Dynamics Of Two Genotypes Of Phaeocystis Globosa During The Blooms In The Beibu Gulf And Comparison Of Their Biological Characteristics

Phaeocystis globosa is a common causative species of harmful algal blooms(HABs)in the form of colonies from temperate to tropical waters worldwide.Since2011,the HABs of P.globosa have been of great concern because of their serious hazards on the coastal ecosystem,tourism,aquaculture,and the safe operation of nuclear power along the coastline.Recent studies have found that genetic differentiation of P.globosa is associated with traits such as the composition of marker pigments and colony size,implying that P.globosa can be divided into multiple genotypes.There are more than four genotypes of P.globosa coexist in the Beibu Gulf,South China Sea(SCS)and the Type I and Type IV are proved to be predominant in the phytoplankton community.In order to reveal the dynamics of the dominant genotypes during the P.globosa blooms and the role of key environmental factors in the Beibu Gulf,and differences in biological characteristics between Type I and Type IV P.globosa and its role in the formation and maintenance of P.globosa blooms,this study developed two genotype-specific qPCR methods to analyze the spatial and temporal distribution patterns of Type I and Type IV P.globosa in two blooms in winters of 2016 and 2018,explored their distribution patterns and the environmental influences,and conducted comparative experiments on the biological characteristics of two genotypes of P.globosa for key environmental factors.The results were as followed:Based on the mitochondrial gene atp8 of P.globosa,two highly sensitive and specific qPCR assays for P.globosa Type I and Type IV were established,respectively,which have good potential for field application.It was found that the mitochondrial gene atp8 of P.globosa has enough intraspecific resolution to distinguish the different genotypes of P.globosa,which is consistent with the results of previous studies on the typing of P.globosa,and could be used as the sequence target for genotype-specific qPCR assays.Specific primers and probes were designed for Type I and Type IV P.globosa to establish qPCR assays for their quantitative detection.Both methods have good specificity and can only specifically amplify the target genotype of P.globosa.Both developed methods had good repeatability and reproducibility(>98%).They had low detection limits(<1.0 cells/qPCR reaction)and wide detection range(6～8 order of magnitudes).The results suggested both developed qPCR methods have the potential to determine dynamics of P.globosa blooms.The newly developed two genotype-specific qPCR assays were applied to determine the dynamics of two P.globosa blooms in the Beibu Gulf.It was found that the spatial distribution patterns of Type I and Type IV were similar,but the abundance of Type IV was much higher than that of Type I.The dominant genotype of P.globosa blooms was confirmed as Type IV,and three possible seed sources were found,revealing the role of key environmental factors such as nitrate,temperature and salinity biological characteristics in the P.globosa blooms.It was noted that the P.globosa blooms in the Beibu Gulf was maintained for a long time(4～5 months).During the two P.globosa blooms in the gulf,the distribution patterns of the two genotypes of P.globosa were similar,mainly distributed in the adjacent waters of Qinzhou Bay and the western waters of Leizhou Peninsula.At climax of the blooms,the abundance of Type IV exceeded 10~5 cells/L,which was greater than that of Type I,indicating that Type IV dominated the blooms in the Beibu Gulf.At the beginning of the P.globosa blooms,P.globosa in the SCS Counter-wind deep current off the southern of Beibu Gulf was imported to the adjacent waters of Qinzhou Bay,the algal cells in the Guangdong coastal waters entered the western waters of Leizhou peninsula through the westward flow of Qiongzhou Strait,and the algal cells in the northwestern upwelling of Hainan Island was supplemented to the peninsula western waters.The dynamics of P.globosa blooms were primarily regulated by some environmental factors,including nitrate,temperature,and salinity.Of which,nitrate was consumed by P.globosa blooms in large amounts,low temperature(<24℃)provided a suitable time window for algal blooms,and salinity affected the spatial distribution of P.globosa.Lastly,it was found that the higher concentration of nitrate and longer low temperature period was resulted in the larger-scale bloom in the winter of 2016 in the term of bloom intensity and duration than that in the winter of 2018.Experiments comparing the differences in biological characteristics such as salinity tolerance,growth(cell and colony),and colony function between Type I and Type IV P.globosa,complemented by transcriptomic data,revealed distinct differences in survival strategies between Type I and Type IV P.globosa,favoring r-strategists and K-strategists,respectively.The optimal growth salinity of Type IV P.globosa(25～40)was wider than that of Type I(30～35).The growth of TypeⅠwas faster,and its highest cell density was 3～5 times greater than that of TypeⅣ;the growth cycle of TypeⅠwas shorter(ranging from 8 to 14 days)than that of TypeⅣ(>20 days).Type I formed high abundance(>1000 colonies/m L)of small colonies(<1 mm),but Type IV could form low abundance(<100 colonies/m L)of large colonies(>5 mm).Type I was mainly asexual and Type IV was capable of both asexual and sexual reproduction during their growth.High nutrient content in the colonies of Type IV and the protective effects of these colonies provided more suitable survival environment for its internal haploid free-living cells.The results implied that the Type I cells tend to be r-strategists,and the Type IV cells tended to be K-strategists,which helped the Type IV cells achieve a competitive advantage during the blooms and prolonged the bloom duration in the Beibu Gulf.In the present study,the dynamics of P.globosa blooms in the Beibu Gulf were studied by newly developed genotype-specific qPCR assays with highly sensitive for two dominate genotypes(Type I and Type IV).It was founded that the Type IV dominated the blooms and the key environmental factors including nitrate and temperature affected the bloom dynamics and interannual variability in the gulf.The survival strategies Type I and Type IV differed:Types I and IV tended to be r-and K-strategists,respectively.These results will profound the understanding of P.globosa blooms in the Beibu Gulf,furnish an important scientific basis for the mechanism of P.globosa bloom,and provide effective technical assays to monitor and early-warning of P.globosa blooms.