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This study describes two novel parasitic dinoflagellates:Haplozoon gracile sp. nov. isolated from a bamboo worm (Maldanidae), 'cf.Petaloclymenesp.'sensuKobayashiet al. 2018; and,H. pugnus sp. nov. isolated fromNicomachesp. andNicomache personata(Maldanidae). Trophonts (feeding stages) were observed with light, scanning, and transmission electron microscopy. Molecular phylogenetic analyses were performed based on 18S rDNA. COI sequences were obtained for host organisms. Trophonts ofH. gracilewere linear (single longitudinal row) and relatively slender with a mean length of 190 mu m, and consisted of a long and narrow trophocyte, rectangular gonocytes (mean width = 10 mu m), and slightly rounded sporocytes. Trophonts ofH. pugnuswere pectinate (1-8 rows of sporocytes in one plane), with a mean length of 179 mu m, consisting of a bulbous trophocyte, rectangular gonocytes (mean width = 25 mu m), and rounded sporocytes. The body of both species was covered with many depressions that overlaid the amphiesmal vesicles. TEM observations of trophocytes inH. gracilerevealed a stylet with a central dense core and rich mitochondria subtending the amphiesma. Furthermore, amphiesmal vesicles appeared to contain thecal plates in both species. Phylogenetic analyses generally resolved aHaplozoonclade, andH. gracileandH. pugnuswere clearly distinguished from other species for which molecular data are available. Based on the morphological and host comparisons with all described species and their molecular phylogeny, we conclude that these two isolates are new species ofHaplozoon, H. gracile sp. nov. andH. pugnus sp. nov.

Rissoella golikovi (Gulbin, 1979) (Figs 3 E–F, 5A–G) Jeffreysina golikovi Gulbin, 1979: 89, figs. 1–2 (holotype ZIN 41388 /1; Vanino Bay, Sea of Japan, Russia); Kantor & Sysoev, 2006: 248, pl. 123, fig. E (paratype); Hasegawa, 2017: 398, 1063, pl. 355, fig. 7. Type material not available for analyses. Material examined. Fourteen mature specimens (ICHUM RT3001, RT 3002, RT3003, RO 3001, RO 3002, RO 3003, RK3001, RM 3001, RM 3002, RM 3003, ROM 3001, RA3001, RA 3002, RA 3003). For information on specimens collection locality and GenBank accession numbers see Table 1. Description. Shell minute, smaller (296–450 µm) in comparison to other rissoellids, thin, extremely fragile, translucent or whitish opaque, skeneiform (width about 150% of length), with deep, widely perforate umbilicus (Fig. 5A). Protoconch smooth, of about one whorl (Figs. 5B, C). Teleoconch smooth with distinct growth lines, slightly deep suture, of about three convex whorls; aperture simple, entire, nearly circular but with margin adjacent to previous whorl flattened. Operculum typical of family (Fig. 5D). Head–foot brown or dark grey with colorless sole; oral lobes short; cephalic tentacles slightly longer than oral lobes; oral lobes and cephalic tentacles proximally having similar