Spatial and Seasonal Variabilities of Picoeukaryote Communities in a Subtropical Eutrophic Coastal Ecosystem Based on Analysis of 18S rDNA Sequences CHEUNG, Man Kit A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Philosophy in Biology © The Chinese University of Hong Kong Sept 2007 The Chinese University of Hong Kong holds the copyright of this thesis. Any person(s) intending to use a part or whole of the materials in the thesis in a proposed publication must seek copyright release from the Dean of the Graduate School. 1 /M/ 2 Sff M jij >g}VNsLIBRARr SYSTEM Thesis/Assessment Committee Professor KWAN, Hoi Shan (Chair) Professor WONQ Chong Kim (Thesis Supervisor) Professor CHU, Ka Hou (Committee Member) Professor QIAN, Pei Yuan (External Examiner) Abstract Picoeukaryotes are eukaryotes smaller than 2-3 |im in diameter. They occur in photic zones worldwide and play fundamental roles in marine ecosystems. Lacking distinctive external features, picoeukaryotes are difficult to be identified by conventional methods such as electron microscopy. Recent studies based on cloning and sequencing of small subunits (SSU) of ribosomal RNA (rRNA) genes directly from environmental samples have revealed high diversity of picoeukaryotes and identified many novel lineages. While numerous studies have been carried out in various ecosystems and geographical regions, information on subtropical coastal waters of the western Pacific is not available. In addition, information on the relationships between picoeukaryotic diversity and environmental factors are crucial for understanding ecosystem functioning. While several studies have been initiated to work on the effects of various environmental variables such as pH and temperature, data relating marine picoeukaryotic diversity and trophic status is still lacking. In this study, the diversity of picoeukaryotes in coastal waters of the western Pacific is characterized for the first time. In addition, spatial and seasonal variations in picoeukaryotic community composition in Tolo Harbour, a semi-enclosed eutrophic bay, and Mirs Bay, an oligoinesotrophic bay strongly influenced by water circulations, were studied. Eight 18S rRNA gene clone libraries were constructed using coastal seawater samples collected at the two study sites in January, April, July and October 2006. About a hundred clones per library were assessed by restriction fragment length polymorphism (RFLP) using the restriction enzyme Hae\\\. Clones showing the same RFLP patterns were grouped into distinct operational taxonomic units (OTUs). Clone representative from each OTU was partially sequenced. I Examination of 733 picoeukaryotic clones revealed 186 different RFLP patterns, representing 186 OTUs. At least 17 higher-level taxonomic groups of picoeukaryotes were observed. Three additional higher-level groups were potentially novel. Alveolates group II, ciliates and stramenopiles comprised 37%, 17% and 11 %, respectively of the picoeukaryote assemblages and represented the most dominant groups in both Tolo Harbour and Mirs Bay. These observations highlighted the importance of parasitism in aquatic ecosystems and the presence of potentially