University of Zagreb FACULTY OF SCIENCE DEPARTMENT OF BIOLOGY Maja Mucko MORPHOLOGY AND PHYLOGENY OF PICOEUKARYOTES AND PLANKTONIC PENNATE DIATOMS IN THE MIDDLE AND SOUTH ADRIATIC SEA DOCTORAL THESIS Zagreb, 2018 Sveučilište u Zagrebu PRIRODOSLOVNO-MATEMATIČKI FAKULTET BIOLOŠKI ODSJEK Maja Mucko MORFOLOGIJA I FILOGENIJA PIKOEUKARIOTA I PENATNIH PLANKTONSKIH DIJATOMEJA U SREDNJEM I JUŽNOM JADRANU DOKTORSKI RAD Zagreb, 2018 This doctoral dissertation was carried out as a part of the postgraduate programme at University of Zagreb, Faculty of Science, Department of Biology – Botany, under the supervision of dr.sc. Zrinka Ljubešić. The research was performed in the frame of the Bio-tracing Adriatic Water Masses (BIOTA) project, supported by the Croatian Science Foundation (project number UIP- 2013-11-6433); project leader dr.sc. Zrinka Ljubešić). The experimental part of the research was carried out in part at Ruđer Bošković Institute, Zagreb, Croatia, while part of the bioinformatic analyses was carried out at University of Arkansas, Fayeteville, USA. Acknowledgments Firstly, I would like to express my sincere gratitude to my supervisor Dr Zrinka Ljubešić for the continuous support of my Ph.D. study and related research, for her patience, guidance, motivation, and immense knowledge. She helped me in all phases of research and writing of this thesis. I could not have imagined having a better supervisor for my Ph.D. study. In addition to my supervisor, I would like to thank my thesis committee: Dr Daniela Marić Pfannkuchen, Dr Sunčica Bosak and Dr Regine Jahn, for their insightful comments and encouragement, but also for the hard questions, which motivated me to widen my research from various perspectives. My sincere thanks also goes to Dr Sandi Orlić and Dr Petra Peharec Štefanić, who enabled me access to the laboratory and research facilities without which I would not be able to examine my samples. I would also like to thank my ‘over the sea’ colleagues and co-authors Dr Teofil Nakov, Dr Elizabeth Ruck, Dr Daniel Vaulot, Dr Romain Gastineau, Dr David G Mann and Dr Rosa Trobajo for their sincere guidance and fruitful discussions. Without they precious support it would not be possible to conduct this research. I thank my fellow lab mates, current and previous, colleagues from abroad, students, interns and colleagues passing through the lab or cafeteria☺. Thank you Sunčica, Maja, Anamarija, Pero, Kora, Tonka, Mirela, Ana, Tihana, Tanja, Mišel, Dorotea, Zuzanna, Chetan, Solenn, Karoline, Mirta, Veronika, Nikola; for the stimulating discussions, for help, support and encouragement, and for all the fun we have had in the last four years. Additionally, I would like to thank the rest of co-authors on research publications involved in this thesis. I would also like to thank my girls, my best friends, who were, are, or will be on the same life journey, professionally and privately, for their help, patience, listening, encouraging, laughing, partying, crying, and experiencing all the other feelings in between… Thank you Petra, thank you Marina, thank you Sany, thank you Franka, thank you Zrinka! Priatel’ce 4ever ☺ I would like to thank my family: my parents Pero and Ljiljanka, for their irreplaceable love and support, to my brother Danijel and my sisters Martina and Petra for supporting me spiritually throughout writing this thesis and my life in general. In the end, I want to thank my husband Antonio, for his overwhelming love, support, for being my soulmate and my rock, for giving me much needed advice, for being just the opposite to my headstrong soul in unpredictable situations, for giving me higher purpose in my life, for being just my love… To Elena… University of Zagreb Doctoral thesis Faculty of Science Department of Biology MORPHOLOGY AND PHYLOGENY OF PICOEUKARYOTES AND PLANKTONIC PENNATE DIATOMS IN THE MIDDLE AND SOUTH ADRIATIC SEA MAJA MUCKO Faculty of Science, Department of Biology The smallest protists, generally called picoeukaryotes, and planktonic pennate diatoms have received limited attention in world oceans, although they are numerous and important for primary production, biogeochemical cycles and for carbon injection into the deep ocean. In this thesis, represented through seven scientific publications, cultivated planktonic pennate diatoms and picoeukaryotes were investigated with traditional methods (light and electron microscopy, morphometry) and with molecular analyses, including multi-gene phylogenies, and were identified as genera Entomoneis, Pseudo-nitzschia, Haslea and Picochlorum. Detailed investigations of genus Entomoneis resulted with description of seven new species. General picoeukaryotic diversity was addressed by eDNA amplicon sequencing V4 variable region of nuclear 18S rRNA gene. Obtained results