coloration to head, gradually becoming transparent in distal portion. Mantle dark brown or black pigmented, with black or darker brown patch on center of dorsal portion of body whorl; another smaller dark patch placed on left of neck (Figs. 3E, F). Radular formula 12–13 × 1.R.1 (Fig. 5E). Central tooth higher than wide (width about 52% of length), with medial narrow ridge, cutting edge with one small central sharp cusp flanked by several larger cusps (Figs. 5F, G). Lateral teeth triangular (width about 41% of length), each with median ridge; cutting edge with larger median cusp, flanked by 4–5 sharp cusps, consecutively decreasing in size (Figs. 5E, F). Distribution and microhabitat. Known from Vanino Bay, as well as middle Kurile Islands, Russia. Material in this study was collected from Hokkaido, Japan: Otaru and Kamoenai (Sea of Japan), near Omu (Sea of Okhotsk), and Akkeshi and Muroran (Pacific). It was found on various algae including the coralline algae Corallina spp. Remarks. The type material of Rissoella golikovi was not examined, due to restrictions on shipping biological material. However, our newly sampled material agrees with its description. The present morphospecies is nearly identical in radula morphology to Rissoella globularis (Forbes & Hanley), which has been reported from France to northern Norway, as illustrated by Sars (1878). Nevertheless, these two species can clearly be distinguished by shell morphology. In R. globularis, the shell is depressed conical, while it is skeneiform in R. golikovi. The skeneiform shell of this species makes it easily distinguishable from other species in the family where shells are either ovate or elongate. Rissoella golikovi was first described by Gulbin (1979) from Vanino Bay, Russia, and was subsequently recorded from the eastern part of Hokkaido (Hasegawa 2017)., Published as part of Chira Siadén, Luis E., Wakeman, Kevin C., Webb, Stephen C., Hasegawa, Kazunori & Kajihara, Hiroshi, 2019, Morphological and molecular diversity of rissoellids (Mollusca, Gastropoda, Heterobranchia) from the Northwest Pacific island of Hokkaido, Japan, pp. 415-431 in Zootaxa 4551 (4) on pages 419-420, DOI: 10.11646/zootaxa.4551.4.2, http://zenodo.org/record/2623029, {"references":["Gulbin, V. V. (1979) A new species of the littoral gastropod Rissoellidae from the Far-eastern seas. Academy of Sciences of the USSR, Vladivostok. Far East Science Center. Marine Biology, 3, 88 - 89. [in Russian]","Kantor, Y. I. & Sysoev, A. V. (2006) Marine and Brackish Water Gastropoda of Russia and Adjacent Countries: An Illustrated Catalogue. KMK Scientific Pres Ltd., Moscow, 371 pp., 140 pls.","Hasegawa, K. (2017) Rissoellidae. In: Okutani, T. (Ed.), Marine Mollusks in Japan. 2 nd Edition. Tokai University Press, Tokyo, pp. 398 + 1063.","Sars, G. O. (1878) Mollusca regionis Arcticae Norvegiae. In: Bidrag til Kundskaben om Norges Arktiske Fauna. Chritiania, Brogger, 466 pp."]}

