Analysis Of Genotypic Diversity And Plasticity Of Symbiodinium In Zooxanthellate Corals On Basis Of Ribosomal DNA Sequences

The symbiosis between corals and their obligate algal partners(dinoflagellates in the genus Symbiodinium colloquially called zooxanthellae)is of profound importance to coral health and functioning of reef ecosystem.In recent decades,anthropogenic global warming and ocean acidification have posed multiple stressors to coral reef ecosystem,leading to more frequent and severe coral bleaching(the disruption of coral-algal symbiosis).It is believed that adaptive shift of algal symbiont is one defense mechanism by which corals respond to environmental disturbations.Thus,understanding the genetic diversity and flexibility of Symbiodinium is key to predicting corals' adaptation and acclimatization to future climate change.Despite worldwide interest,molecular surveys of Symbiodinium in the South China Sea is rather limited,especially from extensive sampling across large geographic distance.This dissertation focuses on three aspects of Symbiodinium genotypes in major scleractinian coral species off the southeastern coast of China that were investigated with high throughput nest generation sequencing(NGS)and quantitative PCR(qPCR)targeting the internal transcribed spacer(ITS2)and/or large subunit gene(28S)of nuclear ribosomal DNA(rDNA):1.Through keywords searching and data mining,ITS2-containing sequences of Symbiodinium in GenBank and several expert reference databases published to date were integrated.Based on the highly conserved sequence and secondary structure motif of the ITS2-proximal stem,the 3'-end of 5.8S and 5'-end of 28S termini flanking the ITS2 were trimmed.The resulting clean ITS2 dataset was used to build a profile Hidden Markov Model(HMM)for the retrieval of additional ITS2 sequences.This HMM-and rRNA secondary structure-based metadata processing pipeline facilitated the identification of neat and clean Symbiodinium ITS2 from the vast majority of poorly annotated sequences in GenBank,and reduced the complexity and redundancy of the original references.Finally,a custom Symbiodinium ITS2 database was constructed using the new dataset,which will provide an urgently needed reference standard for consistent and unambiguous Symbiodinium genotyping and classification.2.High throughput amplicon sequencing of ITS2 of the nuclear ribosomal DNA was applied to evaluate Symbiodinium diversity in 13 species of corals from 5 contrasting reef regions off the southeastern coast of China.The result showed that most corals were associated with prevalent clade C Symbiodinium,with clades A,D,E,F or G at low background abundance.Despite the single clade C dominance and stability in most coral samples across large geographic distance,the genotypic profiling at subclade level revealed Symbiodinium community structure at an even finer scale,which enables the distinction of region-or host-specificity.Moreover,the algal symbiont community exhibited higher levels of diversity and clade D abundance in the latitudinal extremes(i.e.,SY and DS)than in the middle(WZ?SM and GI)of the surveyed regions,suggesting that clade D symbiont may help increase corals' ability to resist both low and high temperature stress.3.Coral species of Pocillopora damicornis harboring distinct Symbiodinium clades(C or D)were used to investigate the effects of elevated temperature on algal symbiont community.Under elevated temperature,coral with dominant clade C symbiont was more susceptible to bleaching and photochemical damaging than its counterpart with dominant clade D symbiont,demonstrating that Symbiodinium belonging to clade D is thermally tolerant.It was also observed that the symbiotic algal community originally with high level clade C eventually switched to be clade D-dominated under high temperature stress,whereas the originally clade D-dominated symbiont community structure was stable with only changes in some subclades of background abundances.This result demonstrated,first in real time,the existence of symbiont shuffling in corals as a mechanism for increased thermal tolerance.In this study,the newly compiled Symbiodinium ITS2 sequence database will provide necessary annotation guide and data reference for molecular systematics of Symbiodinium and other related studies.Additionally,the molecular-level analyses of Symbiodinium community structure of corals under both the natural reef environments and manipulative laboratory conditions,will shed new insight into corals' responses to environmental stress through symbiotic regulation,as well as provide guidance for coral reef protection and remediation under future climate change.