Title Page

Contents

Abstract 13

요약 14

I. Systematic study on Korean and Antarctic seven hypotrich ciliates (Protozoa: Ciliophora) 15

Introduction 15

Materials and methods 18

Sample site and morphological identification 18

SSU rRNA gene sequence 18

Phylogeny analysis 19

I.1. Metaurostylopsis antarctica n. sp. (Figs 2–8; Tables 2, 3) 22

I.1.A. Results 22

I.1.B. Discussion 26

I.2. Pseudourostyla cristatoides n. sp. (Figs 9–16; Tables 4–6) 39

I.2.A. Results 39

I.2.B. Discussion 44

I.3. Cyrtohymena koreana n. sp. (Figs 17–20; Tables 7, 8) 58

I.3.A. Results 58

I.3.B. Discussion 62

I.4. Cyrtohymena lacuna n. sp. (Figs 21, 22; Tables 7, 8) 72

I.4.A. Results 72

I.4.B. Discussion 74

I.5. Apokeronopsis bergeri Li et al., 2008 (Figs 23, 24; Tables 9, 10) 77

I.5.A. Results 77

I.5.B. Discussion 78

I.6. Australocirrus oscitans Blatterer & Foissner, 1988 (Figs 25–27; Table 11) 84

I.6.A. Results 84

I.6.B. Discussion 84

I.7. Neokeronopsis asiatica Foissner et al., 2010 (Figs 28–35; Table 12) 90

I.7.A. Results 90

I.7.B. Discussion 91

II. An approach for analyzing community structure of ciliate using pyrosequencing data 105

Introduction 105

II.1. Development of ciliate-specific marker (Figs 36–41; Tables 13–18) 108

II.1.A. Materials and methods 108

II.1.B. Results 115

II.1.C. Discussion 119

II.2. Ciliate community in littoral zone of the Yellow Sea (Figs 42–46; Tables 19, 20) 138

II.2.A. Materials and methods 138

II.2.B. Results 141

II.2.C. Discussion 145

III. References 162

Table 1. Ciliate list and data investigated in this study. 21

Table 2. Morphometric data on Metaurostylopsis antarctica n. sp. 30

Table 3. Comparison of seven species of Metaurostylopsis. The data was cited and modified from. 31

Table 4. Morphometric data on the protargol-impregnated specimens of Pseudourostyla cristatoides n. sp. 47

Table 5. Morphometric comparison of P. cristatoides n. sp. with well-known Pseudourostyla species. 48

Table 6. Pairwise similarity (%) based on the Kimura (1980) two-parameter distance option of the 18S rRNA gene sequence between Pseudourostyla cristatoides n. sp. and the congeneric species with Hemicycliostyla franzi. 49

Table 7. Morphometric data on the protargol-impregnated specimens of Cyrtohymena koreana n. sp. (ck), C. lacuna n. sp. (cl), C. muscorum (cm). 65

Table 8. Comparison of morphological features in Cyrtohymena koreana n. sp. with closely related species including Rubrioxytricha. 67

Table 9. Morphometric characterization of Apokeronopsis bergeri. 80

Table 10. Pairwise distance of SSU rRNA gene sequences among Apokeronopsis congeners were calculated using the Kimura two-parameter distance. 81

Table 11. Morphometric data on the protargol-impregnated specimens of Australocirrus oscitans. 86

Table 12. Morphometric data on the protargol-impregnated specimens of Neokeronopsis asiatica. 96

Table 13. Primers used for library construction. 124

Table 14. Conservation of CiliV4, a ciliate-specific SNP site found in each ciliate class. All of the complete or nearly complete SSU rDNA sequences in the nominated ciliate sequences were retrieved from the SILVA-ARB database (version 98) and analyzed to determine the... 125

Table 15. Summary of the sequences and OTUs identified using clone libraries and pyrosequencing with CiliV4-based ciliate-specific or eukaryote universal primers. 126

Table 16. Alignment sequences used to find the group-specific SNP sites. Sequences from diverse groups of eukaryotes were retrieved from the NCBI database and aligned using ClustalX.... 127

Table 17. Positions of SNP sites conserved in ciliates. Each SNP position was manually identified based on alignment with diverse eukaryote sequences retrieved from the NCBI. SNPs used for ciliate-specific primer development are shown in the grey boxes. 129

Table 18. List of ciliate species identified in clone libraries (Fig. 38) and pyrosequence data sets (Fig. 40). Species unique in the clone library or universal pyrosequence data sets are shown in bold. 130