Rissoella elatior (Golikov, Gulbin & Sirenko, 1987) (Figs 2 A–D, 3A–H) Jeffreysina elatior Golikov, Gulbin & Sirenko, 1987: 35, pl. 3, fig. 6; Kantor & Sysoev, 2006: 248, pl. 123, fig. D (holotype 36525/1; Moneron Island, Russia). Type material not available for analyses. Jeffreysiella elatior — Hasegawa, 2017: 398, 1063, pl. 355, fig. 6. Material examined. Thirteen mature specimens (ICHUM RT1001, RT 1002, RO 1001, RO 1002, RO 1003, RSH 1001, RSH 1002, RK1001, RK1002, RM 1001, RSU 1001, RSE 1001, and RSE 1002). For information on specimens collection locality and GenBank accession numbers see Table 1. Description. Shell minute (800–1270 µm) but relatively larger if compared to other species described here, thin, fragile, translucent or whitish opaque, elongate (width about 63% of length), with narrow umbilicus, spire of about 25% of total length (Fig. 4A). Protoconch smooth, slightly pointed, of approximately one whorl, without sculpture at suture (Figs. 4B, C). Teleoconch smooth except for faint markings of growth lines; with deep suture; up to 3 ½ convex whorls; aperture simple, entire, semicircular, slightly shorter or almost 50% of total length. Operculum typical of family (Fig. 4D). Head–foot opaque white, with slender oral lobes and longer cephalic tentacles. Mantle brown or black pigmented, with black patch centrally placed on dorsal portion of body whorl. Black patch hardly recognized in specimens with black mantle (Figs. 3 B–D). Radular formula 15–16 × 2.1.R.1.2 (Fig. 4E). Central tooth wide (width about 61% of length), with 7–8 sharp cusps, latter gradually increasing in size from left to right until 6th (or in some cases 7th); right-most cusp slightly smaller than left ones. Group of 10–13 minute secondary cusps encircling upper margin of last right cusp (Figs. 4E, F). Lateral teeth elongate-triangular (width about 78% of length), each with large, sharp, smooth median cusp, and 8–12 smaller cusps along inner and outer margins (Figs. 4E, G). Inner marginal teeth represented by small, curved plates (width about 93% of length), each with large, sharp, smooth median cusp, flanked by 4–5 (along inner margin) or 5–7 (along outer margin) smaller cusps (Figs. 4E, H). Outer marginal teeth reduced, simple, plate-like (width almost 200% of length) (Figs. 4E, H). Distribution and microhabitat. Originally reported from the northern part of the Sea of Japan (Moneron Island) (Golikov et al. 1987), Russia, and subsequently reported to be widely distributed along the Japanese Archipelago from Hokkaido to Miyako Island, Okinawa (Hasegawa 2017). Material in this study was collected from Rumoi to Setana on the Sea of Japan; and on the Pacific coast near Muroran, Japan. It was found on various algae including the coralline algae Corallina spp. Remarks. Although the type specimens of Rissoella elatior have not been examined in this work, our newly sampled material agrees with the original description of this species (Golikov et al. 1987), as well as the photograph of the holotype (Kantor & Sysoev 2006: pl. 123, fig. D). In some specimens (Fig. 3A) the mantle coloration is brighter than others (Figs. 3 B–D), the former being pale brown with yellowish white asymmetrical patches and a brown patch centrally placed on the dorsal portion of the body whorl., Published as part of Chira Siadén, Luis E., Wakeman, Kevin C., Webb, Stephen C., Hasegawa, Kazunori & Kajihara, Hiroshi, 2019, Morphological and molecular diversity of rissoellids (Mollusca, Gastropoda, Heterobranchia) from the Northwest Pacific island of Hokkaido, Japan, pp. 415-431 in Zootaxa 4551 (4) on pages 418-419, DOI: 10.11646/zootaxa.4551.4.2, http://zenodo.org/record/2623029, {"references":["Golikov, A. N., Gulbin, V. V. & Sirenko, B. I. (1987) Prosobranch from Moneron Island shelf (Sea of Japan). I. Orders Patelliformes-Calyptraeiformes. In: Fauna and Distribution of Molluscs, North Pacific and Polar Basin, edited by A. Kafanov. Far Eastern Science Center of the USSR Academy of Science, Moscow, pp. 20 - 40.","Kantor, Y. I. & Sysoev, A. V. (2006) Marine and Brackish Water Gastropoda of Russia and Adjacent Countries: An Illustrated Catalogue. KMK Scientific Pres Ltd., Moscow, 371 pp., 140 pls.","Hasegawa, K. (2017) Rissoellidae. In: Okutani, T. (Ed.), Marine Mollusks in Japan. 2 nd Edition. Tokai University Press, Tokyo, pp. 398 + 1063."]}

