Historical And Geographical Distribution Of The "Brown Tide"-Causing Alga Aureococcus Anophagefferens In The Seas Of China And The Evidences For A Resting Stage In Its Life History

The nonmotile,spherical,picoplanktonic(2-3?m)pelagophyte Aureococcus anophagefferens has caused numerous harmful algal blooms(HABs)("brown tides")along the east coast of the USA since 1985,a few times in South Africa around 1997,and frequently in the coastal waters of the Bohai Sea,China since 2009.As a consequence,the harmful blooms have caused massive losses in aquaculture and coastal ecosystems,particularly mortalities in cultured shellfish.It was hypothesized that A.anophagefferens was introduced to South Africa recently and expanded its distribution via ships'ballast water transport.Therefore,as a relatively novel HAB-forming species in China,whether A.anophagefferens was recently introduced to China via natural/artificial transport or has been an indigenous species has become a question of profound ecological significance and broad interest,which necessitated an extensive investigation on the geographic and historical presence of this species in the seas of China.Moreover,one of the key questions regarding whether a harmful algal species is able to be introduced from another site via ships'ballast water is whether or not there is a resting stage in the life history of the species.Therefore,answering whether A.anophagefferens has a resting stage may help to explain the origin of A.anophagefferens found in China,the apparent geographic expansion of the species around the world,and the mechanisms of annual recurrances of blooms.Since A.anophagefferens cells are too small in size and too simple in morphology to be distinguished microscopically from other small algae,PCR-based molecular detection using species-specific primers became the best option for the extensive survey.In addition,sediment samples,which may provide a much more accurate and comprehensive record because of the nature of sediments in accumulating all seasonal and historical deposits,were used for the detection.In testing sediment samples with PCR amplification and sequencing for partial 18S and 28S r DNA,we discovered that160 of 201 surface sediment samples collected from all four seas of China(Bohai Sea,Yellow Sea,East China Sea and South China Sea)were positive for the presence of A.anophagefferens,a geographic distribution covering a latitude range of 29.841°(i.e.>3000 km),a longitude range of 15.674°(?1500 km at the widest),and a water depth range from 2.5 to 3456 m(temperate to tropical and coastal to open oceans).The positive detection in one sediment core(dated via a combined approach of ²¹⁰Pb and¹³⁷Cs measurements and AMS ¹⁴C dating)proved a presence of the species for>1500years at least in Bohai Sea.These detections extended its southernmost tropical range of presence recorded in the northern hemisphere by 1700 km and disproved it to be an alien species recently-introduced into China.Fifty-two 28S r DNA sequences of A.anophagefferens with a length of 993 bp(covering partial D2 and D3-D6 domains;Gen Bank accession Nos.MG782521-MG782572)were obtained from 12 sediment samples covering all four China Seas.The Maximum Likelihood phylogenetic analysis based on these sequences and 20 sequences of the clonal culture of A.anophagefferens strain CCMP1984(Gen Bank accession Nos.MG782499-MG782520)and a sequence of A.anophagefferens deposited in Gen Bank(Gen Bank accession No.AF289042)demonstrated that the sequences from China belong to one population and are not phylogenetically distant from that the population from the east coast of the USA,suggesting they share a common ancestor and have not been geographically separated for a long age.In order to test whether A.anophagefferens presents in the sediment as resting stage cells,germination experiments were conducted for the sediment sample collected on 8 May,2014 from the station BS106 in the coast of Qinhuangdao,Bohai Sea(39.602°N,119.913°E,water depth 25 m),where brown tides have occurred since 2009.Before being used in the germination experiments,the sediment sample of BS106 had been stored in darkness at 4? for more than one year to make sure that live cells existing in the sediment sample must be cells at resting stage rather than normal vegetative cells.At last,existence of a resting stage in A.anophagefferens was proved through germination experiments,combination of a specific monoclonal antibody conjugated to fluorescein isothiocyanate(FITC-MAb)staining and species-specific PCR identification for germlings,and RT-PCR detection of r RNA in sediment samples.In addition,an intensive study using clonal culture of A.anophagefferens strain CCMP1984 discovered the resting stage cells of A.anophagefferens.Characterization of the cellular morphology and physiological status of the resting stage cells,a time series observation for the germination processes of the resting stage cells and the ability of resting stage cells transforming into vegetative cells after prolonged treatment in the dark(4?)were conducted.With all evidences presented in this study,we are now confident that,by definition,a resting stage does exist in the life history of A.anophagefferens which can survive stressful conditions such as near freezing temperature and darkness for a fairly long period of time(>1 year)in the sediment and recover(or"germinate")to normal vegetative growth within a reasonable time(a few weeks)when they are placed back to a favorable environment.The resting stage cells,therefore,at least provide a facility for A.anophagefferens to seed annual blooms and expand its distribution via either artificial(e.g.ships'ballast water)or natural pathways.Discovery of a resting stage of A.anophagefferens is of vital significance not only in the basic biology but also in the ecology of this notorious HABs-causing pelagophyte.Considering the above-described findings of this work that A.anophagefferens widely distributes from coastal to pelagic waters,a presence in the sediment for>1500years,and the existence of resting stage,A.anophagefferens may perform transoceanic transportation by itself continuously and thus has achieved a possibly global distribution.After searched the supplementary data set of de Vargas et al.(2015),we found that A.anophagefferens was detected in water samples from many stations of the Atlantic Ocean,Indian Ocean,Mediterranean Sea,and the south Pacific Ocean,which clearly confirmed that this species has in fact a global,cosmopolitan distribution.Production of resting stage cells may be an important reason for the global distribution and annual recurrence of the brown tide.The results obtained in this study not only provide a decisive understanding of the origin of A.anophagefferens found in China and the global distribution pattern of this species,but also provide a crucial scientific guidance for further studies on the causes of brown tides,i.e.the causes of the outbreak of brown tide must be explored from the local environmental settings.We believe the approach adopted in this study also provides an exemplary case applicable to other harmful bloom-forming species which have appeared to expand their geographic distribution during the last decades.