Taxonomy of the symbiotic dinoflagellates, transcriptomic analyses of Calvin cycle genes in mixotrophic and heterotrophic dinoflagellates, and an improved real-time PCR method to quantify a red tide dinoflagellate

Abstract

Dinoflagellates are ubiquitous protists and live in marine environments as diverse forms. Although they often cause red tides or harmful algal blooms, they also play such important roles in marine food webs as prey, predator, endosymbiont, and parasite. However, in studying taxonomy, genetics and quantification of some dinoflagellate species, there have been some difficulties and problems in identifying species, quantifying their abundances, and understanding their molecular genetic characteristics. To solve the difficulties in exploring the taxonomy, the trophic mode associated molecular genetic characteristics, and improved qPCR based quantification of small dinoflagellates and mixotrophic protists, this study focused on 1) discovering and distinguishing symbiotic species by molecular and morphological method to set up their morphological standard, 2) comparing the genetic status with the trophic mode of dinoflagellates by analyzing the expressed genes through transcriptome analysis, and 3) developing a fast and accurate method to detect red-tide organisms. Among the dinoflagellate species, the genus Symbiodinium forms symbiosis with a broad diversities of host species such as corals, jellyfish, sea anemones or giant clams. Especially they provide organic photosynthases to host species, mostly coral, and in return, corals provide other nutrients to symbiodinium. Thus, they are crucial components for survivals of corals and for building tropical reef ecosystems. However, despite their ecological and economic importance in marine ecosystems, the taxonomy of Symbiodinium remains limited due to their small size and fragile surface, which has been causing the difficulties in identification of these species for several decade. Especially, type species of genus Symbiodinium have been described to have incomplete morphology, although the morphological and molecular characteristics of type species plays as a overall standard for species belonging to the genus. These incomplete morphological standards have ended with multiple synonyms for a single species. Thus, here, I completed the morphological characterization of Symbiodinium microadriaticum, the type species, and also established a new Symbiodinium species which is symbiotic to the giant clam (Tridanidae), to name it as Symbiodinium tridacnidorum sp. nov.. Furthermore, I clarified the unreported morphological characteristics of two clade B Symbiodinium, Symbiodinium minutum and Symbiodinium psygmorphilum, to complete their plate formula. The dinoflagellate are known to have three different trophic modes

autotrophic, heterotrophic and mixotrophic. Due to these characteristics, dinoflagellates plays diverse roles in marine food webs as prey and predators. However, the difference in molecular genetic characteristics between mixotrophic and heterotrophic dinoflagellates have not been reported so far. Thus, molecular genetic characteristics of mixotrophic and heterotrophic dinoflagellates should be compared to trace evolutionary routes of trophic modes in dinoflagellates. I explored the transcriptome of two closely related dinoflagllates which have high similarity in their morphology, ribosomal DNA, and edible prey species, but have different trophic modes in order to discover any parallellism between the gene expression and the trophic modes. When I compared the expressed gene sets of the mixotrophic dinoflagellate Paragymnodinium shiwhaense and the heterotrophic dinoflagllate Gyrodiniellum shiwhaense by analyzing their transcriptome, the expressed genes exhibited huge dissimilarities, although there were many similar expressed genes, especially in the gene groups related to photosynthesis. Furthermore, based on this comparison analysis, I proved that the mixotrophic dinoflagllate may have more genes that play diverse functional characteristics in genetic level than the heterotropic dinoflagellates because the former must conduct both photosynthesis and phagotrophy in contrast to the latter performing phagotrophy only. Every summer, Korea suffers from harmful algal blooms (HABs) caused by red tide dinoflagellate Cochlodinium polykrikoides. Thus, it is important to detect and quantifying their abundances accurately. However, quantifying the harmful dinoflagellate Cochlodinium polykrikoides in natural samples is not easy due to similarity in morphologies between Cochlodinium polykrikoides and closely related species. Furthermore, qPCR method for C. polykrikoides is also not easy due to potential variations in DNA contents among individual cells. Here, I also developed new species-specific primers and probe for detecting all the 3 ribotypes of ichthyotoxic dinoflagellate Cochlodinium polykrikoides, and comparatively evaluated the efficiencies of the 4 different preparation methods used to determine standard curves. Furthermore, to confirm the accuracy of result, the abundance of C. polykrikoides in the > 500 samples collected from the coastal South Sea of Korea, in 2014 and 2015, were independently determined using all the 4 methods. Standard curves constructed by by extraction of DNA from each of the serially diluted cultures with different concentrations of cultured C. polykrikoides were most accurate followed by the standard curve obtained by extracting DNA from each of serially diluted field sample with different concentrations of C. polykrikoides. Thus, this study provided more accurately modified methods to detect dinoflagellate species from natural sea water samples. The result of this thesis provided complete morphological standard and eventually provided the basis of overall understanding of taxonomy of symbiotic dinoflagellate Symbiodinium species. Furthermore, the result of comparative transcriptome analyses of dinoflagellates possessing different trophic modes obtained in this thesis will provide better understanding of genetic influence to the trophic mode of dinoflagellate species. In addition, the improved qPCR methods and a newly developed set of specific primer and probe set resolving the two recently discovered 2 new ribotypes of Cochlodinium polykrikoides will facilitate simple and automatic estimation of C. polykrikoides abundance.