pico-sized ciliates. Members from Dinophyceae, Prasinophyceae and Cercozoa also showed significant contributions. Spatial variations in composition and diversity of picoeukaryotes were recognized. Non-photosynthetic members were common in both study sites irrespective of trophic status. However, a decrease in the proportion of photosynthetic members was generally observed in eutrophic Tolo Harbour libraries. A hump-shaped pattern between primary productivity and diversity was suggested for marine picoeukaryotes for the first time. Seasonal variations in picoeukaryote composition were more pronounced in the oligomesotrophic Mirs Bay than in the eutrophic Tolo Harbour. Diversity of picoeukaryotes seemed to be affected by water temperature, but other biotic and/or abiotic factors may also pay a role. This study provides the first piece of information on the diversity of picoeukaryotes in the western Pacific, allowing a better understanding of the true dimensions of picoeukaryotic diversity. In addition, data on spatial and seasonal variations of marine picoeukaryotes with different degrees of eutrophication is provided here, creating basis for formulating hypotheses on ecosystem functioning. II 摘要 微微型浮游真核生物是直徑少於2 - 3微米的真核生物。他們出現在全世 界的透光層及在海洋生態系統中扮演著重要的角色。由於它們欠缺獨特的外在 表徵，使用傳統的方法（例如電子顯微鏡）很難把它們辨認出來。最近一些應 用在環境樣本上的核糖體小亞基基因克隆與序列分析硏究’展現了這些生物的 豐富多樣性，並且在當中分辨出很多新的演化系群。雖然很多相關的硏究已應 用於不同的生態系統及地理位置上，但到目前爲止’我們仍然沒有位於亞熱帶 西太平洋海岸的這些生物的資料。另一方面’知道這些生物的多樣性及環境因素 之間的關係，有助我們理解生態系統的工能運作。雖然一些硏究已著手於不同 的環境變數（例如酸驗値及水溫）對這些生物多樣性的影響，但我們仍不清楚 有關營養位階對海洋中的微微型浮游真核生物多樣性的影響。 在這個硏究’我們首次描續了西太平洋海岸微微型浮游真核生物的多樣 性。另外，我們亦分別於富營養的吐露港及貧至中度營養的大鵬灣’硏究了這 些生物的空間及季節性變化。我們在二零零六年的一月，四月，七月及十月， 分別在兩個實驗站採集了海水樣本’並建立了八個18 S核糖體基因克隆文 庫。我們在每個文庫當中抽取了約一百個殖株’並使用限制晦Haelll對它們作 出了限制晦片段長度多型性分析。我們把那些展示出相同限制晦片段長度多型 性形態的殖株歸納作同一個分類運算單位’繼而從每一個分類運算單位當中選 取了殖株代表以作部份序列分析。 我們考察了7 3 3個微微型浮游真核生物殖株，並發現了18 6個不同的 限制腺片段長度多型性形態，分別代表著18 6個不同的分類運算單位。我們 從中發現了最少17個高階分類群’而另外三個高階分類群更可能是新發現 的°第二組囊泡蟲，纖毛蟲及不等鞭毛生物分別佔微微型浮游真核生物群總數 III 的3 7 %，17 %及1 1%，是在兩個實驗站中最豐富的群組。這展示了寄生 性在水域生態系統中的重要性，亦建議了微微型纖毛蟲的存在。另外’橫裂甲 藻綱，綠色鞭毛藻綱及絲足蟲類的成員也佔著相當的比重。 另一方面，我們發現了微微型浮游真核生物成份及多樣性的空間變化。非 光合成員在兩個不同營養位階的實驗站也很普遍，而光合成員佔有的比例在富 營養的吐露港特別少。我們首次發現初級生產及海洋微微型浮游真核生物多樣 性之間可能呈現著峰形形態。相比起富營養的吐露港，這些生物成份的季節變 化在貧至中度營養的大鵬灣中比較比顯。微微型浮游真核生物的多樣性似乎受 著水溫影響，但其他生物的或非生物的因素也許亦有影響。 這個硏究首次提供了西太平洋微微型浮游真核生物多樣性的資料，令我們 對這些生物的真正多樣性有了更佳的了解。另外’這個硏究提供了海洋微微型 浮游真核生物的季節變化，以及對不同營養富度的空間變化’爲生態系統的工 能運作假設的制定建立了基礎。 IV Acknowledgements First of all, I would like to thank my supervisor Prof. C. K. Wong for his guidance throughout the two years of my M.Phil study in The Chinese University of Hong Kong. I would also like to thank Prof. K. H. Chu who taught me basic knowledge of molecular biology that allowed me to finish my M.Phil project smoothly. In addition, I would like to thank my thesis committee members for their valuable opinions that leaded to apparent improvements of my project. 1 want to thank Mr. Y. H. Yung who helped me to collect samples during field trips. I would also like to thank Mr. C. P. Li and Mr. K. C. Cheung for their helpful technical assistance provided. I want to thank all my labmates in Prof. Wong and Prof. Chu's labs who together created a great atmosphere for pursuing scientific knowledge. 