Rissoella sp. 1 (Figs 3J, 3K) Material examined. Two mature specimens: ICHUM RSH 4001, RSH 4002; Shakotan, Hokkaido, Japan, 43°18′06.2″N 140°35′55.6″E, 25 August 2017. Both specimens were used for DNA extraction. For further information on specimens collection locality and GenBank accession numbers see Table 1. Remarks. Shell minute, thin, fragile, translucent or whitish, elongate. Head–foot translucent white; with very short round oral lobes, long cephalic tentacles with tapering tip, translucent as well. Mantle pigmented in light brown with three or four big yellowish asymmetrical patches. Big black patch, with few small whitish blotches inside, centrally placed on the dorsal portion of body whorl. Visceral mass dark brown to black, with several elongate whitish blotches (Figs. 3J, K). Distribution and microhabitat. Found in Shakotan, Hokkaido, Japan; in the subtidal zone on red algae Gelidium spp.

Rissoella sp. 1 (Figs 3J, 3K) Material examined. Two mature specimens: ICHUM RSH 4001, RSH 4002; Shakotan, Hokkaido, Japan, 43��18���06.2���N 140��35���55.6���E, 25 August 2017. Both specimens were used for DNA extraction. For further information on specimens collection locality and GenBank accession numbers see Table 1. Remarks. Shell minute, thin, fragile, translucent or whitish, elongate. Head���foot translucent white; with very short round oral lobes, long cephalic tentacles with tapering tip, translucent as well. Mantle pigmented in light brown with three or four big yellowish asymmetrical patches. Big black patch, with few small whitish blotches inside, centrally placed on the dorsal portion of body whorl. Visceral mass dark brown to black, with several elongate whitish blotches (Figs. 3J, K). Distribution and microhabitat. Found in Shakotan, Hokkaido, Japan; in the subtidal zone on red algae Gelidium spp., Published as part of Chira Siad��n, Luis E., Wakeman, Kevin C., Webb, Stephen C., Hasegawa, Kazunori & Kajihara, Hiroshi, 2019, Morphological and molecular diversity of rissoellids (Mollusca, Gastropoda, Heterobranchia) from the Northwest Pacific island of Hokkaido, Japan, pp. 415-431 in Zootaxa 4551 (4) on page 423, DOI: 10.11646/zootaxa.4551.4.2, http://zenodo.org/record/2623029

