identification to species. Anatomy of collected specimens showed that the cell shape in rhizoidal and basal regions was round and the marginal teeth along the basal and median region were not found. These characteristics were primarily identical to the diagnostic characteristics of Ulva laetevirens Areschoug (Chlorophyta). The plastid-encoding tufA and nucleusencoding ITS1 were used for its molecular identification. Phylogenetic analysis for the tufA gene placed the specimens from Holly Pond in a well-supported clade along with published sequences of U. laetevirens identified early without any sequence divergence. In ITS tree, the sample also formed well-supported clades with the sequences of U. laetevirens with an estimated sequence divergence among the taxa in these clades as low as 1%. These findings confirmed the morpho-anatomical conclusion. Native to Australia, this species was reported in several countries along the Mediterranean coast after the late of 1990s. This is the first time that U. laetevirens is found in the northeast coast of United States and the second record for Atlantic North America.     

A New Molecular Label Applied to the Study of the Yellow Sea Green Tide

From 2007 to 2017, large-scale green tides occurred every year in the Yellow Sea of China, and Ulva prolifera was the main species leading to the green tide. In this study, we used the Polymerase chain reaction and 3' Rapid-amplification of c DNA ends technique to amplify the nr DNA-LSU and IGS sequences in U. prolifera and one species of Blidingia. These techniques showed 3259 bp of nrD NA-LSU and 3388 bp of IGS in U. prolifera and 3282 bp nr DNA-LSU and 3059 bp IGS in Blidingia sp. At the same time, tandem repeats, short dyads, palindromic and multiple simple repeat sequences in the IGS sequence were found by analyzing the structure of the IGS sequence in U. prolifera and Blidingia sp. G + C contents of the IGS sequence in U. prolifera and Blidingia sp. were 52.42% and 53.09%, respectively. We divided the U. prolifera into two types according to the morphological characteristics. Although the specimens of U. prolifera from the Qingdao coastal area, Jiangsu coastal area and the Yellow Sea have different morphologies, their ITS and IGS sequences are almost identical. Therefore, the main species of the green tides in the Qingdao coastal area, Jiangsu coastal area and the Yellow Sea are the same and have the same origin.     

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.     

A new record of a flathead fish (Teleostei: Platycephalidae) from China based on morphological characters and DNA barcoding

A new record of Platycephalus sp.1 (sensu Nakabo, 2002) was documented based on morphological characters and DNA barcoding. We collected 174 specimens of the genus Platycephalus from Chinese coastal waters of Dongying, Qingdao, Zhoushan, and Beihai. Samples were identified as Platycephalus sp.1 morphologically. The coloration, meristic traits, and morphometric measurements are consistent with previously published records. In brief, it is an orange-brown flathead fish with dark brown spots scattered on head and body, lateral line scales 83 to 99 with one or two spine-bearing anteriormost pored scale, no yellow blotch on the caudal fin. Cytochrome oxidase I subunit (COI) gene fragments were sequenced for phylogenetic analysis. The mean evolutionary distance within the species Platycephalus sp.1 was 0.1%. Net evolutionary distances between Platycephalus sp.1 and other species of Platycephalus ranged from 10.8% to 19.7%, which is much greater than the threshold for species delimitation. The COI sequence analysis strongly supports the validity of Platyceohalus sp.1 at genetic level.     

Acute toxicity of live and decomposing green alga Ulva (Enteromorpha) prolifera to abalone Haliotis discus hannai

From 2007 to 2009, large-scale blooms of green algae (the so-called “green tides”) occurred every summer in the Yellow Sea, China. In June 2008, huge amounts of floating green algae accumulated along the coast of Qingdao and led to mass mortality of cultured abalone and sea cucumber. However, the mechanism for the mass mortality of cultured animals remains undetermined. This study examined the toxic effects of Ulva (Enteromorpha) prolifera, the causative species of green tides in the Yellow Sea during the last three years. The acute toxicity of fresh culture medium and decomposing algal effluent of U. prolifera to the cultured abalone Haliotis discus hannai were tested. It was found that both fresh culture medium and decomposing algal effluent had toxic effects to abalone, and decomposing algal effluent was more toxic than fresh culture medium. The acute toxicity of decomposing algal effluent could be attributed to the ammonia and sulfide presented in the effluent, as well as the hypoxia caused by the decomposition process.     

Molecular diversity of Enteromorpha from the coast of Yantai: a dual-marker assessment

We collected nine Enteromorpha specimens from the coast of Yantai and evaluated their diversity based on analyses of their ITS （internal transcribed spacer） and 5S rDNA NTS （non-transcribed spacer） sequences. The ITS sequences showed slight nucleotide divergences between Enteromorpha linza and Enteromorpha prolifera. In contrast, multiple highly variable regions were found in the ITS region of Enteromorpha flexuosa. In general, there were more variable sites in the NTS region than in the ITS region in the three species. The variations in 5S rDNA NTS sequences indicated that the molecular diversity of Enteromorpha from the coast of Yantai is very high. However, a phylogenetic tree constructed using 5S rDNA NTS sequence data indicated that genetic differences were not directly related to geographical distribution.     

