Epiphytic-benthic dinoflagellates from Korean sandy beaches: morphology, phylogeny, and growth and fatty acid contents under different temperature and salinity conditions

다국어 초록 (Multilingual Abstract)

Marine epiphytic-benthic dinoflagellates were isolated from intertidal sandy beaches on the southern coast of Korea, and cultures were established. Morphological observations and molecular phylogenetic analyses based on small subunit （SSU） and large subunit （LSU） rRNA gene sequences were conducted to identify the species and clarify their taxonomic positions. A total of eight species belonging to seven genera were identified, including Amphidinium, Coolia, Paramphidinium, Pseudadenoides, Spiniferodinium, Thecadinium, and Togula. Among them, Pseudadenoides kofoidii was recognized as an unrecorded species in Korea. The number of large pores on the posterior intercalary plates, previously reported as a diagnostic feature of this species, was either two or three among the strains isolated in this study. In addition, Spiniferodinium imaii sp. nov. was separated from related species by distinctive morphological characteristics and formed an independent lineage in both SSU and LSU phylogenies, supporting its recognition as a new species. Two strains showing no mortality in bioassays, Pseudadenoides kofoidii and Togula britannica, were selected for growth experiments and fatty acid analysis. The two strains showed different growth responses to combined temperature and salinity conditions. In P. kofoidii, the highest cell density was observed at 15 and 20℃ with a salinity of 35, whereas the maximum growth rate was recorded at 20℃ and a salinity of 25. In T. britannica, the highest cell density was observed at 15℃ and a salinity of 30, and the maximum growth rate was recorded at 20℃ and a salinity of 30. T. britannica also maintained relatively high growth rates at salinities of 30 to 35 across all temperature conditions tested. Fatty acid composition varied among the temperature and salinity treatments examined, yet both strains maintained relatively stable proportions of major fatty acids across varying temperature and salinity conditions. In both strains, palmitic acid and docosahexaenoic acid （DHA） were identified as the dominant fatty acids. DHA proportions ranged from 22.1 to 26.7% in P. kofoidii across all treatments, while in T. britannica, DHA proportions tended to increase at lower temperatures, reaching up to 48.9% at 10℃ and a salinity of 15. The DHA proportions observed in both strains were substantially higher than those reported for benthic diatoms such as Navicula and Nitzschia, which are commonly used as feed for aquaculture organisms. As epiphytic-benthic dinoflagellates living attached to substrates, and given their high DHA proportions, these strains may have potential as novel live feed for aquaculture organisms that graze on epiphytic-benthic microalgae, although further studies are required to evaluate mass cultivation and ingestion by target organisms.

국문 초록 (Abstract)

한국 남해안 조간대 사질 해안에서 해양 착생성 저서성 와편모조류를 분리하여 배양하였다. 확보한 종의 동정과 분류학적 위치를 파악하기 위해 형태학적 관찰과 소단위（SSU）와 대단위（LSU） rRNA 유전자 염기서열을 이용한 분자계통학적 분석을 수행하였다. 그 결과, Amphidinium, Coolia, Paramphidinium, Pseudadenoides, Spiniferodinium, Thecadinium 및 Togula의 총 7속 8종을 확인하였다. 이 중 Pseudadenoides kofoidii는 국내 미기록종으로 확인되었다. 이 종의 분류 형질로 보고되어 온 후방 삽입판（posterior intercalary plates）의 라지 포어 수가 본 연구에서 분리된 균주에서 2개 또는 3개로 나타났다. 또한, Spiniferodinium imaii sp. nov.는 근연종과 뚜렷한 형태학적 특징으로 구별되었으며, 소단위 및 대단위 계통수 모두에서 독립적인 분기군을 형성하여 신종으로 제안하였다. 생물독성평가에서 사멸이 관찰되지 않은 Pseudadenoides kofoidii와 Togula britannica를 대상으로 성장 실험과 지방산 분석을 수행하였다. 두 균주는 온도와 염분의 복합 조건에 따라 서로 다른 성장 반응을 보였다. P. kofoidii는 온도 15 및 20℃, 염분 35 조건에서 최대 세포밀도가 관찰되었으며, 최대 성장속도는 온도 20℃, 염분 25 조건에서 확인되었다. T. britannica는 온도 15℃, 염분 30 조건에서 최대 세포밀도가 관찰되었으며, 최대 성장속도는 온도 20℃, 염분 30 조건에서 확인되었다. 또한, T. britannica는 모든 온도 조건에서 염분 30~35 범위에 걸쳐 비교적 높은 성장속도를 유지하였다. 지방산 조성은 온도와 염분 처리구에 따라 변화를 보였으나, 두 균주 모두 다양한 온도-염분 조건에서 주요 지방산의 비율을 비교적 안정적으로 유지하였다. 두 균주 모두에서 팔미트산과 도코사헥사엔산 （docosahexaenoic acid, DHA）이 주요 지방산으로 확인되었다. P. kofoidii의 DHA 비율은 모든 처리구에서 22.1~26.7% 범위로 나타났으며, T. britannica는 저온 조건에서 DHA 비율이 높아지는 경향을 보여 온도 10℃, 염분 15 조건에서 최대 48.9%에 도달하였다. 두 균주의 DHA 비율은 양식생물의 먹이생물로 널리 이용되는 저서성 규조류인 Navicula와 Nitzschia에서 보고된 값보다 뚜렷하게 높았다. 두 균주는 기질에 부착하여 생활하는 착생성 저서성 와편모조류로, 높은 DHA 비율을 고려할 때 착생성 미세조류를 섭식하는 양식생물을 위한 새로운 먹이생물로서의 가능성이 있으나, 대량 배양 및 대상 생물의 섭식 여부에 대한 추가 연구가 필요하다.