Rissoella japonica Chira & Hasegawa, n. sp. (Figs 3 G���I, 6A���G) Rissoella sp.��� Hasegawa, 2000: 700 -701, plate 349, fig. Rissoellidae-1; Hasegawa, 2017: 398, 1063, pl. 355, fig. 5. Type material. Holotype: adult, 0.9 mm (ICHUM RK2001); Kamoenai, Hokkaido, Japan, 43��08���10.5���N 140��25���43.1���E, 6 November 2016. Paratypes: 3 specimens (ICHUM RK2002, RO2001, RO2002) from Oshoro Bay, Hokkaido, Japan; 1 specimen (RSH2001); Shakotan, Hokkaido, Japan. For information on specimens collection locality and GenBank accession numbers see Table 1. ZooBank registration: urn:lsid:zoobank.org:act: E3102674-B307-40F6-A700-7696AE32FCA8 Etymology. The species name, Rissoella japonica, refers to the geographical distribution from where the species was found. Diagnosis. Protoconch with rippled sculpture at suture. Radula, central tooth with 10-13 sharp cusps on cutting edge. Lateral teeth narrow, with median ridge becoming basal process, and outer lateral projection on base; cutting edge with major median cusp, flanked by 5-6 (along outer margin) or 7-9 (along inner margin) sharp cusps. Marginal teeth similar in shape to lateral one but smaller, cutting edge with median cusp, flanked by 3-5 smaller sharp cusps on each side. Description. Shell minute (764���1091 ��m), thin, fragile, translucent or whitish opaque, elongate (width about 67% of length), with narrow umbilicus, spire of about 30% of total length (Fig. 6A). Protoconch smooth, of about 1 whorl, with rippled sculpture along suture (Figs. 6B, C). Teleoconch smooth, with distinct growth lines, deep suture, about 2 �� convex whorls; aperture simple, entire, semicircular, slightly longer than 50% of total length. Operculum typical of family (Fig. 6D). Head���foot dark brown with colorless sole; oral lobes and tentacles dark brown. Mantle dark brown or black pigmented, with black patch placed slightly to left on dorsal portion of body whorl (Figs. 3 G-I). Radular formula 11-13 �� 1.1.R.1.1 (Fig. 6E). Central tooth higher than wide (width about 48% of length), cutting edge with 10���13 sharp cusps of different sizes (Figs. 6E, F). Lateral teeth narrow (width about 23% of length), with median ridge becoming basal process, outer lateral projection on base; cutting edge with larger median cusp, flanked by 5���6 (along outer margin) or 7���9 (along inner margin) sharp cusps (Figs. 6E, G). Marginal teeth with similar shape to lateral one but smaller (width about 33% of length); cutting edge with median cusp, flanked by 3���5 smaller sharp cusps on each side (Figs. 6E, G). Distribution and microhabitat. In the Sea of Japan from Otaru to Setana, Japan. It was found in the intertidal zone on various algae including the coralline algae Corallina spp. Remarks. Both R. japonica n. sp. and R. elatior occur sympatrically in some localities, and they might be confused. However, they can be distinguished by the head-foot coloration (being dark brown in R. japonica n. sp. and white in R. elatior) and the radula morphology, as well as by conchological characters such as spire/total length and aperture/total length ratios. Based on radula morphology, R. japonica n. sp. belongs to a group containing the type species of Rissoella s.s., R. diaphana illustrated by Thiele (1929 ���1935; as R. glabra), in having a symmetrical configuration with five teeth per row. Rissoella japonica n. sp. can be distinguished from R. diaphana by the relatively narrower and smaller central tooth., Published as part of Chira Siad��n, Luis E., Wakeman, Kevin C., Webb, Stephen C., Hasegawa, Kazunori & Kajihara, Hiroshi, 2019, Morphological and molecular diversity of rissoellids (Mollusca, Gastropoda, Heterobranchia) from the Northwest Pacific island of Hokkaido, Japan, pp. 415-431 in Zootaxa 4551 (4) on page 423, DOI: 10.11646/zootaxa.4551.4.2, http://zenodo.org/record/2623029, {"references":["Hasegawa, K. (2000) Rissoellidae. In: Okutani, T. (Ed.), Marine Mollusks in Japan. Tokai University Press, Tokyo, pp. 700 - 701.","Hasegawa, K. (2017) Rissoellidae. In: Okutani, T. (Ed.), Marine Mollusks in Japan. 2 nd Edition. Tokai University Press, Tokyo, pp. 398 + 1063.","Thiele, J. (1929 - 1935) Handbuch der systematischen Weichtierkunde. Gustav Fischer, Jena, 1154 pp."]}

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., (主査) 教授 堀口 健雄, 教授 小亀 一弘, 准教授 柁原 宏, 助教 Kevin C. WAKEMAN, 理学院（自然史科学専攻）

(主査) 教授 堀口 健雄, 教授 小亀 一弘, 准教授 柁原 宏, 助教 WAKEMAN, Kevin C., 理学院（自然史科学専攻）

(主査) 教授 堀口 健雄, 教授 小亀 一弘, 准教授 柁原 宏, 助教 WAKEMAN, Kevin C., 理学院（自然史科学専攻）

(主査) 教授 堀口 健雄, 教授 小亀 一弘, 准教授 柁原 宏, 助教 WAKEMAN, Kevin C., 理学院（自然史科学専攻）

(主査) 教授 堀口 健雄, 教授 小亀 一弘, 准教授 Helena Fortunato, 助教 Kevin C. Wakeman, 理学院（自然史科学専攻）

(主査) 教授 堀口 健雄, 教授 小亀 一弘, 准教授 Helena Fortunato, 助教 Kevin C. Wakeman, 理学院（自然史科学専攻）

(主査) 准教授 柁原 宏, 教授 堀口 健雄, 教授 小亀 一弘, 助教 Kevin C. Wakeman, 理学院（自然史科学専攻）

(主査) 准教授 柁原 宏, 教授 堀口 健雄, 教授 小亀 一弘, 助教 Kevin C. Wakeman, 理学院（自然史科学専攻）