Phylogeny and Population Genetics of Acorn Barnacles in Family Chthamalidae (Crustacea: Cirripedia) WU, Tsz Huen A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Philosophy in Biology The Chinese University of Hong Kong July 2011 / ‘.. 1 e SEP zotTji XJvtN JJBRAP.Y S\S\-my<^W Thesis/Assessment Committee Professor WONG, Chong Kim (Chair) Professor CHU, Ka Hou (Thesis Supervisor) Professor ANG, Put O Jr (Committee Member) Professor BARBER, Paul H (External Examinor) Declaration I declare that this thesis represents my own works and that it has not been previously included in a thesis, dissertation or degree, diploma or other qualification. ‘ . Abstract Barnacles in the family Chthamalidae are shallow water inhabitants commonly found in temperate and tropical regions worldwide. Despite their high ecological values that make them suitable model organisms in environmental science, the phylogenetic relationship within this barnacle family is poorly known. In this study, I aim to investigate the intrafamilial relationship of barnacles in the family Chthamalidae using three molecular markers. In addition, the phylogeography of one of the species from the Chthamalidae, Chthamalus mow in the Northwest Pacific region is studied. Phylogenetic trees generated from the sequences of the three markers, the mitochondrial 12S ribosomal RNA gene, nuclear histone 3 gene and nuclear elongation factor 1 alpha region showed some inconsistencies from the current classification in the family Chthamalidae. The subfamily Euraphiinae was polyphyletic with members clustered with either Chthamalinae or Notochthamalinae. The traditional classification on subfamily Notochthamalinae was valid in general, although it was of polyphyletic origin. Apart from the exclusion of a Euraphiinae barnacle, one of the members in this subfamily, Pseudooctomeris sulcata, is distantly related to the rest of the Notochthamalinae barnacles. The relationship of the subfamily Chthamalinae inferred from genetic data matched the traditional taxonomic classification to a certain degree, except the inclusion of Microeuraphia from the Euraphiinae. Apart from revealing the dissimilarity in phylogenetic relationships inferred by traditional and molecular taxonomy, this study does not support the hypothesis that there is a tendency of /eduction in the number of shell plates in the evolution of acom i barnacles. The phylogenetic tree shows that the species with different number of shell plates intermix and position randomly. It is believed that the evolution of the number of shell plates is likely to be a random event. For the study on phylogeography of C. mow, analysis of the mitochondrial cytochrome c oxidase subunit I sequences of over 240 individuals from 13 localities in Northwest Pacific region has revealed three genetically distinct lineages, namely Taiwan lineage, Ogasawara lineage and Ryukyu lineage, with sequence divergence of 4-9%. Using the molecular clock of closely related barnacles in the genus Euraphia, the time of divergence of the three lineages dated back roughly to 1.3-2.9 million years ago, suggesting they originated during Pleistocene glaciations, when lowering of sea level might have caused geographical isolation and diversification. The Pleistocene glaciations might also lead to population decline and rapid postglacial population expansion in the three lineages, as indicated by the mismatch distribution analysis. Bayesian skyline analysis shows that these island inhabiting lineages have experienced demographic expansion at -450,000-250,000 years ago, which is much earlier than the expansion time for the continental barnacle species. Despite the role of Pleistocene glaciations in causing diversifications and affecting the demographic histories in C. mow, ocean circulation patterns and geography in the region have probably shaped the contemporary distribution range of the lineages. The Kuroshio Current starts near northern Philippines and flows along the eastern coast of Taiwan is likely to maintain the genetic connectivity between populations in the Philippines and the majority of the Taiwan lineage. The Kuroshio Current that flows along the Ryukyu Island chain and mainland Japan may homogenize the genetic structure of both the Ryukyu lineage and ii Ogasawara lineage among different localities in the Ryukyu Islands and Ogasawara Islands. While the Kuroshio Current promotes gene flow between different localities, the complicated hydrography in the Kerama Gap in the Ryukyu Islands prevents complete homogenizing between the Taiwan lineage and the other two lineages, and hence maintains a rather discrete boundary between them. Seasonal eddy currents near Ogasawara Islands probably play a role in producing distinct population structure observed in Ogasawara Island, probably retaining larval supply of C. mow there, and reduce the connectivity between Ogasawara Islands and the other localities. iii 摘要 小藤壺科(Chthamalidae)的成員廣泛分佈於全世界之溫帶和熱帶地區的潮間 帶。