Open Research Online The Open University’s repository of research publications and other research outputs Integrated Study Of Dinoflagellate Diversity In The Gulf of Naples Thesis How to cite: Mordret, Solenn (2018). Integrated Study Of Dinoflagellate Diversity In The Gulf of Naples. PhD thesis The Open University. For guidance on citations see FAQs. c 2017 The Author https://creativecommons.org/licenses/by-nc-nd/4.0/ Version: Version of Record Link(s) to article on publisher’s website: http://dx.doi.org/doi:10.21954/ou.ro.0000d900 Copyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copyright owners. For more information on Open Research Online’s data policy on reuse of materials please consult the policies page. oro.open.ac.uk The Open University, London Stazione Zoologica Anton Dohrn, Naples (ARC center) Integrated study of dinoflagellate diversity in the Gulf of Naples Solenn MORDRET Thesis submitted for the degree of Doctor in Philosophy (PhD) in Life and Biomolecular Sciences December 2017 SOLENN MORDRET Integrated study of dinoflagellate diversity in the Gulf of Naples Abstract Dinoflagellates are a diverse lineage of protists known as an essential component of marine planktonic communities. To study their seasonal diversity at the LTER‐MC station in the Gulf of Naples, I used high throughput sequencing (HTS) metabarcoding of the V4 region in the 18S rDNA. To taxonomically identify the metabarcode sequences, I established a database, called DinoREF, of taxonomically verified and nomenclaturally updated 18S reference barcodes with associated metadata. The reference sequences were organised into Superclades based on phylogenetics and higher taxonomic treatment. DinoREF contains 1,671 sequences for 422 species and covering 22% of the described species. The database revealed that the V4 region alone cannot discriminate between some morphologically and genetically distinct species or genera. Moreover, many species and genera were collapsed together when clustered into 98% similarity OTUs. For the metabarcoding, dinoflagellate HTS V4 reads were gathered from 48 environmental DNA samples taken over three years at LTER‐MC. Results of a‐taxonomic cluster analysis showed three principal seasonal clusters, one with winter samples (16% of reads), one with mainly spring‐summer samples (62%) and one with late summer‐autumn samples (22%). Sorting reads into ribotypes and assigning them with DinoREF to taxa showed that reads belonging to the Gyrodinium, Gymnodiniales and Gonyaulacales Superclades were the most abundant. Winter samples were characterised by specific taxa thriving only in cold season. Results revealed 85 new records and detected 26 potentially toxic species for the Gulf of Naples. Many dinoflagellate genera such as Tripos are underrepresented in DinoREF because many species cannot be cultured. I applied single cell imaging, PCR and sequencing to gather 18S and 28S for 22 Tripos species. Using 18S V4 region as barcode I assessed the seasonal abundances of these species. Some were common all year whereas others showed distinct seasonality, mainly occurring in winter. iii ii SOLENN MORDRET Acknowledgements Firstly, I would like to thank to the Stazione Zoologica Anton Dohrn and the Open University for these three years of research. There are a number of people who have guided me and provided me with support to complete this thesis, and without their help this project would not have been possible. I would like to thank my director of studies, Dr. Diana Sarno, for giving me this opportunity, and for her patience and advice throughout this project. Additional thanks to my internal supervisor, Dr. Wiebe Kooistra, who was particularly helpful during the write-up, whilst also providing essential assistance throughout my three years at Stazione Zoologica. I am also grateful to my external supervisor, Dr. Daniel Vaulot, for his valuable comments, suggestions and corrections. Likewise, to Dr. Marina Montresor for our conversations on the wonderful world of dinoflagellates, and for all her helpful comments on my manuscript. Particular thanks are extended to Dr. Adriana Zingone for providing the HTS data, which was supported by the Italian project MIUR-FIRB Biodiversitalia (RBAP10A2T4). Thanks also to the MEDA unit for providing me with LTER MareChiara environmental parameters metadata. Also to LifeWatch- Italy (Bari) for sequencing eDNA. A big thank you to the MareChiara team for providing plankton samples every week, without which this work would not have been possible. In particular I would like to thank the technicians who helped me throughout this project. For the plankton samples and culture, I thank Ferdinando Tramontano, Carmen Minucci and Alessandro Manfredonia. In the Molecular Biology Service, I am grateful to Ellio Biffali, Elvira Mauriello and Raimondo Pannone, and in the electron microscopy group, Annarita Graziano, Franco Iamunno and Giovanna Benvenuto. I would also like to thank Thomas Mollica for being my extra pair of hands and for helping me conduct the experimental work on single cell extraction, amplification and sequencing. Enormous thanks to my friend and colleague Dr. Roberta Piredda for teaching me the bioinformatic techniques required to process my data. I cannot express my gratitude for the daily support and patience that she gave me. Also to my friends and labmates Dr. Chetan Gaonkar and Dr. Katerina Pargana for just being there for me. Many thanks to all my friends at Stazione Zoologica Arianna, Angela, Daniele, Luigi, Isabella, Greta, Laura V., Laura E., MariPa, Eleonora, Isabella, Ennio, Davide, Gauri, Claudia, Christian, Filomena, Ylenia, Ivan and Filip… and also to my friends outside Stazione Zoologica, Elijah, Hristiana, Mariano, Felix, Celia, Michelle, Dina, Neri, Giulia, Giaccio, Elena, Jovana, Danila, Josh, Lucía, Swaraj, Tara, Mark, Paolo and many more I met along the way. Finally, thank you to my family and to Chris for all their love and support. Cette thèse est dédiée à mes parents, Dominique Abguillerm et Guy Mordret, pour leur soutien immense, leur amour et leur patience. v iv SOLENN MORDRET Table of contents Integrated study of dinoflagellate diversity in the Gulf of Naples: Abstract ........................................................................................................... ii Acknowledgements ......................................................................................... iii Table of contents .............................................................................................. v List of Abbreviations ........................................................................................ xi List of Tables .................................................................................................. xiii List of Figures .................................................................................................. xv CHAPTER I: General introduction 1.1. Introduction ............................................................................................... 2 1.2. Cell Biology ................................................................................................ 5 1.2.a. General features of dinoflagellate cells ................................................................ 5 External morphology and morphological diversity ................................................................ 5 Inside a dinoflagellate cell ...................................................................................................... 7 Photosynthetic dinoflagellates special features .................................................................... 9 1.2.b. Special adaptations and complex organelles ....................................................... 11 From eyespots to complex eye‐like structure ....................................................................... 11 Feeding tools ....................................................................................................................... 11 Extrusomes: defensive and prey capture adaptations .......................................................... 12 Scintillons and Bioluminescence .......................................................................................... 13 1.3. Life histories ..............................................................................................14 1.4. Ecology..................................................................................................... 17 1.4.a. Diversity of habitats and ecological adaptations .................................................. 17 1.4.b. Biogeographic distribution ............................................................................... 19 1.4.c. Nutritional strategies ........................................................................................ 21 1.4.d. Symbiotic associations ...................................................................................... 22 Mutualistic interactions....................................................................................................... 22 Parasitic interactions ........................................................................................................... 23 1.4.e. Secondary metabolites .................................................................................... 24 Production of toxins ............................................................................................................ 24 Production of DMSO ..........................................................................................................