Table 19. Environmental features and the pyrosequencing process. 155

Table 20. Species with high similarity (≥ 99%) to the SILVA SSU rRNA database release 106 using USEARCH software. 156

Figure 1. Sampling sites previously investigated in South Korea. The sites are based on taxonomic studies of free-living ciliates from 1938 to 2012. Uncertain site and thesis/dissertation are excluded. 17

Figure 2. Morphology of Metaurostylopsis antarctica n. sp., from life (A–E) and after protargol impregnation (F, G). A – ventral view of a representative individual, arrow indicates contractile vacuole; B–E – ventral (B, D) and dorsal (C, E) views, showing the... 32

Figure 3. Morphogenesis of Metaurostylopsis antarctica n. sp. at early to late stages after protargol impregnation. A, B – ventral view of early dividers, arrowheads indicate the oral primordium and the arrow marks the proter's oral primordium; C – ventral view of a slightly... 33

Figure 4. Ventral and dorsal view of a late divider of Metaurostylopsis antarctica n. sp. after protargol impregnation. Arrows mark the new frontoterminal cirri. Scale bar: 50 μm. 34

Figure 5. Photomicrographs of Metaurostylopsis antarctica n. sp. from life. A, B, D, E – dorsal views, arrow in (D) marks contractile vacuole; C – left side view; F, G, I, J – cortical granules and transverse cirri on dorsal (F, J) and ventral (G, I) views, arrows denote large... 35

Figure 6. Photomicrographs of Metaurostylopsis antarctica n. sp. during interphase (A–E) and morphogenesis after protargol impregnation (F–J). A–C – ventral views of infraciliature of the anterior body portion, arrow in (B) denotes the buccal cirrus, arrows in (C) mark the frontoterminal cirri; D –... 36

Figure 7. Photomicrographs of dividing cells of Metaurostylopsis antarctica n. sp. after protargol impregnation. A, B – middle divider, marginal anlagen replace parental rows; C–E – ventral and dorsal view of a late divider, arrow in (C) denote as two basal bodies ahead of... 37

Figure 8. Phylogeny of Metaurostylopsis antarctica n. sp. inferred from maximum likelihood (ML) and Bayesian inference (BI) based on SSU rRNA gene sequences. PhyML v. 3.0 and MrBayes v. 3.1.2 were used for ML and BI analyses, respectively. TIM2 + G was selected as a best fit model from jModelTest.... 38

Figure 9. Morphology of Pseudourostyla cristatoides n. sp., from life (1, 2) and after protargol impregnation (3–5). 1. Ventral view of a representative individual, arrows indicate contractile vacuoles. 2. Ventral views of the process of forming contractile vacuoles. 3–5. Ventral (3) and dorsal views (4, 5) of the holotype specimen, arrow denotes the anteriormost midventral pair.... 50

Figure 10. Pseudourostyla cristatoides n. sp., dividers after protargol impregnation. 6, 7. Ventral view of early dividers, the buccal cirrus (arrowheads) is dedifferentiated, arrows mark basal body patches left of some postoral midventral cirri, and proter’s oral primordium... 51

Figure 11. Pseudourostyla cristatoides n. sp., dividers after protargol impregnation. 15–18. Ventral and dorsal views of late dividers, the micronuclei are finished division and arrows denote anteriorly migrating frontoterminal cirri. Mi, micronuclei. Scale bars: 100 μm. 52

Figure 12. Pseudourostyla cristatoides n. sp., reorganizers after protargol impregnation. 19–21. Ventral and dorsal views of middle reorganizers, the marginal rows originate by common anlage (left, arrow; right, arrowhead), the dorsal kinety anlagen form intrakinetally, and asterisk marks posteriorly migrating buccal cirrus. 22, 23.... 53

Figure 13. Photomicrographs of Pseudourostyla cristatoides n. sp. from life (bright field illumination, 24–29) and methyl green-pyronin impregnation (30, 31). 24–26. Dorsal views, arrows mark contractile vacuoles.... 54

Figure 14. Photomicrographs of Pseudourostyla cristatoides n. sp. during interphase (32–34, holotype specimen) and morphogenesis (35–42) after protargol impregnation. 32, 34. Ventral views, with arrow and arrowhead indicating macronucleus and micronucleus,... 55

Figure 15. Reorganization in Pseudourostyla cristatoides n. sp. after protargol impregnation. 43, 44. Ventral (43) and dorsal (44) view of middle reorganizer showing left marginal row anlagen (arrow in 43), dorsal kineties anlagen (arrow in 44), extrusomes (arrowhead in 44).... 56

