BIOLOGICAL ASSESSMENT AND MONITORING OF SINGAPORE AQUATIC ENVIRONMENTS USING NGS TOOLS BILGENUR BALOGLU B. Sc. (Hons.), Istanbul Technical University A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2017 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. ___________________ Bilgenur Baloglu 24 AUGUST 2017 i ACKNOWLEDGEMENTS I would like to thank my Ph.D. advisor Prof. Rudolf Meier for supporting me during these past few years, for his motivation, and immense knowledge. You were instrumental in helping me crank out this thesis, all in one month. Thank you for your brilliant comments on several of my drafts. You really helped me get things into perspective. Your mentorship these past four years has been invaluable. I also thank the members of my thesis advisory committee, Asst. Prof. Darren Yeo and Asst. Prof. Frank Rheindt for their very helpful feedback and encouragement. Your questions helped widen my research from various perspectives. My sincere thanks also go to Asst. Prof. Roman Carrasco, for teaching the most useful lecture I took in NUS and for the stimulating discussions regarding R and the related analysis. You are the reason why I love R so much! Thanks also to Prof. Peter Cranston for sharing his boundless chironomid wisdom with me. Thank you NUS for funding my studies through SINGA scholarship, PUB for funding the chironomid project, members of TMSI for collecting all my chironomid samples, providing the environmental data, and for teaching me 100 ways to be patient. Amrita Srivathsan, thank you for answering my bioinformatics related questions, your amazing time-saving scripts, assistance in editing of my manuscript, and the comforting conversations we had whenever I needed. You have been a very supportive colleague and a friend. Gowri Rajaratnam for checking up on me all those times and trying to make me feel better about PhD, physically and mentally. You’ve always been a supportive and caring friend, and your presence helped make the lab bearable. ii Wing Hing Wong for your immense support ever since I first started in the lab and for all the hugs. You have been a big part of EvoLab. Yuchen Ang for teaching me Photoshop and how to do the morphological sorting. Darren for bearing with my random questions and for being such a great friend. Your absence in the student office and the lab is really felt. Maosheng for helping me collect my chironomids and letting me access to the museum collections at all the times I asked for. Jonathan for all of our awesome fish and midge related conversations and your suggestions on the data analysis. Honor, Wan Ting, Jake, Molly, you were the best midge team I could ever ask for! Thank you for all the PCRs, agarose gels, countless plates and making the lab a lot more fun. Theo, thank you so much for making the primer plates, all the lunch invitations and the takeaway food you brought me many times. I survived and felt cared thanks to you. Other members of EvoLab: Ywee Chieh for helping me get used to the lab, nice conversations and many lunches, and Sujatha for helping me edit my manuscript and your caring questions whenever we came across. Diego, Mindy, Kathy, Jinfa, Wan, you all made the lab such a great place. A number of friends, my former flatmates Maedeh and Elham, my first two years in Singapore were so amazing thanks to you. Francesca for constantly inviting me for coffee breaks and the house parties, and listening to me during my several cycles of depression. You’ve been my support system in all this madness. Graham for the cover letter samples and our great conversations. Serap for being the amazing person you are, our Wednesday walks, and your constant support in all these years. Jonathan for being such a great listener and treating me all those dinners. Ahmet for helping me see the bright side of the things. Nesibe, Erhan, Mehmet, and Aysu for helping me keep physically active. Doğan Can for being my climbing partner on Fridays. Duygu, Betül, Begüm, Canan, Tuçe, and Marc, thanks for putting up with me despite the iii strange level of communication in the last few years. Yıldıray Lise and Mustafa, thank you for all the postcards. My forever encouraging and always enthusiastic grandma, thank you for always putting your trust in me and being there since I was a little kid. Special thanks go to my mom, for being the biggest support in my life since day one, for teaching me how to be strong whatever bad happens, for all your love. My late father, an agricultural engineer, even though you left us so early, you and the genetic traits I inherited from you may be why I am a biologist today. Thank you. My little sister Bestenur, thank you for always being there for me. My little brother Eşref, thank you for our humorous conversations. Seda teyze for the food packages you sent and the laptop you gave me without which I could not write my thesis. Thank you for being the perfect aunt since 1990. Ümit abi, for your invaluable support and our comforting phone calls. My dear cousin Bahar, for all the insect questions you have been asking me, and wanting to study insects. Gizem abla, you may be the only one in the family who understands me truly – and scientifically for that matter. Thank you. Meryem teyze, for your moral support whenever I needed and for all the food. Zhewei for sticking with me all these years, for the letters, chocolates, motivational speeches, ideas. For making me remember whenever I am depressed that life is worth living, that there are many mountains to climb, and places to see. Thank you. iv TABLE OF CONTENTS SUMMARY __________ ........................................................................................... ix List of Figures ............................................................................................................. xi List of Tables ............................................................................................................. xii CHAPTER 1 __________General Introduction ......................................................... 13 1.1 A new voyage of biodiversity discovery .............................................................. 13 1.2 Biodiversity assessment and biomonitoring ......................................................... 15 1.3 Invertebrates and their use in biomonitoring research .......................................... 18 1.4 Invertebrates and their use in conservation research ............................................ 18 1.5 Chironomidae (Diptera) as indicator taxon in bioassessment............................... 19 1.6 The need for high-throughput (cheaper) processing of specimens ....................... 21 1.7 Aims and outline of the thesis ............................................................................... 25 CHAPTER 2 _________Dissecting the causes of a nuisance outbreak of chironomid midges with NGS barcodes ......................................................................................... 27 2.1 Abstract ................................................................................................................. 27 2.2 Introduction ........................................................................................................... 29 2.3 Materials and Methods .......................................................................................... 31 2.3.1 Sampling sites and protocols .................................................................... 31 2.3.2 DirectPCR and high-throughput sequencing ........................................... 32 2.3.4 Bioinformatics .......................................................................................... 33 2.3.5 MOTU delimitation and morphological identifications ........................... 33 2.3.6 Statistical analyses ................................................................................... 34 2.4 Results ................................................................................................................... 36 2.4.1 Sequence data analysis ............................................................................. 36 2.4.2 Structure of chironomid diversity ............................................................. 36 v 2.4.3 Adult community at the edge and center sites .......................................... 41 2.4.4 Spatial variation in MOTU richness and environmental parameters ...... 46 2.4.5 Chironomid community at different sampling intervals ........................... 48 2.4.6 Midge community at different life stage and sampling methods .............. 50 2.5 Discussion ............................................................................................................. 51 2.5.1 Dissecting a mass swarming event using NGS barcoding ....................... 51 2.5.2 Rapid bioassessment: How often should midges be sampled? ................. 53 2.5.3 Rapid bioassessment: Which life stage should be sampled? .................... 55 2.6 Conclusion ............................................................................................................ 57 CHAPTER 3 ________ Towards quick and cheap barcoding in the field: A specimen- based MinION barcode pipeline ................................................................................. 58