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Genetic diversity of selected petrosiid sponges Dissertation zur Erlangung des Doktorgrades der Fakültat für Geowissenschaften der Ludwig-Maximilians-Universität München vorgelegt von Edwin Setiawan Aus Surakarta, Indonesien München, 10. September 2014 Betreuer : Prof. Dr. Gert Wörheide Zweigutachter : PD Dr. Dirk Erpenbeck Datum der mündlichen Prüfung : 20.10.2015 Acknowledgements Acknowledgements This project was financed by the German Academic Exchange Service (DAAD) through their PhD scholarship programme, and by the Naturalis Biodiversity Center Leiden (The Netherlands) through their Martin Fellowship programme. In addition, the project received financial support from Prof Dr. Gert Wörheide of the Molecular Palaeobiology Lab of the LMU in Munich. I would like to express my sincere gratitude for their generous support. Also, I would like to thank the following colleagues from my host institute in Indonesia, the Sepuluh November Institute of Technology in Surabaya: Dian Saptarini, Maya Shovitri, Tutik Nurhidayati, Eny Zulaikha, Dewi Hidayati, Awik Pudji Diah Nurhayati, Nurlita Abdulgani and Farid Kamal Muzaki. Furthermore, I would like to thank the employees of several Indonesian education and research institutions, especially Thomas Triadi Putranto from the Geology Department Diponegoro University in Semarang, Indar Sugiarto from the Electrical Engineering Department at the Petra Christian University in Surabaya, the head and staff of Karimun Jawa National Park in Semarang, Buharianto from the Slolop Dive Centre in Pasir Putih Beach in Situbondo. Also, I would like to thank Jean- Francois Flot from the Max Planck Institute for Dynamics and Self-Organisation in Göttingen (Germany), John Hooper, and Merrick Ekins (Queensland Museum, Brisbane, Australia). I am equally thankful to everyone who has contributed to my fieldwork and helped me to complete my thesis. I apologize that I could not mention everybody in this acknowledgement. A special thank you goes to my three supervisors. First of all, I would like to thank my supervisor Dr. Dirk Erpenbeck who not only taught, educated, and broadened my ‘horizon’ on the sponge taxonomy and phylogeny, but also encouraged me to complete my thesis whenever I got depressed or demotivated. Likewise, I would like to thank Dr. Nicole de Voogd, my second supervisor, for her guidance, as well as her ‘super expert’ colleague in Naturalis, Dr. Rob van Soest for his constructive criticisms, comments and discussions. Last but not least, I would like thank my third supervisor, Prof Dr. Gert Wörheide for his criticisms, comments and encouragements, as well as for his financial support for my fieldwork and my participation in three seminars (2nd Young Reef Coral Reefs Meeting, 2012, Berlin, Germany, 2nd EU Systematic Congress, 2013 Vienna, Austria, and 9th World Sponge Conference, 2013, Fremantle WA, Australia). A big thank you also goes to the technicians in the Molecular Palaeobiology Lab (Gabriele Büttner, Simone Schätzle and Alexandra Scherer) for their contributions and help 1 E. Setiawan: Genetic diversity of selected petrosiid sponges with the laboratory work. In addition, I would like to thank all of my Molecular Palaeobiology Laboratory colleagues (especially Ratih Aryasari, Astrid Schuster, Sergio Vargas, Gaurav Shimpi, Angelo Poliseno) and other students for their criticisms, comments and discussions. Another big thank you goes to Prof Dr. Bettina Reichenbacher and her Iranian PhD students, Azad Teimori and Zeinab Gholami for their help and discussions on the morphological analyses in this thesis. I am very grateful for the support I received from my family. First of all, I would like to thank my parents for their support. Special thanks go to my wife Hermin Restyowati and my son Rafael Nicolas Setiawan who accompanied me for two years during my studies in Germany. A special mention goes to my ‘precious biology teacher’ in high school, Mrs. Emy Yuliastuti. I will never forget that she introduced me to biology, which has made my life meaningful. Finally, I would like to say many thanks to all of my Indonesian friends, especially Herri Febriyanto and family, Hardi Sukrisman and family, the Indonesian Catholic Student Communion, Arfan Bachtiar and family, Dhani Lestari and all my colleagues outside of Indonesia, especially my former classmates at the Goethe Institute Freiburg in Germany, who always cheered me up whenever I got bored or stuck during my studies there. 2 Contents Contents Summary .............................................................................................................................. 7 Author contributions General Introduction 1.1. General characteristics of sponges ............................................................................. 15 1.2. The taxonomy and phylogeny of marine haplosclerids .............................................. 18 1.3. Species concepts usually used in delimiting sponges species and their limitations ....... ........................................................................................................................................... 22 1.4. The use of chemical markers for resolving the phylogeny relationships of haplosclerid sponges .............................................................................................................................. 24 1.5. The use of molecular markers for resolving the phylogenetic relationships of haplosclerid sponges .............................................................................................................................. 26 1.6. The use of molecular biology approaches for resolving phylogeography relationships of sponges .......................................................................................................................... 28 1.7. Selected haplosclerid taxa from the Indonesian archipelago...................................... 30 1.8. The Indo-West Pacific (IWP) biodiversity and the paleogeography history of South East Asia ............................................................................................................................. 35 1.9. The aim, objective and outline of the thesis ............................................................... 37 2. Genetic diversity of the Indonesian giant barrel sponge Xestospongia testudinaria (Porifera; Haplosclerida) 2.1. Introduction ................................................................................................................ 39 2.2. Materials and methods 2.2.1. Sampling ...................................................................................................... 41 2.2.2. DNA extraction, amplification and sequencing ........................................... 42 2.2.3. Data analysis ............................................................................................... 43 2.2.4. Spicules analysis on type specimens ............................................................ 43 2.3. Results 2.3.1. Sequence variation and genealogical relationships .................................... 44 2.3.2. Genetic diversity and spatial relationships ................................................. 49 2.4. Discussion 2.4.1. Cytochrome oxidase 1 and barrel sponge species distinction ..................... 51 3 E. Setiawan: Genetic diversity of selected petrosiid sponges 2.4.2. The suitability of cytochrome oxidase 1 to resolve intra-specific relationships ........................................................................................................................................... 52 2.4.3. Implications for Xestospongia testudinaria genetic diversity and structure.... ........................................................................................................................................... 53 2.5. Conclusion .................................................................................................................. 54 3. The utility of the Adenosine Triphosphate Synthase β subunit intron for resolving phylogeographic relationships of the Indo-Pacific giant barrel sponge Xestospongia testudinaria (Porifera; Haplosclerida) 3.1. Introduction ................................................................................................................ 55 3.2. Materials and methods 3.2.1. Specimen collections .................................................................................... 58 3.2.2. DNA extraction, amplification and sequencing ........................................... 58 3.2.3. Specific primer design of ATPS-β intron for X. testudinaria ....................... 59 3.2.4. Data analyses ............................................................................................... 60 3.3. Results 3.3.1. The ATPS- β intron, heterozygosity and amplification success ................... 60 3.3.2. DNA polymorphisms, genetic structures and spatial relationships ............. 61 3.3.3. Phylogenetic analyses .................................................................................. 63 3.4. Discussion 3.4.1. The suitability of the ATPS-β intron for X. testudinaria phylogeography studies and the Pleistocene low sea level scenario................................................ 65 3.4.2. Xestospongia testudinaria species complex ................................................. 66 3.4.3. MtDNA and nucDNA discordance ............................................................... 66 3.5. Conclusion .................................................................................................................
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