School of Doctoral Studies in Biological Sciences UNIVERSITY OF SOUTH BOHEMIA, FACULTY OF SCIENCE Diversity and biogeography of diplonemid and kinetoplastid protists in global marine plankton Ph.D. Thesis M.Sc. Olga Flegontova Supervisor: Aleš Horák, Ph.D. Biology Centre CAS, Institute of Parasitology České Budějovice, 2017 This thesis should be cited as: Flegontova, O, 2017: Diversity and biogeography of diplonemid and kinetoplastid protists in global marine plankton. Ph.D. Thesis. University of South Bohemia, Faculty of Science, School of Doctoral Studies in Biological Sciences, České Budějovice, Czech Republic, 121 pp. Annotation The PhD thesis is composed of three published papers, one manuscript submitted for publication and one manuscript in preparation. The main research goal was investigating the diversity of marine planktonic protists using the metabrcoding approach. The worldwide dataset of the Tara Oceans project including small subunit ribosomal DNA metabarcodes (the V9 region) was used. The first paper investigated general patterns of protist abundance and diversity in a global set of samples from the photic zone of the ocean. Diplonemids, a sub- group of Euglenozoa, emerged as one of the most diverse lineages. The second paper was a mini-review highlighting this unexpected result on diplonemids. The third paper provided a detailed characteristic of the diversity, abundance and community structure of diplonemids and revealed them as the most species-rich eukaryotic clade in the plankton. The submitted manuscript was focused on the same topics related to planktonic kinetoplastids, the sister- group of diplonemids. The manuscript in preparation will describe the creation of a curated reference database of excavate 18S rDNA sequences, including those of diplonemids and kinetoplastids, that will be indispensable for analyses of environmental high-throughput metabarcoding data. Our studies provide essentially the first global survey for diplonemid and kinetoplastids protists, which were overlooked previously in marine biodiversity and ecology studies. Declaration [in Czech] Prohlašuji, že svoji disertační práci jsem vypracovala samostatně pouze s použitím pramenů a literatury uvedených v seznamu citované literatury. Prohlašuji, že v souladu s § 47b zákona č. 111/1998 Sb. v platném znění souhlasím se zveřejněním své disertační práce, a to v nezkrácené podobě elektronickou cestou ve veřejně přístupné části databáze STAG provozované Jihočeskou univerzitou v Českých Budějovicích na jejích internetových stránkách, a to se zachováním mého autorského práva k odevzdanému textu této kvalifikační práce. Souhlasím dále s tím, aby toutéž elektronickou cestou byly v souladu s uvedeným ustanovením zákona č. 111/1998 Sb. zveřejněny posudky školitele a oponentů práce i záznam o průběhu a výsledku obhajoby kvalifikační práce. Rovněž souhlasím s porovnáním textu mé kvalifikační práce s databází kvalifikačních prací Theses.cz provozovanou Národním registrem vysokoškolských kvalifikačních prací a systémem na odhalování plagiátů. České Budějovice, 14.08.2017 ............................................... Olga Flegontova This thesis originated from a partnership of Faculty of Science, University of South Bohemia, and Institute of Parasitology, Biology Centre of the CAS, supporting doctoral studies in the Molecular and Cell Biology and Genetics study programme. Financial support This work was supported by: ERC CZ, grant LL1205 Internal grant of the Biology Centre of the CAS, grant 320/9564 The Grant Agency of the University of South Bohemia, grant 04−088/2014/P Acknowledgements Foremost, I would like to express my sincere gratitude to my supervisor Aleš Horák for his patient guidance of my research, for his contagious optimism, kindness, easy communication, and for providing opportunities to present my research at a number of conferences. Without his help, my research and writing of this thesis would not be possible. Second, I am grateful to my husband, the best friend, colleague and co-author of some of my papers Pavel Flegontov, who motivated me to start my PhD program, supported me extensively throughout my studies, helped me a lot with organizing scientific expeditions and proofread the thesis. Third, I would like to acknowledge Julius Lukeš who gave me the opportunity to became a protistologist and join his team eight years ago, and whose great interest in diplonemids has driven my project forward. I would also like to thank all the people from Jula’s lab, both former and present lab- members, for their help in my work, for creating friendly atmosphere in the institute, and for all the fun and partying nights we have had together in the last eight years. In addition, none of the work presented in this thesis would