identified 95% of the picoeukaryotic community as hetero- or mixotrophic, mostly belonging to parasitic dinoflagellates and radiolarians, while only 5% of the community was represented with photoautotrophic picoeukaryotes belonging to classes of green algae, haptophytes, stramenopiles and cryptophytes. One cultivated Picochlorum sp. strain was subjected to growth rate experiment and pigment and lipid analyses, resulting in interesting and promising data for future biotechnological studies. (52 pages, 3 figures, 232 references, original in English) Keywords: picoeukaryotes, planktonic pennate diatoms, morphology, phylogeny Supervisor: Dr Zrinka Ljubešić, Associated Professor Reviewers: Dr Daniela Marić Pfannkuchen, Scientific collaborator Dr Sunčica Bosak, Assistant Professor Dr Regine Jahn, Scientific advisor Sveučilište u Zagrebu Doktorski rad Prirodoslovno-matematički fakultet Biološki odsjek MORFOLOGIJA I FILOGENIJA PIKOEUKARIOTA I PENATNIH PLANKTONSKIH DIJATOMEJA U SREDNJEM I JUŽNOM JADRANU MAJA MUCKO Prirodoslovno-matematički fakultet, Biološki odsjek Najmanji protisti, poznati kao pikoeukarioti i penatne planktonske diatomeje često su zanemareni u istraživanjima svjetskih oceana, iako predstavljaju brojnu i važnu komponentu za primarne proizvodnje, biogeokemijskih ciklusa i značajni su za protok ugljika u duboki ocean. U sklopu ove disertacije kroz sedam znanstvenih publikacija navedene skupine organizama istraživane su tradicionalnim metodama (svjetlosna i elektronska mikroskopija, morfometrija) i molekularnim analizama koje uključuju filogenetske anlize sa više različitih genskih markera te su identificirane kao rodovi Entomoneis, Pseudo-nitzschia, Haslea i Picochlorum. Detaljna analiza roda Entomoneis rezultirala je opisom sedam novih vrsta za znanost. Bioraznolikost ukupne pikoeukariotske zajednice u južnom Jadranu odredila se sekvenciranjem amplikona okolišne DNA i to prema varijabilnoj V4 regiji nuklearnog 18S rRNA gena. Dobiveni rezultati identificirali su 95% pikoeukariotske zajednice kao hetero- ili miksotrofne organizme, većim dijelom pripadajući parazitskim dinoflagelatima i radiolarijama, dok je samo 5% zajednice zastupljeno fotoautotrofnim pikoeukariotima koji pripadaju razredima zelenih algi, haptofita, stramenopila i kriptofita. Jedan kultivirani soj alge Picochlorum sp. istražen je kroz eksperiment određivanja brzine rasta te analizama pigmenta i lipida, što je rezultiralo zanimljivim i obećavajućim rezultatima koji mogu poslužiti kao okosnica za buduće biotehnološke studije. (52 stranice, 3 slike, 232 literaturna navoda, jezik izvornika: engleski) Ključne riječi: pikoeukarioti, penatne planktonske dijatomeje, morfologija, filogenija Mentor: Izv. prof. dr. sc. Zrinka Ljubešić Ocjenjivači: Dr. sc. Daniela Marić Pfannkuchen, znanstveni suradnik Doc. dr. sc. Sunčica Bosak Dr. sc. Regine Jahn, znanstveni savjetnik Table of content LIST OF PUBLICATIONS .............................................................................................................. I THESIS SUMMARY ........................................................................................................................II PROŠIRENI SAŽETAK.................................................................................................................. V INTRODUCTION ........................................................................................................................ VIII The unseen diversity of marine microbial protists ............................................................................1 Picoeukaryotes: significance and diversity ......................................................................................6 Diatoms: significance and diversity .................................................................................................8 Species concepts in protistology .................................................................................................... 12 Adriatic Sea: dynamic and oligotrophic ecosystem and a natural laboratory ................................. 16 THESIS OUTLINE ......................................................................................................................... 19 INDIVIDUAL PUBLICATIONS ....................................................................................................IX Publication I ..................................................................................................................................... X Publication II ...................................................................................................................................XI Publication III .................................................................................................................................XII