| Large-scale brown tides of Aureococcus anophagefferens have been recorded from 2009 in the coastal waters of Qinhuangdao,Bohai Sea.Feeding activities of cultured scallops were strongly inhibited during the brown tides,and resulted in huge economic losses.During the brown tides,nano-and pico-sized phytoplankton were dominant groups,but the knowledge on the species composition and their dynamics was quite limited.Therefore,it's necessary to perform studies on phytoplankton communities.Recently,the rapid development of high-throughput sequencing technology showed potential advantages in studies on nano-and pico-sized phytoplankton communities.In this study,the high-throughput sequencing method was optimized and used to analyze the phytoplanton samples collected from the brown tide zone in Qinhuangdao coastal waters in 2013,2014 and 2016.The composition and dynamic of phytoplankton communities were systematically investigated,and the major results and findings were listed as followed:1.The method of high-throughput sequencing was optimized for the analysis of phytoplankton samples.The targeted region was selected based on the analysis of corresponding sequences in NCBI,and methods for genome DNA extraction was selected and optimized.Based on the sequence information in NCBI,and the comparison of high-throughput sequencing results of 18 S rDNA V1V2,V4 and V9 in the same phytoplankton sample,the resolution of the three targeted regions was compared.The results show that the V1V2 region and the V4 region have relatively moderate evolution rate,and are capable of resolving most causative species of harmful algal blooms.The V1V2 region,however,could not accurately distinguish A.anophagefferens from other pelagophyte species.Compared with the V1V2 and the V4 regions,the V9 region is too hyper-variable.The V9 sequence of many species overlap with each other,and couldn't be distinguished from each other.Considering that a large pool of 18 S rDNA V4 sequences have been deposited in NCBI,and its capability in distinguishing causative species of harmful algal blooms,the V4 region was selected as the target for high-throughput sequencing of field phytoplankton samples in Qinhuangdao coastal waters.Based on the analysis of simulated and natural phytoplankton samples,three methods(CTAB,SDS and Sucrose)for extraction of genome DNA were compared,and the CTAB method was selected in this study based on the yield,purity and integrity of DNA extracted from phytoplankton samples.2.Based on the high-throughput sequencing analysis of phytoplankton samples from the “brown tide zone” in Qinhuangdao coastal waters from 2013 to 2014,the composition and succession of phytoplankton communities were revealed.The results further confirmed that pelagophyte A.anophagefferens is the major causative species of brown tide in the coastal waters of Qinhuangdao.In this study,22 samples of nano-and pico-sized phytoplankton were analyzed by high-throughput sequencing(18S rDNA V4)in 2013 and 2014,respectively.On average,more than 200,000 tags were obtained per sample and more than 2,000 OTUs(Operational taxonomic units)were found,belonging to 17 groups of phytoplankton,including diatoms,dinoflagellate,pelagophyte and green algae etc.During the brown tide,there was a similar succession pattern of major phytoplankton groups in 2013 and 2014.The main dominant groups of nano-and pico-sized phytoplankton were dinoflagellates,prasinophytes,cryptophytes and diatoms prior to the occurrence of brown tide,which shifted to a community dominated by pelagophytes,and finally a community dominated by more diversified phytoplankton groups including dinoflagellates,pelagophytes,chrysophytes,cryptophytes and prasinophytes etc.For both years,A.anophagefferens was the most dominant species during the bloom period and its maximum relative abundance exceeded 50 percent.The results further confirmed that A.anophagefferens is the major causative species of brown tide.By comparing the results of the two years,the outbreak time of brown tide in 2014 was about one month earlier than that in 2013,and an increasing dominance of dinoflagellates could be observed in the Qinhuangdao coastal waters.By calculating the Pearson correlation coefficient between A.anophagefferens and other phytoplankton species,the species closely related to the dynamics of A.anophagefferens were found,including some rare OTUs(relative abundance < 0.01%).3.A strain of A.anophagefferens isolated from Qinhuangdao coastal waters was identified by morphological observation,pigment analysis and sequencing results.Based on the analysis of partial rDNA sequences of four A.anophagefferens strains from China and the United States,as well as the results of high-throughput sequencing of phytoplankton samples from Qinhuangdao coastal waters,the genetic diversity of A.anophagefferens was revealed.The strain MEL50 was isolated by serial-dilution method in 2015,and strain MEL51 was isolated from MEL 50 by single-cell isolation method.Both strains were identified as A.anophagefferens by morphological observation,pigment analysis and molecular information.The clone libraries of 18 S rDNA V4,28 S rDNA D1D2 and the ITS region were constructed for the four strains MEL 50,MEL 51,CCMP 1984 and CCMP 1850.Genetic diversity were revealed in 18 S rDNA V4 region through high-throughput sequencing of phytoplankton samples collected from the Qinhuangdao coastal waters.Both the Chinese strain MEL50 and the American strains CCMP1984 and CCMP1850 belong to the genotype I.In summary,the composition and dynamic of nano-and pico-sized phytoplankton assemblages in Qinhuangdao coastal waters in 2013 and 2014 were analyzed by highthroughput sequencing.Pelagophyte A.anophagefferens was further confirmed as the causative species of brown tides,and many species related to A.anophagefferens were revealed.Potential genetic diversity was found within A.anophagefferens.The results provide strong evidence to better understand the mechanism of brown tide in the coastal waters of China. |
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