由於它們擁有很高的生態價值，使它們經常應用於各項環境科學研究中，但是 人們對小藤壺科的親緣關係所知甚少。是次研究利用了三個分子標記去探討小藤壺 科內的親緣關係。此外，是次研究亦會探討直背小藤壺…fhamqlus moro, family Chthamalidae)於西北太平洋地區的親緣地理關係。 在是次研究中，由三個分子標記的基因序列（線粒體的12S核糖體RNA基因， 細胞核的組蛋白亞單位3基因和細胞核的延伸因子la基因）所產生的系統演化樹出 現了一些與傳統小藤壺科分類學上不一致的地方。首先，地藤壺亞科(subfamily Euraphiinae)為多源系，這亞科下的兩個藤壶品種分別歸於小藤壺亞科（subfamily Chthamalinae)或樂都小藤壶亞科（subfamily Notochthamalinae)�此外’在是次硏究 中顯示擬肋筋藤壺sulcata (subfamily Notochthamalinae)並不是樂都 小藤壺亞科的近親，所以樂都小藤壺亞科亦為多源糸。對於小藤壺亞科內的親緣關 係，除了系統演化樹所顯示的其中一種地藤壺與它有密切親緣關係外，是次基因排 序的結果與傳統的小藤壶亞科的分類大致上吻合。 是次研究除了揭示與傳統分類學相異的親緣關係之外，亦明顯不支持藤壺穀板. 數目在進化過程中有減少趨勢的假設。糸統演化樹說明，藤壶穀板數目與小藤壺科 的演化史並無明確之關係，而小藤壺科穀板數量的演化可能是一個隨機事件。 iv >! ‘ 關於直背小藤壺的親緣地理學硏究，這次硏究對來自西北太平洋的13個地 區，共超過240個藤壺樣本的線粒體細胞色素C氧化酶亞基I (COI)序列進行分 析，並發現了三個不同的基因族群，分別定名為台灣族群、小笠原族群及琉球族群， 而各族群之間的序列差異為4-9%�跟據與直背小藤壺同源的地藤壺的分子時鐘， 三個直背小藤壺族群的分化時間大致可追溯至1.3-2.9萬年前，即更新世冰期。更新 世冰期時的惡劣氣候，及因為冰冠擴張所引致的海平面下降，都可能是造成族群分 化的主因。另外，硏究中的不配對分佈分析顯示更新世冰期的惡劣氣候導致小笠原 族群和琉球族群數量大幅減少，及冰期完結後族群的迅速擴張。’ 直背小藤壺的族群結構除了受到更新世冰期的影響外，還會受到西北太平洋地 區的海洋環流模式及地理因素影響。例如，流經菲律賓北部及台灣東部沿海的黑潮 維持了菲律賓和其他台灣族群統群間的基因聯繫。另外，流經琉球列島和小笠原群 島的黑潮亦分別混和了小笠原族群及琉球族群在北菲律賓海各個地點的遺傳結 構。相反地，位於琉球群島間的慶良間海槽，因為有著複雜的水流’形成了一個屏 障，阻止了琉球列島以南的台灣族群，與其以北的兩個族群之間的基因交流，因此 形成了它們之間的分佈邊界。另外，小笠原群島附近的季節性過流可能減少小笠原 群島上的直背小藤壺族群和其他地方族群之間的連通性，從而導致小笠原群島族群 與別的族群結構上出現明顯的差異。 V Acknowledgements First and foremost I would like to express my deepest gratitude to my supervisor, Prof Ka Hou Chu, for his warm encouragements and exceptional patience in guiding me throughout this thesis and the master study. I very much appreciate the freedom he grants me in exploring the field of marine biology, and also his generosity in providing me opportunities to broaden my horizon. Being a member in Prof Chu's lab is one of the most thankful and fascinating things I have ever experienced, as it brings me the key to the awesome world of biology. For this I am indebted to him. My appreciation also goes to Dr Benny Chan from Academia Sinica, Taiwan for providing specimens for the research project, and more importantly, fundamental training in barnacle taxonomy and constructive comments on my thesis. I would like to give thanks to my thesis committee, Prof Chong Kim Wong and Prof Put O ANG, Jr. for their insightful questions and valuable advice on my research work, and Prof Paul Barber, for devoting his time serving as my external examiner. It is a pleasure to thank all the staff and colleagues in the Simon FS Li Marine Science Laboratory for providing me with technical support and lots of joy. Special thanks are given to Dr LM Tsang and Ms KY Ma, for teaching me all the lab skills and • assisting in my research work. They are not just good teachers, but also companies who are willing to share my burdens and support me always. It is really a blessing to work with such a friendly and wonderful research team. Finally, I want to extend my gratitude to my beloved family and friends. I must thank my parents for having confidence in me, in all my pursuits, and telling me that no vi matter what I want to do, they are with me and love me always. Thanks go to my brother as well, who always delights me when I am frustrated. I am never a tough girl; without the love, support and understanding from my family and the warm encouragements from friends, I can never go this far, and it would be impossible for me to finish my thesis. For these, I would like to dedicate my thesis to them. vii Content Chapter 1 General Introduction to thesis 1 Chapter 2 Literature review 3 2.1 Introduction 3 2.1.1 General introduction to barnacles 3 2.1.2 Classification of barnacles 4 2.1.3 Importance of barnacles 4 2.2 Molecular phylogenetics of barnacles 6 2.2.1 What is phylogenetics 6 2.2.2 Phylogenetic studies on barnacles 7 2.2.3 Choices on characters for barnacle phylogenetics: Morphological characters molecular characters 11 2.2.4 Choices of molecular markers in phylogenetic and population genetic studies on barnacles 14 2.3 The use of barnacles as model organism in population genetic studies 21 2.5.1 Pleistocene glaciations 22 2.5.2 Oceanographic pattern and habitat availability 23 Chapter 3 Phylogenetic relationship of barnacles in family Chthamalidae 26 3.1 Introduction 26 3.2 Materials and methods 31 3.2.1 Sample collection, DNA extraction and amplification 31 3.2.2 Phylogenetic analyses 34 3.3 Results 35 3.4 Discussion 40 3.4.1 Subfamily Notochthamalinae 40 3.4.2 Subfamily Chthamalinae 42 3.4.3 Subfamily Euraphiinae 42 3.4.4 Phylogenetic relationship in the family Chthamalidae 43 3.4.5 Suggestions on taxonomy of Chthamalidae 45 Chapter 4 Cryptic Diversity and Genetic Structure of the Acorn