목차 (Table of Contents)

• General introduction 1
• Chapter Ⅰ. Morphology and molecular phylogeny of epiphytic-benthic dinoflagellates from Korean sandy beaches 6
• 1. Introduction 7
• 2. Materials and Methods 10
• 2.1. Sampling and cultures 10
• 2.2. Light microscopy (LM) 13
• 2.3. Scanning electron microscopy (SEM) 13
• 2.4. DNA extraction and sequencing 14
• 2.5. Sequence alignment and phylogenetic analyses 16
• 3. Results 19
• 3.1. Morphology of epiphytic-benthic dinoflagellates 19
• 3.1.1. Amphidinium carterae Hulburt 19
• 3.1.2. Coolia malayensis Leaw, P.-T.Lim & Usups 27
• 3.1.3. Paramphidiniuim sp. 31
• 3.1.4. Pseudadenoides kofoidii (Herdman) F. Gómez, R. Onuma, Artigas & Horiguchi 34
• 3.1.5. Thecadinium kofoidii (Herdman) J.Schiller 39
• 3.1.6. Togula britannica (E.C.Herdman) Flø Jørgensen, Sh.Murray & Daugbjerg 43
• 3.1.7. Togula jolla Flø Jørgensen, Sh.Murray & Daugbjerg 47
• 3.1.8. Spiniferodinium imaii sp. nov. D.B. Choi & H.H. Shin 51
• 3.2. Molecular phylogeny of epiphytic-benthic dinoflagellates 57
• 3.2.1. Phylogenetic position of Amphidinium carterae 57
• 3.2.2. Phylogenetic position of Coolia malayensis 61
• 3.2.3. Phylogenetic position of Paramphidinium sp. 65
• 3.2.4. Phylogenetic position of Pseudadenoides kofoidii 69
• 3.2.5. Phylogenetic position of Thecadinium kofoidii 73
• 3.2.6. Phylogenetic position of Togula species 77
• 3.2.7. Phylogenetic position of Spiniferodinium imaii sp. nov 80
• 4. Discussion 84
• 4.1. Morphological characteristics and comparison of Korean epiphytic-benthic dinoflagellates with previously described species 84
• 4.1.1. Amphidinium carterae isolated from sandy sediments of the Haumokdong Port, Jeju Island 84
• 4.1.2. Coolia malayensis isolated from sandy sediments of the Biyangdo Port, Jeju Island 86
• 4.1.3. Paramphidinium sp. isolated from sandy sediments of Geoje Island 88
• 4.1.4. Pseudadenoides kofoidii isolated from sandy sediments of Geoje Island 89
• 4.1.5. Thecadinium kofoidii isolated from sandy sediments of Geoje Island 91
• 4.1.6. Togula species isolated from sandy sediments of Geoje Island 93
• 4.1.7. Spiniferodinium imaii sp. nov. isolated from sandy sediments of Geoje Island 97
• 4.2. Phylogenetic characterization of Korean isolated of epiphytic-benthic dinoflagellates 101
• 4.2.1. Molecular phylogeny of Amphidinium carterae 101
• 4.2.2. Molecular phylogeny of Coolia malayensis 103
• 4.2.3. Molecular phylogeny of Paramphidinium sp 105
• 4.2.4. Molecular phylogeny of Pseudadenoides kofoidii 106
• 4.2.5. Molecular phylogeny of Thecadinium kofoidii 107
• 4.2.6. Molecular phylogeny of Togula species 108
• 4.2.7. Molecular phylogeny of Spiniferodinium imaii sp. nov 110
• Chapter Ⅱ. Growth responses and fatty acid contents of the non-toxic epiphytic-benthic dinoflagellates Pseudadenoides kofoidii and Togula britannica exposed to different temperature and salinity conditions 113
• 1. Introduction 114
• 2. Materials and Methods 116
• 2.1. Strains and culture conditions 116
• 2.2. Toxicity assessment 116
• 2.3. Growth experiments 117
• 2.4. Fatty acid analysis 119
• 2.5. Statistical analysis and data visualization 122
• 3. Results 123
• 3.1. Growth responses of Pseudadenoides kofoidii exposed to different temperature and salinity 123
• 3.2. Growth response of Togula britannica exposed to different temperature and salinity 126
• 3.3. Fatty acid contents of Pseudadenoides kofoidii cultivated under optimal and suboptimal temperature and salinity conditions 135
• 3.4. Fatty acid contents of Togula britannica cultivated under optimal and suboptimal temperature and salinity conditions 136
• 4. Discussion 143
• 4.1. Effects of temperature and salinity on growth of Pseudadenoides kofoidii and Togula britannica 143
• 4.2. Effects of temperature and salinity on fatty acid contents of Pseudadenoides kofoidii and Togula britannica 146
• 4.3. Aquaculture potential of the PUFA-rich dinoflagellates, Pseudadenoides kofoidii and Togula britannca 150
• General conclusion 153
• Reference 155
• Acknowledgement 166