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It is believed that chloroplasts arose through a primary endosymbiotic uptake of a photosynthetic prokaryote by a non-photosynthetic eukaryote, and have spread to a wide range of eukaryotic lineages via secondary endosymbioses. On the other hand, many eukaryotes are also known to have lost the photosynthetic function of their chloroplasts. It is known that in dinoflagellates, multiple losses of chloroplasts had taken place, resulting in the occurrence of large number of heterotrophic species in different lineages. However, the process of reductive evolution of chloroplasts within dinoflagellates is not well investigated. This study aims to understand the process of early stages of reductive evolution of chloroplasts by comparing dinoflagellate species within a single genus, Paragymnodinium, which exhibit varying degrees of dependence on their chloroplasts: mixotrophic species, mostly dependent on phagotrophy, as well as completely phototrophic species. This thesis consists of four chapters. In chapter 1, an overview of chloroplast evolution, characteristics of dinoflagellates and research background relating to the chloroplast reduction are reviewed. For the purpose of this study, I used species of the genus Paragymnodinium. The genus Paragymnodinium was established by Kang et al. (2010), based on a type species, P. shiwhaense, which was newly described from Korea and characterized by mixotrophic nutrition and possession of complex extrusomes, the nematocysts. In chapter 2, four new species of dinoflagellates belonging to the genus Paragymnodinium were described based on observations using light, scanning and transmission electron microscopy, together with molecular analysis. Paragymnodinium stigmaticum was 8.5–15.2 μm long and 6.3–12.4 μm wide and shared many features with P. shiwhaense, including the possession of nematocysts and ingestion of prey cells despite the possession of chloroplasts. However, it was distinguished from P. shiwhaense by its feeding mechanism, its chloroplast ultrastructure, the presence of an eyespot and a benthic lifestyle (P. shiwhaense is planktonic). Paragymnodinium verecundum was 9.4–17.1 μm long and 5.7–13.6 μm wide, and similar to P. stigmaticum in its shape, possession of an eyespot and nematocysts, ingestion of prey, and benthic lifestyle. On the other hand, P. verecundum showed negative phototaxis and possessed a pusule, which were not observed in P. stigmaticum, indicating these two dinoflagellates were different species. Paragymnodinium asymmetricum was 7.9–12.6 μm long and 4.7–9.0 μm wide and did not show feeding behavior and were phototrophically maintainable. Paragymnodinium asymmetricum shared many features with P. shiwhaense, such as nematocysts, absence of eyespot and the planktonic lifestyle. However, P. asymmetricum was distinguished from P. shiwhaense by the asymmetric shape of hyposome and nutritional mode. Paragymnodinium inerme was 15.3–23.7 μm long and 10.9–19.6 μm wide and also did not show feeding behavior. Paragymnodinium inerme was similar to P. shiwhaense in its shape and planktonic lifestyle, but the nutritional mode was different. The presence of incomplete (partly collapsed) nematocysts was also a unique feature in P. inerme. A phylogenetic analysis inferred from concatenated 18S and 28S ribosomal DNA sequences recovered the four dinoflagellates along with P. shiwhaense in a robust clade that was included in the clade Gymnodinium sensu stricto. Therefore, together with the morphological similarities, it was concluded that all of these dinoflagellates should be regarded as new species in the genus Paragymnodinium. The fact that genus Paragymnodinium exhibits various nutritional strategies provides an excellent opportunity to investigate the evolution of the mode of nutrition and the function of the chloroplasts. In chapter 3, I analyzed the growth, pigment composition, absorption spectra, variable chlorophyll a fluorescence, and photosynthetic carbon fixation capabilities of Paragymnodinium stigmaticum, P. asymmetricum and P. inerme. The autotrophic species P. asymmetricum and P. inerme without resorting to any nutrition from prey organisms displayed high photosystem II activity and carbon fixation rates. The pigment compositions of these two species were identical to those of other typical peridinincontaining type dinoflagellates. On the other hand, the phagotrophic species P. stigmaticum showed heterotrophic growth, i.e., addition of cryptomonad Rhodomonas sp. was required for its prey, and the variable chlorophyll a fluorescence properties and carbon fixation rates indicated significantly lower photosynthetic competence relative to those of the above two species. Paragymnodinium stigmaticum also contained peridinin, but pigment content ratios of peridinin, chlorophyll c2 and β-carotene were significantly different from those of other two species. The absorption spectrum analysis revealed a red-shift in the peak of the Qy band of chlorophyll a in P. stigmaticum, presumably due to a change in chlorophyll-protein complexes. Such distinct differences in nutritional strategies between members of the genus Paragymnodinium would provide a platform for the hypothetical loss of photosynthetic function leading to colorless dinoflagellates. In chapter 4, a comparative transcriptomic analysis within the photosynthetic and non-photosynthetic species in the genus Paragymnodinium (P. asymmetricum, P. inerme and P. stigmaticum) was conducted to evaluate differences of the chloroplast-related gene expression which were involved in heme, chlorophyll, isopentenyl diphosphate and carotenoids synthesis pathways, carbon fixation (Calvin cycle) and photosynthesis. Paragymnodinium stigmaticum showed a significant lack of mRNA expressions for photosystem II and its light harvesting complex, in spite of the other components for photosynthetic functions were expressed at the same level to the other phototrophic species. In addition, the transcription of rbcL gene was shown to be absent in P. stigmaticum, whereas the other species actively expressed it. Lacks of expression of a few genes in chlorophyll and carotenoid synthesis pathways were also observed in P. stigmaticum, whereas heme and isopentenyl diphosphate synthesis pathways showed a same level of expression within the three Paragymnodinium species. These results were consistent with the inactivated photosynthesis and carbon fixation in P. stigmaticum, and represented an example for the process of genetic changes during an early transitional stage of loss of photosynthetic capability.
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