Development of high-resolution chloroplast markers for intraspecific phylogeographic studies of Phaeocystis globosa

Phaeocystis globosa is an important harmful algal bloom causative species distributing widely in temperate and tropical coastal waters in the world.The morphological,physiological,and biochemical characteristics are different among geographic strains,which can not be distinguished with nuclear ribosomal DNA markers at present.Therefore,the genetic distance and phylogeographic relationships of nuclear 28S rDNA D1–D2 and ITS regions,and three chloroplast intergenic spacers(petN-trnS1,trnM1-psbA,and rbcS-rpl27)were analyzed and compared among 13 strains of P.globosa isolated from the Pacific Ocean and Atlantic Ocean in this study.In addition,the nucleotide polymorphisms of 28S rDNA D1–D2,ITS,and rbcS-rpl27 regions were evaluated in two P.globosa strains.The various levels of nucleotide polymorphism were in the nuclear 28S rDNA D1–D2 region and ITS region,but no polymorphism was in the chloroplast rbcS-rpl27 intergenic spacer.A reasonable intraspecific phylogeographic relationship was presented by rbcS-rpl27 intergenic spacer,which had the strongest distinction to geographic strains compared to those of 28S rDNA D1–D2 and ITS regions.In the phylogenetic tree of rbcS-rpl27 intergenic spacer,the two strains from the North Sea of the Atlantic Ocean were divided firstly from the species of P.globosa,and then formed an independent clade,while the other Atlantic strains and all of Pacific strains joined up to build the other clade.It was implied that at least two genetically distant populations of P.globosa existed in the Atlantic coastal regions.This study provided a high-resolution chloroplast marker to analyze intraspecific phylogeographic populations of P.globosa,and preliminarily clarified the genetic relationships of the Pacific and Atlantic strains of P.globosa.

Newly recorded bloom-forming dinoflagellate Gymnodinium impudicum in Haizhou Bay,Yellow Sea,China

With the development of industrialization and aquaculture in Jiangsu and Shandong Provinces along the South Yellow Sea coast,China,eutrophication has greatly intensified in the region,resulting in frequent occurrence of diverse harmful algal blooms.An algal bloom formed by a chain-forming dinofl agellate species was recorded in the Haizhou Bay,South Yellow Sea,in September 2020.The causative species was isolated and studied in morphology,molecular phylogeny,pigment profile,presence of paralytic shellfish toxins,and acute toxicity.The loop-shaped apical groove running anticlockwise around the apex,the presence of peridinin as characteristic pigment,as well as a single phylogenic clade of 28S ribosomal DNA(100%posterior probability),defined this species as Gymnodinium impudicum,a non-toxic species that exhibited no obvious biotoxicity to the rotifer Brachionus plicatilis,the copepod Artemia salina,and the shrimp Neomysis awatschensis.Gymnodinium impudicum is typically distributed in coastal waters with high nitrate concentrations,where it reaches a maximum density of 2.6×10~5 cells/L.This is the first report of a G.impudicum bloom in the Yellow Sea;however,G.impudicum blooms may have been misidentified or underreported in Haizhou Bay due to the species morphological similarity with G.catenatum.A combination of multiple methods is recommended to accurately identify new algal bloom species.

Mapping the resting cysts of dinoflagellate Alexandrium catenella along the coast of Qinhuangdao,China

Dinoflagellate Alexandrium catenella is a cosmopolitan bloom-forming species with complex life cycle,the formation and germination of resting cysts are critical for its bloom dynamics.In the coastal waters of Qinhuangdao,A.catenella has been identified as the major causative agent for paralytic shellfish poisoning,but there is little knowledge concerning its resting cysts in this region.In this study,three surveys were carried out along the coast of Qinhuangdao from 2020 to 2021 to map the distribution of A.catenella resting cysts,using a quantitative PCR(qPCR)assay specific for A.catenella.The resting cysts were detected in surface sediments during all the three surveys,and their distribution patterns were similar.High abundance of resting cysts(maximum 1300 cysts/g sediment(wet weight))were found in a region(119.62°E-119.99°E,39.67°N-39.98°N)northeast to the coastal waters of Qinhuangdao,where surface sediments were mainly composed of clay and silt(percentage above 50%).Prior to the formation of the A.catenella bloom in March 2021,the abundance of A.catenella vegetative cells in seawater had extremely significant positive correlation with the abundance of resting cysts in surface sediments,reflecting the important role of resting cysts in the initiation of A.catenella blooms.As far as we know,this is the first report on the distribution of A.catenella cysts along the coast of Qinhuangdao.The results will of fer a sound basis for the future monitoring and mitigation of toxic A.catenella blooms and paralytic shellfish poisoning events in this region.