Figure 16. Phylogenetic tree of 18S rRNA gene sequences showing the position of Pseudourostyla cristatoides n. sp. based on maximum likelihood (ML) and Bayesian inference (BI).... 57

Figure 17. Cyrtohymena koreana n. sp. from life (A) and after protargol impregnation (B, C).––A. Ventral view of a representative specimen, arrow denotes contractile vacuole. ––B, C. Ventral (B) and dorsal (C) view of holotype specimen.... 68

Figure 18. Cyrtohymena koreana n. sp. from life (A–E) and after protargol impregnation (F–K).––A, B, D, E. Dorsal views of representative specimens. ––B, C. Ventral (B) and dorsal (C) view. ––C.... 69

Figure 19. Cyrtohymena koreana n. sp., dividers after protargol impregnation.––A–F. Ventral (A–E) and dorsal (F) view of early dividers. Arrows denote basal body patches and the buccal cirrus (arrowhead) is dedifferentiated. ––G, H.... 70

Figure 20. Cyrtohymena koreana n. sp., dividers after protargol impregnation.––A–D. Ventral (A, C) and dorsal (B, D) views of late dividers. Ma, macronucleus nodules; Mi, micronuclei.... 71

Figure 21. Cyrtohymena lacuna n. sp. from life (A) and after protargol impregnation (B, C).––A. Ventral view of a representative specimen, arrow denotes contractile vacuole. ––B, C. Ventral (B) and dorsal (C) view of holotype specimen.... 75

Figure 22 Cyrtohymena lacuna n. sp. from life (A–E) and after protargol impregnation (F–K).––A, D. Ventral views of representative specimens. ––B, C, E. Dorsal views showing cortical granules and dorsal bristles.... 76

Figure 23. Morphology and infraciliature of Apokeronopsis bergeri from live (A-D) and after protargol impregnation (E, F). A-F, Apokeronopsis bergeri: A, Ventral view of live, arrowindicates the contractile vacuole; B, Two types of densely distributed granules, arrow... 82

Figure 24. Morphology and infraciliature of Apokeronopsis bergeri from live (A-D) and after protargol impregnation (E-G). Dorsal (A, B) and ventral (C, D) views of live, (B) arrow indicates the contractile vacuole; C, Body shape slightly contractile and flexible; D,... 83

Figure 25. Australocirrus oscitans from life (A) and after protargol impregnation (B, C).––A. Ventral view of a representative specimen. ––B, C. Ventral (B) and dorsal (C) view of representative specimen.... 87

Figure 26. Australocirrus oscitans from life (A–D) and after protargol impregnation (E–G). ––A–C. Ventral views of representative specimens, arrow denotes contractile vacuole. ––D. Dorsal view showing dorsal bristles.... 88

Figure 27. Phylogenetic tree of 18S rRNA gene sequences showing the position of Australocirrus oscitans based on maximum likelihood (ML) and Bayesian inference (BI). Both bootstrap values for ML and posterior probability values for BI are represented on... 89

Figure 28. Neokeronopsis asiatica from life (A–F, H) and after protargol impregnation (G).––A. Ventral view of a representative specimen. ––B, C. Dorsal (B) and ventral (C) view showing cortical granules.... 97

Figure 29. Neokeronopsis asiatica, infraciliature after protargol impregnation.––A, B. Ventral (A) and dorsal (B) view of a representative specimen. Asterisk in (B) denotes anterior fragmentation of dorsal kinety 1.... 98

Figure 30. Neokeronopsis asiatica, dorsal kineties after protargol impregnation. Asterisks denote fragmentation in dorsal kinety 1. More than half of cells examined (10/18) were fragmented at posterior position. Scale bars 200 μm. 99

Figure 31. Neokeronopsis asiatica, divider after protargol impregnation.––A, B. Ventral (A) and dorsal (B) view showing a middle divider. Parental midventral cirri between the cirral anlagen of proter and opisthe are resorbed.... 100

Figure 32. Neokeronopsis asiatica from life.––A, B. Dorsal views showing contractile vacuole (CV) and entire body shape. ––C. Ventral view of anterior body. Buccal horn (BH) is recognized at between the distal end of paroral membrane (PM) and adoral zone of... 101

Figure 33. Neokeronopsis asiatica, protargol-impregnated specimens of interphasic specimen (A–D), mid-divider (E, F), and late reorganizer (G, H).––A–D. Ventral (A, D) and dorsal (B, C) views of a representative specimen showing infraciliature.... 102