originate without numerous collaborators who helped us to generate data and provided advice, namely, Shruti Malviya, Chris Bowler, Colomban de Vargas, and Kasia Piwosz. Last but not least, I thank my parents and close friends, who have supported me throughout my life and made my life in Ceske Budejovice happy, easy and unforgettable. List of papers and author’s contribution The thesis is based on the following papers (listed chronologically): I. de Vargas C, Audic S, Henry N, Decelle J, Mahé F, Logares R, Lara E, Berney C, Le Bescot N, Probert I, Carmichael M, Poulain J, Romac S, Colin S, Aury JM, Bittner L, Chaffron S, Dunthorn M, Engelen S, Flegontova O, Guidi L, Horák A, Jaillon O, Lima- Mendez G, Lukeš J, Malviya S, Morard R, Mulot M, Scalco E, Siano R, Vincent F, Zingone A, Dimier C, Picheral M, Searson S, Kandels-Lewis S; Tara Oceans Coordinators, Acinas SG, Bork P, Bowler C, Gorsky G, Grimsley N, Hingamp P, Iudicone D, Not F, Ogata H, Pesant S, Raes J, Sieracki ME, Speich S, Stemmann L, Sunagawa S, Weissenbach J, Wincker P, Karsenti E (2015) Eukaryotic plankton diversity in the sunlit ocean. Science. 348(6237):1261605 (IF = 37.205). Olga Flegontova participated in data analysis and interpretation. II. Lukeš J, Flegontova O, Horák A. (2015) Diplonemids. Current Biology. 25(16):R702-704 (IF = 8.851). Olga Flegontova prepared figures and edited the manuscript. III. Flegontova O*, Flegontov P*, Malviya S*, Audic S, Wincker P, de Vargas C, Bowler C, Lukeš J, Horák A (2016) Extreme diversity of diplonemid eukaryotes in the ocean. Current Biology. 26(22):3060-3065 (IF = 8.851). Olga Flegontova participated in study design, analyzed and interpreted the data and contributed to writing of the manuscript. IV. Flegontova O, Flegontov P, Malviya S, Poulain J, de Vargas C, Bowler C, Lukeš J, Horák A (submitted manuscript) Neobodonids are dominant kinetoplastids in the global ocean. Olga Flegontova participated in study design, analyzed and interpreted the data and contributed to writing of the manuscript. V. Flegontova O*, Karnkowska A*, Kolisko M*, Lax G*, Maritz JM*, Panek T*, Carlton JM, Cepicka I, Horak A, Keeling PJ, Lukes J, Simpson AGB, Tai V (manuscript in preparation). Excavata in EukRef, a novel curated database of small subunit rRNA gene sequences. Olga Flegontova participated in data analysis and interpretation and contributed to writing of the manuscript. * These authors contributed equally ___________________________________________________________________________ Co-author agreement Aleš Horák, the supervisor of this Ph.D. thesis and co-author of all presented papers, fully acknowledges the contribution of Olga Flegontova. …………………………………………. Aleš Horák, Ph.D. Contents 1 Introduction…………………………………………………………………………. 1 1.1 Environmental metabarcoding: a new tool for biodiversity studies……………1 1.2 Composition of Euglenozoa and their morphological and molecular traits...… 7 1.3 Phylogeny of kinetoplastids…………………………………………………. 12 1.4 Phylogeny of diplonemids………………………………………………...… 16 1.5 Environmental studies of kinetoplastids and diplonemids………….…..…… 18 2 Research objectives………………………………………………………………. 23 3 Summary of results and discussion…………………………………..…...….... 24 4 References………………………………………………………………………..... 29 5 Original publications…………………………………………………...………... 40 3.1 Paper I…………………………………………………………...…………... 40 3.2 Paper II………………………………………………………………………. 55 3.3 Paper III……………………………………………………………………… 59 3.4 Manuscript I…………………………………………………………………..78 3.5 Manuscript II………………………………………………………………...106 6 Curriculum vitae………………………………………………………...………. 118 1 Introduction 1.1 Environmental metabarcoding: a new tool for biodiversity studies High-throughput sequencing of environmental DNA isolated from whole communities has revolutionized many scientific fields, from microbial ecology to archaeology (Lozupone et al. 2012, Worden et al. 2015, del Campo et al. 2016, Pedersen et al. 2016, Seersholm et al. 2016). Studies of microbes relying on environmental DNA fall into two major types: sequencing whole community genomes or transcriptomes (termed metagenomes and metatranscriptomes) (Bragg & Tyson 2014) or sequencing short highly variable amplicons termed tags or barcodes (Taberlet et al. 2012). The former approach reveals metabolic potential of the community and is especially useful for studies focused on prokaryotes: organisms with relatively uniform morphology and behavior, but with extremely diverse metabolic capacities (Keeling & del Campo 2017). The latter approach reveals organism diversity and community composition at an unprecedented resolution, if a large number of tags is screened against a reference database allowing taxonomic assignments. Sequencing of full-length small-subunit ribosomal RNA genes has further revealed previously