The distribution of green algal micro-propagules and macroalgae at the early stage of green tide in the coastal area of South Jiangsu Province in 2014

An integrated investigation was carried out during late April to late May of 2014 in the coastal area of South Jiangsu Province to understand the early development of green tide in the Yellow Sea and discover the temporal and spatial distribution of green algal micro-propagules and macroalgae at the early stage of green tide. The results showed that green algal micro-propagules distributed in all stations from late April to late May, and micro-propagules of high density( 300 ind. L~(-1)) concentrated in the adjacent Porphyra aquaculture area. Floating macroalgae were initially observed in the northern survey areas, and increased gradually. The dominant species was U. prolifera in the floating macroalgae, ranging between 90% and 100%. The seawater surface temperature and salinity in the above mentioned time period were suitable for the growth of macroalgae. This work can help government to strengthen management to reduce the blooms of macroalgae in the coastal area of South Jiangsu Province and also facilitate the decision-making for managers at the early stage of green tide.     

Morphology and molecular identification of Ulva forming green tides in Qingdao, China

Green tides are caused by the proliferation of chlorophytes under suitable hydrographic conditions.These blooms lead to environmental degradation and negatively impact the waters and seagrass beds,as well as fishing and other recreational activities in the bay.A comprehensive ecological understanding of the bloom dynamics,including the origin and persistence,is needed to foster management decisions.The algae in the great majority of green tide blooms usually belong to two genera of Ulvophyceae,Ulva and Enteromorpha.Ulva has been observed more often in recent years.In China,green tides occurred for the first time in the middle area of the Yellow Sea in 2007,and a large-scale algae blooming broke out in the middle and southern areas of the Yellow Sea in late May 2008.We identified them as Ulva prolifera by comparative analysis of the rDNA internal transcribed spacer 1 (ITS1),5.8S and ITS2 sequences in combination with microscopic observation.Morphological differences were found between the free-floating algae and the attached thalli.Various reproduction patterns of the free-floating algae include sexual,asexual and vegetative propagations,which played important roles in the long-term green tide persistence in China.The ITS sequences of the blooming algae were identical to those of the samples from the Lianyungang sea area but were different from the attached samples from the Qingdao sea area.The results infer that the blooms are originated from other sea areas rather than from the local attached populations.     

Spatiotemporal variation in community structure of marine benthic ciliates in the Yellow Sea during and after macroalgal and giant jellyfish blooms

The annual bloom of the green macroalgal Ulva prolifera from May through July since 2008 and another of giant jellyfish Nemopilema nomurai from June through September have been frequent events in the Yellow Sea. However, the patterns of benthic ciliate communities during and after the blooms are still not known. In combination with analyses of benthic environmental factors, we investigated the distribution and community composition of benthic ciliates in the Yellow Sea in July and November 2011. In July, ciliates had high standing crops and diversity in the northern Yellow Sea, and in the inshore area off the southern Shandong Peninsula, where large numbers of green macroalgae accumulated. In November, the abundance, biomass and diversity of ciliates were high in the sea areas off the Shandong Peninsula and Changjiang estuary, where a large quantity of jellyfish occurred in August. Neither the abundance nor the biomass had significant difference between seasons, or between different compartments of the Yellow Sea. The species number, and both Margalef and Shannon-Wiener indices of ciliates were all significantly higher in November than in July. In both seasons, prostomateans and karyorelicteans consistently constituted the first and second most important ciliate groups in biomass; and carnivorous ciliates constituted the primary feeding type in terms of biomass as well as species richness, followed by bacterivores, algivores and omnivores. Compared with that in June 2007 when no macroalgae occurred, the percentage of small-sized bacterivores (e.g. Metacystis spp., Euplotes spp. and scuticociliates) increased in July 2011. The proportion of carnivorous ciliates increased in November, and this increased dominance of carnivorous ciliates may be a response to the increase in predominance of heterotrophic nanoflagellates, which might in turn be ascribed to an effect of green macroalgal and giant jellyfish blooms in the Yellow Sea.     

