Assessing Soil Micro-Eukaryotic Diversity Using High-Throughput Amplicons Sequencing: Spatial Patterns from Local to Global Scal
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Faculty of Science Institute of Biology Laboratory of Soil Biodiversity Assessing soil micro-eukaryotic diversity using high-throughput amplicons sequencing: spatial patterns from local to global scales and response to ecosystem perturbation. Christophe Victor William Seppey For the degree of: Doctor in Science in Biology Thesis committee: Prof. Edward A.D. Mitchell (Supervisor) University of Neuchâtel (Switzerland) Dr. Enrique Lara (Co-supervisor) University of Neuchâtel (Switzerland) Dr. David Bass (Jury) Natural History Museum of London (United Kingdom) Centre for environment Fisheries & Aquaculture Science (United Kingdom) Dr. Micah Dunthorn (Jury) University of Kaiserslautern (Germany) Dr. Stefan Geisen (Jury) Netherlands Institute of Ecology (Netherlands) Wageningen University (Netherlands) July 4, 2017 Faculté des Sciences Secrétariat-décanat de Faculté Rue Emile-Argand 11 2000 Neuchâtel ± Suisse Tél : + 41 (0)32 718 21 00 E-mail : [email protected] IMPRIMATUR POUR THESE DE DOCTORAT La Faculté des sciences de l'Université de Neuchâtel autorise l'impression de la présente thèse soutenue par Monsieur Christophe SEPPEY Titre: —Assessing soil micro-eukaryotic diversity using high-throughput amplicons sequencing: spatial patterns from local to global scales and response to ecosystem perturbation“ sur le rapport des membres du jury composé comme suit: x Prof. Edward Mitchell, directeur de thèse, Université de Neuchâtel, Suisse x Dr Enrique Lara, co-directeur de thèse, Université de Neuchâtel, Suisse x Dr Micah Dunthorn, University of Kaiserslautern, Allemagne x Dr David Bass, Natural History Museum, Londres, UK x Dr Stefan Geisen, Netherlands Institute of Ecology, Wageningen, NL Neuchâtel, le 3 juillet 2017 Le Doyen, Prof. R. Bshary Imprimatur pour thèse de doctorat www.unine.ch/sciences Choose a job you love, and you will never have to work a day in your life. Confucius Résumé Les micro-eucaryotes exhibent une immense diversité qui remplie plusieurs fonctions essentielles dans les écosystèmes colonisés. Ces micro-organismes sont impliqués dans tout les niveaux trophique microbiens, interagissent entre eux ainsi qu’avec d’autre groupes d’organismes tel les procaryotes ou les macro-organismes, et influencent les cycles d’éléments comme ceux du carbone ou de l’azote. Les diversité et écologie des micro-eucaryotes sont étudiées à partir de la morphologie de ces organismes et de plus en plus avec des méthodes moléculaire devenant plus abordable que jamais. Le séquençage haut débit de fragments d’ADN donnant une information taxonomique prise directement de l’environnement est maintenant le standard pour établir les communautés microbiennes et pratiquement saturer la diversité microbienne. Cette thèse profite des avancées dans cette technique pour étudier l’écologie des micro- eucaryotes des sols, organismes qui représentent la base de la plupart des écosystèmes terrestre et sont impliqué dans de critique questions écologique comme les changement climatique ou l’approvisionnement alimentaire. Les cinq chapitres suivent des communautés contraintes par différent niveaux de stress ou perturbation et distribuées autant sur de petite surfaces que sur le globe. Des analyses écologique classique et innovante sont utilisées dans ce travail pour couvrir des questions à propos de bioindication, fonctions, niveaux trophique, distribution spatiale et diversité de ce groupe de micro-organismes peu connu à l’immense diversité. Mots clefs: Micro-eucaryotes . Sols . ADN environnementale . Séquençage haut débit Illumina . Metabarcoding . Petite sous unité du gène de l’ARN ribosomique Summary Micro-eukaryotes exhibit a huge diversity which fulfils many essential functions in the colonized ecosystems. These micro-organisms are involved in every level of microbial trophic networks. They interact with each other and with other biota like prokaryotes or macro-organisms, and influence element cycles like the carbon or nitrogen cycle. The diversity and ecology of micro-eukaryotes are studied based on morphological analyses and more and more with molecular methods which are increasingly affordable. High-throughput sequencing of taxonomically informative DNA fragments taken directly from the environment is now the golden standard to assess microbial communities and virtually saturate the microbial diversity. This thesis takes advantage of the advances in this technique to study the ecology of micro-eukaryotes in soils, which represent the basis of most terrestrial ecosystems and are involved in critical ecological issues like climate changes or food supply. The five chapters follow communities constrained by different levels of stress or perturbation and distributed from very limited areas to global ecosystems. Classical and innovative ecological analyses are used in this work to cover questions about the bioindication, functions, trophic networks, spacial distributions and diversity of these hyper-diverse and largely unknown micro-organisms. Keywords: Micro-eukaryotes . Soils . Environmental DNA . Illumina high-throughput sequencing . Metabarcoding . SSU rRNA gene Contents Résumé ............................................. ........ v Summary............................................ ......... v Contents vii Introduction 1 1 Micro-eukaryoticcommunities. ................ 1 2 Soils ............................................. ....... 4 3 Thesisaims ........................................ ........ 7 1 Euglyphids response in cadaver impacted soil 9 1.1 Introduction .................................... ............ 9 1.2 Materialsandmethods ............................. ............. 10 1.3 Results......................................... .......... 12 1.4 Discussion ...................................... ........... 13 2 Co-occurrence network of micro-eukaryotes in peatlands 19 2.1 Introduction .................................... ............ 19 2.2 Materialsandmethods ............................. ............. 20 2.3 Resultsanddiscussion. ............... 22 3 Spatial modelling of soil protist diversity in the Swiss western Alps 33 3.1 Introduction .................................... ............ 34 3.2 Materialsandmethods ............................. ............. 34 3.3 Results......................................... .......... 35 3.4 Discussion ...................................... ........... 37 4 Algivory: an alternative pathway for the soil microbial loop 59 4.1 Introduction .................................... ............ 59 4.2 Materialsandmethods ............................. ............. 60 4.3 Results......................................... .......... 62 4.4 Discussion ...................................... ........... 65 5 Comparison between soils and planktonic micro-eukaryotic communities 81 5.1 Introduction .................................... ............ 82 5.2 Materialsandmethods ............................. ............. 83 5.3 Resultsanddiscussion. ............... 84 Discussion and conclusion 97 1 Background ........................................ ........ 97 2 Achievements ...................................... ......... 98 3 Perspectives ...................................... .......... 99 Bibliography 101 Curriculum Vitae 111 Remerciements / Acknowledgements 115 vii Introduction Christophe V.W. Seppey1∗ 1Laboratory of Soil Biodiversity, University of Neuchâtel, Rue Émile-Argand 11, 2000 Neuchâtel, Switzerland ∗Corresponding author: [email protected] Abstract: Micro-eukaryotes exhibit a huge diversity which fulfils many essential functions in the colonized ecosystems. These micro-organisms are involved in every level of microbial trophic networks. They interact with each other and with other biota like prokaryotes or macro-organisms, and influence element cycles like the carbon or nitrogen cycle. The diversity and ecology of micro-eukaryotes are studied based on morphological analyses and more and more with molecular methods which are increasingly affordable. High-throughput sequencing of taxonomically informative DNA fragments taken directly from the environment is now the golden standard to assess microbial communities and virtually saturate the microbial diversity. This thesis takes advantage of the advances in this technique to study the ecology of micro-eukaryotes in soils, which represent the basis of most terrestrial ecosystems and are involved in critical ecological issues like climate changes or food supply. The five chapters follow communities constrained by different levels of stress or perturbation and distributed from very limited areas to global ecosystems. Classical and innovative ecological analyses are used in this work to cover questions about the bioindication, functions, trophic networks, spacial distributions and diversity of these hyper-diverse and largely unknown micro-organisms. Keywords: Micro-eukaryotes . Soils . Environmental DNA . Illumina high-throughput sequencing . Metabarcoding . SSU rRNA gene 1 Micro-eukaryotic communities Mulot et al., 2017). Fungi form the second main group of micro- eukaryotes. Indeed, although many Basidiomycota and What is a micro-eukaryote? Ascomycota produce macroscopic carpophores, many Micro-eukaryotes, include any eukaryotic organism small species are only found in a microscopic form like yeasts enough to be invisible to the naked eye and are found in (e.g. Saccaromyces) (Masinova et al., 2017; Hittinger, all super-groups of eukaryotes (Pawlowski et al., 2012). 2013). In addition, most of their biomass - at least in soils These micro-organisms encompass