Barcoding And Molecular Ecology Of Oligotrich And Choreotrich Ciliates (Ciliophora, Spirotrichea) In China Seas

Marine planktonic ciliates are abundant and important component of planktoniccommunities and play a crucial role in the functioning of microbial food webs. Theyare unicellular and eukaryotic protists mainly comprised by subclass Choreotrichiaand Oligotrichia. Despite their ecological importance, the diversity and characteristicsof the community structures of these two groups in coastal Yellow sea and Bohai Sea,a unique marine area in the world, is poorly characterized. They have long beenknown to be the least studied organisms.In order to provide fundamentical data on understanding the planktonic ciliatecommunity dynamics and their relationships with environmental conditions,molecular phylogenetics and ecological surveys, incluingfingerprint approaches,rDNA copy number statistics, molecular biodiversity analyses, biogeographyresearches, spatial and temporal distribution surveys of the functional groups onplanktonic ciliates, were carried out in this work during an annual cycle betweenOctober2010and December2011, in Bohai Sea, semi-enclosed Jiaozhou bay, EastSea, Qingdao bathing beach and the sea shore of Yantai, northern China.We carried a comprehensive phylogenetic study to the ecologically importanttintinnids, with158new rRNA sequences from sing cells of83marine species/isolatesfrom northeast China seas, covering three rRNA gene regions (SSU, ITS-5.8S-ITS2and LSU); We Used single cell sequencing and QPCR approach to estimate rDNAcopy number and sequence variation; The molecular diversity and distributionpatterns of nanoplanktonic oligotrich and choreotrich ciliates were investigated forsamples in surface water, standard layer water and surface sediments of three neriticbasins of northern China, the South Yellow Sea (SYS), North Yellow Sea (NYS) and Bohai Sea (BS).The following conclusions could be drawed:1. Barcoding and Phylogenetic study of Choreotrichia and Oligotrichia(Ciliophora, Spirotrichea) inferring three rDNA genes, under a widersampling strategy in China seas1) Three genes consistently supported the monophyly of tintiniids genera:Eutintinus, Amphorellopsis, Tintinnidium, Strombidinopsis and Rhabdnella;2) The deviation of Favella species to the revised genus Favella and genusSchmidingerella in the previous studies was supported by the updated SSUgene phylogeny, as well as the LSU gene and5.8S-ITS gene trees;3) Genera Metacylis, Stenosemella and Tintinnopsis were suggested to beparaphyletic;4) Nine highly supported subclades of Tintinnopsis were exhibited in themost species-abundant18S gene trees, which could be used as a profile forthe DNA based identifications of Tintinnopsis species;5)5.8S-ITS region had a highest discrimation capacity among the three genesfor tintiniids groups, suggesting a potential candidate for barcoding genemarkers; LSU was the most divergent gene, while SSU shared the lowestaverage divergent number;6) Potential genotypes were indicated by the SSU rDNA sequences for themorphospecies of choreotrichids.2. Quantification of rDNA copy number and sequence polymorphisms ofoligotrich and peritrich ciliates using single-cell analysis1) The rDNA copy numbers per cell were extremely high and differentamong even congeners, with the highest record of about310000;2) There was substantial intraindividual haplotype diversity and nucleotidediversity for the rDNA markers, with sequence differences primarilycharacterized by single nucleotide polymorphisms; 3) Haplotype and nucleotide diversity were positively correlated to the rDNAcopy number.3. SSU rDNA sequence diversity, spatiotemporal distribution ofnanoplanktonic oligotrich and choreotrich ciliates in surface water ofBohai and Yellow Seas1) T-RF richness and Shannon diversity were generally higher in the SYSthan in the BS, and negatively correlated with the molar ratio of P to Si;2) Multidimensional scaling and permutational multivariate analysis ofvariance of the community composition demonstrated a distinct seasonalpattern (P<0.01) but no diel and spatial differentiation (P>0.05) across allsamples. Nevertheless, a spatial pattern was recognized in the winterdataset (P<0.01);3) Mantel tests showed that the environmental factors, P:Si ratio, watertemperature (Temp) and concentration of dissolved oxygen (DO)determined the pattern observed for all samples (P<0.05); bothbiogeography and environment shaped the community in winter, withTemp and DO are the most important factors (P<0.01);4) Analyses of three clone libraries of SSU rRNA genes revealed a largenovel ribotype diversity, of which many were rare phylotypes belonging tothe subclass Oligotrichia, but divergent from described morphospecies.4. Molecular diversity, vertical and seasonal distribution of nano-sizedoligotrich and choreotrich ciliates in the South Yellow Sea Cold WaterMass1） South Yellow Sea Cold water mass revealed a large group of nano-oligotrichs ciliates distribution;2）The diversity parameters of the winter samples showed a much highernumber than summer samples; 3）MDS and ANOSIM results showed the South Yellow Sea Cold Water Massshared the same seasonal distribution pattern (P<0.01);4） BIOENV analyse results showed that Salinity, pH, DO were the keyenvironmental factors in shaping the diversity and community ofnanociliates (P<0.01);5）Spearman correlation results showed the DO and Temp had a negativecorrelation with mosts diversity indexs, whereas the PO4-P showed apostive relation with the diversity indices;6）CCA results indicated that the environment parameters salinity, DO andNO3-N had a significant positive relation with nanociliates communitystructure in SYSCWM (P<0.01), whereas the temperature showed apositive correlation with nanociliates (P<0.05).5. Biogeography of nano-sized oligotrich and choreotrich ciliates insurface sediment across Bohai and Yellow Seas1）Three clone libraries showed a total of269sequences which wereassigned to37OTUs; A large amounts of these OTUs affiliated toChoreotrich ciliates group;2）A number of novel groups existed in the sediment samples from thepylogenetic tree results;3）Diversity estimates showed a higher diversity indices in site B47S thanother two sites;4） MDS and ANOSIM results did not find obvious seasonal or spatialdistribution pattern among the the surface sediment samples;5）The T-RF number and Richness indices showed a negative correlation tothe environmental Chl-a;6） No siginificant correlation was found between geographic distance andsimilarity of T-RF communities, which indicated the communitiescompositions were not significantly effected by their specialbiogeographical location or season; 7）CCA result indicated that only DO had a significant positive relation to thenanociliates community structures in the sampling area (P<0.05).