Metabarcoding Dissection And Comparative Genomic Analysis Of Harmful Algal Bloom Species In The East China Sea

The East China Sea(ECS)is one of the four seas of China,where harmful algal blooms(HABs)occurred frequently.With the increasing impact of human activities and global climate change,HAB species in the coastal regions of China are becoming more diverse,with smaller cell sizes and more harmful species.Traditional species identification methods are inadequate for species identification and quantitative analysis of HAB species in the ECS.Furthermore,morphology-based methods cannot adequately process large quantities of samples obtained from scientific expeditions.This Ph.D.thesis focused on sanalyzing the biodiversity and geographic distribution of HAB species in the ECS,using the amplicon sequence variants(ASVs)-based metabarcoding method.This project aimed to test the hypothesis that the frequent occurrences of diverse HABs in the ECS are dictated by the presence of rich HAB species in this region.In this study,we have constructed the mitochondrial genomes(mt DNA)of two HAB species Pseudo-nitzschia pungens and Pseudo-nitzschia cuspidate for the first time,as well as one non-HAB species Pseudo-nitzschia micropora.A high resolution molecular marker of P.pungens was developed,and comparative genomics was used to analyze the differences in mitochondrial genomes between HAB and non-HAB species of the genus Pseudo-nitzschia.The main results were as follows:1.Composition of HAB species in the red tide event recorded in the ECSThe 18S r DNA V4 regions of the samples were amplified and sequenced using high-throughput sequencing method,resulted in the identification of 3,966 amplicon sequence variants(ASVs),which was annotated to belong to 35 classes in six phyla/divisions of phytoplankton.Different phytoplankton communities were found to have unique geographical distribution patterns in the area.237 potential phytoplankton species were annotated based on the PR~2 database and Genbank database,including 58 HAB species,of which 23 HAB species had not been reported previously in the ECS.47 HAB species were found in the blooming site S05-1,with Prorocentrum donghaiense,Lebouridinium glaucum and Noctiluca scintillans substantially elevated,suggesting that the site was experiencing a multiple-species HAB event.Such high diversity of HAB species in this region may be responsible for the high occurrences of diverse HABs in the ECS.This study has demonstrated that the ASV-based metabarcoding dissection could be applied to the identification of algae species with small cell size and those species that are fragile during fixation.Temperature and salinity may be the two most important environmental factors affecting the phytoplankton community structure and the distribution of dominant HAB species in this area,while species-species interactions may have also contributed to the development of the HABs.2.Composition and distribution of HAB species in the ECS in springFor the frequent occurrence of HABs in the ECS in spring,a census of HAB species was conducted in the continental shelf of the ECS within 100 m depth in May2019.Metabarcoding dissection based on the ASVs was also used to analyze the phytoplankton community in the ECS,and the results shown that Dinoflagellata was the first dominant division in spring,with the highest richness and relative abundance.249 potential phytoplankton species were identified in this study,of which 178 had never been reported in the ECS.For the 71 HAB species,31 had never been reported in the ECS.As for the 10 dominant HAB species,8 were Dinoflagellates and only 2were belonged to Ochrophyta.The dominant HAB species of Dinoflagellates were more likely distributed in the nearshore stations of each section,while the relative abundance of dominant HAB species of Ochrophyta were high in the Changjiang estuary and adjacent waters of Hangzhou Bay.The environmental factors showed significant regional differences in the ECS,and significantly correlated with phytoplankton communities and some dominant HAB species.By analyzing the Spearman correlations between plankton communities,there existed significant correlations between phytoplankton and zooplankton,and between different phytoplankton communities.The ECS has complicate hydrological conditions,and HAB species with high biodiversity were widely distributed in the sea area,all of these HAB species may be blooming with suitable environmental conditions.It is not only an important reason for the frequent occurrence of HABs in this sea,but also an important challenge for the monitoring and control of HABs in the ECS.3.Development of a high-resolution molecular marker of Pseudo-nitzschia pungensNine Pseudo-nitzschia pungens strains were isolated from the Bohai Sea,Jiaozhou Bay and the ECS by capillary separation,and the first mitochondrial genome(mt DNA)of P.pungens(strain CNS00141)was constructed.It was a circular mt DNA with 40,380 bp in length,encoding 35 protein-coding genes(PCGs),24 transfer RNA(t RNA)genes,2 ribosomal RNA(r RNA)genes,and 2 open reading frames(orfs)of unknown functions.The mt DNA contained a group II intron in cox1 and a 2,000bp-long repetitive segment.Comparative genomic analysis was performed with related species(Pseudo-nitzschia multiseries and Fragilariopsis kerguelensis),and the results showed that most regions of the genome were conserved.A molecular marker ppmt1 was developed through comparative analysis of mt DNAs of nine P.pungens strains.The marker showed higher resolution and specificity than common markers,which had great application prospects for tracking genetic diversity of the HAB species P.pungens.4.Comparative genomic analysis of the genus Pseudo-nitzschiaBy capillary separation,one P.cuspidata strain(CNS00150)was collected from the ECS and one P.micropora strain(CNS00133)was collected from the Jiaozhou Bay.The mt DNAs of P.cuspidata and P.micropora were constructed for the first time,using the mt DNA of P.pungens as reference sequence.The completed mt DNA of P.cuspidate was 37,203 bp in length and P.micropora was 38,792 bp in size,and both of them encoded 35 PCGs,24 t RNA genes,2 r RNA genes and 1 orf157.The mt DNAs of the two species were much shorter than that of P.pungens(40,380 bp)and P.multiseries(46,283 bp).Most of the sequences of the three newly constructed genomes were conserved through comparative genomic analysis,and the genomic structures of P.cuspidate and P.micropora were very similar with no introns.Each of the four mt DNAs of Pseudo-nitzschia species contained a repeatitive sequence in the non-genetic coding region that was different among different mt DNAs.The mt DNAs of the HAB species(P.pungens and P.cuspidata)and non-HAB species(P.micropora)show high colinearity.P.cuspidata and P.micropora clustered closely,while P.pungens and P.multiseries clustered closely in the phylogenetic tree.In summary,this study applied ASV-based metabarcoding analysis approach in exploring the biodiversity and biogeographic distribution characteristics of the HAB species in the ECS for the first time.This study also applied bioinformatics methods to explore how environmental factors and species-species interactions influenced phytoplankton communities and HAB species distribution.Furthermore,this study constructed mt DNAs of three Pseudo-nitzschia species for the first time,and in particular,developed a high-resolution molecular marker ppmt1 for P.pungens,which can be used to track genetic diversity of this HAB species.Finally,this study analyzed the mt DNAs of HAB species and non-HAB species of the genus Pseudo-nitzschia,which confirmed the phylogenetic relationships of four Pseudo-nitzschia species.Results of this study set up a platform for exploring the biodiversity and dynamics of HABs in the ECS,and in particular for exploring the genetic diversity and dynamics of P.pungens.