Biodiversity of Marine Heterobranchia (Gastropoda) around North Sulawesi Indonesia Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch Naturwissenschaftlichen Fakultät der Rheinischen-Friedrich-Wilhelms-Universität Bonn vorgelegt von NANI INGRID JACQULINE UNDAP aus Tomohon Indonesien Bonn, Mai 2020 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn. Die Arbeit wurde am Zoologischen Forschungsmuseum Alexander Koenig in Bonn durchgeführt. 1. Gutachterin: Prof. Dr. Heike Wägele Zoologisches Forschungsmuseum Alexander Koenig 2. Gutachter: Prof. Dr. Thomas Bartolomaeus Institut für Evolutionsbiologie und Ökologie Tag der Promotion: 13.07.2020 Erscheinungsjahr: 2020 ii “Life is like riding a bicycle. To keep your balance you must keep moving” Albert Einstein Acknowledgements First of all I would like to thank my supervisor Prof. Dr. Heike Wägele for providing and supervising this work. I am very grateful for the good time in her working group and her always open door. She supported me in all ways related to my work and beyond it. I was always impressed of her leadership abilities and networking skills. Thank you so much for your time and your patience. You allowed me to grow and to increase my scientific networks during this PhD. Hopefully, I was able to adopt some by her features during my years as PhD student. Further, I want to thank Prof. Dr. Thomas Bartolomaeus for being the second referee for this thesis as well as two further referees. I thank the Federal Ministry of Education and Research (BMBF) and the German Academic Exchange Service (DAAD) for funding my PhD project. I also need to thank Dr. Astrid Gärdes and Matthias Birkicht of the Leibniz Center for Tropical Marine Research (ZMT) Bremen. I really appreciate working at their laboratory and am thankful all help they gave me. Many thanks to the members of the molecular laboratory section in the Zoological Research Museum Alexander Koenig (ZFMK), in particular to Claudia Etzbauer and Sandra Kukowka for the intensive help in the laboratory. I sincerely thank Prof. Grevo Gerung, Prof. Farnis Boneka, Prof Markus Lasut of the Faculty of Fisheries and Marine Science, Sam Ratulangi University, Manado for iii their support and help in the ABS paper work to follow the Nagoya Protocol during my field trips. I would also thank Dr. Gregor Christa who offered advice and suggestion whenever asked. Thanks to AG-Mollusca members: Dr. André Koch, Fatemeh Maniei, Dario Karmeinski, Dorothea Schillo, Adelfia Papu, Corinna Sickinger, Nilz Arenz, Antonia Schönleber for charming discussion and for all the fun we have had in the last four years. Many of the above and some who are not named here, but all went with me through that times, are still connected to me in friendship, even if sometimes separated by distance and time, and I thank all these people that they have been at my side. I am so grateful to all the friends and friendly people who gave me support. I want to thank for my parents, my brothers and sisters (Yani, Ully, Meiny and Nathan) for your pray, support throughout writing this thesis, for believing in me and for being there for me. I dedicate this thesis to my lovely husband, Heffry, and my sons, Derrelyo and Hiroyuki. Thanks for giving me patience, strength, motivating, and being my inspiration. iv Declaration Declaration I hereby declare that I am the sole author of this thesis and that no other sources or learning aids, other than the ones listed, have been used. Furthermore, I declare that I have acknowledged the work of others by providing detailed references of said work. Hereby I also declare, that this thesis has not been prepared for another examination or assignment, neither wholly nor partially. Erklärung Hiermit versichere ich, dass ich diese Arbeit selbständig verfasst habe, keine anderen als die angegebenen Quellen/Hilfsmittel verwendet habe und alle Stellen, die wörtlich oder sinngemäß aus veröffentlichten Schriften entnommen wurden, als solche kenntlich gemacht habe. Ich versichere außerdem, dass ich die beigefügte Dissertation nur in diesem und keinem anderen Promotionsverfahren eingereicht habe und, dass diesem Promotionsverfahren keine entgültig gescheiterten Promotionsverfahren vorausgegangen sind Bonn, _____________________ _________________ v Summary North Sulawesi province is one of the most potential areas in terms of natural marine resources. In North Sulawesi there are many interesting tourist places with high marine biodiversity and many objects are currently under development, including around Bunaken National Marine Park (BNP), Lembeh Strait, in the island of Sangihe, and Bangka Archipelago. Coastal areas of North Sulawesi are considered as the most diverse habitats in the world with high species diversity in coral reefs, but also in adjacent sea grass beds and mangroves. The high diversity also includes marine heterobranch sea slugs, which are known by an extremely large number of species of up to probably 2000, with many undescribed ones. My study in Sangihe Island is the first survey of marine heterobranch around this island and included in the collection 23 species, with Phyllidiidae showing the highest dominance (Chapter 3). The amount of species is far lower than in the studies around BNP, or the Bangka Archipelago, probably due to unfavorite weather conditions. The Chromodorididae is a large and colourful family of nudibranch sea slugs distributed across the tropical and temperate world’s oceans. These sea slugs are known by many divers because of their beauty and by many pharmacists because of their high diversity in natural compounds. For studying the Chromodorididae, 375 specimens were collected around North Sulawesi in 2015, 2016 and 2017. Chapter 4 and chapter 5 focus on this family. The phylogenetic hypothesis based on two mitochondrial genes, CO1 and 16S, is the only subsequent study after the first study published in 2012 by Johnson & Gosliner. The major result of my analyses is the confirmation of the results obtained by Johnson & Gosliner 2012 at that time. Chromodoris is a genus of colourful nudibranchs that feed on sponges and is found across the Indo-Pacific. Chapter 5 in this study focuses on four species of this genus (i.e. C. annae, C. dianae, C. willani, C. lochi), of which hundreds of specimens were vi collected. The results about Chromodoris species clearly show wide spread cryptic speciation. Biodiversity is related to water quality, which was also addressed in this thesis. The results with regard to water quality indicate that all sampling points are within the range of normal values and no specific pollution can be seen (Chapter 6). vii Table of Contents Summary ..................................................................................................... vi Chapter 1 - General Introduction .............................................................. 1 North Sulawesi, Indonesia .......................................................................................... 1 Heterobranch sea slugs ............................................................................................... 2 Indonesian marine heterobranch................................................................................. 3 Aims of the present study ........................................................................................... 4 Chapter 2 - Materials and Methods .......................................................... 6 Collection of specimens ............................................................................................. 6 DNA extraction and amplification ............................................................................. 6 Sequence analysis and alignment ............................................................................... 7 Chapter 3 - First Survey of Heterobranch Sea Slugs (Mollusca, Gastropoda) from the Island Sangihe, North Sulawesi, Indonesia ........ 8 Abstract: ..................................................................................................................... 9 Introduction .............................................................................................................. 10 Materials and Methods ............................................................................................. 12 Results ...................................................................................................................... 16 Systematics ............................................................................................................... 32 Chapter 4 - Biodiversity of Chromodorididae (Anthobranchia, Nudibranchia) around North Sulawesi ................................................... 57 Introduction .............................................................................................................. 57 Materials and Methods ............................................................................................. 59 Results ...................................................................................................................... 62 Discussion ................................................................................................................ 78 Chapter 5 - Chromodoris of North Sulawesi ........................................... 82 viii Introduction .............................................................................................................. 82 Materials and methods .............................................................................................