1 want to thank my parents who gave born to me. Last but not least, special thanks should be given to my girlfriend who gave me plenty of supports when I got exhausted in cases of experimental failure. V Table of Contents Page Abstract (English) i Abstract (Chinese) m Acknowledgements V Table of contents VI List of figures IX List of tables XI List of Appendices XII Chapter 1. General introduction l 1.1. Picoeukaryotes 1 1.2. Conventional characterization techniques 1 1.3. Cloning and sequencing approach 3 1.3.1. Applications in prokaryotic plankton 3 1.3.2. Applications in eukaryotic picoplankton 3 1.4. Variations in diversity with environmental factors 5 1.5. Study site 6 1.6. Objectives 8 Chapter 2. Materials and methods 9 2.1. Study site 9 2.2. Sample collection 9 2.3. DNA extraction and 18S rRNA gene amplification \ \ VI 2.4. Clone library construction and screening 12 2.5. Sequencing and phylogenetic analysis 13 2.6. Statistical analyses 14 Chapter 3. Results 15 3.1. Hydrological parameters of study site 15 3.2. Clone libraries 15 3.3. Higher-level taxonomic distribution 21 3.4. Phylogenetic affiliations of OTUs 22 3.4.1 Alveolates 35 3.4.2 Stramenopiles 36 3.4.3 Rhizaria 36 3.4.4 Other lineages 37 3.4.5 Novel higher-level groups 38 3.5. Diversity estimates of picoeukaryotes 39 Chapter 4. Discussion 42 4.1. Picoeukaryotic diversity 42 4.1.1 Overall diversity 42 4.1.2 Diversity of individual taxonomic groups 44 4.1.2.1 Most represented lineages 44 4.1.2.2 Other photosynthetic lineages 52 4.1.2.3 Other non-photosynthetic lineages 55 4.1.2.4 Novel higher-level lineages 56 VII 4.2. Spatial and seasonal variations of picoeukaryotes 58 4.2.1 Spatial variations 59 4.2.1.1 Compositional variations 60 4.2.1.2 Variations in diversity 61 4.2.2 Seasonal variations 65 4.2.2.1 Compositional variations 65 4.2.2.2 Variations in diversity 66 4.3. Methodological aspects 67 4.3.1 Sample collection 67 4.3.2 PGR amplification 68 4.3.3 Cloning and RFLP screening 69 4.3.4 Statistical estimates 71 4.3.5 Future directions 71 Chapter 5. General conclusion 73 References 81 Vlll List of figures Figure Page Fig. 1. 10 Map showing the sampling locations of the current study. Fig. 2. 17 Gel photo showing 16 restriction fragment length polymorphism (RFLP) patterns from library THOl. Fig. 3. 19 Rank abundance curve for the pooled dataset of 733 picoeukaryote clones, representing 186 OTUs. Fig. 4. 20 Histogram of GenBank BLAST similarities of sequences obtained in this study. Fig. 5. 23 Relative abundance of the six most represented picoeukaryote groups in the eight clone libraries. Fig. 6. 24 Relative abundance of the photosynthetic and non-photosynthetic groups in the eight clone libraries. IX Fig. 7. 25 Maximum-likelihood (ML) phylogenetic tree of 18S rDNA sequences from all major picoeukaryotic groups observed in the current study. Fig. 8. 27 ML phylogenetic tree of alveolate 18S rDNA sequences. Fig. 9. 31 ML phylogenetic tree of stramenopile 18S rDNA sequences. Fig. 10. 33 ML phylogenetic tree of 18S rDNA sequences of rhizaria. Fig. 11. 34 ML phylogenetic tree of 18S rDNA metazoan sequences. Fig. 12. 41 Rarefaction curves for the eight clone libraries.