Laboratory simulation of dissolved oxygen reduction and ammonia nitrogen generation in the decay stage of harmful algae bloom

To evaluate how the decay of bloom-forming algae affect the coastal dissolved oxygen,a laboratory simulation was conducted in terms of three typical harmful algae,Alexandrium catenella,Prorocentrum donghaiense,and Skeletonema costatum.Algae of same biomass(55μg/mL)were conducted in lightproof columns,and the cell density,dissolved oxygen(DO),and ammonia nitrogen of different layers were monitored at certain time series.Results show that the decomposition of algae significantly decreased the DO,and increased the ammonia nitrogen in all layers;and significant deference between different species was observed.The A.catenella treatment showed the lowest DO(average concentration of 3.4 mg/L)and the highest ammonia nitrogen(average concentration of 0.98 mg/L)at the end of test,followed by P.donghaiense;and the S.costatum showed relatively high DO and low ammonia nitrogen due to slow decay rate.Results indicate that decomposition of harmful bloom algae,especially dinoflagellate,would cause significantly DO depletion and toxic ammonia nitrogen increase,which will detrimentally affect both pelagic and benthic ecosystem.

Information standardization for typical toxic and harmful algae in China's coastal waters——a case study of Karenia mikimotoi

To assist the researches of toxic and harmful algae and provide government workers with judgment basis for decision-making related events,we established a biological information management system for toxic and harmful algae in China’s of fshore to assist relevant research.In this study,Karenia mikimotoi was studied as a typical toxic and harmful algae species,and the basic biological information and biogeographic distribution information of K.mikimotoi were systematically studied and collected.In the part of basic biological information,the name,toxin,and molecular characteristic sequence of K.mikimotoi were sorted out by literature searching and website browsing.Through experimental means,the relevant information of morphological identifi cation,pigment composition,and lipid composition were obtained.In the part of biogeographic distribution information,this study sorted out the information of K.mikimotoi,analyzed the characteristics of its occurrence,and completed the standardized construction of biogeographic distribution information.Through the collation of basic biological information and biogeographic distribution information of K.mikimotoi,the standardization of related information was completed,which provided template and method reference for information collection of other toxic and harmful algae species,which was benefi cial to the database analysis and design.

Screening for lipophilic marine toxins and their potential producers in coastal waters of Weihai in autumn,2020

Lipophilic marine toxins(LMTs)produced by some microalgae in the sea could accumulate in shellfish and pose potential threats to the health of seafood consumers.Phytoplankton and shellfish samples were collected from coastal waters of Weihai in Shandong Peninsula,China in autumn,2020,and screened for lipophilic marine toxins and their potential producers using liquid chromatography-tandem mass spectrometry(LC-MS/MS)analysis and high throughput sequencing of partial DNA(V4 region of the 18S rRNA gene)extracted from phytoplankton.Pectenotoxin-2(PTX2),trace amounts of azaspiracid(AZA1 or AZA40),and 13-desmethyl spirolide C(13-DesMe-C)were detected in phytoplankton samples,while PTX2 and gymnodimine(GYM)were detected in shellfish samples.The toxin content in shellfish samples was much lower than the regulatory limit or values reported previously.Results suggest that lipophilic marine toxins should have low risk in coastal waters of Weihai in autumn.Based on the data of high throughput sequencing,the OTUs were assigned to 5 identified species of Alexandrium,including A.ostenfeldii capable of producing 13-DesMe-C and GYM.Two OTUs were found closely related to the toxic species in genus Dinophysis,but it is impossible to assign them to any identified species due to the low resolving power of the V4 region for Dinophysis.The OTUs could not be assigned to any identified species in the genus Azadinium,suggesting the existence of unidentified species in this region.

Dynamics of Phaeocystis globosa bloom and implications for its seed sources in the Beibu Gulf,China

Algal blooms of haptophyte Phaeocystis globosa have been recorded in the Beibu Gulf,South China Sea(SCS)since 2011.Recent evidence suggests that TypeⅠand TypeⅣare the two dominant genotypes of P.globosa coexisting in this area,and their marker pigment profiles,colony sizes,and genetic information differ.Two genotype-specific quantitative polymerase chain reaction(qPCR)assays were used to clarify the distribution and dynamics of P.globosa bloom in the Beibu Gulffrom November 2018 to February 2019.Results show that the patterns of spatial-temporal distribution of TypeⅠand TypeⅣwere similar,the abundance of TypeⅣwas higher than that of TypeⅠduring the algal bloom,and thus TypeⅣcells dominated the P.globosa bloom in winter 2018.The relationships between environmental factors and P.globosa abundance indicated that temperature and nitrate were critical to the distribution and dynamics of P.globosa blooms in this area.The distribution of P.globosa cells in different water masses before the bloom revealed that the westward water via the Qiongzhou Strait,the SCS bottom water,and the current in the northwestern waters of Hainan Island could transport P.globosa cells into the Beibu Gulf serving as the seed sources triggering the intense blooms in the Beibu Gulf.