Molecular Ecological Study On Benthic Microbial Eukaryotes In Sediments Of The Seagrass Meadow And The Yellow Sea Of China

Microbial eukaryotes consist of protist and single-celled fungi,which cover a wide spectrum of cell sizes,shapes and taxonomic affiliations.As the main component of microbial food web,microbial eukaryotes play an important role in energy flowing and matter cycling.In the past decades,culture-independent molecular methods have been widely applied in characterizing microeukaryotic diversity in marine environments.However,studies of benthic microeukaryotes are still rare.This work involved three aspects of characterizing benthic microeukaryotes in marine environments as follows:The diversity and community structure of micro-eukaryotes in vegetated and unvegetated sediment samples of seagrass ecosystem were investigated using clone library and Miseq high-throughput sequencing.It was found that Apicomplexa was a major group of which the average relative abundance of rDNA sequences was about 83.2%.Phylogenetic analysis indicates these apicomplexians were closely related to Lecudina polymorpha,a parasitic species previously detected in marine inveterbrates.Richness of microeukaryotes was significantly and negatively correlated with the concentrations of several heavy metals(such as Cd and As).There were significant differences in the community structure of microeukaryotes between vegetated and unvegetated sediments,which were probably due to changes in water salinity and ammonium concentration in these sediments,as shown by RDA ordination.Furthermore,Fungi was the largest contributor to that the community differences.Specific primers targeting 18 S rDNA of Lecudina polymorpha were newly designed and showed good specificity.Quantitative real-time PCR(qPCR)assays indicated that the 18 S rDNA gene copy number of Lecudina polymorpha was ten times higher in the vegetated sediments than those in the unvegetated sediments.Across these samples,the gene copy number of L.polymorpha was significantly and negatively correlated with the concentration of porewater ammonium,and positively with the sediment grain size and the concentration of Chl-a in overlying waters.With pyrosequencing of 18 S rDNA genes of benthic microeukaryotes and characterized environmental factors,the sequences of ciliates were extracted,which allowed to investigate the community structure and biogeographic distribution of benthic ciliates in the three basins,the Bohai Sea,North and South Yellow Seas.The results showed that the sequences of the classes Spirotrichea,Oligohymenophorea and Litostomatea accounted for a large proportion in the ciliate dataset.The variation of ciliate community showed significant differences between these basins rather than among the summer and winter seasons.The relative abundance of the orders Urostylida and Apostomatida was strongly correlated with the concentrations of nitrate and nitrite.Partial Mantel tests indicated that water depth rather than geographic distance was the key factor determining benthic ciliate community structure in these regions.CCA showed that temperature,pH,salinity,and Zn2+ were the major environmental factors driving ?-diversity of ciliates.Specific primers were the keys to molecularly investigate the diversity and quantity of microeukaryotic populations in marine habitats.In this study,two peritrich-specific PCR primers were newly designed to amplify a fragment including the internal transcribed spacer(ITS)region of ribosomal rDNA from environmental samples.The primers showed high specificity in silico,and in tests with peritrich isolates and environmental DNA.Application of these primers in clone library construction and sequencing yielded exclusively sequences of peritrichs for water and sediment samples.We also found the ITS1,ITS2,ITS,D1 region of 28 S rDNA,and ITS+D1 region co-varied with,and generally more variable than,the V9 region of 18 S rDNA in peritrichs.The ITS2 region was the most variable locus,suggesting its great potential in use for metabarcoding of peritrich ciliates.The newly designed specific primers thus provide additional tools to study the molecular diversity,community composition,and phylogeography of these ecologically important protists in different systems.