Figure 34. Phylogenetic tree of 18S rRNA gene sequences showing the position of Cyrtohymena koreana n. sp., C. lacuna n. sp., Neokeronopsis asiatica based on neighbor joining (NJ, first number) maximum likelihood (ML, second number) and Bayesian... 103

Figure 35. A cladistic (Hennigian) argumentation scheme for species in Cyrtohymena undulating membranes pattern, based on the morphology, ontogeny, molecular. This cladistics is mainly based on the 18S rRNA gene tree.... 104

Figure 36. Group-specific SNPs located in the conserved area between the V4 and V5 variable regions of the SSU rRNA gene were identified based on a multiple alignment retrieved from the NCBI database. Colored narrow boxes indicate the specific SNPs of the ciliates (adenine at the 968th position in T. canadensis), dinoflagellates (guanine and adenine at the 943rd and 1,018th positions, respectively), diatoms (adenine at...(이미지참조) 132

Figure 37. SPAT strategy. 133

Figure 38. A neighbor-joining phylogenetic tree constructed based on the V2–V5 region of the SSU rRNA gene including sequences from the NCBI (black), and representative OTUs from ciliate-specific (red) and universal (blue) clone libraries.... 134

Figure 39. Relative histogram of planktonic and non-planktonic ciliates in the clone (Fig. 38) and pyrosequencing libraries (Fig. 40). Life forms of the ciliates were identified based on the criteria established by Lynn (2008). 135

Figure 40. Multiple-comparative tree created with the MEGAN based on BLASTN taxonomy. Ciliate-specific and eukaryote universal reads determined by pyrosequencing are shown in red and blue, respectively. 136

Figure 41. Rarefaction curve of pyrosequencing reads using mothur software (3% cutoff). Ciliate-specific library showed slightly saturated curve than universal one. 137

Figure 42. Schematic illustration of sampling sites in the littoral zone. 157

Figure 43. Distribution pattern of operational taxonomic units among sampling sites. 158

Figure 44. Rarefaction curves and similarities between pyrosequencing reads and the SILVA database. 159

Figure 45. Jarccard tree produced using mothur software. Based on the OTUs of the unique reads estimated based on the 3% cutoff pairwise distance, their relationship among the sampling sites was represented as a Jaccard tree.... 160

Figure 46. USEARCH result visualized using MEGAN software. (A–C) The MEGAN trees were based on the identical USEARCH result and expanded. 161

남극의 킹조지섬과 한국으로부터 7종의 저서 섬모충류가 채집되었고 4종의 신종이 포함되어 있으며 이는 다음과 같다: 남극 킹조지섬–Metaurostylopsis antarctica n. sp., Neokeronopsis asiatica Foissner et al., 2010; 한국–Pseudourostyla cristatoides n. sp., Cyrtohymena koreana n. sp., Cyrtohymena lacuna n. sp., Apokeronopsis bergeri Li et al., 2008, 그리고 Australocirrus oscitans Blatterer & Foissner, 1988. 이들의 형태와 분자계통이 연구되었으며, C. koreana, M. antarctica, 그리고 P. cristatoides의 형태형성과정 또한 기재하였다. 또한 위와 같은 전통적인 형태분류학과 더불어 pyrosequencing 기술을 사용하여 표준화된 섬모충류 모니터링 기술 개발을 위해 섬모충류 특이 프라이머를 제작하였으며 4개의 황해연안 환경시료에 적용하였다. 그 결과, 섬모충류 특이 프라이머는 80.9–99.2% 특이성을 보여주었으며, 3% 기준을 적용하였을 때 171–569개의 분자작동단위(OTU)를 나타냈다. 이들 분자작동단위는 singleton에 의해 영향을 많이 받았으며 대략 두 배의 분자작동단위를 갖게 만들었지만 beta diversity에는 영향을 끼치지 않았다. 이번 결과를 통해 연안의 섬모충류 분포 패턴을 규명하기에는 시료의 수가 부족하였지만 이들 결과를 통해 다음과 같은 결론을 도출할 수 있었다. 이번 연구에서 제작된 섬모충류 프라이머는 다양한 환경시료에 적용 가능할 것으로 보이며 높은 섬모충류 특이성을 나타낼 것으로 예상된다. 또한, 이를 통해 신뢰할 만한 데이터를 얻기 위해서는 singleton이 제거되어야 한다.