Cylindrotheca closterium Is a Species Complex as Was Evidenced by the Variations of rbcL Gene and SSU rDNA

The genus Cylindrotheca consists of a small group of marine diatoms with a few species described. Eleven isolates of diatoms identified as Cylindrotheca closterium morphologically were obtained from Jiaozhou Bay with their nuclear-encoded small-subunit ribosomal RNA （SSU rDNA） and chloroplast-encoded rbcL gene sequences determined in this study. Interestingly, very high sequence divergences of SSU rDNA and rbcL gene were found among these isolates, and numerous nucleotide variation of rbcL gene caused relatively few variation of deduced amino acid sequence. Phylogenetic analyses based on SSU rDNA and rbcL gene, respectively, grouped the isolates into 6 clades. Phylogenetic tree of SSU rDNA placed all the Cylindrotheca isolates together, separating them into two lineages clearly. LineageⅠ was composed of the eleven C. closterium isolates obtained in this study together with another C. closterium isolate, but some clades were not well supported. LineageⅠwas contained two C. closterium isolates and one C. fusiformis isolate. Phylogenetic analysis of rbcL gene also separated the Cylindrotheca isolates into two well-defined lineages. The eleven C. closterium isolates formed a lineage and all clades were supported strongly. Statistical comparisons of SSU rDNA indicated that the average distance within lineageⅠwas significantly higher than that of other microalgae species （P 〈0.01）. These results suggested the existence of cryptic species within C. closterium.     

Applications of three DNA barcodes in assorting intertidal red macroalgal flora in Qingdao, China

This study is part of the endeavor to construct a comprehensive DNA barcoding database for common seaweeds in China. Identifications of red seaweeds, which have simple morphology and anatomy, are sometimes difficult solely depending on morphological characteristics. In recent years, DNA barcode technique has become a more and more effective tool to help solve some of the taxonomic difficulties. Some DNA markers such as COI (cytochrome oxidase subunitⅠ) are proposed as standardized DNA barcodes for all seaweed species. In this study, COI, UPA (universal plastid amplicon, domain V of 23S rRNA), and ITS (nuclear internal transcribed spacer) were employed to analyze common species of intertidal red seaweeds in Qingdao (119.3 -121 E, 35.35 -37.09 N). The applicability of using one or a few combined barcodes to identify red seaweed species was tested. The results indicated that COI is a sensitive marker at species level. However, not all the tested species gave PCR amplification products due to lack of the universal primers. The second barcode UPA had effective universal primers but needed to be tested for the effectiveness of resolving closely related species. More than one ITS sequence types were found in some species in this investigation, which might lead to confusion in further analysis. Therefore ITS sequence is not recommended as a universal barcode for seaweeds identification.     

Molecular phylogenetic analysis of attached Ulvaceae species and free-floating <Emphasis Type="Italic">Enteromorpha</Emphasis> from Qingdao coasts in 2007

Based on the sequence data of the nuclear ribosomal DNA internal transcribed spacer (ITS) 1, 5.8 S, and ITS 2, the molecular phylogeny was analyzed on Ulvaceae species collected from Qingdao coasts in summer of 2007, including 15 attached Ulva and Enteromorpha samples from 10 locations and 10 free-floating Enteromorpha samples from seven locations. The result supported the monophyly of all free-floating Enteromorpha samples, implying the unialgal composition of the free-floating Enteromorpha, and the attached Ulvaceae species from Qingdao coasts were grouped into other five clades, suggesting that they were not the biogeographic origin of the free-floating Enteromorpha in that season. Supported by NSFC (40506030); the Innovative Key Project of the Chinese Academy of Sciences (KZCX2-YW-209); Science & Technology Project of Qingdao City (06-2-2-12-JCH)     

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 analysis of floating Enteromorpha prolifera in the Yellow Sea with AFLP marker

Extremely large accumulation of green algae Enteromorpha prolifera floated along China’coastal region of the Yellow Sea ever since the summer of 2008.Amplified Fragment Length Polymorphism (AFLP) analysis was applied to assess the genetic diversity and relationships among E. prolifera samples collected from 9 affected areas of the Yellow Sea.Two hundred reproducible fragments were generated with 8 AFLP primer combinations,of which 194 (97%) were polymorphic. The average Nei’s genetic diversity, the coefficiency of genetic differentiation (Gst), and the average gene flow estimated from Gst in the 9 populations were 0.4018, 0.6404 and 0.2807 respectively. Cluster analysis based on the unweighed pair group method with arithmetic averages (UPGMA) showed that the genetic relationships within one population or among different populations were all related to their collecting locations and sampling time. Large genetic differentiation was detected among the populations.The E. prolifera originated from different areas and were undergoing a course of mixing.     

Effects of nitrogen specification and culture method on growth of Enteromorpha prolifera

Eutrophication, which is the enrichment of a water mass with inorganic and organic nutrients that support plant growth, is a key factor in stimulating phytoplankton growth. In this study, we determined the effects of various nitrogen sources, different nitrogen concentrations in the culture medium, and two culture methods on the growth of the green alga, Enteromorpha prolifera. The relationship between the specific growth rate of E. prolifera and NO3--N concentration was consistent with that estimated using the Monod equation (R2 = 0.9713, P < 0.01). In the NO3--N medium, the maximum specific growth rate was calculated to be 0.1634/d and the semi-saturation constant was calculated to be 16.86 μmol/L. Our results show that E. prolifera can effectively utilize NH4+-N, NO3--N, and NO2--N and urea-N in the range of 5 to 50 μmol/L. NH4+-N was preferentially assimilated by E. prolifera, and urea-N was favorable for long-term growth.     

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.